A new validated SPE-HPLC method for monitoring crocetin in

Transcription

Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 563–568
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis
journal homepage: www.elsevier.com/locate/jpba
A new validated SPE-HPLC method for monitoring crocetin in human
plasma—Application after saffron tea consumption
Dimitra G. Chryssanthi a , Fotini N. Lamari a,∗ , Constantine D. Georgakopoulos b , Paul Cordopatis a
a
b
Laboratory of Pharmacognosy & Chemistry of Natural Products, Department of Pharmacy, Greece
Department of Ophthalmology, School of Medicine, University of Patras, 26504 Rion, Greece
a r t i c l e
i n f o
Article history:
Received 20 September 2010
Received in revised form 11 February 2011
Accepted 14 February 2011
Available online 21 February 2011
Keywords:
Crocus sativus
Saffron
Crocetin
HPLC
Solid-phase extraction
Human plasma
a b s t r a c t
Saffron (stigmas of Crocus sativus L.) is a well-known spice with many attributed therapeutic uses throughout centuries. Although studies have demonstrated that crocetin and crocins from saffron have various
biological functions, issues concerning the route and way of saffron administration, the absorption and
metabolism of saffron carotenoids in humans have not been answered yet. In the present study, an
isocratic reversed-phase liquid chromatographic method was developed and validated for the determination of crocetin in plasma. Samples were pre-treated by solid phase extraction (recoveries >72%)
and were chromatographed on a Luna C-18 column (4.6 mm × 250 mm, 5 ␮m) with a mobile phase consisting of methanol–water–trifluoroacetic acid (75.0:24.5:0.5, v/v/v) at a flow rate of 1.0 mL min−1 . The
HPLC method developed resulted in sharp peaks at 10.7 (trans-crocetin) and 18.6 min (cis-crocetin),
whereas the calibration curve of total crocetin in plasma displayed a good linearity for concentrations of
0.020–20 ␮M (R2 = 0.999). Specificity, precision, accuracy and stability were studied with spiked plasma
samples and were acceptable. The developed method was applied to the determination of crocetin levels in plasma of four healthy human volunteers before and after consumption of one cup of saffron tea
(200 mg of saffron in 80 ◦ C water for 5 min). Results showed that the concentration of crocetin was high
after 2 h (1.24–3.67 ␮M) and still determined after 24 h (0.10–0.24). Interestingly, the percentage of the
cis-isomer ranges from 25 to 50%, suggesting in vivo isomerization.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Saffron, the dried stigmas (the term styles is also used by certain
botanologists [1]) of Crocus sativus flowers, is a spice important in
the Mediterranean, Indian and Chinese diet and has been continuously used in folk remedies for more than 90 diseases for over
3000 years [2]. Recent research on its biomedical properties supports its use for many of these indications, although the great
majority of the studies are conducted in vitro and in experimental
animals [3]. Its composition is also quite unique: the main constituents are hydrophilic carotenoids, the crocins, which are monoand diglycosyl-esters of crocetin and are responsible for the characteristic golden yellow color they confer.
Besides culinary uses, saffron is consumed as a herbal tea per se
or is added in other herbal infusions for its unique flavor characteristics and its attributed medicinal uses. According to the WHO
monograph issued in 2007, daily doses of up to 1.5 g of saffron are
∗ Corresponding author at: Department of Pharmacy, Laboratory of Pharmacognosy & Chemistry of Natural Products, University of Patras, 26504 Rion, Greece.
Tel.: +30 2610 969335; fax: +30 2610 993278.
E-mail address: fl[email protected] (F.N. Lamari).
0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jpba.2011.02.018
thought to be safe; at doses of 5.0 g or more, Stigma Croci may cause
serious adverse reactions and overdose (12.0–20.0 g/day) may be
fatal [3]. Intraperitoneal administration of saffron ethanolic extract
to Wistar rats at the high doses of 0.35, 0.70 and 1.05 g kg−1 body
weight for two weeks caused mild to severe hepatic and renal tissue
injuries, weight loss, significant reductions in the haemoglobin and
haematocrite levels and total red blood cell counts; intraperitoneal
median lethal dose (LD50) value of ethanolic extracts was found
to be 3.5 g kg−1 body weight [4]. Modaghegh et al. showed that
consumption of 200 and 400 mg saffron tablets by healthy humans
for 7 days is safe [5]. Administration of 200 mg saffron capsules
to men with erectile dysfunction for 10 days was safe and had a
positive effect on sexual function [6], whereas administration of
30 mg capsules to patients with mild-to-moderate Alzheimer’s disease [7] and to women with symptoms of premenstrual syndrome
[8] had a favorable outcome. A reduction in lipoprotein oxidation in
20 human subjects was also reported after administration of 50 mg
saffron in milk twice a day for 3–6 weeks [9].
However, issues concerning the route and way of saffron administration, the absorption and metabolism of saffron carotenoids
in humans have not been answered. Experiments conducted in
rodents, showed that orally administered crocins are hydrolysed
to crocetin before being incorporated into blood circulation and
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D.G. Chryssanthi et al. / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 563–568
that crocetin is likely to be metabolized to glucuronide conjugates
both in the intestinal mucosa and in the liver of mice [10,11]. The
aim of this study was the development and validation of a chromatographic analytical method for the determination of crocetin
in human plasma and application of that method to the determination of plasma crocetin levels after saffron tea consumption by
healthy volunteers. In this context, we screened saffron tea composition to investigate if tea preparation induces changes in saffron
components, i.e. carotenoid isomerization.
CA, USA). The Column Compartment (TCC-3100) was stabilized at
40 ◦ C and UV-detection was performed with a diode array detector
(DAD), Ultimate DAD-3000. Data were collected, stored and integrated on a Chromeleon v 6.80 Systems software. Separation of
analytes was performed on a Luna C-18 reversed-phase column
(250 mm × 4.6 mm I.D., 5 ␮m particle size) from Phenomenex (Torrance, CA, USA). Elution was performed with methanol–water–TFA
(75.0:24.5:0.5, v/v/v) for 30 min. The flow rate of the mobile phase
was 1 mL min−1 . Spectra were monitored by the diode array detector.
2. Materials and methods
2.5. Method validation
2.1. Reagents and chemicals
Methanol (MeOH) and acetonitrile (AcCN) of HPLC grade were
supplied from Merck KGaA (Darmstadt, Germany). Dimethylsulphoxide (DMSO), ammonia solution, hydrochloric acid and
solid reagents (ammonium acetate, sodium chloride and sodium
phosphate), of analytical grade were also purchased from
Merck. Trifluoroacetic acid (TFA) and butylated hydroxytoluene
were obtained from Sigma–Aldrich (St. Louis, USA) whereas
trans-crocetin [(2E,4E,6E,8E,10E,12E,14E)-2,6,11,15-tetramethyl2,4,6,8,10,12,14-hexadecaheptaenedioic acid] (purity >98% for the
sum of both isomers) was purchased from Extrasynthese (Genay
Cedex, France). Saffron in the form of dried styles was supplied from
Cooperative de Saffron (Krokos Kozanis, Greece). The dried plant
material was identified by Professor Gregoris Iatrou, Department
of Biology, University of Patras, Greece.
2.2. Preparation of crocetin standards
Stock solutions of crocetin were prepared in DMSO at an initial concentration of 10 mM and diluted to working concentrations
(0.005–100 ␮M) in methanol. Methanolic solutions of the analyte
were kept at −20 ◦ C in a dark place, until further use. The solutions
were stable for at least one month. Pooled plasma, obtained from
young healthy volunteers (25–35 years old), was used for the preparation of spiked plasma standards. For spiked solutions of crocetin
in plasma, 25 ␮L of crocetin was diluted in plasma to a final volume
of 500 ␮L.
2.3. Plasma sample pretreatment
During the optimization of plasma sample pretreatment, samples were submitted to solid phase extraction (SPE) procedure for
the removal of matrix interferences during the analysis. Reversed
phase Strata-X cartridges (200 mg/3 mL) consisting of a surface
modified with styrene–divinylbenzene polymer were obtained
from Phenomenex (Torrance, CA, USA). The cartridges were conditioned with methanol and equilibrated in loading buffer. In the
proposed method, 500 ␮L of plasma sample was diluted in 500 ␮L
of MeOH:CH3 COOH (25 mM) containing 10% NaCl (w/v) (10:90,
v/v), centrifuged and the supernatant was loaded onto the cartridges. Washing was performed with 1 mL of MeOH–CH3 COOH
(25 mM) (5:95, v/v). The retained crocetin was eluted with 6 mL
NH3 –MeOH–water (5:90:5, v/v/v). Finally, the eluate was evaporated to dryness in a speed Vac system (Labconco Corp., Kansas
city, MO, USA). The residues were stored at −20 ◦ C until the next
day. Dry residues were redissolved in 100 ␮L of the HPLC mobile
phase.
2.4. HPLC determination of crocetin in plasma
The chromatographic system consisted of an Ultimate 3000
Pump (Pump LPG-3400 A, Dionex Corporation, Sunnyvale, CA, USA)
with a 20 ␮L Rheodyne 8125 injector (Rheodyne, Ronhert Park,
The proposed method was validated according to the official
guidelines of FDA for bioanalytical methods [12]. Selectivity was
investigated by analysis of blank samples (plasma) from at least
6 healthy individuals. The calibration curve was obtained by plotting the sum of peak areas (y) of trans- and cis-crocetin versus their
concentration (x). Nine different concentrations (0.002, 0.02, 0.2,
0.5, 1, 2, 5, 10, 20 ␮M) were used for total crocetin. Good linearity was determined through the correlation coefficient (R2 ), which
should be better than 0.99. The LLOD was determined as the analyte concentration yielding signal with a signal-to-noise (S/N) ratio
of 3:1, whereas the LLOQ was defined as the analyte concentration yielding signal with S/N ratio 10:1. The noise was evaluated as
the largest deviation of detector signal of baseline. Intra-day accuracy and precision were estimated with five analyses on the same
day of plasma samples spiked with crocetin at three different concentration levels: 0.15, 3, 15 ␮M. The accuracy was calculated by
comparison of each concentration measured to the nominal concentration of the standard solution and was expressed as % of the
relative error values whereas precision was numerically expressed
by relative standard deviation (RSD) of values. Inter-day accuracy
and precision of the method were estimated with three analyses of
the plasma samples spiked with crocetin (0.15, 3, and 15 ␮M) ten
days later. Precision and accuracy should be less than 15% except for
the LLOQ where the values should not exceed 20%. The stability of
crocetin is determined by analysis of the low (0.15 ␮M) and the high
(15 ␮M) spiked plasma standards after three freeze (at −20 ◦ C and
−70 ◦ C) and thaw cycles, and after 24-h storage at room temperature. SPE recovery of total crocetin was calculated by comparison
of the determined concentrations obtained from analysis of spiked
plasma samples after SPE to those calculated from the analysis of
the respective concentrations of crocetin standards in methanol (5
times the concentration of spiked plasma samples).
2.6. HPLC analysis of various saffron infusions
Saffron infusion was prepared by adding 150 mL of hot water
(80 ◦ C) to 200 mg of saffron and leaving the stigmas steep for 5 min.
The infusion was centrifuged and the supernatant was used directly
for HPLC analysis. In order to compare the HPLC profile to that
of the standard aqueous methanol extract, saffron was extracted
with methanol:water (1:1, v/v) (3 mL/50 mg) for 4 h at 25 ◦ C with
continuous stirring. The extract was centrifuged, filtered through
a 0.2-␮m filter and evaporated to dryness. The residue was stored
at −20 ◦ C until further use and redissolved in methanol:water (1:1,
v/v). All procedures were performed in the absence of light.
Chromatographic analysis was performed on a Supelcosil C18
(5 ␮m, 25 cm × 4.6 mm, Sigma–Aldrich) column on a Mod.10 AKTA
instrument (Amersham Biosciences, Piscataway USA) as described
previously by minor modifications [13,14]. Elution was performed
with methanol:water (20:80, v/v) for 2 min, a gradient of methanol
(20–70%) for 50 min, with a gradient of methanol (70–100%) for
5 min and 100% methanol for 1 min with a flow rate 0.7 mL/min.
Both solvents (water and methanol) contained 1% (v/v) acetic
565
acid. Detection wavelength was 440 nm. Semi-quantification was
carried out by multiplying the peak area of each crocin with
the respective molecular coefficient absorbance value (89,000 for
trans- and 63,350 for cis-crocins) and expressed as the percentage
of each crocin in relation to the total crocin content [15].
2.7. Method application: study design and biologic material
For the preparation of saffron infusions, hot water (80 ◦ C, about
150 mL) was added to one cup containing 200 mg saffron stigmas which were allowed to steep for 5 min. Four young (25–35
years old, 3 female and 1 male, normal BMI) healthy volunteers
participated in the study and their written informed consent was
obtained. The collection of the biologic material and the procedure
followed conformed to the ethical standards of the Helsinki Declaration of 1975 and its latest revisions. The study was approved
by the local Bioethics Committee of the University of Patras and all
procedures took place in the University Hospital of Patras under
medical supervision. All volunteers drank the saffron tea at about
09:00–10:00 a.m. after overnight fasting. Blood was collected in
EDTA coated tubes immediately before (0 h), 2 and 24 h after tea
consumption. Plasma was prepared by centrifugation of blood at
1000 × g for 15 min at 4 ◦ C and then kept at −20 ◦ C till analysis (next
day).
3. Results and discussion
3.1. Method development
Initial experiments were performed with pure crocetin standards in methanol. For the determination of the mobile phase, a
number of elution systems were examined. The use of a gradient
of AcCN:water (15–75% of the organic phase in 25 min) showed
a very broad peak at 5 min. Addition of ammonium acetate at the
final percentage of 0.1% (w/v) in both solvents resulted in two broad
and tailing peaks (a main one at 13 and a minor at 20 min). Better HPLC profiles with good resolutions but still broad and tailing
peaks (main peak at 9 and minor at 20 min) were observed with
isocratic elution with MeOH–water–acetic acid (75:23:2, v/v/v),
as earlier suggested [11]. Decrease in the percentage of acetic
acid led to longer elution times. Replacing acetic acid with TFA
to the optimum mobile phase composition of MeOH–water–TFA
(75.0:24.5:0.5, v/v/v) resulted in sharp peaks at 10.7 and 18.6 min
(Fig. 1). A higher amount (>0.5%) of TFA causes broadening of the
peaks and increase in the peak area of the late-eluting substance.
Monitoring of spectra of the two peaks by the diode array detector showed that both of them share maximum double peaks at
420–460 nm, characteristic of the carotenoids; the earlier eluting
peak has another maximum at 250 nm whereas the late-eluting
peak has two maxima at 250 nm and 310–330 nm (Fig. 1). Thus, the
peak at 10.7 min corresponds to trans-crocetin and the second one
at 18.6 min to cis-crocetin.
This is the first report stressing that during HPLC analysis of
trans-crocetin (high purity standard both commercially available
and that prepared by alkaline hydrolysis of saffron extract as earlier
described [10,16,17]), a small amount (about 10%) of the cis-isomer
always appears irrespective of the matrix (i.e. in pure crocetin standards or in plasma spiked with crocetin), and is well resolved from
the trans-isomer, i.e. it elutes much later; in our optimum conditions it elutes 8 min later. The existence of the cis-isomer is probably
a result of the susceptibility of carotenoids to various factors, e.g.
light and acids. Earlier HPLC studies on determination of plasma
crocetin by Xi et al. and Asai et al. do not report the existence of the
cis-isomer [10,11].
Fig. 1. Crocetin determination. HPLC chromatogram of plasma spiked with crocetin
(upper panel, I: trans-crocetin, II: cis-crocetin) and UV–visible absorption spectra
(lower panel). Absorbance maximum of peak I appear at approximately 423 nm
whereas peak II displays additional maxima at 320 nm.
Purification and condensation of plasma samples were accomplished via SPE. All experiments were conducted with plasma
samples spiked with crocetin. Having in mind the acid–base
balance of the carboxylic groups of crocetin at different pH
values, reversed phase SPE columns were loaded with 500 ␮L
plasma diluted in water (1:1, v/v), washed with MeOH–phosphate
buffer (50 mM, pH 3.0) (20:80, v/v) (2 mL), and eluted with 6 mL
NH3 –MeOH–water (5:90:5, v/v/v). About 50% of crocetin was
recovered but nearly 30% of crocetin was lost in the washing procedure, probably because of strong interactions with plasma proteins
eluted at that step. Indeed, repetition of the procedure without
MeOH in the washing step had no difference on recovery values. Non-specific binding of crocetin to human serum albumin via
H-bonding, the stability of those complexes and the changes on
protein secondary structure have been earlier shown by Kanakis
et al. [18]. In order to disrupt those complexes, a high concentration of salt, a small percentage of methanol and low pH values
were used. In the proposed method, 500 ␮L of plasma sample
was diluted in 500 mL of MeOH–CH3 COOH (25 mM) containing
10% NaCl (w/v) (10:90, v/v). Samples were loaded on cartridges
after centrifugation, and washing was performed with 1 mL of
MeOH–CH3 COOH (25 mM) (5:95, v/v). The retained crocetin was
eluted with 6 mL NH3 –MeOH–water (5:90:5, v/v/v), as in previous tests. Those changes led to high recovery values; 94.8% at the
concentration of 0.2 ␮M, 83.3% at 0.5 ␮M and 72.3% at 20 ␮M.
3.2. Method validation
Method’s quality parameters were studied under optimum separation parameter conditions using plasma spiked with crocetin
at final concentrations ranging from 0.002 to 20 ␮M. The calibration curve of total crocetin (sum of areas of two peaks) displayed
a good linearity for concentrations of 0.020–20 ␮M (R2 = 0.999,
y = 1.131x − 0.054). In detail, calibration curves were linear from
566
Table 1
Accuracy and precision data (intra- and inter-day) for analysis of plasma samples
spiked with crocetin expressed as relative error (% RE) and relative standard deviation (% RSD), respectively.
Intra-day
Inter-day
Concentration (␮M)
Accuracy (% RE)
Precision (% RSD)
0.15
3
15
0.15
3
15
5.55
−1.25
5.72
12.73
−0.07
6.52
6.34
2.11
1.05
16.64
3.56
1.32
Table 2
Percentages (%) of crocins extracted by methanol–water (1:1, v/v) and by hot water
(infusion).
Crocins
50% aqueous
MeOH for 4 h
Trans-crocin-4
Trans-crocin-3
Cis-crocin-4
Trans-crocin-2
Cis-crocin-3
Cis-crocin-1
50.1
21.1
9.1
3.8
7.8
1.8
±
±
±
±
±
±
1.4
0.5
2.9
1.3
2.2
0.4
Infusion in 80 ◦ C
water for 5 min
59.5
21.0
5.0
1.3
8.2
1.1
±
±
±
±
±
±
0.2
0.4
0.1
0.2
0.2
0.3
Crocin-4, crocetin-di-(␤-d-gentiobiosyl) ester; crocin-3, crocetin–(␤-d-glycosyl)(␤-d-gentiobiosyl) ester; crocin-2, crocetin (␤-d-gentiobiosyl) ester; crocin-1,
crocetin (␤-d-glycosyl) ester.
0.020 to 20 ␮M for trans-crocetin (R2 = 0.999) and from 0.569 to
10 ␮M for cis-crocetin (R2 = 0.978). LLOD values are 0.002 and
0.170 ␮M for trans- and cis-crocetin, and the respective LLOQ values
are 0.020 and 0.569 ␮M.
The selectivity of the method was determined by analysis of
blank plasma samples of six different healthy individuals. HPLC
analysis showed no endogenous peaks at the retention times of
the crocetin isomers (Fig. 2A).
The intra and inter-day accuracy and precision for the total
amount of crocetin were less than 15% and at the LLOQ less than 20%
(Table 1). Results are presented as sum of all isomers. These values
show that the proposed methodology is reproducible and suitable
for the quantitative determination of crocetin in human plasma
samples. Peak asymmetry and theoretical plates for trans-crocetin
are 1.249 ± 0.134 and 3966 ± 408, respectively, whereas for ciscrocetin the respective values are 1.126 ± 0.093 and 4327 ± 437.
The short-term (up to 24 h in room temperature) and
freeze–thaw (after three cycles) stability was also studied by comparing the mean back-calculated concentrations of treated samples
to those of freshly prepared ones. Low-concentration samples
(0.15 ␮M) were not stable at room temperature for 24 h and after
three freeze (at −20 ◦ C)–thaw cycles since differences were around
30 and 48%, respectively. High concentration samples (15 ␮M) were
stable in all conditions (differences < ± 15%).
3.3. Analysis of saffron infusions
Saffron infusions were analyzed by HPLC and the fingerprint obtained is in complete agreement with previous reports
[14,15,19]. Ten peaks could be identified: two absorbing at 250 nm
(picrocrocin and HTCC) and eight absorbing at 440 nm (transcrocin-4, trans-crocin-3, trans-crocin-2 , cis-crocin-5, cis-crocin4, trans-crocin-2, cis-crocin-3, cis-crocin-2). Semi-quantification
showed that saffron tea preparation does not induce crocin isomerization and hydrolysis (Table 2). Indeed, in saffron infusions,
the only noteworthy changes concern trans- and cis-crocin-4:
content of trans-crocin-4 is higher and of cis-crocin-4 is lower.
The lower percentage of trans-crocin-4 in aqueous methanolic
extracts might be explained by the higher extractability of less
polar crocins in the presence of methanol; the low-degree forma-
Fig. 2. Plasma analysis before and after saffron tea consumption. HPLC chromatograms of human plasma before saffron administration (A) after 2 h (B) and
after 24 h (C). I: trans-crocetin, II: cis-crocetin.
tion of methyl esters in the presence of methanol has been put
forward [20] although Kanakis et al. showed the requirement of
non-aqueous conditions for the preparation of dimethylcrocetin
[18].
Table 3
Concentration (␮M) of total crocetin determined by SPE-HPLC-DAD analysis in
human plasma samples and % percentage of cis- isomers.
Sample
1
2
3
4
a
Total crocetin (␮M)
Cis-crocetin/total
crocetin (%)
2h
24 h
2h
24 h
3.67
2.00
1.24
1.27
0.12
0.10
0.24
0.13
49.4
24.1
27.5
24.5
NDa
NDa
NDa
NDa
ND, cis-crocetin was not detected.
3.4. Application to determination of plasma crocetin after saffron
tea consumption
The developed SPE-HPLC analytical methodology was applied
for the quantification of crocetin in plasma of healthy humans.
Plasma was taken before (0 h) and after (2 and 24 h) saffron tea
consumption. The results show that, after consumption of a cup of
tea from 200 mg saffron, crocetin is detected in blood stream and
trace amounts are found even after 24 h (Fig. 2, Table 3).
This is the first report showing crocetin concentrations in human
plasma after saffron consumption. These results confirm previous
studies performed in rodents, which showed that orally administered crocetin is quickly absorbed into blood plasma whereas
crocins are hydrolysed to crocetin in the gastrointestinal tract
before entering blood circulation [10,11]. Asai et al. observed a peak
in crocetin concentration at 30 min after administration of a mixed
micelle solution (0.2 mL) containing crocetin or crocins (40 nmol
each) [10] whereas Xi et al. showed that plasma concentrations of
crocetin in rats began to decline after 1 h [11]. In the study of Asai
et al., crocetin glucuronides are also identified probably indicating
the partial metabolism of crocetin to the glucuronide conjugates in
the intestinal mucosa, in the liver, or in both [10]. However, in our
experiments, spectra monitoring of early eluting peaks does not
reveal the existence of glucuronides in the chromatograms. This
does not contradict earlier findings since glucuronides might be lost
during SPE. Treatment with glucuronidase before SPE as suggested
by Asai et al. will aid future pharmacokinetic studies.
Both isomers were detected in blood plasma 2 h after saffron
tea drinking, whereas the cis-isomer was not detected after 24 h
because it was below LLOD. In three out of four individuals, the
cis-isomer accounted for about 25% of total crocetin, whereas in
the other one it accounts for nearly 50%. The higher determined
percentages of the cis-isomer in blood samples after saffron tea
consumption compared to the usual 10% we observed in pure
trans-crocetin standards and spiked plasma samples may be the
combined result of in vivo isomerization and hydrolysis of ingested
cis-crocins (about 15%) present in saffron infusion in the gastrointestinal tract. Various isomers of lycopene (mainly cis- and trans-)
are also found in blood samples, with the ratio of the cis-isomer
reaching the 60–80% of total lycopene [21] even though lycopene
in foods occurs mainly in the all-trans form. The mechanisms underlying the formation, interconversion or biological roles of various
crocetin isomers remain enigmatic.
4. Concluding remarks
An analytical SPE-HPLC method was developed for total crocetin
determination in human plasma, which displays good selectivity, linearity, sensitivity, accuracy and precision. The method was
applied to the determination of crocetin in blood plasma of healthy
human individuals before and after saffron tea consumption. Crocetin was not detectable before tea drinking, but its concentration
was high after 2 h (1.24–3.67 ␮M) and still determined after 24 h
567
(0.10–0.24). This first study in humans confirms findings of previous pharmacokinetic studies in rodents showing that orally
ingested crocins are hydrolysed to crocetin before entering blood
circulation. This is the first report showing the determination of the
cis-isomer of crocetin in small percentages (10%) in pure crocetin or
spiked plasma samples due to interconversion of the trans-isomer
during analysis to a small degree. The significance of this observation is manifested in the determination of crocetin in healthy
human individuals after saffron tea consumption; percentages of
the cis-isomer range from 25 to 50% suggesting in vivo isomerization. At last, we showed that saffron tea preparation does not
induce changes in saffron composition concerning the presence of
individual crocins. This HPLC method enables the determination
of crocetin concentrations in humans and the knowledge of actual
concentrations and the percentages of the trans- and cis-isomers
after saffron consumption will aid future nutritional or pharmacokinetic studies.
Acknowledgements
The authors acknowledge with thanks the Research Committee of the University of Patras for financial support under the “K.
Karatheodoris” Grant No. C178 and Professor Gregoris Iatrou for
his kind assistance.
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[11] L. Xi, Z. Qian, P. Du, J. Fu, Pharmacokinetic properties of crocin (crocetin digentiobiose ester) following oral administration in rats, Phytomedicine 14 (2007)
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JOURNAL OF MEDICINAL FOOD
J Med Food 14 (9) 2011, 1060–1064
# Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2010.0187
Differential Antioxidant Effects of Consuming Tea from Sideritis clandestina
subsp. peloponnesiaca on Cerebral Regions of Adult Mice
Zacharoula I. Linardaki,1 Catherine G. Vasilopoulou,1 Caterina Constantinou,1,*
Gregoris Iatrou,2 Fotini N. Lamari,3 and Marigoula Margarity1
1
Laboratory of Human and Animal Physiology, Department of Biology; 2Division of Plant Biology,
Department of Biology; 3Laboratory of Pharmacognosy & Chemistry of Natural Products,
Department of Pharmacy; University of Patras, Patras, Greece.
ABSTRACT Oxidative stress is involved in the pathophysiology of neurodegenerative diseases and aging. Many species of
the genus Sideritis (mountain tea) are widely consumed in the Mediterranean region as herbal tea. This study evaluated the
effect of supplementation of mice with herbal tea from Sideritis clandestina subsp. peloponnesiaca on the antioxidant status of
different brain regions. To select the most bioactive herbal tea, the polyphenolic content (Folin–Ciocalteu method) and the
antioxidant properties (ferric reducing antioxidant power [FRAP] and 2,2-diphenyl-1-picrylhydrazyl assays) of several taxa
and different populations of the S. clandestina infusions were measured in vitro. Male adult mice had ad libitum access to
water (control) or the herbal tea (4% w/v) for 6 weeks. At the end of the treatment period we assessed the total antioxidant
power (FRAP assay) and the levels of malondialdehyde (indicator of lipid peroxidation) and reduced glutathione in the
cerebral cortex, cerebellum, and midbrain. These biochemical measures have also been determined in liver samples used as a
comparative reference peripheral tissue. Consumption of 4% herbal tea increased the total antioxidant power of the midbrain
by 72% (P < .05); a significant (P < .05) decrease in malondialdehyde levels and increase in reduced glutathione content of the
cerebellum (78% and 27%, respectively) and midbrain (59% and 32%, respectively) were also observed. These findings
indicate that mountain tea consumption enhances the antioxidant defense of the adult rodent brain in a region-specific manner.
KEY WORDS: antioxidant capacity brain reduced glutathione lipid peroxidation mountain tea Sideritis
A moderate in vitro antioxidant action of extracts of
Sideritis species has been described elsewhere,6 but to our
knowledge no in vivo studies have examined their effect.
Mammalian brain is particularly vulnerable to oxidative
stress,8 a factor that is considered to play a pivotal role in the
pathogenesis of several age-related neurodegenerative disorders.9 However, the brain areas exhibit differential vulnerability to oxidative damage, probably because of
differences in their endogenous antioxidant defense system.10 Therefore, we sought to investigate the antioxidant
effect of consumption of tea composed of 4% S. clandestina
on the cerebral cortex, cerebellum, and midbrain of adult
normal mice. We also assessed the effects on the mice’s
livers, a reference peripheral tissue. This plant was chosen
after in vitro evaluation of the polyphenolic content and the
antioxidant properties of infusions of several taxa and different populations of S. clandestina, grown in different regions of the Peloponnese.
INTRODUCTION
S
ideritis L. (Lamiaceae), growing mainly in the
Mediterranean region, comprises approximately 150
species worldwide. The dried aerial parts of Sideritis species
are widely consumed in Greece as an herbal tea, popularly
termed mountain tea.1 It has been used extensively in folk
medicine as a calmative agent and as a treatment for inflammation, cough, and gastrointestinal disorders. Recent
research has demonstrated that Sideritis species have antiinflammatory2 and antimicrobial3 activities, which have
been attributed to their chemical composition. Indeed, many
reports indicated that these plants contain essential oils,4
polyphenols5 (especially flavonoids6), and diterpenoids,7
which are of particular pharmacologic and nutritional
interest.
*Current Address: Molecular and NMR Surgical Laboratories, Massachusetts General
Hospital and Shriners Hospital for Children, Harvard Medical School, Boston, Massachusetts, USA
MATERIALS AND METHODS
Manuscript received 21 July 2010. Revision accepted 20 October 2010.
Plant material and preparation of herb extracts
Address correspondence to: Marigoula Margarity, Laboratory of Human and Animal
Physiology, Department of Biology, University of Patras, 26500 Patras, Greece, E-mail:
[email protected]
S. clandestina is a species endemic in Greece (specifically, the Peloponnese). It is taxonomically divided into 2
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ANTIOXIDANT ACTION OF GREEK MOUNTAIN TEA IN MOUSE BRAIN
subspecies: S. clandestina subsp. clandestina prospers in the
mountains of the southern Peloponnese, and S. clandestina
subsp. peloponnesiaca prospers in the mountains of the
northern Peloponnese. S. clandestina subsp. clandestina was
collected between July and August 2008 from the Taygetos
(southern Peloponnese), Parnon (eastern Peloponnese), and
Mainalo (Central Peloponnese) mountains; S. clandestina
subsp. peloponnesiaca was collected from the Kyllini
Mountain (northern Peloponnese). Taxonomic identification
was performed at the Division of Plant Biology, Department
of Biology, University of Patras, where herbarium vouchers
are deposited. Green tea (blended and packed in Sri Lanka
under the direction of St. Dalfour, Freres & Cie, Cour
Cheverny, France), in 2-g packs, was used as standard.
Infusions were prepared with addition of 4 g of equal
amounts of leaves, flowers, and stems of Sideritis taxa to
100 mL boiling water for 5 minutes. After 5 minutes, the
solution was filtered and the filtrate volume was adjusted to
100 mL. Green tea was also prepared in the same way. For
the animal studies, the infusions were prepared daily. For
the in vitro studies, deionized distilled water was used. The
weight of the dry extract was recorded after lyophilization in
a Labconco FreezeZone 6 freeze-dry system.
Animals
Adult (3–4 months old) male Balb-c mice were kept in the
same room under a constant temperature (23–25C) with
alternating 12-hour light/dark cycles. The mice were allowed free access to food. Mice were managed according to
the Greek National Laws (Animal Act, PD 160/91).
The animals were divided into 2 groups consisting of 8
animals each. Group 1 mice served as controls and received
water ad libitum. Group 2 mice had ad libitum access to 4%
(w/v) tea infusion of S. clandestina subsp. peloponnesiaca
for 6 weeks (40 days), based on the protocol of Choudhary
and Verma11 with slight modifications. Body weight was
measured weekly in both groups and did not significantly
differ between them at the end of the treatment period.
On the completion of the 40-day treatment period, all
animals were sacrificed by light ether anesthesia. Liver and
brain were excised immediately, and cerebral cortex, cerebellum, and midbrain were separated from the whole brain.
The tissue samples were kept at -75C until use.
Preparation of tissue homogenates
Brain regions and liver samples were weighed and homogenized (10% w/v) with a glass-Teflon homogenizer in
ice-cold 1.15% KCl.12 Tissue homogenates were centrifuged for 5 minutes at 15000 g, and the supernatants were
used to determine the antioxidant activity (by ferric reducing antioxidant power [FRAP] assay) and malondialdehyde
(MDA) and reduced glutathione levels.
1061
Aldrich), in the range of 25–800 mg/L, were used as reference standards, and total phenol content was expressed as
gallic acid equivalents.
2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical–scavenging
capacity assay. The antioxidant properties of the infusions
were evaluated by the DPPH free-radical method. This assay
is based on the measurement of the reducing ability of antioxidants toward DPPH by measuring the decrease of its absorbance at 515 nm.15 The radical-scavenging activity of the
samples tested, expressed as percentage inhibition of DPPH ,
was calculated according to the following formula:16 Inhibition (%) = [(AB - AA)/AB] · 100, where AB is the absorbance
of the blank sample and AA is the absorbance of the antioxidant tested. IC50 values of the plant extracts, defined as the
concentration of the sample that causes 50% inhibition of
DPPH , were also calculated from the kinetic curve of the
percentage inhibition of DPPH toward different concentrations of each sample and were expressed in mg/mL.
FRAP assay. The antioxidant activity of the plant extracts and the tissue samples was determined by FRAP assay.12 Aqueous solutions of known FeII concentration (range,
5–1000 lM FeSO4) were used as standard solutions. The data
were expressed as lmol of FeII equivalents per mg dry extract
for the tea infusions and as nmol of FeII equivalents per mg
wet tissue weight for the brain and liver tissues (FRAP value).
Lipid peroxidation assay. Lipid peroxidation in the brain
regions and liver was assessed fluorometrically at 553 nm
(emission wavelength) and 515 nm (excitation wavelength)
by measuring the formation of MDA.17 Solutions of known
MDA concentration (0.05–10 lM) were used as standards.
Results are reported as lmol MDA per g of protein.
Reduced glutathione determination. Reduced glutathione content of the tissue samples was estimated fluorometrically according to the procedure of Mokrasch and
Teschke.18 Standard solutions of reduced glutathione were
prepared in a range of 0.5–100 lM. The tissue reduced glutathione content was expressed as lmol reduced glutathione
per g of protein.
Protein determination. Total protein concentration was
determined by the Bradford method,19 using bovine serum
albumin as standard.
Statistical analysis
The results are expressed as mean – standard error. Statistical analysis was performed with GraphPad Instat 3
software using the nonparametric Mann–Whitney test for
evaluating statistically significant differences (P < .05) between the experimental groups.
Methods
Total phenolics assay. Total polyphenols were determined spectrophotometrically by using Folin–Ciocalteu
reagent.13,14 Aqueous solutions of gallic acid (Sigma-
RESULTS AND DISCUSSION
To select the most bioactive population of all S. clandestina samples among those collected, their polyphenolic
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LINARDAKI ET AL.
content and antioxidant activity were determined in vitro.
Table 1 presents the total polyphenol content of all Sideritis
taxa infusions tested and their solid residue contents. All tea
infusions of S. clandestina taxa and populations had significantly (P < .05) lower polyphenolic composition than
Camellia sinensis leaves, which were used as the standard.
The polyphenolic content of 4 of 5 populations of S. clandestina subsp. clandestina was significantly lower than that
of S. clandestina subsp. peloponnesiaca. The in vitro antioxidant properties of the plant extracts were measured by
DPPH and FRAP assays (Table 1). The infusion of S.
clandestina subsp. peloponnesiaca displayed significantly
(P < .05) stronger in vitro antioxidant properties than the
other taxa of S. clandestina. However, the in vitro antioxidant properties of S. clandestina subsp. peloponnesiaca
were lower than those of green tea. Similarly, Triantaphyllou et al.20 showed that the water extract of China black
tea displayed higher total phenol content and antioxidant
activity than that of S. reaseri collected from a mountainous
region of northern Greece.
Another study showed that not only the species but also
many environmental factors,21 such as the soil type, sun
exposure, and rainfall, have a strong effect on polyphenol
composition. This finding explains the variation observed
among the different populations of S. clandestina subsp.
clandestina. All S. clandestina infusions had significantly
lower antioxidant activity than C. sinensis, in accordance
with their lower polyphenolic content. Tunalier et al.,5 in
their study of the antioxidant properties and phenolic composition of 27 Sideritis species grown in Turkey, indicated a
linear relationship between these 2 measures. Thus, the infusion of S. clandestina subsp. peloponnesiaca displayed
high polyphenolic content and strong antioxidant properties
in vitro, rendering it suitable for the further in vivo study.
To this end, the biological effects of 4% (w/v) herbal tea
from S. clandestina subsp. peloponnesiaca consumption for
6 weeks by adult control mice were further investigated. We
assessed the total antioxidant activity, the extent of lipid
peroxidation, and reduced glutathione content of 3 brain
regions (cerebral cortex, cerebellum, and midbrain) and a
peripheral tissue (liver) in control and treated mice. As
shown in Table 2, there is a hierarchy in antioxidant/reducing capacity and MDA levels of the examined brain
regions in control animals: cerebral cortex > cerebellum >
midbrain; the differences reached significance (P < .05) between all brain areas. However, with respect to their reduced
glutathione content, the cerebellum ranks higher than the
cerebral cortex. In accordance with our observation, Feoli
et al. also reported a similar hierarchy in total antioxidant
reactivity levels, as estimated by total antioxidant reactivity
assay.22 Other studies also reported greater lipid peroxidation in the cerebral cortex than in the cerebellum or midbrain
of young control rats23,24 and higher reduced glutathione
content in rat cerebellum than in the cortex and midbrain.10
These regional differences could be attributed to the differences in structure and physiology of the brain areas,
leading to differences in vulnerability to oxidative stress and
activity of their endogenous antioxidant defense system.
It is known that the brain regions vary with respect to their
fatty-acid composition because white matter rich in myelin
contains fewer polyunsaturated fatty acids (the major targets
of free radicals) than the grey matter.25 In this context, the
midbrain, a heavily myelinated region, is expected to be
more resistant to lipid peroxidation than cerebral cortex and
cerebellum10,26 and thus demanding the development of
lower degree of endogenous antioxidant defense. As shown
in Table 2, comparison of the above biochemical measures
among liver and the brain regions of the controls revealed
significantly higher total antioxidant capacity, significantly
lower reduced glutathione content, and a moderate level of
lipid peroxidation in liver, in accordance with findings from
a previous study in normal rats.27
Consumption of the tea significantly increased (P < .05)
the antioxidant capacity of midbrain by 72% but did not
significantly influence the antioxidant power of the cerebral
cortex, cerebellum, and liver (Table 2). Furthermore, herbal
Table 1. Total Polyphenol Content and Antioxidant Activity of Sideritis Taxa Water Extracts
Antioxidant Activity
Sideritis samples
S. clandestina subsp. clandestina (Taygetos I)
S. clandestina subsp. clandestina (Taygetos II)
S. clandestina subsp. clandestina (Parnon)
S. clandestina subsp. clandestina (Taygetos III)
S. clandestina subsp. clandestina (Mainalo)
Green tea (Camellia sinensis)
S. clandestina subsp. peloponnesiaca (Kyllini)
Solid residue content of Concentration of polyphenols DPPH assay—IC50 FRAP assay
(lg gallic acid/mg)b
(lmol FeII/mg)d
(mg/mL)c
extracts (mg/100 mL)a
302
332
404
262
500
744
330
36.8 – 0.9**
34.6 – 1.8**
30.3 – 2.2**
44.7 – 0.8
23.7 – 0.5**
165.0 – 8.8
45.6 – 1.7
7.75 – 0.55**
7.31 – 0.39**
7.58 – 1.12**
16.18 – 1.69**
8.90 – 1.55**
0.38 – 0.12
2.79 – 0.21
Values are the mean of 4 or 5 independent tea preparations – standard error.
a
Expressed as mg dry extract per 100 mL of infusion.
b
Expressed as lg gallic acid equivalent per mg of dry extract.
c
Expressed as IC50 values corresponding to the aqueous extract concentration (mg/mL) causing 50% inhibition of DPPH radical.
d
Expressed as lmol FeII per mg dry extract.
**P < .05 compared with S. clandestina subsp. peloponnesiaca.
DPPH, 2,2-diphenyl-1-picrylhydrazyl; FRAP, ferric reducing antioxidant power; IC50, 50% inhibitory concentration.
0.43 – 0.07**
0.49 – 0.12**
0.56 – 0.09**
0.27 – 0.07**
0.33 – 0.03**
4.84 – 0.33
1.19 – 0.09
12.560 – 0.683
20.612 – 1.109* ([ 27%)a
18.737 – 0.919* ([ 32%)a
6.836 – 0.159
12.472 – 0.430
16.265 – 1.085**
14.145 – 0.493
7.271 – 0.144****
Values are expressed as mean – standard error of 8 values corresponding to 8 animals.
a
Percentage change ([ increase, Y decrease) compared with control mice.
*P < .05 compared with control mice.
**P < .05 compared with cerebral cortex of the control mice.
***P < .05 compared with cerebellum of the control mice.
****P < .05 compared with each brain region of the control mice.
FRAP, ferric reducing antioxidant power; GSH, reduced glutathione; MDA, malondialdehyde.
3.273 – 0.273
1.957 – 0.203**
1.381 – 0.112**,***
1.659 – 0.136**
Cerebral cortex
Cerebellum
Midbrain
Liver
6.018 – 0.408
3.404 – 0.378**
2.176 – 0.281**,***
17.222 – 0.512****
6.047 – 0.172
3.744 – 0.389
3.743 – 0.391* ([ 72%)a
18.717 – 1.976
2.802 – 0.144
0.426 – 0.011* (Y 78%)a
0.555 – 0.047* (Y 59%)a
1.524 – 0.077
Treated mice (4% herbal tea)
Control mice
Control mice
Control mice
Mouse tissue
GSH (lmol/g protein)
MDA (lmol/g protein)
FRAP (nmol FeII/mg wet tissue)
Table 2. Effect of Herbal Tea Intake on Biochemical Measures on Oxidant/Antioxidant Status of Adult Mouse Brain and Liver
ANTIOXIDANT ACTION OF GREEK MOUNTAIN TEA IN MOUSE BRAIN
1063
tea intake led to a significant reduction in MDA levels
(Table 2) in the cerebellum (78%) and midbrain (59%), with
simultaneous significant elevation in reduced glutathione
content of these regions (Table 2) by 27% and 32%, respectively, compared with the control mice. Conversely, in
accordance with their total antioxidant power, the MDA and
reduced glutathione levels of the cerebral cortex and liver
remained unchanged after tea consumption. These findings
clearly show that consumption of the tea infusion reinforces the antioxidant defense system of the mouse brain in a
region-specific manner.
The diverse pattern of antioxidant effects of tea consumption obtained in different brain areas may be attributed
to differences in regional antioxidant defense. This region
specificity of antioxidant reinforcement in the brain by
polyphenol-rich plant extracts has been also observed in
previous studies.24, 28 Ultimately, the cerebral cortex seems
to have a stronger antioxidant defense system, resulting in
resistance to changes in its oxidant/antioxidant status. The
comparison between the brain regions and liver reveals that
the cerebral cortex and liver share the same antioxidant response pattern in response to the herbal tea consumption. As
in the cortex, the high antioxidant power of liver, shown in
the present and previous studies,27 might render this tissue
insensitive to changes in its oxidant/antioxidant status. This
finding is somewhat expected because liver executes several
vital functions of the organism.
Our findings conclusively indicate that the consumption
of the herbal tea from Sideritis species induces a significant
region-specific antioxidant reinforcement in the brain, with
the cerebellum and midbrain the most affected brain areas.
Although further studies should address the specific bioactive ingredients of Sideritis infusions, their absorption and
bioavailability, and their mechanism of action, the present
data strongly suggest that this herbal tea could be a good
source of natural antioxidants. The tea might help prevent
the age-related deficits and neurodegenerative disorders
related to oxidative damage of the specific brain regions.
ACKNOWLEDGMENT
This research was partially supported by BIOFLORA, a
network of the University of Patras.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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Available online at www.sciencedirect.com
Journal of Nutritional Biochemistry 23 (2012) 961 – 965
Wild blueberry (V. angustifolium)-enriched diets alter aortic glycosaminoglycan
profile in the spontaneously hypertensive rat☆
Aleksandra S. Kristoa , Christina J. Malavakib , Fotini N. Lamaric , Nikos K. Karamanosb , Dorothy Klimis-Zacasa,⁎
a
b
Food Science and Human Nutrition, University of Maine, Orono, ME 04469, USA
Laboratory of Biochemistry, Department of Chemistry, University of Patras, Greece
c
Department of Pharmacy, University of Patras, Patras, Greece
Received 1 December 2010; received in revised form 28 April 2011; accepted 2 May 2011
Abstract
Glycosaminoglycans (GAGs) are essential polysaccharide components of extracellular matrix and cell surface with key roles on numerous vascular wall
functions. Previous studies have documented a role of wild blueberries on the GAG profile of the Sprague–Dawley rat with a functional endothelium as well as in
the vascular tone of the spontaneously hypertensive rat (SHR) with endothelial dysfunction. In the present study, the effect of wild blueberries on the
composition and structure of aortic GAGs was examined in 20-week-old SHRs after 8 weeks on a control (C) or a wild blueberry-enriched diet (WB). Aortic tissue
GAGs were isolated following pronase digestion and anion-exchange chromatography. Treatment of the isolated populations with specific GAG-degrading lyases
and subsequent electrophoretic profiling revealed the presence of three GAG species, i.e., hyaluronic acid (HA), heparan sulfate (HS) and galactosaminoglycans
(GalAGs). A notable reduction of the total sulfated GAGs and a redistribution of the aortic GAG pattern were recorded in the WB as compared to the C group: a
25% and 10% increase in HA and HS, respectively, and an 11% decrease in GalAGs. Fine biochemical analysis of GalAGs at the level of constituent disaccharides
with high-performance capillary electrophoresis revealed a notable increase of nonsulfated (18.0% vs. 10.7%) and a decrease of disulfated disaccharides (2.2% vs.
5.3%) in the WB aorta. This is the first study to report the redistribution of GAGs at the level of composition and their fine structural characteristics with
implications for the endothelial dysfunction of the SHR.
© 2012 Elsevier Inc. All rights reserved.
Keywords: Wild blueberry; SHR; Aorta; GAGs; Heparan sulfate; Chondroitin sulfate
1. Introduction
Numerous studies suggest a protective role of dietary polyphenols
against cardiovascular disease (CVD) [1–3], due to direct antioxidant
activity [4,5] and/or signaling effects [6,7]. Wild blueberries, a rich
source of anthocyanins and other phenolics [8–10], have demonstrated beneficial properties for various vascular functions [11–13].
Glycosaminoglycans (GAGs) are linear polysaccharides composed of
repeating disaccharide units of hexosamine, D-galactosamine or Dglucosamine, and uronic acid (UA), D-glucuronic acid or L-iduronic
acid. Four major classes of GAG molecules, determined by the
Abbreviations: C, control; CVD, cardiovascular disease; CS, chondroitin
sulfate; DS, dermatan sulfate; ECM, extracellular matrix; GalAG, galactosaminoglycan; GAG, glycosaminoglycan; HA, hyaluronic acid; HPCE,
high-performance capillary electrophoresis; HS, heparan sulfate; PG,
proteoglycan; SD, Sprague–Dawley; SHR, spontaneously hypertensive
rat; UA, uronic acid; WB, wild blueberry.
☆
This work was supported by a Wild Blueberry Association of North
America grant to D.K.-Z. and a fellowship by the Greek State Scholarship
Foundation to A.S.K.
⁎ Corresponding author. Tel.: +207 581 3124.
E-mail address: [email protected] (D. Klimis-Zacas).
0955-2863/$ - see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.jnutbio.2011.05.002
hexosamine and UA type as well as sulfation pattern, are present in
the vascular wall: (a) hyaluronic acid (HA); (b) galactosaminoglycans
(GalAGs), comprised of chondroitin sulfate (CS) and dermatan sulfate
(DS); (c) heparan sulfate (HS) and (d) keratan sulfate [14].
Hyaluronic acid is the only GAG molecule that is not sulfated and
not linked to a protein core, whereas HS and GalAGs occur as part of
proteoglycan (PG) molecules that may contain more than one GAG
type, such as HS- and CS-containing syndecan, glypican or CS- and DScontaining versican, decorin and bigycan [15].
Glycosaminoglycans, as prevalent components of the extracellular
matrix (ECM) and cell surface, interact with numerous proteins and
ligands, modulating crucial biological processes such as cell growth
and development [16]. In particular, the glycocalyx associated with
the vascular endothelium is increasingly gaining appreciation as a
determinant of signaling, mechanotransduction, vascular permeability and inflammatory response and thereby the development of
endothelial dysfunction and CVD [17,18]. Additionally, the initiation
of atherosclerotic process by lipid accumulation and retention in the
ECM is largely mediated by CS/DS proteoglycans [19].
Abnormalities in GAG synthesis, such as CS elevation and
increased sulfation, observed in the vasculature of the spontaneously
hypertensive rat (SHR) are among the early events that contribute
to the vascular pathology of this animal model of endothelial
962
A.S. Kristo et al. / Journal of Nutritional Biochemistry 23 (2012) 961–965
dysfunction [20–24]. In comparison with its normotensive genetic
control, the Wistar Kyoto (WK) rat, the SHR aorta is characterized by a
thicker subendothelial matrix and a higher CS synthesis leading to a
higher concentration of total GAGs and elevated peripheral resistance
and blood pressure [22]. The increased sulfate incorporation in PG
molecules in the resistance arteries of the SHR [23] is another
hallmark of endothelial dysfunction in this animal model [24].
Although the role of bioactive dietary ingredients on ECM and GAG
metabolism has not been thoroughly examined to date, various plantderived micronutrients [25], vitamins E and C [26], flavonoids [27],
tea polyphenols [28], sorghum phenolics [29] and genistein [30] have
been reported to positively affect signaling pathways and enzymatic
processes related to ECM and GAG metabolism. Wild blueberries
consumed with diet have been documented to elicit reduced sulfation
and population redistribution of aortic GAGs in the Sprague–Dawley
(SD) rat with a normal endothelium [31]. Hence, in the present study,
the effect of a wild blueberry diet on the aortic GAG profile and fine
structural characteristics was examined in the SHR model of
endothelial dysfunction after 8 weeks of diet consumption.
2. Methods and materials
2.1. Chemicals
Pure NaCl, KCl, CaCl2, MgSO4, KH2PO4, NaHCO3 and dextrose for the physiologic salt
solution (PSS) as well as heparinase I, II and III, chondroitinase AC and ABC, standards
for HA, HS and CS/DS analysis and standard preparations of sulfated D-disaccharides
from CS/DS [Δ-di-di(2,6)S, Δ-di-di(2,4)S, Δ-di-mono6S, Δ-di-mono4S and Δ-di-nonS]
were purchased from Sigma (St. Louis, MO, USA). Chondroitinase AC I was purchased
from Seikagaku America (Ijamsville, MD, USA), the Blyscan assay kit from Accurate
Chemical (Westbury, NY, USA) and PRONASE Protease from Calbiochem (San Diego,
CA, USA).
2.2. Animal model and diets
The animal welfare and the experimental protocols conformed to the Animal Care
and Use Committee of the University of Maine standards (IACUC Protocol A2008-20062005). Twenty male SHRs (Charles River Laboratories, Wilmington, MA, USA) at the age
of 12 weeks were randomly assigned to one of two diets: control diet (C) (modified
AIN-76, n=10) and wild blueberry diet (WB) (control diet+8% wt/wt freeze-dried
wild blueberry powder substituting for dextrose, n=10), for a period of 8 weeks. The
animals were housed in the Small Animal Facility at the University of Maine in
individual stainless-steel mesh-bottomed cages in a room controlled for temperature
(22°C) and light conditions (12:12-h light:dark cycle). Tap water and food were
provided ad libitum. Food consumption was measured daily; and body weights,
weekly. The diets were prepared in our laboratory from purified diet ingredients,
stored at 4°C and used within 5 to 7 days. The diet ingredients, dextrose, egg white
solids, vitamin mix (A.O.A.C. Special Vitamin Mixture), DL-methionine, biotin, corn oil,
were purchased from Harlan Teklad (Madison, WI, USA); custom-made mineral mix
was purchased from MP Biomedicals (Solon, OH, USA). Wild blueberries, provided as a
composite by Wyman's (Cherryfield, ME, USA), were freeze-dried and powdered with
standard procedures by FutureCeuticals (Momence, IL, USA). Twenty-one different
anthocyanins were detected in the wild blueberry powder with the main anthocyanins,
malvidin 3-galactoside and peonidin 3-glucoside, representing approximately 13% of
the total anthocyanin content (1.6±0.2 mg/100 mg) [8].
4°C, samples were centrifuged for 5 min at 12,000g, supernatant was decanted, and the
precipitate was dissolved in 2× distilled H2O, 5 μl/mg aortic tissue.
2.5. Uronic acid and total sulfated GAG determination
Carbazole reaction modified by Bitter and Muir was applied on aliquots from each
sample to determine UA content [32]. Individual samples were also analyzed
colorimetrically for total sulfated GAGs (sGAGs) with Blyscan.
2.6. GAG fractionation and identification
The intra-differences in UA and total sGAGs within each diet group were not
significant. Therefore, all GAG samples obtained from each diet group (C, n=10 and
WB, n=10) were pooled due to limited tissue availability from each rat and applied to
ion-exchange chromatography on a DEAE-Sephacel column (7×1.6 cm i.d.), eluted
with 0.1 M NaCl (3 volumes) and a NaCl linear gradient (0.1 to 0.9 M, 10 volumes), as
previously described [31]. Fractions of 0.8 ml were collected and analyzed for UA.
Uronic acid recovery from the DEAE column was higher than 90% in both C and WB
pooled samples. The UA-positive DEAE fractions containing distinct GAG populations
were pooled, precipitated overnight with 4.5 volumes of absolute ethanol at 4°C and
centrifuged at 12,000g for 15 min. The precipitate was then dissolved in 2× distilled
H2O at the final concentration of 1 μl/μg UA. Glycosaminoglycan populations obtained
by DEAE fractions were identified by cellulose acetate membrane electrophoresis using
known standards of HA, HS and CS/DS and a running buffer of 0.1 M pyridine–0.1 M
formic acid, pH 3.1, at a constant current of 0.5 mA/cm of membrane width for 50 min.
Toluidine blue in 15% vol/vol aqueous methanol was applied for a 10-min membrane
staining. Membranes were rinsed with water and scanned immediately [31].
2.7. GAG degradation with specific lyases
Digestions of pooled samples with a mixture of chondroitinases AC and ABC (0.01
units/10 μg UA) in 0.1 M sodium acetate–0.1 Tris-HCl buffer, pH 7.3, and a mixture of
heparin lyases I, II and III (0.05 units/25 μg UA) in 20 mM acetate buffer, pH 7.0, with
1 μmol calcium acetate were performed overnight at 37°C and stopped by transfer of
samples in 100°C for 1 min [31].
2.8. High-performance capillary electrophoresis (HPCE) analysis
Following enzyme digestions, GAG profile analysis was conducted as previously
described [31,33] on an HP3DCE instrument (Agilent Technologies, Waldronn,
Germany) with a built-in diode array detector set at 232 nm, using uncoated fusedsilica capillary tubes (50 μm i.d., 64.5 cm total and 56 cm effective length) at 25°C.
Phosphate buffer, 50 mM, pH 3.0, was used as a running buffer. NaOH 0.1 M (1 min), 2×
distilled H2O (1 min) and operating buffer (5 min) were used before each run to wash
the capillary tube. Migration of Δ-disaccharides from the cathode to the anode by
electrophoretic mobility and against the electroosmotic buffer flow was achieved by
applying samples at the cathodic end under 50 mbar and 30 kV for 5 s.
2.9. Statistical analysis
Student's t test was used for comparisons of food consumption, body weights, and
liver and aorta weight between the diet groups. Two-way analysis of variance was
applied in ranked observations of UA and sGAG concentration of individual aortic
specimens. Statistical analysis was performed with Sigmastat Statistical Program
version 2.0 (SPSS Inc., Chicago, IL, USA). Values are given as mean±standard deviation
(S.D.) or mean±standard error of mean (S.E.M.); differences are considered
statistically significant at P≤.05.
3. Results
2.3. Aortic sample collection and preparation
3.1. Food intake and animal growth
After 8 weeks of dietary treatment, rats were anesthetized with 95%CO2/ 5%O2; the
thoracic aorta was excised and transferred in PSS, composed of NaCl 118, KCl 4.7, CaCl2
2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 12.5 and dextrose 11.1 mM. Aortas were cleaned
of the adherent connective and fat tissue, frozen in liquid N2 and stored at −80°C. After
all samples were collected, frozen specimens were powdered under liquid N2, defatted
twice with methanol/chloroform solution (1:1, vol/vol), incubated for 16 h at 4°C,
rinsed with acetone and vacuum dried.
The daily food intake was similar between the control and the wild
blueberry diet-fed animals: 20±0.4 g in both diet groups. Growth
rate, assessed by weekly measurement of body weight, did not differ
significantly between the two diet groups (data not shown). As
2.4. Tissue digestion and separation of GAGs
Dry defatted aortas were rehydrated overnight at 4°C with 2× distilled H2O,
30 μl/mg aorta. The rehydrated samples were digested overnight at 37°C with
PRONASE (1.6 units/mg aortic tissue) in 50 mM Tris-HCl, pH 8.0. The reaction was
stopped with 0.6 M NaCl and transfer of vials in 100°C for 1 min. Aortic digests were
centrifuged for 5 min at 12,000g, and the collected supernatant was diluted in 4.5
volumes of ethanol solution with 2.5% sodium acetate. After overnight incubation at
Table 1
Body weight (BW), liver weight and aortic tissue weight a of the two diet groups
Diet group
BW (g)
Liver (g)
Aorta (mg) b
C
WB
343.0±2.7
353.0±5.3
13.2±1.0
12.8±0.9
10.4±2.4
10.0±2.2
a
b
Mean±S.D., n=10 rats per diet group.
Aorta dry weight.
presented in Table 1, no significant difference between diet groups
was detected in the final body weights or liver wet weight.
Furthermore, thoracic aorta dry weight did not differ between the
two diet groups.
3.2. Wild blueberry diet affects total content of aortic sulfated GAGs
Aortic samples were analyzed for UA and total sGAGs content after
tissue defatting and proteolytic digestion. No significant differences
were found in the UA concentration of the aortas isolated from either
diet group: 5.2±0.3 vs. 4.6±0.3 μg/mg aorta in the WB and C group,
respectively (Table 2). Notably, the concentration of total sGAGs was
significantly lower in the WB group (12.6±0.2) as compared to the
control (13.7±0.2 μg/mg aorta, Pb.05, Table 2).
3.3. Distribution of GAG content is substantially affected in WB
vs. control rats
Fractionation, isolation and identification of GAGs were performed
as earlier described [31]. In brief, UA-positive populations were
separated with ion-exchange chromatography of C and WB aortic
samples. The identity of these populations was confirmed with
cellulose acetate electrophoresis before and after treatment with
specific GAG-degrading lyases (not shown). Glycosaminoglycan
concentration (μg/mg aortic tissue) estimated by the amount of
recovered UA is presented in Table 3. In both diet groups, HS was
quantitatively the major GAG population, followed by GalAGs and HA.
Although the total GAG content between the two groups tested was
similar, the distribution of GAG populations was notably different
between the diet groups. Hyaluronic acid concentration was
significantly higher (25% increase) in the WB (154.2 vs. 123.6 μg/mg
in control group). Heparan sulfate was also 10% higher in the WB
(1370.3 vs. 1242.2 μg/mg), whereas GalAGs content was 11% lower in
the WB group (960.8 vs. 1081.4 μg/mg, Table 3, Fig. 1).
3.4. Wild blueberry diet increases nonsulfated GalAGs
disaccharide content
Analysis of the isolated aortic GalAG population with HPCE after
combined digestion with chondroitinases AC and ABC showed that
Δdi-mono4S was the main GalAG disaccharide in both diet groups
(52.4% in C and 49.5% in WB). The other monosulfated disaccharide,
Δdi-mono6S, was also slightly lower in the WB group (30.3% vs.
31.6%). Notably, as shown in Table 4, the content of GalAGs in
nonsulfated disaccharides increased twofold in WB group (18.0% vs.
10.7%), and that in disulfated disaccharides (Δdi-di(2,6)S) decreased
by 60% (2.2% vs. 5.3%).
963
Table 3
Glycosaminoglycan-derived UA concentration a in each population isolated from
pooled aortic samples with DEAE-Sephacel anion-exchange chromatography in the
two diet groups
HA
HS
GalAGs
Total GAGs
C
WB
123.6 (5.1%)
1242.2 (50.8%)
1081.4 (44.2%)
2447.3
154.2 (6.2%)
1370.3 (55.1%)
960.8 (38.7%)
2485.4
a
μg UA/mg of dry defatted aortic tissue. Percentages of each type of GAG are
provided in parentheses.
was observed in the SHR thoracic aortas after 8 weeks of wild
blueberry consumption (Table 2). Although the total aortic GAG
content was similar with only a slight increase (2%) in the WB group,
wild blueberry consumption resulted in a redistribution of GAG
populations. The levels of HA and HS were 25% and 10% higher and the
GalAGs 11% lower in the aortic tissue isolated from the WB group
(Table 3, Fig. 1). The GalAG disaccharide analysis revealed that the
disulfated and monosulfated GalAG disaccharides were reduced due
to the wild blueberry diet, whereas a higher percentage of
nonsulfated GalAG disaccharides was observed in the SHR aortas
from the WB group (Table 4).
The elevated peripheral resistance and blood pressure of the SHR
are related to an increase of vascular PGs [22]. Furthermore, in
comparison with the parental WK strain, the SHR aorta is characterized by a thicker subendothelial matrix and a higher CS synthesis
leading to a higher concentration of total GAGs [22]. With most CSPGs
contained in the media layer of the vascular wall, hypertrophy of this
layer in small and large SHR vessels agrees with the elevated CS in the
SHR [34]. Hypertrophy of the aortic media observed in young SHRs
before the development of hypertension is most likely responsible for
the lower compliance and distensibility of the SHR aortic wall [35].
Additionally, GalAGs seem to promote atherogenesis, since CS and
DS are highly involved in low-density lipoprotein (LDL) binding [36],
and especially biglycan is a critical CS/DS-containing PG for LDL
retention [37]. Subendothelial retention of apoB100 lipoprotein, the
major apolipoprotein of human LDL, is an early step in atherogenesis,
with the atherogenicity of the LDL particle linked to its binding
affinity for arterial PGs [19,38]. Another indicator of the role of GalAG
in atherosclerosis is the recently documented predictability for the
postoperational CS4 levels in patients undergoing coronary artery
bypass surgery based on preoperational apoB and apoE levels [39].
In the present study, we observed lower GalAGs concentration
after 8 weeks of WB treatment, although it was not determined
whether the lower GalAGs reflected a decrease in CS or DS or both
types. The decrease of GalAGs due to wild blueberry consumption was
accompanied by higher concentrations of both HA and HS in our adult
4. Discussion
1600
Table 2
Uronic acid a and total sGAGs b concentration in aortic tissue of the two diet groups
Diet group
UA (μg/mg) a
sGAGs (μg/mg) b
C
WB
4.6±0.3
5.2±0.3
13.7±0.2
12.6±0.2 ⁎
Individual aortas were analyzed for uronic acid concentration with carbazole reaction
and sGAG content with Blyscan. Values are given in μg/mg of dry defatted aortic tissue.
a
Mean±S.E.M., n=6.
b
Mean±S.E.M., n=10.
⁎ Statistically significant at P≤.05.
1400
µg UA/mg aorta
This is the first study to document that a wild blueberry-enriched
diet elicited multiple alterations of aortic GAGs in the SHR model of
endothelial dysfunction. A significant reduction of total sulfated GAGs
C
WB
+10%
1200
-11%
1000
800
600
400
200
+25%
0
HA
HS
GalAGs
Fig. 1. Glycosaminoglycan-derived UA concentration (μg UA/mg of dry defatted aortic
tissue) in each population isolated by DEAE-Sephacel anion-exchange chromatography
on pooled aortic samples from the two diet groups.
964
Table 4
Galactosaminoglycan disaccharide composition as determined by HPCE analysis of
pooled aortic samples after combined digestion with chondroitinases AC and ABC a in
the two diet groups
Disaccharide
C
WB
Δdi-di(2,6)S
Δdi-di(2,4)S
Δdi-mono6S
Δdi-mono4S
Δdi-nonS
5.3
–
31.6
52.4
10.7
2.2
–
30.3
49.5
18.0
Dashes indicate nondetectable disaccharides.
a
Percentage of total recovered Δ-disaccharides.
SHRs. In young SHRs, an increase of HA aortic content after diuretic
treatment was positively correlated with total systemic compliance
[40]. Hyaluronic acid of the arterial glycocalyx is necessary for
activating flow-induced nitric oxide (NO) release from the endothelial cells by sensing and transferring the shear stress to the
endothelium [41]. Furthermore, the hydrophilicity of HA is crucial
for the structural stability of the glycocalyx [17]. Heparan sulfate
localized on the luminal surface is another mechanosensor that
mediates NO synthesis in response to shear stress [42]. In addition to
the key role of HS on vascular tone, several antiatherogenic
characteristics, such as inhibition of oxidized LDL and monocyte
binding to the ECM and inhibition of smooth muscle cell proliferation,
have been attributed to HSPGs and especially perlecan [43]. As
opposed to GalAGs, HS is not involved in LDL binding [36], and
perlecan, the main HS-containing subendothelial PG, does not colocalize with human apoB and therefore appears reduced in human
atherosclerotic lesions [37]. The greater amount of HA and HS in the
SHR aorta observed in the WB-fed rats further supports the beneficial
role of wild blueberries on CVD.
Higher HA and HS levels might be related to our recent findings
that documented an NO pathway-mediated attenuation of the
vascular tone in response to adrenergic stimuli in the adult SHRs
treated with wild blueberries [44]. The effect of wild blueberries on
vascular tone, acting through the endothelium and more specifically
on the NO pathway, has been also documented in the SD rat [45,46].
Besides the beneficial redistribution of GAG populations, the wild
blueberry diet caused a significant decrease in GAG sulfation in the
SHR aorta. Reynertson et al. have reported a higher sulfate
incorporation into aortic PG in the SHR compared to the WK rat
[23], as well as in other experimental models of hypertension [47].
The higher sulfation of PG molecules from vascular smooth muscle
cells of mesenteric resistance arteries observed in the young
prehypertensive SHRs indicates that alterations of PG synthesis
occur before the establishment of vascular dysfunction and hypertension in this model [24]. The degree of sulfation is also positively
correlated with LDL binding affinity to PGs [37]. A lower LDL retention
induced by statin treatment was attributed to the decreased sulfation
incorporation into smooth muscle cell PGs [48].
The attenuation of sulfation in the SHR aorta after 8 weeks of wild
blueberry consumption in the present study suggests that dietary
wild blueberries play an important role in aortic GAG remodeling and
may improve structural characteristics of the vascular wall related to
the development and progress of CVD. This is an important finding,
especially in the light of the emerging role of GAGs as vascular agents
for prevention of atherosclerosis and CVD, with special attention
given to their sulfation pattern [49].
The reduced sulfation in the SHR due to the wild blueberry diet is
consistent with past observations in aortic tissue from SD rats [31].
Wild blueberries, other than being a rich source of bioactive
polyphenols and anthocyanins, are a good source of manganese
[50], a trace element documented to decrease sulfation of HS in SD rat
aorta [51].
Whereas further studies are required to elucidate the mechanisms
of actions of wild blueberries on the aortic GAG profile, the possibility
cannot be excluded that consumption of wild blueberries alters GAG
synthesis and turnover through an effect on enzymatic processes and
related signaling pathways. Phenolics from sorghum bran were
reported to inhibit hyaluronidase activity, with implications in the
balance between HA synthesis and degradation in the ECM and hence
connective tissue remodeling and homeostasis [29]. Flavonoid
compounds can inhibit the activity of HA-splitting enzymes based
on the number of their hydroxyl groups [27]. Plant-derived
compounds (ascorbic acid and quercetin) and tea extract induced
alterations of the ECM composition leading to reduction of monocyte
adhesion in the endothelium [25]. High concentrations of genistein
can inhibit GAG synthesis through an effect on epidermal growth
factor-dependent pathway in patients with mucopolysaccharidoses, a
condition of excessive GAG accumulation in lysosomes [30]. Signaling
processes initiated by vitamins E and C [26] or tea catechins [28] that
modulate ECM environment with effects on vascular remodeling and
maintenance of vascular wall function also indicate a positive role of
dietary and bioactive compounds on vascular integrity.
Current biomedical research has been targeting the endothelium
in an attempt to uncover vascular-directed agents for the prevention
of atherosclerosis and CVD [49]. Glycosaminoglycans are rendered as
such molecules, with special attention given to their chain elongation
and sulfation patterns [48]. In the present study, wild blueberries
were involved in primary aspects of GAG metabolism such as
population redistribution and sulfation in the SHR aorta. We are the
first to document the role of a wild blueberry diet on arterial GAG
remodeling in the SHR. Wild blueberries modulate structural
characteristics that potentiate cardiovascular risk, but most importantly enhance the protective features of GAG molecules associated
with endothelial dysfunction and vascular pathology in the SHR.
Therefore, wild blueberries may be employed as a component of
endothelium-targeted nutrition to prevent endothelial dysfunction
and CVD development.
Acknowledgments
The authors would like to express their gratitude to Maria I.
Krevvata for her significant contribution to this project, the Wild
Blueberry Association of North America (WBANA) for contributing
the wild blueberries and FutureCeuticals (Momence, IL) for processing them. This work was supported by a WBANA grant to D.K.-Z. and
a fellowship by the Greek State Scholarship Foundation to A.S.K. This
is Maine Agricultural and Forest Experiment Station Publication
Number 3157.
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PHYTOTHERAPY RESEARCH
Phytother. Res. 26: 956–963 (2012)
Published online 15 November 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.3670
Cell-Line Specific Protection by Berry Polyphenols
Against Hydrogen Peroxide Challenge and Lack
of Effect on Metabolism of Amyloid Precursor
Protein
Magdalini A. Papandreou,1 Maria Tsachaki,2 Spiros Efthimiopoulos,2 Dorothy Klimis-Zacas,3
Marigoula Margarity1 and Fotini N. Lamari4*
1
Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Greece
Division of Animal and Human Physiology, Department of Biology, University of Athens, Greece
3
Department of Food Science and Human Nutrition, University of Maine, Orono, ME 04469, USA
4
Laboratory of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, University of Patras, Greece
2
Amyloid precursor protein (APP) altered metabolism, Ab-overproduction/aggregation and oxidative stress are
implicated in the development of Alzheimer’s disease pathology. Based on our previous data indicating that
administration of a polyphenol-rich (PrB) blueberry extract (from wild Vaccinium angustifolium) is memory
enhancing in healthy mice and in order to delineate the neuroprotective mechanisms, this study investigated
the antioxidant effects of PrB in H2O2-induced oxidative damage, Ab peptide fibrillogenesis and APP metabolism.
PrB suppressed H2O2-initiated oxidation (DCF assay) and cell death (MTT assay) in SH-SY5Y cells. Protective
effects were observed on Chinese hamster ovary (CHO) cells overexpressing APP770 carrying the mutation
Val717Phe only at high concentrations, while further damage on HEK293 cells was induced after co-treatment with
250 mM H2O2 and PrB in comparison with H2O2 alone. Using the thioflavine T assay, blueberry polyphenols inhibited Ab-aggregation (~70%, 15 mg/mL) in a time-dependent manner, while in the CHOAPP770 cells it had no effect
on APP metabolism as assessed by western blot. The results suggest that blueberry polyphenols exhibit antioxidant
and/or pro-oxidant properties according to the cellular environment and have no effect on APP metabolism.
Copyright © 2011 John Wiley & Sons, Ltd.
Keywords: antioxidant; amyloid fibrillogenesis; Vaccinium angustifolium; SH-SY5Y; CHOAPP770; APP metabolism.
INTRODUCTION
Alzheimer’s disease, a form of senile dementia, is an
irreversible, progressive neurodegenerative disorder,
resulting from a complex pathological cascade that
includes the accumulation of beta-amyloid (Ab) peptide
aggregates or aberrant amyloid precursor protein
(APP) processing. The disease is manifested years after
its initiation with serious cognitive and neuropsychiatric
deficits but a clear understanding of its pathogenetic
factors is lacking, thus limiting the development of
adequate prevention and treatment strategies. Multiple
cellular/biochemical events (inflammation, oxidative
damage, aberrant signaling, high metal concentrations,
aberrant tau/APP processing) are known to contribute
to cognitive deficits by disruption of neuronal signal
transduction pathways involved in memory and finally
neuronal and synaptic loss. There is still no effective
therapy for Alzheimer’s disease and pleiotropic drugs
or cocktails have been proposed to have a better chance
for halting disease progression (Cole and Frautschy,
2010). In that aspect, natural products either individually
* Correspondence to: Fotini N. Lamari, Department of Pharmacy, University
of Patras, 26 504 Rion, Greece.
E-mail: fl[email protected]
or in plant extracts are good candidates since pleiotropy
is their inherent characteristic.
Blueberries (fruits of various Vaccinium species) contain a wide range of compounds, including the red and
blue colored anthocyanins, the flavonols and ellagic acid
derivatives, and have the highest antioxidant capacity
among fruits and vegetables. A number of studies have
documented that rodent diet supplementation with
berries is effective in reducing not only oxidative stress
but also neuronal, behavioral and cognitive defects
associated with aging, inflammation, ischemia and
Alzheimer’s disease (Shukitt-Hale et al., 2008; Galli et al.,
2006; Joseph et al., 1998, 1999, 2003). In particular,
blueberry-fed (4 months of age) APP + PS1 transgenic
mice showed no deficits in Y-maze performance (at
12 months of age) with no alterations in Ab burden
(Joseph et al., 2003). Enhancement of cognitive functions
and antioxidant protection were also observed by Joseph
and his co-workers after dietary supplementation for
8 weeks of aged Fischer 344 rats with blueberry extracts
(Joseph et al., 1998, 1999), or of rats subjected to
ischemia/reperfusion, with the latter exhibiting a reduction
in the volume of infarction in the cerebral cortex and an
increase in post-stroke locomotor activity, while they
also had significantly lower caspase-3 activity in the
ischemic hemisphere (Wang et al., 2005). Additional
experiments have shown that blueberry supplementation for 10 weeks of young and old rats subjected to an
Received 20 May 2011
Revised 01 September 2011
Accepted 02 September 2011
ANTIOXIDANT AND ANTI-AMYLOID EFFECTS OF BLUEBERRY POLYPHENOLS
in vitro inflammatory challenge (LPS) restored the ability of the aged hippocampal cells to respond to an inflammatory challenge with a large production of
inducible heat shock protein 70 (HSP70) (Galli et al.,
2006). It was shown previously that a 6-day intraperitoneal administration of a polyphenol-rich wild blueberry extract (PrB) enhanced cognitive performance
of healthy mice, brain antioxidant markers and
inhibited total brain acetylcholinesterase (Papandreou
et al., 2009). Thus, blueberry extracts either through
diet or administered via other routes seem to prevent
characteristic cognitive deficits of senescence and
Alzheimer’s disease and confer antioxidant protection
in rodents.
Some mechanisms have been postulated for the
efficacy of the blueberry extract and its constituents,
but many issues concerning their protective effects
remain unresolved. Correspondingly, polyphenol and
anthocyanin treatment were also shown to be
effective against diverse oxidative stressors, such as
H2O2-induced reactive oxygen species (ROS) generation in red blood cells (Youdim et al., 2000a,
2000b) and Ab-induced toxicity in COS-7 cells (Joseph
et al., 2002). It has been recently documented that
blueberry extracts significantly enhance microglial
clearance of Ab, inhibit aggregation of Ab1-42, and
suppresses microglial activation, all via suppression
of the p44/42 MAPK module (Zhu et al., 2008).
Additionally, the whole blueberry extract and many
fractions of it (i.e. anthocyanin, pre-C18, post-C18,
proanthocyanidins, chlorogenic acid, etc) have been
studied for their protective ability against dopamine-,
Ab42- and LPS-induced decrements in calcium
buffering in primary hippocampal cells and the degree
of protection differed according to the degree of fractionation and the stressor applied (Joseph et al.,
2010). Oxidative stress induces the expression and
misprocessing of APP, leading to the generation of
amyloidogenic fragments that readily form aggregates.
This process can result in a potentially vicious cycle
whereby oxidative stress leads to Ab production, and this
production in turn results in increased oxidative stress,
neuronal dysfunction and ultimately neuronal death
(Miranda et al., 2000). The effect of blueberry polyphenols on APP metabolism has not yet been studied.
The aim of the present study was to examine the
effects of the extract rich in blueberry polyphenols
(PrB) against H2O2-induced cell death in different
neuronal and non-neuronal cell lines, which have not
been previously studied, and furthermore to study
the effect of PrB on APP metabolism and Abaggregation in vitro. For this reason, the study used
(a) the human neuroblastoma SH-SY5Y cell line, a
well known model cell system for studying neuronal
cell death induced by oxidative stress (Ruffels et al.,
2004); (b) Chinese hamster ovary (CHO) cells stably
transfected to express mutant human APP770 carrying
the Val717Phe mutation (CHOAPP770) and thus release
considerable amounts of the toxic, amyloidogenic
Ab (Sun et al., 2002); and (c) human embryonic
kidney HEK293 cells. Despite the fact that the
HEK293 cell line was derived by transformation of
primary human embryonic kidney cell and thus are
considered a model system of peripheral cells, it has
been shown that they have unexpected neuronal
progenitor properties (Shaw et al., 2002).
957
METHODS AND MATERIALS
Plant material and extraction. Wild blueberries were
purchased as a composite from Wyman’s (Cherryfield,
ME), freeze-dried with standard procedures by American
Lyophilizer Inc. (Bridgeport, PA, USA) and powdered.
Polyphenols were extracted from the above as described
previously (Lohachoompol et al., 2004; Papandreou et al.,
2009). In brief, 2 g of blueberry powder was extracted in
the dark, under magnetic stirring with 15 mL/g of methanol, acetic acid and distilled water at a ratio of 25:1:24,
respectively, for 2 h. The extract was evaporated to dryness
and the dry residue was stored at 20 C until further use.
The dry residue was re-dissolved with 1 mL of 3%
formic acid in water (w/v), centrifuged and the supernatant
was absorbed on a C18 Sep-Pak cartridge. The cartridge
was washed with methanol, equilibrated with 5 mL of
3% formic acid in water (w/v) and eluted with 5 mL 3%
formic acid in 50% methanol (w/v). The polyphenols
eluted from the cartridge were evaporated under vacuum
until dryness and kept at 20 C until use.
Total phenolics were measured with the Folin-Ciocalteu
reagent method (Singleton and Rossi, 1965). The total
polyphenolic content was expressed as gallic acid equivalents (GAE), using a standard curve with 50–600 mg/L
gallic acid. Total anthocyanin content was estimated
according to Giusti and Wrolstad (2001) by UV-visible
spectroscopy at 538 nm after dissolution in a mixture of
methanol and 0.1 M HCl at a ratio of 85:15. The total
anthocyanin content was expressed as cyanidin 3-rutinoside
equivalents.
Cell culture and drug treatment. Human neuroblastoma
SH-SY5Y and human embryonic kidney HEK293 cell
lines were obtained from the American Type Culture
Collection (ATCC), while the Chinese Hamster Ovary
(CHO) control cells (wild type, wt) or CHOAPP770 cell,
stably transfected to express mutant human APP770
carrying the Val ! Phe mutation at residue 717, were a
kind gift of Professor Leonidas Stefanis from the Biomedical Research Foundation of the Academy of Athens
(BRFAA). All cultures were grown in DMEM containing
10% heat-inactivated fetal bovine serum, 4.5 g/L glucose,
2 mM L-glutamine, 100 mg/mL penicillin and 100 mg/mL
streptomycin sulfate. Confluent cultures were subcultured
using 0.5 g/L trypsin/0.2 g/L EDTA into 96-well plates at a
density of 1.5 104 cells/well for SH-SY5Y cells and 5 103 cells/well for HEK293 and CHOAPP770 cells. All
experiments were carried out 24 h after the cells were
seeded. Prior to all treatments, the cells were incubated
in serum-free media for 24 h. In a pilot investigation, the
cells were treated with H2O2 at concentrations ranging
from 50 to 1000 mM, for 18 h, and then examined for cell
viability. Finally, the cells were co-incubated with 250 mM
H2O2 and the tested phytochemical (1–250 mg/mL PrB),
or vehicle as control (medium containing max. 0.1% v/v
DMSO), for 18 h.
Cell extracts were prepared by treating cells with
50 mM Tris, 150 mM NaCl, 2 mM EDTA, pH 7.6 containing protease inhibitors Complete (Roche Applied
Science) and 1% Triton X-100 for 30 min on ice. The
lysates were then centrifuged at 10000 g for 30 min at
4 C. The supernatants were collected and the amount
of total protein was measured using the bicinchoninic
assay (Pierce, Bonn, Germany).
Phytother. Res. 26: 956–963 (2012)
958
M. A. PAPANDREOU ET AL.
Assessment of cell viability and reactive oxygen species
levels. Cell viability was estimated using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
(MTT reagent) (Plumb et al., 1989). Briefly, on completion of the treatment, the cells were washed with
pre-warmed phosphate-buffered saline (PBS) and
incubated with serum-free media containing MTT at a
final concentration of 2.5 mg/mL for 150 min. Supernatants were carefully aspirated and the resultant formazan
product was dissolved in DMSO and the absorbance was
measured using a UV-Vis spectrometer (Denley WellScan,
West Sussex, UK) at 545 nm. Wells without cells were used
as blanks and were subtracted as background from
each sample. The results were expressed as a
percentage of control untreated cells.
Intracellular formation of ROS was assessed using the
probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA)
(Sigma-Aldrich Corporation, St Louis, MO, USA),
which was used as a substrate (Wang and Joseph,
1999). All cell lines (SH-SY5Y: 1.5 104 cells/well,
HEK293/CHOAPP770: 5 103 cells/well), growing in
microtiter 96-well plates, were washed two times with
pre-warmed PBS, and then incubated with 50 mM
DCF-DA in PBS for 30 min at 37 C. Fluorescence
was then quantified in a microplate-reader (Bio-Tek
Instruments Inc., Ville St Laurent, Que, Canada) at
an excitation wavelength 485 nm and emission wavelength
530 nm.
Assessment of malondialdehyde levels. CHOAPP770 cells
were incubated in serum-free media, for 24 h, and then
treated with PrB extract (10–50 mg/mL) for 48 h. The cells
were then lysed, centrifuged and the collected supernatants were subjected to malondialdehyde (MDA)
analysis. The concentration of MDA, a compound that
is produced during lipid peroxidation was determined
fluorometrically by the thiobarbituric acid (TBA)
method, as previously described (Jentzsch et al., 1996;
Papandreou et al., 2009). The assay showed good linearity
(0.05–5.00 mM, y = 11.488x + 0.7415, R2 = 0.9976) and
sensitivity. The level of MDA was expressed as nmol/mg
protein in order to correct for any variability in cell
number dictated by cell treatment.
Thioflavine T assay. Thioflavine T (ThT) measurement
was performed according to the method described
previously (Tjernberg et al., 1999; Papandreou et al.,
2006). For Ab aggregate-formation assay, Aß1-40
(10 mg/mL) was diluted in PBS (10 mM, 150 mM NaCl,
pH 7.4) to 40 mM and incubated at room temperature
for 5 and 30 days, with various concentrations of the
PrB extract or without (control) (Papandreou et al.,
2006). Within 30 min after addition of thioflavine T
(10 mM), the fluorescence intensity was measured with a
RF-1501 spectrofluorometer (Shimadzu), using an excitation filter of 435 nm and an emission filter of 482 nm,
respectively. The percentage inhibition was calculated
by comparing these fluorescence values with those found
in control solutions with no PrB.
Western blot analysis of amyloid-precursor protein
(APP) metabolism. CHOAPP770 cells were treated with
the plant extract (10–50 mg/mL for PrB) for 48 h. The cells
were then lysed, centrifuged and the obtained supernatant, as well as the cell culture media, were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Proteins were separated on gels (12% for
media and 16% for cell lysates) and transferred to nitrocellulose membranes, which were incubated overnight at
4 C with the primary antibodies (1:1000) (6E10: residues
Ab1-16 monoclonal; R1(57): anti-APP C-terminal polyclonal; 71-192wt-1147: residues APP590-596 (Parisiadou
and Efthimiopoulos, 2007; Parisiadou et al., 2008). Bound
antibodies were visualized using peroxidase-conjugated
secondary antibodies (anti-rabbit used in 1/2000 for R1
(57) and 1/1000 dilution for 71-192wt-1147; anti-mouse
used in 1/1,000 dilution for 6E10), followed by ECL+
detection (ChemBio Ltd).
Statistical analysis. Data are presented as mean SE for
at least ten replicates. Statistical analysis was performed
with GraphPad Instat 3 software (GraphPad Instat
Software, Inc. USA) using the nonparametric MannWhitney test. In all tests, a criterion of p ≤ 0.05 (twotailed) was considered necessary for statistical
significance.
RESULTS
H2O2-induced toxicity on SH-SY5Y, HEK293 and
CHOAPP770 cell lines
To determine the effective concentrations of H2O2, all
cell lines were treated with various concentrations
(50–750 mM) of H2O2 for 18 h and the viability of the
cells was assessed by the MTT assay. Treatment with
50 mM and 100 mM H2O2 had virtually no effect on the
viability of all cell lines under the conditions employed,
but the viability of cells exposed to concentrations
higher than 100 mM showed a dose-dependent decrease.
In the SH-SY5Y cell line, the cell viability decreased by
43.0 0.5%, 56.0 0.5% and 60.0 0.9%, compared
with untreated cells after treatment with 250 mM,
500 mM and 750 mM H2O2, respectively. Similarly, in the
HEK293 cells, in the presence of 250 mM and 500 mM
H2O2, the percentage of cell viability decreased by
30.0 2.0% and 48.0 0.9%, respectively, while at
750 mM H2O2, by 69.0 0.8%. Treatment of CHOAPP770
cells with 250 mM, 500 mM and 750 mM H2O2, resulted in
35.0 1.2%, 40.0 0.8% and 49.0 1.2% cell death,
respectively. There was a significant injury in all three
cell lines tested, with the H2O2-treated cells becoming
round and some of them detaching from the plate.
Moreover, the H2O2-induced oxidative damage to cells
was also measured as ROS formation, as assayed by
the conversion of the probe DCFH-DA to DCF. ROS
accumulation increased significantly in all cell lines
tested after incubation with H2O2 (250 mM), as indicated
by the increase in relative fluorescence units (SH-SY5Y,
73400 4585; HEK293, 38529 2220; CHOAPP770,
48999 1086 DCF/MTT) compared with the untreated
(control) cells (SH-SY5Y, 58114 2901; HEK293,
18406 8433; CHOAPP770, 40861 1381 DCF/MTT
values), respectively.
PrB content in polyphenols and anthocyanins
The PrB extract, obtained from solid-phase extraction
on Sep-Pak C18, had a total polyphenolic content of
Phytother. Res. 26: 956–963 (2012)
13.01 0.06 mg GAE/g dry weight and a total anthocyanin content of 3.78 0.05 mg/g dry weight, as reported
previously (Papandreou et al., 2009). Comparison of
this composition with that reported for freeze-dried
blueberry powder by others (Mazza et al., 2002) shows
that the solid-phase extraction concentrated the extract
in polyphenols about four times.
PrB rescues viability and decreases ROS levels in
H2O2-treated SH-SY5Y cells
As illustrated in Fig. 1A, treatment of SH-SY5Y cells
with PrB extract (1–250 mg dry extract/mL) significantly reduced the cell death caused by H2O2 in a
non-concentration-dependent fashion. No difference
was seen in cell viability between cells treated with
PrB alone and controls (data not shown). Moreover,
co-incubation of SH-SY5Y cells with PrB extract and
H2O2 not only completely abolished H2O2-induced
ROS accumulation but further reduced ROS levels
by 57–63% compared with the untreated cells.
PrB confers additional toxicity in H2O2-treated
HEK293 cells
Co-treatment of HEK293 cells with the PrB extract and
H2O2 (250 mM) further reduced the viability of HEK293
cells by 12–35% in comparison with the H2O2-treated
cells (Fig. 2A). In addition, the ROS levels were significantly higher (28.4–146.0%) in the presence of PrB
and H2O2 in comparison with H2O2 alone (Fig. 2B).
959
or untreated cells even though viability was not different
than that of the H2O2-treated cells (Fig. 3).
Furthermore, the effect of PrB on membrane lipid
peroxidation in CHOAPP770 cells was investigated. As
shown in Fig. 4, CHOAPP770 cells producing considerable amounts of Ab species, showed an increase in lipid
peroxidation (~88%), in comparison with the wild-type
CHO cells, as evidenced by determination of MDA
levels (0.160 0.005 nmol/mg protein vs 0.091 0.002
nmol/mg protein). However, PrB prevented peroxidation of lipids compared with the control, by 51%, 25%
and 31%, at PrB concentrations of 1, 10 and 50 mg/mL,
respectively.
APP metabolism is not affected by PrB
When CHO APP770 cells were treated with PrB for 48 h,
no alterations were observed either on sAPPa/b levels
secreted into their conditioned media (Fig. 5A, B), or
on cellular APP expression (Fig. 5C), as evidenced by
western blot, using b-actin as an internal control.
Inhibition of Αb aggregation in vitro by PrB
The effect of PrB on the Ab1-40 propensity to form
aggregates was studied by the standard ThT-based
fluorescence assay. The PrB extract significantly inhibited
Ab-aggregation in vitro in a time-dependent manner
(Fig. 6). Notably, the inhibitory effect at the lowest
concentration (15 mg dry extract/mL) was 67% and 62%,
at 5 and 30 days, respectively.
Antioxidant effects of PrB in CHOAPP770 cells
DISCUSSION
The PrB treatment significantly reduced the cell death
of CHOAPP770 cells treated with H2O2 only at the highest
applied concentrations of PrB (125 and 250 mg/mL) with
a concomitant low (7–9%) but statistically significant
decrease in ROS levels in comparison with H2O2-treated
cells (Fig. 3). However, at concentrations of 1–50 mg/mL
of PrB in the presence of 250 mM H2O2, the ROS levels
were significantly higher than those of the H2O2-treated
Hydrogen peroxide has been used extensively as an
inducer of oxidative stress in in vitro models. Cotreatment of cells with H2O2 and PrB showed that the
PrB effects varied among the types of cell lines tested
(Figs 1–3). In particular, in the human neuroblastoma
SH-SY5Y cell line, the PrB extract reduced H2O2induced cell death and generated ROS, to levels lower
than those of the untreated cells; whereas in the
Figure 1. SHSY5Y viability and ROS levels. Cells were co-treated with 250 mM H2O2 and PrB (1–250 mg dry extract/mL), for 18 h, and both cell
viability (A) and ROS levels produced (B) were measured by MTT and DCF assay, respectively. *p ≤ 0.05 vs H2O2-treated cells. Percentages
indicate the percentage decrease observed after incubation with PrB in the presence of H2O2. There was a statistically significant difference
(p ≤ 0.05) in viability and ROS levels of all treated cells from control untreated cells.
Phytother. Res. 26: 956–963 (2012)
960
Figure 2. HEK293 viability and ROS levels. Cells were co-treated with 250 mM H2O2 and PrB (1–250 mg dry extract/mL), for 18 h, and both cell
viability (A) and ROS levels produced (B) were measured by MTT and DCF assay, respectively. *p ≤ 0.05 vs H2O2-treated cells. Percentage
indicates the percentage of cell death in the presence of the H2O2. There was a statistically significant difference (p ≤ 0.05) in viability and
ROS levels of all treated cells from control untreated cells.
Figure 3. CHOAPP770 viability and ROS levels. Cells were co-treated with 250 mM H2O2 and PrB (1–250 mg dry extract/mL), for 18 h, and both
cell viability (A) and ROS levels produced (B) were measured by MTT and DCF assay, respectively. *p ≤ 0.05 vs H2O2-treated cells. 1p ≤ 0.05
vs untreated control.
Figure 4. Effect of PrB extract on lipid peroxidation (MDA) levels.
CHOAPP770 cells were incubated with PrB (1–250 mg/mL) for 48 h.
*p ≤ 0.05 vs untreated control, #p ≤ 0.05 vs wild CHO cells.
HEK293 cells it exacerbated H2O2 cytotoxicity. Since
CHOAPP770 cells overproduce APP and Ab in turn, they
can be considered as more stressed in comparison with
wild type cells; MDA measurements confirm this
phenomenon. Treatment of those cells with PrB
reduced the lipid peroxidation levels significantly. This
is in agreement with reports whereby blueberry
polyphenols protected primary hippocampal neuronal
cells (HNCs) against Ab-induced stress (Joseph et al.,
2010). In our experiment, when CHOAPP770 cells were
additionally stressed with H2O2, PrB further increased
ROS levels (the higher the concentration, the lower
the increase), while cell viability was enhanced in
comparison with H2O2-treated cells, only at the highest
applied concentrations. The current findings of PrBinduced increases in ROS levels in H2O2-stressed
CHOAPP770 and HEK293 cells are in line with the
recently published data by Joseph and his co-workers
(Joseph et al., 2010), whereby treatment of HNC cells
with whole blueberry extract or certain fractions of it
(e.g. proanthocyanidins, POST-C18 and to some extent
PRE-C18), resulted in increases in ROS levels, even
in the absence of the stressors (i.e. dopamine, LPS
or Ab42).
This type of polyphenol-induced cytotoxicity is not
uncommon in cell culture experiments and has been
largely ascribed to the nature of the phenolic
compounds, which can act either as pro-oxidants or as
cytotoxic agents, in certain conditions (Sergediene et al.,
1999; Shamim et al., 2008). Recent findings have also
implicated certain phenolic compounds (e.g. delphinidin)
in the induction of H2O2, within commonly used cell
culture media, possibly due to their unstable nature
and their ability to undergo chemical transformations,
resulting thus in the formation of by-products capable
Phytother. Res. 26: 956–963 (2012)
961
Figure 5. Effect of PrB extract on secretion of soluble amyloid precursor protein (sAPP) a- (A) and b- (B), and on whole APP (C) in CHOAPP770 cells.
Figure 6. In vitro inhibition of Ab1-40 fibrillogenesis in the presence
of various concentrations of PrB extract at 5 and 30 days, as assayed
with the thioflavine T fluorescence assay. The fluorescence absorbance of aged Ab1-40 alone was considered as 100%. PrB exhibited
statistically significant (p ≤ 0.05) inhibition of Ab1-40 fibrillogenesis at
all concentrations.
of causing cell-specific toxicity (Long et al., 2010). In
our experiments, however, treatment of cells only with
PrB did not affect viability, as assessed by MTT,
although it is more difficult to delineate on the changes
of the phenolic compounds contained within the extract,
due to the experimental procedure followed, i.e. coadministration with H2O2.
In addition, the lack of dose-dependency observed
in either cell-viability or ROS production after cotreatment of all three cell lines in the particular concentration range is not uncommon in studies of natural
products. For instance, treatment of normal epithelial
cells and cells derived from the salivary gland with
epigallocatechin gallate, resulted in a dose-independent
effect in ROS production (Yamamoto et al., 2003).
The fact that PrB is an extract with many different
polyphenol entities, which act by a variety of mechanisms, implicates the principles of synergy, antagonism and pleiotropy in explaining this phenomenon.
Taking all these data into consideration, it is concluded that the PrB extract selectively protected
SH-SY5Y cell viability from H2O2 stress and significantly decreased ROS levels, in contrast to HEK293
cells and partly CHOAPP770 cells. The observed differences in response could be ascribed to the existence
of distinct cell surface receptors, cellular membrane
composition and intracellular retention transport. For
example, although quercetin-3-glucoside protects both
SH-SY5Y and HEK293 cells against H2O2-induced
viability loss, the underlying mechanisms are not the
same: it induces sterol regulatory element-binding
protein-2-mediated cholesterol biosynthesis only in
SH-SY5Y cells (Soundararajan et al., 2008).
Oxidative stress and ROS accumulation, apart from
their implication in cellular damage, are also involved,
at least in part, in the toxicity of Ab-peptide and its
aggregation/accumulation (Miranda et al., 2000), and
thus, with the pathogenesis of neurodegeneration in
AD. The active form of b-amyloid consists of soluble
oligomeric fragments, composed of 39–43 amino acids;
Ab1-40 and Ab1-42, are the predominant forms. The
latter peptidergic fragments are derived from the
proteolytic processing of the b-amyloid precursor
protein (APP) via two pathways, each resulting in
distinct cleavage products: (1) a non-amyloidogenic
which involves cleavage of APP to soluble APP (sAPP)
by the a-secretase; and (b) a formation of amyloidogenic
b peptides by the b- and g-secretases. As the proportion
of APP processed by b-secretase vs a-secretase may
affect the amount of Ab produced, regulation of these
two pathways may be critically important to the pathogenesis of AD.
The examination of the PrB extract on CHOAPP770
cells, stably transfected to express human APP, revealed
that the tested phytochemical had no effect on APP
metabolism, as evidenced by western blot analysis
(Fig. 5). The former observation might provide an
explanation for previous experiments performed by
Joseph et al. (2003) showing no effect on Ab peptide
production, deposition or amyloid load on transgenic
PS1-APP mice brains, after supplementation with blueberries extracts (2% of diet) from 4 to 12 months of
age. Similar results were also observed after treatment
of both transfected murine N2A (Swedish mutant form
of APP, SweAPP N2a) and primary neuronal cells
derived from transgenic APPsw mice with the catechins,
()-epigallocatechin and ()-epicatechin (Rezai-Zadeh
et al., 2005).
However, the PrB extract significantly inhibited
in vitro Ab-fibrillogenesis (up to 78.79 3.63%, at a
concentration of 0.1 mg/mL, after 5 days of incubation)
Phytother. Res. 26: 956–963 (2012)
962
(Fig. 6) as evidenced by the thioflavin T assay. Inhibition
of Ab1-42 aggregation by blueberry polyphenols was also
reported by Zhu et al. (2008) and Fuentealba et al.
(2011), whereas Guo et al. (2010) have reported a
marginal inhibitory effect using two different in vitro
tests. Silver staining after gel electrophoresis showed
that a blueberry-enriched polyphenol extract significantly inhibited the formation of small aggregates (e.g.
10, 40 kDa), which are considered to be toxic (Fuentealba
et al., 2011). Considering the in vitro results and the results
in transgenic PS1 + APP rodents (Joseph et al., 2003), it
seems that although in vitro tests document antiaggregant
properties of blueberry polyphenols, these might not be
enough to prevent amyloid aggregation in vivo or might
be more potent against the formation of small soluble
aggregates (not observed with the dye techniques usually
used for visualization of amyloid load). Questions on the
metabolism, bioavailability in humans and the ability to
prevent amyloid deposits at the onset of the disease
remain to be answered.
In conclusion, our results demonstrated that the PrB
extract of wild V. angustifolium protect or further
damage cells against H2O2-induced oxidative stress
according to the cellular environment and characteristics
of the particular cell line investigated. Measurements
of cell viability and scavenging of ROS production
after co-treatment with Η2Ο2 and various concentrations of PrB showed that the PrB extract significantly
protected SH-SY5Y at all concentrations tested and
CHOAPP770 only at high concentrations. On the
contrary, co-treatment of HEK293 with H2O2 and
PrB exacerbated further the oxidative damage to cells.
Beyond antioxidant effects in CHOAPP770 cells (it
lowered MDA levels in untreated CHOAPP770 cells
compared with wild CHO cells), PrB extract did not
induce changes in APP processing. These findings
contribute significantly to the ongoing search for compounds with pleiotropic actions against Alzheimer’s
disease and in particular to study of the neuroprotective
properties of blueberries.
Acknowledgement
The authors would like to kindly acknowledge Professor Leonidas
Stefanis from Biomedical Research Foundation of the Academy of
Athens (BRFAA) for providing the human neuroblastoma SH-SY5Y
and stably transfected Chinese Hamster Ovary CHOAPP770 cell lines
and the Wild Blueberry Association of North America for providing
the wild blueberry powder.
Conflict of Interest
The authors have declared that there is no conflict of interest.
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Phytochemical composition of “mountain
tea” from Sideritis clandestina subsp.
clandestina and evaluation of its
behavioral and oxidant/antioxidant effects
on adult mice
Catherine G. Vasilopoulou, Vassiliki
G. Kontogianni, Zacharoula I. Linardaki,
Gregoris Iatrou, Fotini N. Lamari,
Alexandra A. Nerantzaki, et al.
European Journal of Nutrition
ISSN 1436-6207
Eur J Nutr
DOI 10.1007/s00394-011-0292-2
1 23
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Eur J Nutr
DOI 10.1007/s00394-011-0292-2
ORIGINAL CONTRIBUTION
Phytochemical composition of ‘‘mountain tea’’ from Sideritis
clandestina subsp. clandestina and evaluation of its behavioral
and oxidant/antioxidant effects on adult mice
Catherine G. Vasilopoulou • Vassiliki G. Kontogianni • Zacharoula I. Linardaki
Gregoris Iatrou • Fotini N. Lamari • Alexandra A. Nerantzaki •
Ioannis P. Gerothanassis • Andreas G. Tzakos • Marigoula Margarity
•
Received: 20 September 2011 / Accepted: 5 December 2011
Ó Springer-Verlag 2011
Abstract
Purpose The goals of this study were to monitor the
effect of drinking of herbal tea from Sideritis clandestina
subsp. clandestina for 6 weeks on behavioral and oxidant/
antioxidant parameters of adult male mice and also to
evaluate its phytochemical composition.
Methods The phytochemical profile of the Sideritis tea
was determined by liquid chromatography-UV diode array
coupled to ion-trap mass spectrometry with electrospray
ionization interface. The effects of two doses of the herbal
infusion (2 and 4% w/v, daily) intake on anxiety-like state
Catherine G. Vasilopoulou and Vassiliki G. Kontogianni contributed
equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00394-011-0292-2) contains supplementary
material, which is available to authorized users.
C. G. Vasilopoulou Z. I. Linardaki M. Margarity (&)
Laboratory of Human and Animal Physiology,
Department of Biology, University of Patras, 26504 Rio, Greece
e-mail: [email protected]
V. G. Kontogianni A. A. Nerantzaki I. P. Gerothanassis
Department of Chemistry, Section of Organic Chemistry and
Biochemistry, University of Ioannina, 45110 Ioannina, Greece
G. Iatrou
Department of Biology, University of Patras, 26504 Rio, Greece
F. N. Lamari
Department of Pharmacy, University of Patras,
26504 Rio, Greece
A. G. Tzakos (&)
Human Cancer Biobank Center, Department of Chemistry,
Section of Organic Chemistry and Biochemistry,
University of Ioannina, 45110 Ioannina, Greece
e-mail: [email protected]
in mice were studied by the assessment of their thigmotactic behavior. The oxidant/antioxidant status of brain
(-Ce), liver and heart of adult male Balb-c mice following
the consumption of Sideritis tea was also evaluated via the
measurement of malondialdehyde (MDA) and reduced
glutathione (GSH) levels using fluorometric assays. Our
study was further extended to determine the antioxidant
effects of the herbal tea on specific brain regions (cerebral
cortex, cerebellum and midbrain).
Results The identified compounds were classified into several natural product classes: quinic acid derivatives, iridoids,
phenylethanol glycosides and flavonoids. Our results showed
that only the 4% Sideritis tea exhibited anxiolytic-like properties as evidenced by statistically significant (p \ 0.05)
decrease in the thigmotaxis time and increase in the number of
entries to the central zone in comparison with the control
group. Consumption of both tea doses (2 and 4% w/v) elevated
GSH (12 and 28%, respectively, p \ 0.05) and decreased
MDA (16 and 29%, p \ 0.05) levels in brain (-Ce), while liver
and heart remained unaffected. In regard to the effect of herbal
tea drinking (2 and 4% w/v) on specific brain regions, it caused
a significant increase in GSH of cerebellum (13 and 36%,
respectively, p \ 0.05) and midbrain (17 and 36%, p \ 0.05).
Similarly, MDA levels were decreased in cerebellum (45 and
79%, respectively, p \ 0.05) and midbrain (50 and 63%,
respectively, p \ 0.05), whereas cerebral cortex remained
unaffected.
Conclusions Mountain tea drinking prevents anxiety-related behaviors and confers antioxidant protection to rodent’s
tissues in a region-specific, dose-dependent manner, and its
phytochemical constituents are shown for the first time.
Keywords LC-MS/MS Mountain tea Sideritis
clandestina subsp. clandestina Oxidant/antioxidant
parameters Thigmotaxis test
123
Eur J Nutr
Abbreviations
MDA
GSH
TBA
BHT
TCA
LC/DAD/ESI-MSn
NMR
Malondialdehyde
Reduced glutathione
Thiobarbituric acid
Butylated hydroxytoluene
Trichloroacetic acid
Liquid chromatography/diode array
detection/electrospray ion-trap
tandem mass spectrometry
Nuclear magnetic resonance
Introduction
The neurotoxic effects of oxidative stress and the consequent neurodegeneration in specific brain areas have been
proposed as causal factors in Alzheimer’s disease, Parkinsonism and aging process [1] The brain is the most
susceptible organ to oxidative damage due to its high
oxygen demand, high lipid content, especially polyunsaturated fatty acids, the abundance of redox-active transition
metal ions, the low activity of antioxidant defense system
and the reduced capacity for cellular regeneration [1].
Polyphenolic compounds supplied by nutritional sources
[2] such as fruits, vegetables, wine and tea have displayed a
wide range of beneficial biological actions [3], many of
which have been attributed to their strong antioxidant
properties [4].
Anxiety is a psychological and physiological state
characterized by somatic, emotional, cognitive and
behavioral components. Recent studies demonstrate a link
between oxidative stress and anxiety-related behavior [5].
Some of these studies suggest that oxidative stress causes
anxiety-related behaviors in humans with panic disorders
and especially that oxidative metabolism can affect the
regulation of anxiety [6], and others support a cause–effect
relationship in humans and animal models but do not
explain the underlying mechanisms [7].
The genus Sideritis (Lamiaceae) comprises about 140
species distributed in several countries of the Mediterranean region [8]. Sideritis species have been reported to
present an array of biological activities, including antiinflammatory and antinociceptive [9], antimicrobial [10]
and antiulcer [11] activities. Interestingly, aqueous extracts
derived from Sideritis species have been proposed to form
the basis to design ‘‘functional foods’’ for the prevention of
osteoporosis [12]. Furthermore, in vitro studies have shown
moderate antioxidant activity for S. javalambrensis,
S. raeseri, S. euboea, S. perfoliata subsps. perfoliata and
S. libanotica ssp. linearis that was mainly attributed to
flavonoids [13–17]. Rats drinking a tea from S. caesarea
123
for 50 days were protected against chemical-induced oxidative injury [18]. We have previously found that consumption of a herbal tea from a different Sideritis taxon
(clandestina subsp. peloponnesiaca) enhanced the antioxidant defense of the adult rodent brain in a region-specific
manner; however, the phytochemical composition remained
unknown [19].
The aims of the present study are (1) to characterize the
composition of the aqueous extract of the herbal tea from
Sideritis clandestina subsp. clandestina using LC/DAD/
ESI-MSn analysis, (2) to evaluate the effect of a 6-week
consumption of 2 and 4% w/v of a herbal tea (commonly
consumed tea doses) from the same taxon on the levels of
anxiety and (3) to determine the oxidant/antioxidant
activity of peripheral and brain tissues of adult mice by
measuring malondialdehyde (MDA, index of lipid peroxidation) and reduced glutathione (GSH). This is the first
study on the phytochemical composition of S. clandestina
subs. clandestina ever, the first study on the composition of
the aqueous and not organic solvent extract of the herbal
tea from this taxon and one of the few studies to investigate
its antioxidant effects in vivo after a period of drinking.
With regard to the latter, we aimed to investigate whether
the beneficiary effects, recorded after the consumption of
another Sideritis taxon [19], would present a similar pattern
or are taxon specific and whether these are dose dependent.
Materials and methods
Plant material, reagents and standards
The plant Sideritis clandestina subsp. clandestina used in
the study was collected from Mainalo mountain (Central
Peloponnese) between July and August (2008). Taxonomic
identification of the plant material was carried out in the
Division of Plant Biology, Department of Biology, University of Patras, by Prof. G. Iatrou, and a voucher specimen has been deposited in the local Herbarium.
MDA, GSH, TBA, BHT, TCA, o-phthalaldehyde and
Chromasolv Plus acetonitrile and water for HPLC (99.9%)
were obtained from Sigma–Aldrich. Analytical-grade acetic acid was provided by Merck (Darmstadt, Germany), and
quinic acid (98%) was obtained from Aldrich (Steinhem,
Germany).
Sample preparation and extraction
All parts of dry material (flowers, leaves and stems) in
equal quantities were used for the extraction. For the
preparation of the tea infusions, 2 or 4 g of leaves, flowers
and stems of Sideritis taxa was extracted with 100 mL of
boiling water for 5 min, in order to simulate actual brewing
Eur J Nutr
conditions for tea consumed by human adults, and filtered
after cooling. The filtrate was made to the volume of
100 mL. For the animal studies, the infusions were prepared daily. For the characterization of the phytochemical
composition of the herbal tea, it was lyophilized and a
residue (nice green powder) of 0.294 g (for the 2 g) was
obtained.
Assignment of compounds present in the extract was
determined on the basis of 2D 1H–1H COSY, 1H–13C
HSQC and HMBC spectra. Relevant compound quantification was performed on the basis of the recorded integrals
of resonance absorptions that are characteristic for specific
compounds in the 1D 1H-NMR spectrum of the extract.
Animals
LC-MS analysis
To evaluate the phytochemical composition of the aqueous
Sideritis extract, we used liquid chromatography-UV diode
array coupled to ion-trap mass spectrometry with electrospray ionization interface (LC/DAD/ESI-MSn). All LCMSn experiments were performed on a quadrupole ion-trap
mass analyzer (Agilent Technologies, model MSD trap SL)
retrofitted to a 1,100 binary HPLC system equipped with a
degasser, autosampler, diode array detector and electrospray ionization source (Agilent Technologies, Karlsruhe,
Germany). All hardware components were controlled by
Agilent Chemstation Software.
The Sideritis extract was dissolved in acetonitrile–water
50–50% to the desired concentration (4 mg mL-1 of
extract). A 20-lL aliquot was filtered (0.45 lm) and
injected into the LC-MS instrument. Separation was
achieved on a 25 cm 9 4.6 mm i.d., 5 lm Altima C18
analytical column (Alltech, Deerfield, USA), at a flow rate
of 0.6 mL min-1, using solvent A (water/acetic acid, 99.9:
0.1 v/v) and solvent B (acetonitrile). The gradient used in
this experiment was as follows: 0–5 min, 95–92% A;
5–15 min 92% A; 15–20 min 92–75% A; 20–40 min
75–50% A; 40–50 min 50–20% A; 50–60 min 20–95% A.
The UV/Vis spectra were recorded in the range of
200–400 nm, and chromatograms were acquired at 254,
280 and 330 nm.
Both precursor and product (MS2 and MS3) ions scanning of the phytochemicals were monitored between m/z 50
and m/z 1,000 in negative polarity. The ionization source
conditions were as follows: capillary voltage, 3.5 kV;
drying gas temperature, 350 °C; nitrogen flow and pressure, 12 L min-1 and 12 psi, respectively. Maximum
accumulation time of ion trap and the number of MS repetitions to obtain the MS average spectra were set at 30 ms
and 3, respectively.
NMR analysis
Nuclear magnetic resonance (NMR) experiments were
performed at 298 K on a Bruker AV-500 spectrometer
equipped with a TXI cryoprobe (Bruker BioSpin, Rheinstetten, Germany). Samples were dissolved in 0.5 mL
DMSO-d6 and transferred to 5-mm NMR tubes. The NMR
system was controlled by the software TopSpin 2.1.
Male, 3- to 4-month-old Balb-c mice (25–30 g BW) were
kept in polyacrylic cages (38 cm 9 23 cm 9 10 cm) (8
per cage) under constant temperature (23–25 °C) and relative 50–60% humidity, with alternating 12-h light and
dark cycles and ad libitum access to food. The mice were
randomly divided into three groups consisting of sixteen
animals each. Group I mice (n = 16) served as controls
and received water ad libitum. Group II and III mice
(n = 16/group) had ad libitum access to the tea from
S. clandestina subsp. clandestina 2 and 4% (w/v), respectively, for a period of 6 weeks. Each of the above groups
was divided into two subgroups (n = 8/subgroup), which
were used for the isolation of a. brain, liver, heart and b.
specific brain tissues (cerebral cortex, cerebellum and
midbrain), respectively. The body weight, food and water
or infusion intake were measured weekly in all the experimental groups throughout the treatment period. All procedures were in accordance with the Greek National Laws
(Animal Act, PD 160/91).
Thigmotaxis test
At the end of the treatment period, animals were submitted
to the open-field behavioral thigmotaxis test in order to
evaluate the effects of herbal tea consumption on anxietyrelated responses [20]. Thigmotaxis (walking close the
walls of the apparatus), which was assessed by the time that
mice spent close to the walls (\8 cm away from the walls),
and the number of entries in the central zone of the open
field are considered as an index of anxiety. All training and
testing sessions were carried out during the light phase
between 08:00 and 14:00 a.m.
Tissue preparation
One day after the behavioral test, all mice were killed by
light ether anesthesia and brain (-Cerebellum) [referred as
brain (-Ce)], liver, heart and specific brain regions (cerebral
cortex, cerebellum and midbrain) were rapidly removed,
weighted and stored at -80 °C until use. All tissues were
homogenized (10% w/v) using a glass-Teflon homogenizer
(Thomas, Philadelphia, USA, No B 13957) in ice-cold
30 mM Na2HPO4, pH 7.6. The homogenates were then
centrifuged for 20 min at 15.000g at 4 °C in a Heal Force,
123
Eur J Nutr
Neofuge 13R centrifuge. Supernatants were collected and
used for the determination of GSH and MDA levels. Protein levels were evaluated using the Bradford assay.
treated group from control and between the two treated
groups.
Determination of MDA
Results
MDA, one of the better-known secondary products of lipid
peroxidation, was determined fluorometrically after reaction with thiobarbituric acid (TBA), as previously described [21]. The assay showed good linearity (0.05–10 lL,
y = 6.6034 9 -0.5959, R2 = 0.9993) and sensitivity. The
level of MDA was expressed as lmol/g of tissue protein.
All determinations were carried out at least three times and
in triplicates.
Phytochemical profile and identification of the main
constituents of aqueous Sideritis extract
Determination of GSH
GSH content of the brain and peripheral tissues was estimated fluorometrically after reaction with o-phthalaldehyde, as previously described [22]. A good linearity
was obtained for GSH in the range of 0.5–100 lM
(y = 1.2353 9 -3.46, R2 = 0.9969). The levels of GSH
were expressed as lmol/g of tissue protein. All determinations were carried out at least three times and in
triplicates.
The results are expressed as mean ± SE. Statistical analysis was performed with the GraphPad Instat 3 software
using the nonparametric Mann–Whitney test for evaluating
statistically significant differences (p \ 0.05) of each
Fig. 1 Total ion chromatogram of Sideritis clandestina aqueous extract
1 Quinic acid derivative, 2 Quinic acid derivative, 3 Melittoside
derivative, 4 Melittoside derivative, 5 b-Hydroxyverbascoside or
b-Hydroxyisoverbascoside, 6 Apigenin 7-O-allosyl(1 ? 2)glucoside,
7 Isoscutelarein 7-O-[6000 -O-acetyl]-allosyl(1 ? 2)glucoside, 8 Apigenin 7-O-[6000 -O-acetyl]-allosyl(1 ? 2)glucoside, 9 Apigenin 7-O-[600 -O-
123
The total ion chromatogram of the investigated extract is
illustrated in Fig. 1. The LC/DAD/ESI-MSn analysis of the
aqueous extract led to the separation and identification of
the majority of the extract components, seventeen (17) in
total, that belong to several classes of phytochemicals:
quinic acid, melittoside, phenylpropanoids and flavonoid
derivatives. Specifically, two quinic acid derivatives along
with two melittoside derivatives, two phenylethanoid glycosides (b-hydroxyverbascoside or b-hydroxyisoverbascoside is described for the first time in Sideritis species) and
one flavonoid 7-O-diglycoside were identified along with
six acetylated flavonoid 7-O-diglycosides of apigenin and
isoscutellarein and four isomers of apigenin 7-O-(coumaroyl)glucopyranoside (See Table 1 for a detailed list of
compounds and Figures S1–S9 for representative MS
spectra of selected compounds).
The identification of the compounds was based on their
fragmentation pattern as has been documented in the literature [23–29]. On the basis of this methodology, we were
not only able to determine the presence of well-known
phenolic compounds in the extract, but also able to identify
the two iridoid glycosides and quinic acid derivatives that
could not be attributed to any known compound.
acetyl]-allosyl(1 ? 2)glucoside, 10 Martynoside, 11 Isoscutellarein
7-O-[2000 ,6000 -O-diacetyl]-allosyl(1 ? 2)glucoside, 12 Apigenin 7-O[2000 ,6000 -O-diacetyl]-allosyl(1 ? 2)glucoside, 13 Apigenin 7-O-acetylcoumaroyl-allosyl(1 ? 2)glucoside, 14–17 Apigenin 7-O- (coumaroyl)glucopyranoside isomers
Eur J Nutr
Table 1 Compounds identified in Sideritis clandestina aqueous extract
Peak
Rt (min)
[M–H]- (m/z)
–MS2 [M–H]- (m/z) (%)
–MS3 [base peak]- (m/z) (%)
Compounds
1
4.2
421
191 (100)
389 (100), 127 (17), 173 (14)
Quinic acid derivative
2
4.2
533
191 (100)
173 (100), 127 (64), 150 (67)
Quinic acid derivative
3
5.9
654, 583, 523
523 (100), 179(6), 565 (8)
179 (100), 361 (30), 343 (24), 463 (11)
Melittoside derivative
4
11.9
583, 523, 665
523 (100), 565 (17), 179 (8)
179 (100), 343 (29), 361 (24), 463 (14)
Melittoside derivative
10
26.7
449
389 (100), 167 (16), 329 (11)
329 (100), 161 (30), 179 (15), 251 (11)
Unknown
5
27.1
639
621 (100), 459 (8)
459 (100), 179 (13)
b-Hydroxyverbascoside or
b-hydroxyisoverbascoside
20
28.8
579
339 (100), 327 (44)
324 (100), 161 (30), 179 (15)
Unknown
6
29.3
593
269 (100), 431 (21)
269 (100)
Apigenin 7-O-allosyl(1 ? 2)
glucoside,
7
29.8
651
609 (100), 285 (4)
285 (100), 447 (28)
Isoscutelarein 7-O-[6000 -O-acetyl]allosyl(1 ? 2)glucoside
8
30.7
635
593 (100), 269 (7)
269 (100), 431 (24)
Apigenin 7-O-[6000 -O-acetyl]allosyl(1 ? 2)glucoside
9
31.9
431, 635
269 (100), 593 (51),
515 (8), 431 (7)
269 (100), 431 (26)
Apigenin 7-O-[600 -O-acetyl]allosyl(1 ? 2)glucoside
30
32.1
783, 665
607 (100), 651 (53), 737 (41)
329 (100)
Unknown
10
33.6
651
475 (100), 505 (31), 457 (18)
161 (100), 329 (84)
Martynoside
11
34.1
693
651 (100), 633 (59)
285 (100), 489 (58), 609 (14)
Isoscutellarein 7-O-[2000 , 6000 -Odiacetyl]-allosyl(1 ? 2)
glucoside
40
34.5
595
269 (100), 474 974), 513 (19)
269 (100)
Unknown
50
35.4
733
698 (100), 714 (92), 634 (39)
714 (100), 673 (59)
Unknown
12
36.5
677
635 (100), 269 (63)
269 (100), 593 (31), 473 (67)
Apigenin 7-O-[2000 , 6000 -Odiacetyl]-allosyl(1 ? 2)
glucoside
13
37.6
781
739 (100), 721 (69),
269 (53), 635 (40), 575 (12)
269 (100), 593 (64), 577 (16), 431 (14)
Apigenin 7-O-acetyl-coumaroylallosyl(1 ? 2)glucoside
14
38.7
577
269 (100), 431 (1), 413 (1)
225 (100), 269 (71), 149 (59), 117 (26)
Apigenin 7-O-(coumaroyl)
glucopyranoside
15
39.2
577
269 (100)
225 (60), 269 (100), 149 (43)
glucopyranoside
16
40.4
577
269 (100)
225 (100), 269 (64), 149 (43)
glucopyranoside
17
42.5
577
269 (100), 431 (15), 413 (15)
225 (42), 269 (43), 149 (100)
glucopyranoside
Specifically, the unknown iridoid glycosides were compounds 3 and 4 (relevant peak fractions in Fig. 1). Compound 3 gave deprotonated molecular ions [M–H]- at m/z
654 (100%), 583 (90%), 613 (76%) and 523 (67%) (Figure
S3). The fragmentation at m/z 583 showed the presence of
one main fragment at m/z 523 (100%), 565 (8%) and 179
(6%). The MS3 spectra of the ion at m/z 523 gave ions at
m/z 179 (100%), 361 (30%), 343 (24%), 463 (11%) and 161
(11%). The ion at m/z 523 could belong to melittoside, and
compound 3 could be attributed to a melittoside derivative.
In the MS3 spectra, the ion at m/z 361 corresponds to the
loss of 162 amu (glucose) and the ion at m/z 179 corresponds to the loss of 182 amu, which are characteristic
neutral losses for iridoid glycosides [25]. Melittoside has
been isolated from different species of genus Sideritis [30],
while recently a new melittoside derivative was isolated
from an extract of the species Sideritis lanata L. [31].
Similarly, compound 4 eluting at 11.9 min with isocratic
conditions gave deprotonated molecular ions [M–H]- at
m/z 583 (100%) and 523 (64%) (Figure S4). The fragmentation at m/z 583 showed the presence of one main fragment at m/z 523 (100%), 565 (17%) and 179 (8%) again,
while the MS3 spectra of the ion at m/z 523 gave ions at m/z
179 (100%), 343 (29%), 361 (25%), 463 (14%), 439 (10%)
and 161 (9%). Compounds 3 and 4 must have related
structures, as they show similar product ions.
In order to ascertain the structure of compound 3, as it
was the main constituent in the extract and since there is
lack of available literature data regarding the fragmentation
pattern of melittoside and its derivatives, we isolate it
123
Eur J Nutr
through preparative liquid chromatography. NMR investigation (2D 1H–1H COSY, 1H–13C HSQC and 1H–13C
HMBC) of the isolated compound confirmed that the base
configuration was melittoside (Table S1), in accordance
with literature data [32]. The full assignment is not completed due to extensive resonance overlapping problems.
Furthermore, the compound apigenin 7-O-acetylcoumaroyl-allosyl(1 ? 2)glucoside (compound 13) was
identified and was characterized in the extract for the first
time. The deprotonated molecular ion [M–H]- of compound 13 was detected at m/z 781 (Figure S9). After MS2
of the compound, the base peak at m/z 739 (100%) was
detected corresponding to the loss of an acetyl group, along
with peaks at m/z 721 (69%), 269 (53%), 635 (40%) and
575 (12%). The MS3 spectra of the ion at m/z 721 yielded
the ion at m/z 269 (100%), 593 (64%), 577 (16%) and 431
(14%). The peak at m/z 593 was probably derived from the
loss of a coumaroyl or rhamnosyl moiety (146 amu). The
fragment at m/z 431 (14%) related to [M–H-308]- was
derived from the loss of a glucose esterified with coumaric
acid. The base peak [(M–H)-324]- that was detected
indicated an O-glycosylation on a phenolic hydroxyl with a
dihexoside ([Y70]-) [35]. The occurrence of loss of 162 amu
(m/z 577, 16%) was indicative of 1 ? 2 glycosylation
between both sugars. This compound could be tentatively
characterized as apigenin 7-O-acetyl-coumaroyl-allosyl(1 ? 2)glucoside, and it is detected for the first time.
Among the aforementioned compounds, the main constituents in the extract were found to be melittoside and quinic
acid derivatives (Fig. 1). This was evident from the relevant integrals of characteristic resonance absorptions for
the specific compounds in the 1H-NMR spectrum of the
crude herbal tea (Figure S10).
with the controls. The first 5 min of the testing session in
both treated animal groups displayed no difference in the
values of the above parameters when compared with the
control group, as this time interval is a period of accommodation [20].
Evaluation of the antioxidant effects of Sideritis
infusion intake on adult mice brain and peripheral
tissues
Differences in the MDA and GSH levels among brain (-Ce)
and peripheral tissues as well as among the three brain
regions were observed in control mice. These differences
are in accordance with previous studies [19, 33] and show
that tissues exhibit a significant variation in the distribution
of antioxidant defense [34] being closely related to the
composition and the functional role of each tissue.
As illustrated in Table 2, the consumption of both doses
(2 and 4%, w/v) of Sideritis herbal tea caused a significant
decrease in MDA levels (16 and 29%, respectively) and an
enhancement in the GSH levels (12 and 28%, respectively)
in the brain (-Ce) in comparison with the control group,
whereas liver and heart remained unaffected. Moreover,
Sideritis-treated group that received the higher tea dose
(4% w/v) exhibited significant further alterations in the
Evaluation of the anxiolytic-like effects of Sideritis
infusion intake on adult mice
Due to the well-studied beneficial effects in human health
of the basic structures of the main phytochemical constituents revealed in the studied extract, we could hypothesize
that the Sideritis clandestina subsp. clandestina extract
could also confer anxiolytic behavioral effects and also
antioxidant defence in brain. Figure 2a and b illustrates the
measurement of the thigmotaxis time and the number of
central entries to the open field at 5-min time intervals
during the 30-min testing session (except of the first
5 min). From this, it became evident that mice receiving
4% (w/v) herbal tea displayed statistically significant
decrease (10–13%) in the thigmotaxis time and increase
(70–180%) in the number of entries in the central zone
compared with the control group. Similar but not statistically significant alterations in the above parameters were
observed in Sideritis 2% (w/v)-treated group in comparison
123
Fig. 2 Evaluation of a the thigmotaxis time and b number of entries
in the central area during the first 30 min in the open field.
Immediately after the introduction of naive mice into the open field
of thigmotaxis test, the thigmotaxis time and number of central entries
were measured during six consecutive periods of 5 min each. Data are
means ± SE. of 8 mice per group. *Statistical difference between
control and Sideritis 4%-treated mice
Eur J Nutr
above oxidant/antioxidant indices of the brain (-Ce) in
comparison with the treated mice that received the lower
dose (2% w/v).
Taking into consideration that brain was the only tissue,
which was influenced by the Sideritis tea intake, we further
extended our study to determine the antioxidant effects of
the herbal tea on specific brain regions, such as the cerebral
cortex, cerebellum and midbrain. Our results showed that
drinking of the herbal tea from S. clandestina subsp.
clandestina for 6 weeks affected significantly the oxidant/
antioxidant status of mice brain regions in a region- and
dose-dependent manner. Particularly, as presented in
Table 3, intake of both doses of the herbal tea decreased
significantly the MDA levels of cerebellum (45 and 79%,
respectively) and midbrain (50 and 63%, respectively), in
comparison with the control mice, whereas cerebral cortex
MDA levels remained unaffected. The consumption of the
high dose (4%) of Sideritis infusion caused significant
further reduction in midbrain and cerebellum lipid peroxidation levels, as compared to the low dose (2%). Furthermore, consumption of the low and high herbal tea dose
increased significantly the GSH levels of cerebellum by 13
and 36% and of midbrain by 17 and 36%, respectively, in
comparison with the control group. Neither of two tea
doses affected the GSH content in the cerebral cortex.
Regular measurement of animal liquid and food intake
exhibited no difference between the treated animal groups
and the controls. Similarly, there was no difference in
animal body weight between control and experimental
group throughout the 6-week treatment period.
Discussion
The current study confirmed that drinking of the herbal tea
from S. clandestina subsp. clandestina for 6 weeks affected
significantly the oxidant/antioxidant status of mice brain
regions in a region- and dose-dependent manner. Specifically, the significant alterations in MDA and GSH levels of
the mice brain that accompanied the herbal tea intake
exhibited a region specificity as cerebellum and midbrain
were significantly affected in contrast to the cerebral cortex
that showed remarkable stability. Significant stability in
their oxidant/antioxidant status also displayed the liver and
the heart. Accordingly, in our previous study, mice drinking a herbal tea (4% w/v) from another Sideritis taxon,
Table 2 Effect of herbal tea consumption (2 and 4% [w/v]) on MDA and GSH levels of mice tissues
Tissues
lmol MDA/g protein
lmol GSH/g protein
Control mice
Sideritis 2%
w/v mice
Sideritis 4%
w/v mice
Control mice
Sideritis 2%
w/v mice
Sideritis 4%
w/v mice
Brain (-Ce)
5.24 ± 0.17
4.42 ± 0.13*
3.74 ± 0.16*, 11.80 ± 0.47
13.18 ± 0.41*
14.90 ± 0.38*, (;16%)
(;29%) [;15%]
(:12%)
(:28%) [:13%]
Liver
1.59 ± 0.16
1.52 ± 0.09
1.48 ± 0.09
7.06 ± 0.28
6.91 ± 0.17
6.93 ± 0.19
Heart
1.19 ± 0.16
0.95 ± 0.09
0.96 ± 0.11
5.05 ± 0.28
5.11 ± 0.15
4.77 ± 0.28
Data are mean ± SE (n = 8). Statistical analysis was performed by Mann–Whitney test. Percentage decrease (;) of MDA values or increase (:)
of GSH values as to the control group. Percentage decrease [;] of MDA values or increase [:] of GSH values as to the Sideritis 2%-treated group
* Statistically significant difference between control and Sideritis-treated mice (p \ 0.05)
Statistically significant difference between Sideritis 2% and Sideritis 4%-treated mice (p \ 0.05)
Table 3 Effect of herbal tea consumption (2 and 4% [w/v]) on MDA and GSH levels of mice brain regions
Tissues
lmol MDA/g protein
Control mice
Sideritis 2%
w/v mice
lmol GSH/g protein
Sideritis 4%
w/v mice
Control mice
Sideritis 2%
w/v mice
Sideritis 4%
w/v mice
Cerebral cortex
3.13 ± 0.22
2.95 ± 0.09
2.70 ± 0.10
12.47 ± 0.42
12.21 ± 0.41
12.28 ± 0.61
Cerebellum
2.10 ± 0.13
1.15 ± 0.04*
0.43 ± 0.008*, 15.54 ± 0.70
17.52 ± 0.75*
21.07 ± 0.98*, Midbrain
1.45 ± 0.08
(;45%)
(;79%) [;62%]
0.72 ± 0.035*
0.53 ± 0.04*, (;50%)
(;63%) [;35%]
13.95 ± 0.44
(:13%)
(:36%) [:20%]
16.27 ± 0.43*
19.01 ± 0.86*, (:17%)
(:36%) [:17%]
Data are mean ± SE (n = 8). Statistical analysis was performed by Mann–Whitney test. Percentage decrease (;) of MDA values or increase (:)
of GSH values as to the control group. Percentage decrease [;] of MDA values or increase [:] of GSH values as to the Sideritis 2%-treated group
* Statistically significant difference between control and Sideritis-treated mice (p \ 0.05)
Statistically significant difference between Sideritis 2% and Sideritis 4%-treated mice (p \ 0.05)
123
Eur J Nutr
S. clandestina subsp. peloponnesiaca, exhibited lower
MDA and higher GSH levels in midbrain and cerebellum
compared with their control littermates; cortex and liver
remained unaffected [19]. The remarked stability of the
cerebral cortex toward changes in its oxidant/antioxidant
status has been also documented in the study of Haque
et al. [35], where green tea catechins orally administered to
rats exerted strong antioxidant effects in the hippocampus
but not in the cerebral cortex. Conclusively, drinking of the
herbal tea exerted a dose-dependent depletion of brain
(cerebellum and midbrain) MDA levels with a simultaneous significant elevation of its GSH content. Since MDA,
the end product of tissue lipid peroxidation, is highly
reactive and responsible for cytotoxic effects, whereas
glutathione is a tripeptide, which acts as a free radical
scavenger preventing tissue damage, our findings support a
neuroprotective action of the herbal tea through the inhibition of brain oxidative damage and the enhancement of
its endogenous antioxidant defense.
In addition, thigmotaxis-testing results clearly show for
the first time that Sideritis tea (4% w/v) consumption by
adult mice exerts anxiolytic-like effects, although additional behavioral assessment is necessary. Therefore, the
observed significant reduction of the anxious behavior that
followed Sideritis tea consumption was accompanied by a
significant antioxidant reinforcement of the mouse brain.
There are also other reports that establish a strong link
between oxidative stress and anxiety-like behaviors in
rodents, but the underlying mechanisms are still unclear
[5–7]. Despite the physiological role of cerebellum and
midbrain in the control of motor functions, the anxiolyticlike effects of the herbal tea in the present study were
combined with a significant elevation in the antioxidant
capacity of the specific brain areas, while cerebral cortex
remained unlikely stable. Nevertheless, the implication of
cerebellum and midbrain in anxiety disorders has been
recently demonstrated [36, 37]. Since behavior constitutes
a complex brain process, future studies examining multiple
biochemical indices of brain oxidant status and neurotransmitter systems may extend the current knowledge
about anxiolytic-like and antioxidant properties of Sideritis
clandestina tea.
In order to get a deeper understanding of the mechanism
of action of the Sideritis clandestina tea, the network of
interactions between individual constituents and cellular
components needs to be characterized [38]. However, the
identification of specific basic structures of phytochemicals, for which their activities have been investigated in the
past, clearly supports the determined capacity of the studied extract to confer enhanced antioxidant activity, cognition-enhancing activities and neuroprotective actions. The
biological and pharmacological properties of numerous
derivatives bearing the same basic structure, as the main
123
constituents identified in the current extract (quinic acid,
melittoside (iridoid) and apigenin (flavonoid)), have been
extensively studied in the literature. For instance, iridoids
have shown to enhance the antioxidant capacity in primary
hippocampal neurons by upregulating the antioxidative
enzyme heme oxygenase-1 via the PI3 K/Nrf2-signaling
[39]. Furthermore, another iridoid glycoside, loganin, was
very recently found to improve learning and memory
impairments induced by scopolamine in mice [40]. The
iridoids E-harpagoside and 8-O-E-p-methoxycinnamoylharpagide significantly improved the impairment of
reference memory in scopolamine-treated mice through
both anti-acetylcholinesterase and antioxidant mechanisms
[41]. The iridoid 8-O-E-p-methoxycinnamoylharpagide
and its aglycone, harpagide, were very potent to protect
primary cultured neurons against glutamate-induced oxidative stress primarily by acting on the antioxidative
defense system and on glutamatergic receptors, respectively [42]. Concerning the activities of phytochemicals
bearing the quinic acid basic structure, it has been shown to
move through the blood–brain barrier and to present cognition-enhancing activities [43]. They have also demonstrated the neuroprotective effects through the upregulation
of PGK1 expression and ATP production activation [44] as
well as due to their metal chelation properties for metallotoxins (aluminum) [45]. Furthermore, quinic acid derivatives displayed neuroprotective action on amyloid Abinduced PC12 cell toxicity and neurotrophic activity by
promoting neurite outgrowth in PC12 cells [46]. Moreover,
they could effectively inhibit the formation of advanced
glycation end products (AGEs) and rat lens aldose reductase
[47]. In addition to these phytochemicals, the identified basic
structure of apigenin in the extract has been shown to possess
a variety of pharmacological actions on the central nervous
system as well as antidepressant-like behavioral and neurochemical effects [48]. It also protects brain neurovascular
coupling against amyloid-b25–35-induced toxicity in mice
[49] and inhibits the production of nitric oxide (NO) and
prostaglandinE2 (PGE2) in microglia and inhibits neuronal
cell death in a middle cerebral artery occlusion–induced focal
ischemia mice model [50]. Furthermore, apigenin was found
to inhibit the oxidative stress–induced macromolecular
damage in N-nitrosodiethylamine (NDEA)-induced hepatocellular carcinogenesis in rats [51], and also it can confer a
protective role in the status of lipid peroxidation and antioxidant defense against hepatocarcinogenesis in rats [52].
In conclusion, the remarked anxiolytic-like effects and
in vivo antioxidant capacity of Sideritis clandestina subsp.
clandestina infusion accompanied by the phytochemical
analysis of its aqueous extract support the beneficial role of
regular drinking of herbal mountain tea from Sideritis taxa
in the prevention of neurobehavioral diseases and the deleterious effects of aging.
Eur J Nutr
Acknowledgments We are grateful to Dr. Garbis S. for helpful
discussions and comments. This work was partially supported by
BIOFLORA, network of University of Patras and Esthir-Gkani
Foundation, Ioannina. Special thanks are given to the Mass Spectrometry Unit of University of Ioannina for providing access to LCMS/MS facilities and Mrs. Theodora Lamari for collecting the plant
material. NMR data were recorded in the NMR center of the University of Ioannina.
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Article
pubs.acs.org/JAFC
Saffron as a Source of Novel Acetylcholinesterase Inhibitors:
Molecular Docking and in Vitro Enzymatic Studies
George D. Geromichalos,*,† Fotini N. Lamari,*,§ Magdalini A. Papandreou,⊗ Dimitrios T. Trafalis,⊥
Marigoula Margarity,⊗ Athanasios Papageorgiou,‡ and Zacharias Sinakos△
†
Department of Cell Culture−Molecular Modeling and Drug Design, Symeonidion Research Center, Theagenion Cancer Hospital,
Thessaloniki, Greece
§
Laboratory of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, University of Patras, Rion 26504,
Greece
⊗
Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Patras 26504, Greece
⊥
Department of Pharmacology, Medical School, University of Athens, Athens, Greece
‡
Department of Experimental Chemotherapy, Symeonidion Research Center, Theagenion Cancer Hospital, Thessaloniki, Greece
△
Honored Professor of Haematology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece
S Supporting Information
*
ABSTRACT: Inhibitors of acetylcholine breakdown by acetylcholinesterase (AChE) constitute the main therapeutic modality
for Alzheimer’s disease. In the search for natural products with inhibitory action on AChE, this study investigated the activity of
saffron extract and its constituents by in vitro enzymatic and molecular docking studies. Saffron has been used in traditional
medicine against Alzheimer’s disease. Saffron extract showed moderate AChE inhibitory activity (up to 30%), but IC50 values of
crocetin, dimethylcrocetin, and safranal were 96.33, 107.1, and 21.09 μM, respectively. Kinetic analysis showed mixed-type
inhibition, which was verified by in silico docking studies. Safranal interacts only with the binding site of the AChE, but crocetin
and dimethylcrocetin bind simultaneously to the catalytic and peripheral anionic sites. These results reinforce previous findings
about the beneficial action of saffron against Alzheimer’s disease and may be of value for the development of novel therapeutic
agents based on carotenoid-based dual binding inhibitors.
KEYWORDS: acetylcholinesterase, dual inhibitors, in silico, saffron, mixed inhibition, crocetin, safranal
■
INTRODUCTION
Alzheimer’s disease (AD) is a multifactorial dementia
characterized by cerebral accumulation of extracellular
amyloid-β (Aβ) protein aggregates (senile plaques) and
intraneuronal hyperphosphorylated twisted filaments of tau
protein (neurofibrillary tangles) in brain areas associated with
learning and memory (e.g., cortex, hippocampus, nucleus
basalis of Meynhert),1 resulting, thus, in profound memory
disturbances and irreversible impairment of cognitive function.
The latter was ascribed primarily to the loss of cholinergic
neurons in those areas, due to Aβ and tau buildups, and led to
the formation of “the cholinergic hypothesis” of AD.2,3
The abnormalities in cholinergic metabolism seen in AD are
not viewed as the cause of the disorder, but cholinergic
involvement is significant because it is universal, correlates with
cognitive defects, and is one of the few pathophysiologic
phenomena that can be addressed with currently approved
treatment options, such as cholinesterase inhibitors (ChEIs)
[e.g., tacrine (THA), rivastigmine, and galanthamine (GNT)].
ChEIs enhance cognitive function by inhibiting the catabolism
of the neurotransmitter acetylcholine (ACh) by acetylcholinesterase (AChE) increasing the action of ACh in the synapse.
Although various clinical studies document the beneficial effects
of current AChE inhibitors on cognition and behavior, their
benefits are modest, suffer from short half-lives, and show
© 2012 American Chemical Society
serious side effects caused by activation of peripheral
cholinergic systems.4−6 Potent AChE inhibitors belong to the
class of alkaloids, due to the requirement for positively charged
quaternary ammonium in the structure, such as GNT and
physostigmine.
AChE, apart from its involvement in cholinergic synaptic
transmission, is also known to accelerate the aggregation of Aβ
during the early stages of AD, primarily via interactions through
its peripheral anionic binding site (PAS).7−9 In light of these
data, the search for new AChE inhibitors for AD now includes
the search of dual binding molecules, being able to interact
simultaneously with both the PAS and the catalytic subsite of
AChE.10−12
Advances in X-ray crystallography and site-directed mutagenesis have enabled the detailed mapping of AChE’s active
center. Human AChE (EC 3.1.1.7) is a globular protein
containing a 20 Å deep groove (gorge), which includes the
following loci: (1) the acyl-binding pocket Phe295(288) and
Phe297(290) (values in parentheses represent the amino acid
numbering positions in Torpedo californica AChE) at the base
Received:
Revised:
Accepted:
Published:
6131
February 9, 2012
May 29, 2012
June 1, 2012
June 1, 2012
dx.doi.org/10.1021/jf300589c | J. Agric. Food Chem. 2012, 60, 6131−6138
Journal of Agricultural and Food Chemistry
Article
Figure 1. Chemical structures of AChE’s substrate (acetylthiocholine, ATCI) and inhibitors (tacrine, THA, and galanthamine, GNT) and saffron’s
bioactive constituents/metabolites (crocin, CRC; crocetin, CRT; dimethylcrocetin, DMCRT; and safranal, SFR).
molecular modeling studies to be conducted using the
coordinates of the published eel AChE X-ray structure, as
stated by Khabid et al.27 The chemical structures of all saffron
constituents tested, that is, crocin (CRC), crocetin (CRT),
dimethylcrocetin (DMCRT)m and safranal (SFR), as well as of
AChE’s substrate acetylthiocholin iodide (ATCI) and of AChE
known inhibitors THA and GNT, are shown in Figure 1. CRC,
a natural carotenoid, is the diester formed by the disaccharide
gentiobiose and CRT. CRT is the main metabolite in humans,
whereas the synthetic analogue of DMCRT was used to
augment the study of carotenoid structure−activity relationships, that is, to investigate the role of CRT carboxyl groups.
SFR is a monoterpene aldehyde and the main component of
the essential oil of saffron.
of the gorge; (2) the esteratic locus, which consists of two
subsites, the oxyanion hole [Gly120(118), Gly121(119), and
Ala204(201)] and the active site catalytic serine hydrolase triad
[His447(440), Glu334(327), and Ser203(200)]; (3) the
quaternary ammonium binding locus [Trp86(84)]; and (4)
the PAS site [Tyr72(70), Tyr124(121), and Trp286(279)],
situated >10 Å above the active site triad and near the gorge
entrance.13,14
Saffron, derived from the stigmas of Crocus sativus, is a wellknown spice with many reputed therapeutic uses, including its
use as a tonic, nerve sedative, and antidepressant and its use
against dementias.15,16 Its composition is rare because it is a
source of crocins, that is, mono- and diglycosides of crocetin
(CRT). We have shown that the extract has powerful
antioxidant properties and that the crocin constituents inhibit
amyloid aggregation in vitro.17 After oral consumption of a cup
of saffron infusion, CRT is determined in blood plasma as the
main metabolite in humans.18 Administration of saffron extract
to aged mice has been demonstrated to counteract age-related
memory impairments and improve cerebral antioxidant
markers, in addition to the above effects of saffron
administration to adult mice of significantly decreased AChE
activity.19 Beneficial effects against scopolamine-induced
recognition memory deficits have also been documented in
rats.20−22 In accordance, two recent phase II clinical studies
provide preliminary evidence of a possible therapeutic effect of
saffron extract in the treatment of patients with mild to
moderate AD.16,23,24 However, the effect of saffron constituents/metabolites on AChE activity has not been studied,
despite its promising in vivo effects.
In the present work, in silico molecular docking studies and
in vitro enzymatic kinetic studies have been undertaken as an
attempt to explore the ability of saffron constituents to act as
potent inhibitors of AChE and to elucidate the possible
mechanism of action. AChE from electric eel was used because
(a) its oligomeric forms are structurally similar to those in
AChE of vertebrates, nerve, and muscles25,26 and (b) it allows
■
Plant Material and Extraction. Commercially available pure red
Greek saffron (stigmas of C. sativus) was kindly provided by the
Cooperative Association of Krokos in Kozani, in West Macedonia,
Greece. Saffron was extracted with methanol/water 1:1 v/v, as
previously described.17 CRT (purity > 98%) was prepared by
saponification of saffron extract, whereas DMCRT was prepared by
saponification of saffron extract in the presence of methanol, as
previously described,28,29 and its final purity was >98%. SFR (purity >
88%) was purchased from Sigma-Aldrich Corp., St. Louis, MO, USA.
In Vitro AChE Inhibition Assay. The assay for AChE activity was
performed with the colorimetric method of Ellman et al.,30 utilizing
ATCI as a substrate and AChE from the electric eel (Sigma-Aldrich
Corp.). Briefly, in the 96-well plates were added 50 μL of 50 mM TrisHCl, pH 8.0, containing 0.1% bovine serum albumin (BSA), 25 μL of
the tested samples (0−8 mM CRT, DMCRT, and SFR dissolved in 50
mM Tris-HCl, pH 8.0) or GNT (1−256 μM), 25 μL of 15 mM ATCI
(dissolved in water), and 75 μL of 5 mM 5,5′-dithiobis(2-nitrobenzoate) (DTNB) (dissolved in 50 mM Tris-HCl, pH 8.0, 0.1 M
NaCl, 0.02 M MgCl2·6H2O). Absorbance was measured at 405 nm
after 5 min of incubation at room temperature, in the dark, to subtract
these values from the values after AChE addition and correct for
nonenzymatic change of reagent absorbance. Then, 25 μL of 0.22 U/
mL of AChE from electric eel (dissolved in 50 mM Tris-HCl, pH 8.0,
containing 0.1% w/v BSA) was added, and the absorbance was
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Article
Table 1. Enzyme Kinetics of the Tested Compounds on AChE Activitya
Km (μM)
Vmax (μmol/L/min)
23.01 ± 2.70
(conf interval: 17.0−29.0)
65 ± 3.3
1.93 ± 0.05
26.3 ± 3.02
safranal (SFR)
21.09 ± 0.17
ATCI
crocetin (CRT)
ATCI
dimethylcrocetin (DMCRT)
substrate
inhibitor
ATCI
control
ATCI
galanthamine (GNT)
ATCI
Ki (μM)
type of inhibition
65 ± 3.3
3.4 ± 0.3
competitive
18.5 ± 23.9
27 ± 2.4
90.6 ± 2.0
mixed
96.33 ± 0.11
19.30 ± 2.6
39 ± 2.2
28.0 ± 2.3
mixed
107.1 ± 0.11
16.5 ± 1.9
38 ± 1.8
16.5 ± 2.4
mixed
IC50 (μM)
a
The values of Vmax, Km, and Ki were calculated by monitoring hydrolysis of substrate (ATCI) by AChE at different substrate concentrations (0.125−
64 μM) in the presence or absence of samples (mean results of 4 experiments, run in duplicate) (0.15−34 μM).
measured again after 5 min of incubation at room temperature in the
dark. GNT (1−32 μM, final concentrations) (Sigma-Aldrich) was used
as a reference inhibitor. In all experiments the final organic solvent
percentage was never above 6% v/v; control experiments with the
same percentage of organic solvent without sample were also
conducted, and the values were subtracted. All determinations were
carried out at least four times, and in duplicate, at each concentration
in 96-well microplates, using a UV−vis microplate reader (Molecular
Devices). Results were reported as percentage of inhibition of AChE
activity in the absence of inhibitor/tested compounds (positive
control). The concentrations of the tested extracts that inhibited the
hydrolysis of substrate (ATCI) by 50% (IC50) were determined by
GraphPad Prism4.0 (GraphPad Software, Inc., USA) from sigmoidal
dose−response curves obtained by plotting the percentage of
inhibition (Y) versus the log concentration (x, μM) of the tested
samples, using the equation
quantum-chemical calculations by utilizing the ab initio Hartree−
Fock method with the 6-31G* basis set.
Docking calculations were carried out via the BioMedCAChe
program, which is part of the CAChe package (CAChe WorkSystem
Pro version 7.5.0.85, Fujitsu). Docking experiments employed full
ligand flexibility and partial protein flexibility focused at the ligand
binding site. AChE complex with various inhibitors has provided
valuable knowledge of the interactions that mediate inhibitor
binding.31 To identify the molecular determinants responsible for
the binding mode of the studied compounds with AChE protein, we
used the determined X-ray crystallographic structure of T. californica
AChE in complex with the AChE inhibitor, THA (PDB accession no.
1ACJ).32 X-ray structure was obtained from the Brookhaven Protein
Data Bank (RCSB Protein Data Bank, operated by the Research
Collaboratory for Structural Bioinformatics).33 The produced
compound−protein complexes were ranked by the energy score,
including their binding conformations. 3D models of the above protein
crystal structures have been developed after the deletion of the
cocrystallized bound inhibitor. The docking procedure provides the
treatment of ligand flexibility within the protein binding site by means
of a four-point chiral pharmacophoric comparison between the ligand
and the site. The final output of the docking procedure is a set of
solutions ranked according to the corresponding scoring function
values, each defined by the 3D coordinates of its atoms and expressed
as a PDB file. The accuracy of BioMedCAChe has been shown to
successfully reproduce experimentally observed binding modes, in
terms of root-mean squared deviation (rmsd). BioMedCAChe
provided excellent result as was observed for the low value of rmsd
(best docked solution of 0.43 Ǻ ) between experimental and docked
structures (this is also shown by superimposition of the above
structures). The ability to accurately predict the binding conformation
of AChE inhibitors, THA and GNT, to AChE, gave confidence that
the BioMedCAChe could also predict, with a similar accuracy, the
binding conformations of saffron’s bioactive constituents utilized in the
current study. The PyMol34 molecular graphics system was used to
visualize the molecules and the results of the docking experiments.
Statistical Analysis. Data are presented as the mean ± SE.
Statistical analysis was performed with GraphPad Instat 3 software
(GraphPad Instat Software, Inc., USA) using the nonparametric
Mann−Whitney test (p < 0.05). In all tests, a criterion of p ≤ 0.05
(two-tailed) was considered to be necessary for statistical significance.
Y = 100/(1 + 10(LogIC50 − x ))
Kinetic Analysis of AChE Inhibition. Microplate wells were filled
with 50 μL of 50 mM Tris-HCl, pH 8.0, containing 0.1% BSA, 25 μL
of the tested phytochemicals (0−256 μM CRT and DMCRT and 0−
120 μM SFR) or GNT (0−120 μM), 25 μL of AChE (0.22 U/mL),
and 75 μL of 3 mM DTNB (dissolved in 50 mM Tris-HCl, pH 8.0,
containing 0.1 M NaCl, 0.02 M MgCl2·6H2O). The mixture of enzyme
and inhibitor/or samples was mixed, and absorbance was read at 405
nm after 5 min of incubation at room temperature, in the dark.
Immediately after addition of the substrate (25 μL ATCI, 1−512 μM
concentrations, dissolved in water), the change of absorbance at 405
nm was monitored for 5 min, and the reaction rate was calculated
according to the method of Ellman et al.30 Inhibition studies were
analyzed using GraphPad Prism4.0 software. The analysis of the type
of inhibition of AChE activity was determined by the Lineweaver−
Burk (LB) plot, whereas the kinetic parameters Km and Vmax were
obtained by curve fitting according to the classical Michaelis−Menten
equation. The inhibition constants (Ki) of the tested samples were
calculated according to GraphPad Prism v4.0, from linear regression
analysis of LB plots (S, hyperbolic; I, hyperbolic mixed model).
In Silico Computational Methods (Molecular Modeling and
Docking Calculations). Molecular models of all compounds
described in this work were built in 3D coordinates and their best,
most stable (lower energy) conformations were detected by
geometrical optimization of their structure in the gas phase, as
implemented in the Spartan ‘08 Molecular Modeling program suite
(Spartan ‘08 v. 1.2.0, Wave Function Inc., Irvine, CA, USA). The
molecules’ structures were initially optimized by conformational search
using the Monte Carlo method with the MMFF94 molecular
mechanics model. Geometry optimization was accomplished via
■
RESULTS
In Vitro Effects on AChE Activity. To study if the tested
phytochemicals directly inhibit AChE, the effects of saffron
extract and of CRT, DMCRT, SFR, and GNT (positive
control) on electric eel AChE activity were tested in vitro, and
6133
Article
Figure 2. Lineweaver−Burk plots in the absence (a) and presence of standard GNT (b) and saffron’s constituents (c−e): 1/Vmax versus 1/[ATCI].
The topography of the calculated binding sites of the studied
compounds provides a clearer understanding of their potency
differentiation in the inhibition of AChE.
The global energy of interaction (in kcal/mol) for each
docking experiments is given as follows: CRT, −32.24;
DMCRT, −36.07; CRC, −30.63; SFR, −18.43; THA,
−18.26; ATCI, −19.65; and GNT, −32.89. The ligand binding
contacts of best docked poses of the known AChE inhibitors
GNT and THA and the AChE substrate, ATCI, with key amino
acid residues and water molecules in the active site of AChE,
are shown in Tables 2−4 in the Supporting Information.
CRT docked very close to ATCI (at a nearby binding
position). Its stabilization to the protein’s binding pocket was
mainly attributed to its formed hydrophobic contacts (23 total
contacts): (a) by the hydrophobic residues Phe284, Phe290
(acyl-binding pocket), Phe330, Phe331, Ile287, Leu358; (b) by
the acidic and basic charged polar residues Asp285 and Arg289;
and (c) by the polar noncharged residues Tyr121 and Ser286
(Table 5 in the Supporting Information). Additionally, CRT
was localized inside the binding cavity, making two hydrogen
bond contacts with Tyr121 and Asp285. Water molecule
wat634 also contributes to the ligand binding by forming a
hydrogen bond with O13 of CRT. The binding contacts of
CRT with Phe330 and wat634 were found to be common with
those of ATCI, THA, and GNT, whereas Tyr121, Phe331, and
the acyl-binding residue Phe290 contributed also to the binding
of THA and GNT inhibitors. As illustrated from the ligand
binding site architecture (Figure 3), CRT was inserted into the
narrow aromatic gorge of the crystal structure of AChE,
oriented in a way that the molecule’s polyene backbone could
the results are presented in Table 1 and in Figure 3 in the
Supporting Information. Saffron showed moderate AChE
inhibitory activity (up to 30%). Dose-dependent inhibition of
AChE was observed for GNT (Figure 3a of the Supporting
Information), a standard AChE inhibitor, with an IC50 of 1.93
μM, as well as for the pure compounds, in the order SFR >
CRT ≥ DMCRT (Figure 3c−e in the Supporting Information).
The determined IC50 value of GNT is in agreement with
previous results.31,33
To elucidate the mechanism of AChE inhibition, kinetic
studies of enzyme activity were performed. The relationship
between substrate concentration and reaction velocity was in
good agreement with Michaelis−Menten kinetics. In the
absence of inhibitors, the average Km for the substrate
(ATCI) was 23.01 ± 2.70 μM. When the slopes or the
intercepts in the Lineweaver−Burk plots were drawn in
GraphPad Prism 4.0 as a function of inhibitor concentration
and the kinetic standards determined (Figure 2), CRT,
DMCRT, and SFR showed mixed-type inhibition; the α values
of CRT and DMCRT are indicative of a mixed-type,
uncompetitive inhibition, whereas the α values of SFR show
a mixed-type competitive inhibition (Table 1).
In Silico Protein−ligand Docking Study. In silico
molecular docking studies were also undertaken to gain insight
into the interaction of CRT, DMCRT, CRC, and SFR with the
residues of the ligand binding site of the AChE protein and to
investigate the underlying mechanism(s) of action(s). The
docking simulation studies on the crystal structure of target
protein AChE aimed to explore the ability of the studied
compounds to act as potent inhibitors of the AChE enzyme.
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Figure 3. Ligand binding site architecture of AChE target protein
(PDB accession no. 1ACJ) with the superimposed docked molecules
CRT, ATCI, THA, and GNT. AChE protein is illustrated as a cartoon
colored according to chainbow, with additional depiction of selected
contacting amino acid residues rendered in line mode (a). The binding
envelope of CRT in the target protein (b) is structured with
interacting amino acid residues in stick model colored according to
chainbow. Interacting water634 molecule is depicted as a blue-dotted
sphere. Docked molecules are represented in stick model and colored
according to atom type (CRT carbon atoms in light blue (a) or light
magenta (b); ATCI, THA, and GNT carbon atoms in yellow, hot
pink, and white, respectively). Hydrogen atoms are omitted from all
molecules for clarity (final structure was ray traced).
Figure 4. Docking of DMCRT (shown in stick representation and
colored according to atom type with light pink C atoms) in the crystal
structure of AChE protein cocrystallized with its known inhibitor THA
(hot pink C atoms) (PDB accession no. 1ACJ). (a) Target protein is
depicted as semitransparent ray-traced cartoon colored by secondary
structure (cylindrical helices, red; plank-sheet, yellow; loop, green). A
closeup view of the ligand binding pocket is illustrated in
semitransparent surface colored by chain (in blue) (b) depicting the
binding interactions of DMCRT (light magenta C atoms) with
selected amino acid residues in contact highlighted yellow. Hydrophobic and H-bond interactions are shown as dotted lines. Hydrogen
atoms are omitted from all molecules for clarity (final structure was ray
traced).
be stabilized via hydrophobic contacts with the amino acid
residues of the wall of this prolonged cavity. CRT penetrated
into the central cavity of the protein, where the catalytic active
site is located, with one of its carboxylic ends to be positioned
near the catalytic site triad and near the position occupied by
ATCI, THA, and GNT. The other end of the molecule
(situated near the opening of the aromatic groove at a distance
∼20 Å from the catalytic active site) protrudes from the
protein’s molecule and is stabilized in its position via two Hbond contacts with the amino acid residue Asp285 and the
water molecule wat634. Hydrophobic contacts between CRT’s
atoms of the hydrocarbon skeleton and amino acid residues of
the peripheral anionic site contributed further to the attachment of the molecule.
DMCRT superimposed with THA inhibitor is shown in
Figure 4 to be anchored inside the ligand binding pocket of the
protein. H-bond and hydrophobic interactions between the
DMCRT compound and the amino acid residues of the ligand
binding cavity of the target protein are shown in Figure 4b. The
polyene backbone moiety of the compound is localized inside
the anionic binding groove, making contacts with the following
amino acid residues: Phe284, Tyr121 (PAS), Trp279 (PAS),
and Tyr334. In contrast, Phe330 and Asp72 residues mainly
contribute to the attachment of the molecule into the catalytic
binding site (Figure 4b). The DMCRT molecule is stabilized in
the ligand binding cavity mostly through hydrophobic
interactions with residues Asp72, Phe330, Tyr334, Trp279,
Phe284, and Asp285 (Table 6 in the Supporting Information).
Phe330 was a common binding residue with the ATCI
substrate, whereas Phe330 and Tyr121 were common with
THA and GNT. The total number of binding contacts found
between DMCRT and AChE protein was 21, of which 5 were
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H-bonds, 1 was a bridged H-bond, and the rest were
hydrophobic.
Docking studies of CRC on AChE demonstrated that the
ligand was complexed with the protein with the incorporation
of both H-bond and hydrophobic contacts. The docking studies
revealed that the CRC molecule anchors inside the aromatic
groove, but in a different binding locus from that of THA
(binding is stabilized at the peripheral site located at the gorge
rim, which encompasses binding sites for allosteric ligands).
CRC has a unique orientation along the active-site gorge,
extending from the anionic subsite of the active site at the
bottom, to the peripheral anionic site at the top, via aromatic
stacking interactions with various aromatic acid residues. The
superimposed structures of CRC and THA on the target
protein are illustrated in Figure 5a. The best possible docking
binding mode of CRC was mediated through residues Asn230,
His398, Pro232, Asp285, Ser235, and Trp524 and water
molecules wat640 and wat650 (11 H-bond contacts) and
Arg289, Pro361, Cys357, Asp285, and Phe284 (17 hydrophobic contacts) (Figure 5b; Table 7 in the Supporting
Information). No common binding residues with ATCI, THA,
or GNT were observed.
The docking procedure positioned SFR into the AChE
binding site almost at the same place occupied by THA (Figure
6a,b). Through interaction analysis, it was shown that SFR
makes one hydrogen bond contact with the hydroxyl group of
wat634, whereas the stabilization of the compound into the
pocket was additionally attributed to seven hydrophobic
contacts formed by residues Phe330, Gly118, His440, and
Trp84 (Figure 6c; Table 8 in the Supporting Information). All
binding contacts were found to be common with the
corresponding AChE−ATCI/THA complexes, whereas
Gly118, His440, and Trp84 were common with the AChE−
GNT complex. The former amino acid contacts of SFR are
important anchoring residues for the inhibitor and are the main
contributors to the inhibitor’s interaction. (Data concerning
ATCI, GNT, and THA are not shown in the text or tables; they
are provided as additional Supporting Information available
online.)
■
DISCUSSION
Our results showed that the crude aqueous methanolic saffron
extract exhibited low dose-independent inhibitory values.
Analysis of the in vitro effect of CRT, DMCRT, and SFR on
AChE inhibition revealed a dose-dependent inhibitory profile,
but the IC50 values were higher than that of GNT. Kinetic
analysis confirmed that GNT exerts a competitive type of
inhibition, with a Ki value of 3.36 μM in electric eel enzyme, but
the majority of the tested saffron phytochemicals exhibited
higher Ki values and a mixed type of inhibition.
Molecular docking studies revealed that CRT and DMCRT
were not docked at the same place as ATCI, TCH, and GNT
inhibitors, but bound simultaneously to the catalytic and
peripheral anionic sites of AChE. CRC, due to its bulky
glycosidically linked sugar moieties, was unable to be inserted in
depth to the aromatic gorge (did not reach the active site that is
found deep in the pocket) and attached to a different locus
from that of ATCI, THA, and GNT. The fact that the presence
of glycosidically linked sugars alters/impairs binding characteristics might provide an explanation for the rather low inhibitory
activity of the saffron extract in vitro. The ability of CRT to
span the two “anionic” sites, being ∼20 Å apart, and bind to
both PAS and the catalytic site of the AChE enzyme opens new
Figure 5. Bound CRC ligand (illustrated in stick mode and colored
according to atom type with light pink C atoms) docked in crystal
structure of AChE protein (PDB accession no. 1ACJ) depicted as
spectrum-colored cartoon (a) or solid surface colored by chain (in
purple-blue) with highlighted interacting amino acid residues of the
ligand binding cavity (b) in complex with THA (hot pink C).
Highlighted residues interacting through H-bond: Asn230, light
orange; His398, pale green; Ser235, teal; and Trp524, yellow.
Hydrogen-bonded water molecule wat650 is rendered as a light blue
sphere. Hydrogen bond and hydrophobic interactions are shown as
yellow-dotted lines. Hydrogen atoms are omitted from all molecules
for clarity (final structure was ray traced).
possibilities for the design and synthesis of novel dual binding
AChE inhibitors, which would beneficially affect cholinergic
transmission and Aβ aggregation.
On the other hand, SFR seems to be a more potent inhibitor
(IC50 = 21.09 ± 0.17, Ki = 90.6 ± 2.0) than the carotenoids, but
both kinetic and in silico data show that it completely enters
and interacts only with the catalytic center. Similar weak
inhibitory action has been reported for many other
monoterpenoids,31 and it is attributed to interactions of the
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hydrocarbon skeletal terpenoids with the AChE hydrophobic
active center.
The hypothesis derived from the in vitro experiments
revealed that the majority of the tested phytochemicals
exhibited a mixed type of inhibition, as supported by the
adopted in silico studies, where all saffron constituents (apart
from SFR) were found to be complexed with the enzyme at a
different locus (allosteric binding site) from that of ATCI and
the tested inhibitors (THA and GNT). These findings showed,
for the first time, that CRT, the main metabolite of CRCs in the
living organism, inhibits AChE by binding at two different loci,
the catalytic center and the PAS site. This finding may partly
explain the beneficial effects of saffron in clinical trials against
AD and provides a basis for the design and development of
novel pleiotropic AChE inhibitors. The presence of glycosidically linked sugars impairs this activity. Overall, our results
showed that the in vitro kinetic analysis, in conjunction with
molecular modeling and docking predictions, could be
important initial steps toward the development of novel
AChE inhibitors.
■
ASSOCIATED CONTENT
S Supporting Information
*
The ligand binding contacts of best docked poses of the known
AChE inhibitors GNT and THA, the AChE substrate ATCI,
and saffron constituents CRT, DMCRT, CRC, and SFR, with
key amino acid residues and water molecules in the active site
of AChE, are shown in Tables 2−8. The effectiveness of GNT,
saffron, and its constituents in inhibiting AChE activity is
shown in Figure 3. IC50 or pIC50 (when converted to the −log
IC50 scale) represents the concentration of the tested
phytochemical(s) that is required for 50% inhibition in vitro
and is calculated by converting the log values back to μM. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*(G.D.G.) Department of Cell Culture-Molecular Modeling
and Drug Design, Symeonidion Research Center, Theagenion
Cancer Hospital, 2 Al. Symeonidi str., 54007 Thessaloniki,
Greece. Phone: +30 231 0898237. Fax: +30 321 0845514. Email: [email protected]. (F.N.L.) Department of Pharmacy, University of Patras, Patras 26504, Greece. Phone:+30
261 0969335. Fax: +30 261 0997714. E-mail: fl[email protected].
Figure 6. Cartoon diagram (a) of AChE protein with bound SFR
molecule in complex with ATCI (rendered as sticks with yellow
carbon atoms) and the known inhibitors THA and GNT (rendered as
sticks with hot pink and blue carbon atoms, respectively). SFR
(depicted as a stick with light pink carbon atoms) is docked into the
binding pocket of the protein (illustrated as cartoon colored according
to secondary structure) at almost the same place occupied by the
cocrystallized THA and GNT inhibitors (PDB ID no. 1ACJ)
(dockings of all ligands were performed individually). A closeup
view (b) of the ligand binding cavity of the protein is represented as
semitransparent surface colored by chain with interacting amino acid
residues highlighted in magenta. Wat634 molecule interacting via Hbond with SFR is depicted as a blue-dotted sphere. Interactions of
bound SFR and THA ligands (depicted as sticks with light pink and
hot pink carbon atoms, respectively) docked in crystal structure of the
active site of AChE target protein are colored as chainbow (c). Amino
acid residues of the binding pocket are depicted in stick model and
colored according to atom type (only the contacts of SFR are shown).
The Wat634 molecule is depicted as a magenta-dotted sphere.
Hydrogen atoms are omitted from all molecules for clarity (final
structure was ray traced).
Funding
The present study is cofinanced by the EU European Social
Fund (80%) and the Greek Ministry of Development-GSRT
(20%) and by GlaxoSmithKline.
Notes
The authors declare no competing financial interest.
■
ABBREVIATIONS USED
Aβ, amyloid-β peptide; ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; ATCI, acetylthiocholine
iodide; BSA, bovine serum albumin; ChAT, choline acetyltransferase; ChEIs, cholinesterase inhibitors; CRC, crocin;
CRT, crocetin; DMCRT, dimethylcrocetin; DTNB, 5,5′dithiobis(2-nitrobenzoate); GNT, galanthamine; PAS, peripheral anionic binding site; PDB, Protein Data Bank; SAR,
structure−activity relationship; SFR, safranal; THA, tacrine.
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Food and Chemical Toxicology 52 (2013) 163–170
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
Investigation of the neuroprotective action of saffron (Crocus sativus L.)
in aluminum-exposed adult mice through behavioral and neurobiochemical
assessment
Zacharoula I. Linardaki a, Malvina G. Orkoula b, Alexandros G. Kokkosis a, Fotini N. Lamari c,
Marigoula Margarity a,⇑
a
Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, 26504 Patras, Greece
Laboratory of Instrumental Pharmaceutical Analysis, Department of Pharmacy, University of Patras, 26504 Patras, Greece
c
Laboratory of Pharmacognosy & Chemistry of Natural Products, Department of Pharmacy, University of Patras, 26504 Patras, Greece
b
a r t i c l e
i n f o
Article history:
Received 21 September 2012
Accepted 8 November 2012
Available online 17 November 2012
Keywords:
Aluminum
Saffron
Memory
Cholinesterases
Monoamine oxidase
Oxidative stress
a b s t r a c t
In the present study, the possible reversal effects of saffron against established aluminum (Al)-toxicity in
adult mice, were investigated. Control, Al-treated (50 mg AlCl3/kg/day diluted in the drinking water for
5 weeks) and Al + saffron (Al-treatment as previously plus 60 mg saffron extract/kg/day intraperitoneally
for the last 6 days), groups of male Balb-c mice were used. We assessed learning/memory, the activity of
acetylcholinesterase [AChE, salt-(SS)/detergent-soluble(DS) isoforms], butyrylcholinesterase (BuChE, SS/
DS isoforms), monoamine oxidase (MAO-A, MAO-B), the levels of lipid peroxidation (MDA) and reduced
glutathione (GSH), in whole brain and cerebellum. Brain Al was determined by atomic absorption spectrometry, while, for the first time, crocetin, the main active metabolite of saffron, was determined in brain
after intraperitoneal saffron administration by HPLC. Al intake caused memory impairment, significant
decrease of AChE and BuChE activity, activation of brain MAO isoforms but inhibition of cerebellar
MAO-B, significant elevation of brain MDA and significant reduction of GSH content. Although saffron
extract co-administration had no effect on cognitive performance of mice, it reversed significantly the
Al-induced changes in MAO activity and the levels of MDA and GSH. AChE activity was further significantly decreased in cerebral tissues of Al + saffron group. The biochemical changes support the neuroprotective potential of saffron under toxicity.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Aluminum (Al) is the third most abundant element in nature,
making human exposure unavoidable (Verstraeten et al., 2008).
The main entry sites of Al into the body are the gastrointestinal
and respiratory tract and the skin (Verstraeten et al., 2008), enabling distribution and accumulation in several tissues, like spleen,
lungs, liver, kidneys, heart, bone and brain (Kumar and Gill, 2009),
through systemic circulation. It has been demonstrated that Al
crosses the blood–brain barrier, implicating metal binding to
Abbreviations: AChE, acetylcholinesterase; ATCh, acetylthiocholine iodide;
BuChE, butyrylcholinesterase; BuTCh, butyrylthiocholine iodide; ce, cerebellum;
ChE, cholinesterase; DS, detergent-soluble; GSH, reduced glutathione; i.p., intraperitoneal injections; IL, initial latency; MAO, monoamine oxidase; MDA, malondialdehyde; SS, salt-soluble; STL, step-through latency.
⇑ Corresponding author. Tel.: +30 2610 997430; fax: +30 2610 969273.
E-mail addresses: [email protected] (Z.I. Linardaki), [email protected]
(M.G. Orkoula), [email protected] (A.G. Kokkosis), fl[email protected]
(F.N. Lamari), [email protected] (M. Margarity).
0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2012.11.016
transferrin in the blood and a subsequent transferrin receptormediated mechanism of brain Al influx (Yokel et al., 1999).
High brain levels of Al induce cognitive deficiency and dementia
and, thus, Al is a widely accepted neurotoxin (Kawahara and
Kato-Negishi, 2011). Its implication in the pathogenesis of
neurodegenerative diseases has been suggested decades ago, but
is seriously debated till now (Kawahara and Kato-Negishi, 2011;
Tomljenovic, 2011). However, some researchers consider that the
model of chronic Al-induced neurotoxicity best describes
Alzheimer’s disease, since it manifests many of the pathobiological
hallmarks (Walton and Wang, 2009; Yokel, 2006; Zhang et al.,
2003).
Al neurotoxicity is manifested through several behavioral and
neurochemical alterations, which display diversity depending on
the animal species in question, the administration route and the
chemical form of Al administered (Erasmus et al., 1993; Kumar
and Gill, 2009). It has been demonstrated that Al promotes
oxidative stress in the cerebral cortex and hippocampus of young
and aged rats, and damage of lipids, membrane-associated proteins
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Z.I. Linardaki et al. / Food and Chemical Toxicology 52 (2013) 163–170
(Na+-K+ ATPase and protein kinase C) and endogenous antioxidant
enzymes (Sethi et al., 2008; Sharma et al., 2009; Tripathi et al.,
2009). Negative impacts of Al administration have been also observed in neurotransmitter systems of rodent brain regions, including serotoninergic (Kumar, 2002) and cholinergic systems (Julka
et al., 1995). The effects of Al on AChE activity remain controversial
as both inhibition and activation have been reported (Julka et al.,
1995; Sharma et al., 2009), whereas the respective data concerning
BuChE activity are limited, though BuChE enzyme is considered to
play a supportive functional role in acetylcholine hydrolysis (Lane
et al., 2006). Finally, behavioral deficits assessed with different
behavioral tasks, have been observed in rats following Al exposure
(Julka et al., 1995; Sethi et al., 2008; Tripathi et al., 2009).
Crocus sativus L. (Iridaceae) is a cultivated plant species in many
countries including Greece, since its styles (saffron) constitute an
exquisite spice. Interestingly, saffron has been used as a medicinal
agent for millenia (Schmidt et al., 2007). In recent years beneficial
effects of saffron have been demonstrated in models of neuronal,
cardiovascular, respiratory and other disorders (Bathaie and
Mousavi, 2010), and are related to its unique composition; it is rich
in crocins, which are mono- and di-glycosides of crocetin. Previously, we have reported the inhibitory activity on amyloid betapeptide fibrillogenesis and protective action against H2O2-induced
toxicity in human neuroblastoma cells of saffron and its crocin
constituents in vitro (Papandreou et al., 2006, 2011). Furthermore,
i.p. administration of saffron (60 mg/kg body weight) to normal
and aged mice for 7 days significantly improved learning and
memory as assessed by step-through passive avoidance test and
this was correlated with the significant cerebral antioxidant protection conferred (Papandreou et al., 2011). Other studies have also
demonstrated neuroprotective effects of saffron and its constituents in vitro and in rodent models of brain disorders (amnesic
and ischemic) (Hosseinzadeh and Sadeghnia, 2005; Ochiai et al.,
2007; Pitsikas and Sakellaridis, 2006). The first small-scale clinical
trials of saffron against depression and mild Alzheimer’s disease
have brought forward promising results, although many issues remain unresolved (Akhondzadeh et al., 2005, 2010; Noorbala et al.,
2005). Yoshino et al. (2011) demonstrated the distribution of
crocetin in brain of stroke-prone spontaneously hypertensive rats
90 min after a single oral administration of pure crocetin, but it
has not been proven yet whether crocetin, the main crocin metabolite, crosses the blood–brain barrier after saffron administration.
Taking into consideration the increasing evidence about saffron
neuroprotective role we further investigated the possible
neuroprotective action of saffron extract in a model of chronic
Al-induced neurotoxicity. Shati et al. (2011) demonstrated the
ameliorative effects of aqueous saffron extract administration
against Al-induced neurotoxicity, by presenting changes of brain
antioxidant enzymes, serum tumor markers and brain expression
of genes (especially BCL-W, R-spondin and inositol polyphosphate
4-phosphatase). However, the potential of saffron extract to reverse the changes accompanying Al neurotoxicity has not been
investigated. Therefore, the present study based on a different
experimental design (i.p. administration of saffron extract only
for the last 6 days of the Al treatment period) starts from
behavioral level, including learning and memory process, and is
extended to subcellular level, focusing on determination of brain
cholinergic, monoaminergic and oxidant/antioxidant indices and
cerebral Al and crocetin levels.
2. Material and methods
2.1. Plant material and extraction
Greek saffron (pure red styles of C. sativus) was kindly provided by the Cooperative Association of Krokos in Kozani, in West Macedonia, Greece and a sample has
been deposited at the herbarium of the Department of Biology. Dried styles of C.
sativus were extracted with methanol:water (50:50, v/v, 1 g in 60 mL) for 4 h in
the dark, under magnetic stirring, as previously described (Papandreou et al.,
2006). The extract was centrifuged and evaporated to dryness in a Speed Vac system. The composition was screened with HPLC on a Supelcosil C-18 column as previously described (Papandreou et al., 2006; Tarantilis et al., 1995). The dry residue
was stored at 30 °C until use.
2.2. Animals
Male adult (4 months-old) Balb-c mice were used in this study. The animals
were housed in groups in standard laboratory cages (5 mice per cage) in a temperature controlled room (20 ± 2 °C) with a 12 h light/dark cycle. Food in form of dry
pellets (feed composition: grain and grain by-products, oil seed products, minerals,
vitamins and trace elements from Altromin Spezialfutter GmbH & Co. KG, Lage, Germany) was available ad libitum. Animals were randomly divided into three groups
(n = 10/group). Control group: mice had access to normal drinking water over the
experimental period. Al treated group: mice received orally AlCl3 (aluminum chloride anhydrous, purity >99%, Sigma–Aldrich, St. Louis, USA) (50 mg/kg body weight/
day) dissolved in normal drinking water for a period of 5 weeks. Body weight and
liquid intake were measured regularly to adjust the dose and achieve a constant intake of Al. Al + saffron treated group: mice received orally AlCl3 (50 mg/kg body
weight/day) dissolved in normal drinking water for 5 weeks and saffron extract
(60 mg/kg body weight/day, 30 lL injection volume) intraperitoneally for the last
6 days of the 5-week Al treatment.
AlCl3 solution was freshly prepared every 4 days over the experimental period,
while saffron solution was prepared fresh daily, just before administration, by dissolving the dry extract in saline. During the 6-day saffron treatment, control and Altreated mice received intraperitoneally 30 lL of saline. Al and saffron dosage and
schedule of exposure were based on the literature (Jyoti and Sharma, 2006) and
on our previous study (Papandreou et al., 2011), respectively. All procedures were
in accordance with Greek National Laws (Animal Act, PD 160/91).
2.3. Behavioral testing: step-through passive avoidance task
This test is based on negative reinforcement to examine long-term memory
(Kaneto, 1997). A two-compartment passive avoidance apparatus (white/dark, separated by a guillotine door) was used. Mice were subjected to single trial passive
avoidance task according to previously described procedures (Kaneto, 1997; Otano
et al., 1999; Papandreou et al., 2009). Briefly, on day 5 of saffron treatment an acquisition trial was performed 1 h after a 100 s-long habituation trial, while a retention
trial was performed 24 h after the training session. Results were expressed as the
mean initial (IL, max. time 120 s) and step-through (STL, max. time 300 s) latency
time values recorded once mice crossed into the dark compartment, before and
24 h after the delivery of a mild foot shock to their paws. All training and testing
sessions were carried out during the light phase (08:00–14:00 h), one hour after
intraperitoneal administration of saffron.
2.4. Preparation of tissue homogenates
On the completion of the 5-week treatment period, all animals were sacrificed
by cervical dislocation. Brain was excised immediately, and cerebellum was separated from the remaining whole brain. Liver was also removed and used as a reference peripheral tissue. All tissues were rinsed with saline immediately. From the
tissue samples isolated, only six tissues per group were used for the biochemical
assessments. The others were assayed for Al content by atomic absorption spectrometry. The tissues for biochemical measurements and crocetin determination
were weighed and homogenized (10%, w/v) with a glass-Teflon homogenizer in
ice-cold 30 mM Na2HPO4, pH 7.6. The homogenates were then centrifuged at
15,000g for 20 min at 4 °C. Supernatants constituted the salt-soluble (SS) fractions
and were stored at 75 °C. The pellets were re-suspended in an equal volume of 1%
(w/v) Triton X-100 (in 30 mM Na2HPO4, pH 7.6) and then centrifuged at 15,000g for
20 min at 4 °C. Supernatants, the detergent-soluble (DS) fractions, were collected
and stored at 75 °C. This double-extraction method was performed in order to recover the cytosolic (SS fraction) and membrane-bound (DS fraction) isoforms of
cholinesterases (Das et al., 2005). Protein concentrations were determined by the
Bradford assay (Bradford, 1976).
2.5. Aluminum determination by atomic absorption spectrometry
2.5.1. Digestion of tissues
The whole brain (-ce), cerebellum and liver samples from control, Al-treated
and Al + saffron treated mice were analyzed for Al concentration. Glassware was
not used during sample preparation in order to avoid the possible contamination.
Eppendorf tubes were used after immersing in a solution of nitric acid (HNO3): ethanol (1:9, v/v) for 48 h and washing with ultra-pure water (Missel et al., 2005).
Sample preparation was performed as previously described (Gulya et al., 1995) with
modifications. The dry weight of the tissues was measured after heating at 100 °C
for 20 h. Then, the samples were digested with 1 mL/100 mg wet tissue (for whole
brain) and 1 mL/25 mg wet tissue (for cerebellum and liver) of nitric acid: sulphuric
acid (HNO3:H2SO4, 1:1 v/v) at room temperature overnight. Mixtures were centrifuged at 10,000g for 10 min to remove insoluble material. Al levels were determined in the supernatants.
2.5.2. Atomic absorption spectrometry
Al content in the supernatants was measured employing Atomic Absorption
Spectrometry (Perkin Elmer, AAnalyst 300 equipped with a HGA 700 Graphite Furnace and an AS-70 autosampler). The temperature of the furnace was programmed
as described elsewhere (Johnson and Treble, 1992). Absorbance was measured at
309.3 nm.
Preparation of samples for Al analysis: All samples were prepared in eppendorf
tubes and analyzed immediately after preparation. A 5% v/v HNO3 and 0.1% v/v
Mg solution prepared by proper dilutions from HNO3 solution (65%, Carlo Erba)
and Mg stock standard solution (1000 lg/mL Mg2+ ±2 lg to 5% HNO3, Chem-Lab),
was used as a matrix modifier in order to stabilize Al in the furnace.
The standard addition method was employed for the analysis. Four samples
were prepared for each aliquot, adding known amounts (spikes) of the analyte
(Al) from a stock standard solution (1000 ppm in 1 M HNO3 Fisher Scientific, UK).
The spiked concentration ranged from 0 (unspiked sample) to 60 ppb. The volume
was kept equal for all samples by means of addition of the matrix modifier. Preliminary tests assured that the overall concentration of Al was within the linear
range of the technique.
Analysis: Absorbance for each sample was measured three times. A calibration
curve for each supernatant aliquot was constructed (OriginPro 8). The results are
expressed as lg Al/g wet tissue.
2.6. HPLC determination of crocetin in brain
Crocetin determination in whole brain (-ce) of Al + saffron treated mice was
performed by an isocratic reversed-phase liquid chromatographic method developed by Chryssanthi et al. (2011) for plasma analysis, with slight modifications.
In detail, a high volume (3.5 mL) of the whole brain homogenate (100 mg/mL, SS
fraction) was submitted to solid phase extraction on reversed phase Strata-X cartridges (200 mg/3 mL) consisting of a surface modified with styrene–divinylbenzene polymer that were obtained from Phenomenex (Torrance, CA, USA). The
crocetin eluate was evaporated to dryness in a speed Vac system and the dry residue was redissolved in 30 lL of the HPLC mobile phase. The samples were chromatographed on a Luna C-18 column (4.6 250 mm, 5 lm) with a mobile phase
consisting of methanol–water–trifluoroacetic acid (75.0:24.5:0.5, v/v/v) at a flow
rate of 1.0 mL min 1. Crocetin was quantified using spiked brain standards at the
concentrations of 0.5, 1.0, 1.5, 2.0 and 3.0 lM of total crocetin (Chembiotin, purity
>98%). Results are expressed as nmol of total crocetin/g wet tissue.
2.7. Biochemical assays
2.7.1. Determination of cholinesterase activity
The activity of SS and DS isoforms of AChE and BuChE in brain and liver samples,
was determined spectrophotometrically based on Ellman’s assay and previously described protocols (Ellman et al., 1961; Lassiter et al., 1998; Papandreou et al., 2009).
Total ChE activity was measured by acetylthiocholine iodide (ATCh, Sigma–Aldrich,
UK) as substrate, whereas BuChE activity was determined by S-butyrylthiocholine
iodide (BuTCh, Sigma–Aldrich, Switzerland) as substrate, since BuTCh is hydrolyzed
only by BuChE (Lassiter et al., 1998). AChE activity was then calculated by subtracting BuChE activity from total ChE activity. Enzyme activity is expressed as lmol of
substrate hydrolyzed/min/g of tissue.
2.7.2. Determination of monoamine oxidase activity
The activity of both isoforms of MAO, MAO-A and MAO-B, was assessed in
mouse whole brain (-ce) and cerebellum by a fluorimetric assay based on previously described protocols (Mahmood et al., 1994; Xu et al., 2010). Since MAO is a
mitochondrial membrane-bound enzyme, the DS fractions of brain tissues homogenates were used. Briefly, 50 lL of brain sample was preincubated at 37 °C for
20 min with 100 lL of either 1 lM R-( )-Deprenyl hydrochloride (Deprenyl,
MAO-B inhibitor, Sigma–Aldrich, USA) or 1 lM N-Methyl-N-propargyl-3-(2,4dichlorophenoxy)propylamine hydrochloride (Clorgyline, MAO-A inhibitor, Sigma–Aldrich, USA) in 100 lL 30 mM Na2HPO4, pH 7.6. Then 10 lL of 3.07 mM kynuramine dihydrobromide (Sigma–Aldrich, USA) was added to the reaction mixture as
substrate. The mixture was then incubated at 37 °C for 15 min again. After incubation, the reaction was terminated by adding 100 lL of 0.6 M perchloric acid and
centrifuging the mixture at 1500g for 10 min to remove precipitated proteins. An
aliquot of 0.3 mL of the supernatant was added to 2 mL of 1 N NaOH. The fluorescence intensity of the produced 4-hydroxyquinoline was detected at an excitation
wavelength of 315 nm and an emission wavelength of 380 nm, using a fluorescence
spectrometer. The concentration of product was estimated from a corresponding
standard fluorescence curve of 4-hydroxyquinoline (1–100 lM) (Sigma–Aldrich,
USA). MAO activity is expressed as nmol of 4-hydroxyquinoline formed/g tissue/
min.
165
2.7.3. Estimation of lipid peroxidation and reduced glutathione levels
Malondialdehyde (MDA) levels, an index of tissue lipid peroxidation, were measured in cerebral and liver samples (SS fractions of tissue homogenates) following a
previously described fluorimetric assay (Papandreou et al., 2009). Lipid peroxidation levels are expressed as lmol MDA/g of tissue protein.
Reduced glutathione (GSH) content of brain and liver samples (SS fractions of
tissue homogenates) was determined fluorometrically according to the procedure
of Mokrasch and Teschke (1984). GSH levels are expressed as lmol/g of tissue
protein.
2.8. Statistical analysis
Data are presented as mean ± S.E.M. Statistical analysis was performed with
GraphPad Instat 3 software using the nonparametric Mann–Whitney test for evaluating statistically significant differences (p < 0.05) between the experimental
groups.
3. Results
3.1. General observations
HPLC analysis showed that saffron extract contains a variety of
crocins: trans-crocin-4 constitutes 46% of total crocin content of
the crude extract, trans-crocin-3, 26%; cis-crocin-4, 12%; and
trans-crocin-2, 7%, as previously described (Papandreou et al.,
2006; Tarantilis et al., 1995).
All animals developed normally during the experimental period
and no mortality was recorded during Al treatment. Also, there
were no differences on body weight gain and liquid intake, between the experimental groups.
3.2. Effect of saffron on learning and memory ability of Al-exposed
mice
Passive avoidance task was performed to examine long-term
memory. The initial latency (IL) time, measured at training trial,
is an indicator of motor activity, and step-through latency (STL)
time, measured at testing trial, indicates memory of the aversive
experience. Al intake resulted in impaired long-term memory of
mice, as evidenced by the significantly lower (59%) mean STL time
of Al-treated animals compared to mean STL time of the controls
(Fig. 1). The lack of difference of mean STL time between Al +
saffron- and Al-treated groups (Fig. 1), shows that saffron extract
co-administered with Al during the last 6 days of the treatment
period was ineffective in reversing Al-induced memory
Fig. 1. Effect of saffron administration on initial (IL) and step-through (STL) latency
time of Al-exposed adult mice in step-through passive avoidance task. Al treated
mice displayed lower learning/memory capacity during the testing session of the
task, compared to the controls, whereas saffron co-treatment was ineffective. Each
horizontal line represents the mean latency time of ten animals in each group
(n = 10). Individual values are presented as squares for control mice, triangles for Al
treated and circles for Al + saffron treated ones. ap < 0.05 vs. STL time of the controls
(non-parametric Mann–Whitney test).
166
impairment. The mean IL time values were not significantly different among all experimental groups (Fig. 1), implying no difference
in motor activity of mice in training session.
3.3. Al and crocetin levels in cerebral tissues
The levels of Al in whole brain (-ce), cerebellum and liver of
control, Al-treated and Al + saffron treated mice are presented in
Table 1. Al was not detected in whole brain (-ce) of control mice,
whereas it was determined in high amounts in cerebellum. In
all animal groups Al levels in cerebellum were significantly
(p < 0.05) higher than those of the remaining brain. Also, Al concentrations were significantly (p < 0.05) increased in both cerebral
tissues and liver of Al-treated mice compared to the controls and
did not differ significantly (p < 0.05) in the cerebral and liver tissue
of Al + saffron treated mice, compared to Al-treated group.
Pooled whole brain (-ce) homogenates of Al + saffron treated
and control mice were analyzed by HPLC. HPLC chromatogram
(Supplementary data) of Al + saffron treated brain (chromatogram
B) shows a peak at 12.4 min, which is not detected in control brain
(chromatogram A), and corresponds to trans-crocetin. UV–visible
absorption spectrum of the peak presents a maximum double peak
at 420–450 nm, characteristic of the carotenoids. Crocetin concentration, was 2.0 ± 0.1 nmol total crocetin/g tissue (n = 2) in whole
brain of mice treated with saffron extract and not detected in brain
of control mice.
and 21%) and cerebellum (23%, 15% and 22%, 19%) compared to
the controls, except of the activity of DS-BuChE of whole brain
(-ce) that remained unchanged. Further significant (p < 0.05)
reduction of the SS- and DS-AChE activity in whole brain (-ce)
(15%, 28%) and cerebellum (24%, 27%) was observed in Al + saffron
treated mice in comparison with Al treated group, whereas saffron
treatment did not further affect the activity of cerebral BuChE.
Also, Al-treated mice displayed significantly (p < 0.05) decreased
liver BuChE (11% SS, 21% DS) and SS-AChE (25%) activities
compared to the controls, while Al + saffron treated mice displayed
further inhibition of liver BuChE (27% SS, 19% DS) and SS-AChE
(31%) compared to Al-treated mice.
3.5. Effect on brain MAO activity
The effect of administration of either Al alone or in the presence
of saffron extract at the end of the treatment period, on MAO isoforms activity of mouse whole brain (-ce) and cerebellum, is presented in Fig. 2. Al-treated mice displayed significantly (p < 0.05)
higher activities of MAO-A (19%) and MAO-B (10%) in their whole
brain (-ce) and significantly lower MAO-B activity (14%) in cerebellum, compared to the controls. Short-term co-administration of
saffron restored MAO-A and MAO-B activity of whole brain (-ce)
(decrease by 14% and 10%, respectively) and MAO-B activity (17%
increase) of cerebellum, to normal levels.
3.6. Effect on brain and liver oxidant/antioxidant indices
3.4. Effect on brain and liver ChE activity
The impact of Al or saffron treatment on the activities of both
isoforms (SS, DS) of AChE and BuChE in whole brain, cerebellum
and liver of adult mouse is presented in Table 2. Al intake decreased significantly (p < 0.05) the activity of both SS and DS isoforms of AChE and BuChE in mouse whole brain (-ce) (26%, 17%
Table 1
Al concentrationsa in cerebral tissues and liver of control, Al-treated and Al + saffron
treated adult mice.
Mice groups
Whole brain (-ce)
Cerebellum
Liver
Control
Al treated
Al + saffron treated
ND
1.30 ± 0.45**
0.97 ± 0.12**
7.77 ± 0.62
11.67 ± 0.63*
10.17 ± 1.47
3.97 ± 0.15**
7.62 ± 0.53*,**
10.22 ± 0.98*
ND, not detected.
a
Al concentrations are expressed as lg/g wet tissue. Data are the mean ± SEM
(n = 4 animals/group).
*
p < 0.05 vs. the control group.
**
p < 0.05 vs. cerebellum of the respective group (non-parametric Mann–Whitney
test).
MDA and GSH levels, as indicators of lipid peroxidation and cellular antioxidant defense, respectively, were measured in mouse
brain tissues and liver of all experimental groups. As shown in
Fig. 3a, Al intake increased (18%) significantly (p < 0.05) MDA levels
only in whole brain (-ce) in comparison with controls, while the
subsequent short-term co-administration of saffron restored
(decrease by 22%) MDA levels to normal. Short-term coadministration of saffron also improved the antioxidant status of
cerebellum and liver by significantly lowering the levels of MDA,
even though these tissues remained unaffected by Al treatment
(Fig. 3a). Additionally, Al-treated mice displayed remarkable
reduction in GSH content of whole brain (-ce) (44%), cerebellum
(16%) and liver (29%), compared to the control animals, whereas
Al + saffron treated mice exhibited elevated levels of GSH (29%,
14% and 22%, respectively) compared to Al-exposed group (Fig. 3b).
4. Discussion
The present study investigates the neuroprotective potential of
a 6-day i.p. administration of saffron extract to adult mice that
Table 2
Activity of SS- (salt soluble) and DS- (detergent soluble) isoforms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in brain and liver of control, Al treated and
Al + saffron treated adult mice.
Mice groups
Whole brain (-ce)
SS-ChE
AChE activity (lmol/min/g tissue)
Control
11.17 ± 0.42
Al treated
8.26 ± 0.20*
6.99 ± 0.28*,#
BuChE activity (lmol/min/g tissue)
Control
1.78 ± 0.08
Al treated
1.40 ± 0.07*
1.50 ± 0.07*
Cerebellum
Liver
DS-ChE
SS-ChE
DS-ChE
76.72 ± 0.99
63.47 ± 3.57*
45.43 ± 1.32*,#
6.32 ± 0.37
4.88 ± 0.14*
3.69 ± 0.18*,#
16.29 ± 0.61
13.87 ± 0.22*
10.06 ± 0.17*,#
5.93 ± 0.14
4.47 ± 0.36*
3.10 ± 0.34*,#
1.53 ± 0.06
1.24 ± 0.04*
1.13 ± 0.07*
26.95 ± 0.68
23.86 ± 0.80*
17.42 ± 0.42*,#
2.49 ± 0.07
2.50 ± 0.05
2.40 ± 0.06
Data are the mean ± SEM (n = 6 animals/group).
*
p < 0.05 vs. the control group.
#
p < 0.05 vs. Al treated group (non-parametric Mann–Whitney test).
2.47 ± 0.09
1.93 ± 0.12*
1.84 ± 0.07*
SS-ChE
DS-ChE
1.07 ± 0.31
0.90 ± 0.15
0.68 ± 0.17
9.64 ± 0.29
7.60 ± 0.26*
6.17 ± 0.25*,#
Fig. 2. Effect of saffron administration on monoamine oxidase activity (MAO-A and
MAO-B) of Al-exposed adult mouse brain. Al-induced activation of both MAO
isoforms in whole brain (-ce), inhibition of MAO-B in cerebellum and reversal
effects of saffron are evident. Data are the mean ± SEM (n = 6 animals/group).
⁄
p < 0.05 vs. the control group, #p < 0.05 vs. Al treated group (non-parametric
Mann–Whitney test).
were previously exposed to high Al intake through their drinking
water for a 5-week period. To this end, cognitive behavior and
brain cholinergic, monoaminergic and oxidative indices were assessed. Our findings showed that long-term Al intake induced
learning/memory decline in adult mice. Impaired performance of
167
rats on passive avoidance task has been shown by Bhalla et al.
(2010) after long-term administration of a double dose of AlCl3
in drinking water, while Sethi et al. (2008) showed declined spatial
learning abilities of rats in Morris-water maze test after administration of the same dose of AlCl3 (50 mg/kg/day) in drinking water
for 6 months. Saffron co-administration at the end of the treatment
period failed to reverse Al-induced cognitive deficits. In our previous study, short-term saffron administration enhanced learning/
memory in healthy adult and aged mice in passive avoidance task
(Papandreou et al., 2011). However, in the current report the
administered dosage of saffron extract and/or the duration of exposure (which are the same as in the study of Papandreou et al., 2011)
were insufficient to counteract Al-induced memory deficit.
Al levels were determined in whole brain (-ce), cerebellum and
liver of all animal groups prior to biochemical evaluation. The values of Al found in cerebral tissues, are comparable with those of
other studies that use Al as neurotoxic agent, although different
Al forms, doses and routes of administration are investigated
(Esparza et al., 2005; Kaizer et al., 2008; Sánchez-Iglesias et al.,
2007). Our findings showed high concentrations of Al in cerebellum of control mice (higher than liver), but none or below detection limits in whole brain (-ce), in accordance with Bellés et al.
(1998). In the current study, long-term AlCl3 intake through drinking water resulted in significantly increased Al concentrations in
whole brain, cerebellum and liver of adult mice. Our results are
Fig. 3. Effect of saffron administration on (a) lipid peroxidation (MDA) and (b) reduced glutathione (GSH) levels of Al-exposed adult mouse brain and liver. Al-induced
increase in MDA levels of whole brain (-ce), decrease in GSH levels of whole brain (-ce), cerebellum and liver, and reversal effects of saffron are evident. Data are the
mean ± SEM (n = 6 animals/group). ⁄p < 0.05 vs. the control group, #p < 0.05 vs. Al treated group (non-parametric Mann–Whitney test).
168
in accordance with previous studies where AlCl3 is administered to
rodents (Bhalla and Dhawan, 2009; El-Maraghy et al., 2001). The
entry of Al into the brain through the blood–brain barrier has long
been established (Yokel et al., 1999) and is also confirmed by our
results. The accumulation of Al in brain is consistent with the observed impaired learning/memory ability of mice in passive avoidance test. However, cerebral Al levels did not differ significantly
between Al- and Al + saffron treated mice, showing that short-term
saffron administration in the end of the treatment period did not
affect Al bioavailability.
Additionally, for the first time, we determined crocetin, the major metabolite (aglycon) of the bioactive crocin glycosides of saffron, in whole brain of Al + saffron treated mice, which was
absent from the brain of control mice. The detection of crocetin
in mouse brain demonstrates for the first time that this compound
crosses the blood–brain barrier when saffron extract is administered for a short term through intraperitoneal route. In recent report (Chryssanthi et al., 2011), the presence of crocetin in human
plasma was shown after 2 and 24 h of saffron tea consumption,
while only Yoshino et al. (2011) have demonstrated the distribution of crocetin in rat brain after a single oral administration of
pure crocetin, up to now.
The fact that saffron short-term co-administration failed to reverse the cognitive decline raises questions on the dosage, the period and the route of administration, before definite conclusions can
be drawn about its effectiveness. However, our study presents
some remarkable brain biochemical alterations that followed saffron co-treatment.
Determination of ChE activity showed that the DS-AChE was
predominant in cerebral tissues, while approximately equal values
of the activity of the two BuChE isoforms were recorded. These
observations are consistent with previous findings (Das et al.,
2005; Fernández-Gómez et al., 2008). In accordance with other
studies (Lassiter et al., 1998; Li et al., 2000), AChE activity constitutes the major fraction of total ChE activity in whole brain (-ce)
and cerebellum, whereas BuChE activity predominates in liver. In
the current work, Al intake resulted in significant decrease of the
activity of AChE and BuChE isoforms in mouse whole brain and cerebellum. Region-specific inhibition of the activity of rat cerebral
membrane-bound AChE and BuChE after Al intoxication, has been
shown by others (Julka et al., 1995). Previous studies have shown
necrotic rat hippocampal ultrastructural changes following the
same Al administration protocol (Jyoti and Sharma, 2006), providing an explanation for our cholinotoxic and memory impairment
results. However, Al has been suggested to interfere with the action of metabotropic glutamate receptors, enhancing excitotoxicity
and neuronal injury (Blaylock, 2012). Also, Al impairs hippocampal
long-term potentiation (LTP) in rats (Platt et al., 1995). Thus, besides the cholinotoxic effects of Al in the present study, the
involvement of possible Al-induced glutamatergic neurotransmission disturbances in the observed memory decline, should also
be considered. In liver, both BuChE isoforms’ activity (the main liver ChE fraction) was significantly decreased following Al administration, while only SS-AChE activity was decreased significantly.
However, increased activity of soluble and membrane-bound
forms of rat liver BuChE after Al intake through diet for 100–
115 days (Dave et al., 2002), has been previously demonstrated,
but the Al dose and period of exposure are different.
Saffron short-term co-administration caused further significant
reduction of cerebral AChE and liver BuChE isoforms activity. Cerebral BuChE activity was not further affected and only SS-AChE
activity of liver was further significantly decreased after saffron
co-treatment. The inhibitory effect of saffron on the activity of
brain SS- and DS-AChE in adult healthy mice has been demonstrated in our previous study (Papandreou et al., 2011). However,
the impact of saffron administration on brain BuChE and liver
ChE activities, has not been studied before. The considerable higher
percentage of inhibition of DS-AChE in whole brain of Al + saffron
treated mice compared to SS-AChE, suggests greater susceptibility
of the enzyme’s DS isoform to saffron treatment. Geromichalos
et al. (2012) have recently demonstrated, for the first time, that
crocetin inhibits AChE by binding at two different loci, the catalytic
center and the peripheral anionic sites, as assessed by in vitro enzymatic and molecular docking studies. Thus, the observed significant cerebral AChE inhibitory activity conferred by saffron shortterm co-administration, may be attributed to a direct interaction
of crocetin with the enzyme, since crocetin was detected in brain
of Al + saffron treated mice and was absent in controls’ brain; however, it remains to be investigated whether the crocetin concentration is sufficient for such an effect. Furthermore, other indirect
mechanisms cannot be excluded.
Monoamine oxidase (MAO, types A and B) is a mitochondrial
membrane-bound enzyme which catalyzes the oxidative deamination of monoamines, thus regulating monoaminergic neurotransmission. Increased brain MAO activity has been recorded in
neurodegenerative process (Hanish Singh et al., 2011; Mallajosyula
et al., 2008). Our results showed significant increase of whole brain
MAO isoforms activity after Al intoxication, and an opposite effect
on cerebellum MAO-B activity. Bhalla et al. (2010) have also presented increased and decreased MAO activity in rat cerebrum
and cerebellum, respectively, after long-term oral administration
of AlCl3. Activation of MAO isotypes in rat brain by prolonged intake of Al has been also previously reported (Huh et al., 2005),
but the underlying mechanisms have not yet been clarified.
Saffron co-treatment completely reversed Al-induced activation
of brain MAO isoforms and inhibition of cerebellar MAO-B. The effect of saffron administration on cerebral MAO activity has not
been studied before. MAO inhibitors have long been well characterized for their antidepressant properties (Bortolato et al., 2008).
The first small-scale clinical trials of saffron in the treatment of
mild to moderate depression, have shown significant benefits in
the mood of patients after a 6-week treatment (Akhondzadeh
et al., 2005; Noorbala et al., 2005). The antidepressant efficacy of
saffron could, at least in part, correlated with brain MAO inhibitory
activity of saffron extract and the presence of crocetin in brain that
are presented in our current study.
Oxidative stress has long been implicated in the initial and later
stages of neuronal degeneration (Melo et al., 2011). In the present
study, Al intake elevated significantly lipid peroxidation in whole
brain and decreased GSH content in both cerebral tissues and liver.
Other reports have also demonstrated increased lipid peroxidation
levels and decreased GSH content in rat brain regions after longterm oral administration of AlCl3 (Jyoti and Sharma, 2006; Nehru
and Bhalla, 2006). The resistance of liver to Al-induced lipid peroxidation, indicated by us and other studies (Kaneko et al., 2004), is
expected, as it constitutes the major site of detoxification in living
organism. Although Al is not a redox-active metal, there is extensive experimental evidence on oxidative stress-mediated Al neurotoxicity (Kumar and Gill, 2009). It has been demonstrated that Al
induces oxidative stress in cultured rat hippocampal neurons by
potentiating iron-mediated oxidative injury (Xie et al., 1996). Also,
in vitro studies (Oteiza, 1994) have shown that Al stimulates ironinduced lipid peroxidation in isolated membrane fractions through
binding to the membrane and promotion of changes in the
arrangement of membrane lipids. Considering the byproduct
(hydrogen peroxide) of MAO-mediated reactions, Al-induced activation of brain MAO isoforms provided by our results could, at
least in part, contribute to the observed increased brain oxidative
stress.
Reversal effects of saffron short-term co-administration were
recorded against Al-induced brain lipid peroxidation and GSH content reduction in cerebral tissues and liver. In our previous study
(Papandreou et al., 2011), saffron administration significantly
decreased MDA levels and increased GSH content in the brain of
healthy adult and aged mice. Shati et al. (2011) also showed that
intraperitoneal co-administration of saffron aqueous extract with
AlCl3 ameliorated the disturbances in mouse brain lipid peroxidation and antioxidant enzymes’ activity induced by the metal when
injected alone. Additionally, in our previous in vitro work (Papandreou et al., 2011) both saffron extract and its crocetin component
provided strong protection against H2O2-induced toxicity in
human neuroblastoma SH-SY5Y cells, by repressing reactive
oxygen species production. It has been reported that crocin, the
di-gentiobiosyl ester of crocetin, promotes mRNA expression of
c-glutamylcysteinyl synthase, the rate-limiting enzyme of GSH
synthesis, in PC12 cells under hypoxic conditions (Ochiai et al.,
2007). Accordingly, crocetin may mediate the observed brain antioxidant protection of saffron extract under Al neurotoxicity.
5. Conclusions
Our findings show that long-term intake of a relative high dose
of AlCl3 through drinking water resulted in metal accumulation in
adult mouse brain tissues and exerted neurotoxic effects, as evidenced by the declined learning and memory capacity, metalinduced inhibition of ChEs, changes of MAO and the production
of oxidative damage. On the other hand, short-term co-administration of saffron extract at the end of the treatment period beneficially affected mouse brain oxidative stress and antioxidant
status markers and MAO activity that were disturbed by Al. The
bioavailability of crocetin in mouse brain after saffron extract
administration through intraperitoneal route demonstrated in the
present study, supports the implication of this bioactive component in the observed neurochemical alterations. However, the particular saffron treatment scheme was ineffective in reversing
cognitive defects induced by the metal, although it had beneficial
action on biochemical markers of brain function. The findings
support further investigation of the potential of saffron and its
crocin constituents as neuroprotective agents.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was partially supported by BIOFLORA network of
University of Patras. Also, partial support from the University of Patras Research Committee is greatly acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.fct.2012.11.016.
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Structure–Activity Studies of lGnRH-III Through Rational Amino Acid
Structure–Activity
of lGnRH-III Through
Substitution and Studies
NMR Conformational
Studies Rational Amino Acid
Substitution and NMR Conformational Studies
Eleni V. Pappa,1 Aikaterini A. Zompra,1 Zoi Diamantopoulou,2 Zinovia Spyranti,1 George Pairas,1
Fotini N. Lamari,1 Panagiotis Katsoris,2 George A. Spyroulias,1 Paul Cordopatis1
1
Department of Pharmacy, University of Patras, Patras 26504, Greece
2
Department of Biology, University of Patras, Patras 26504, Greece
Received 3 April 2012; revised 31 May 2012; accepted 2 July 2012
Published online 17 July 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22123
the well-defined b-turn conformation of GnRH-I.
ABSTRACT:
#
Lamprey gonadotropin-releasing hormone type III
2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98:
(lGnRH-III) is an isoform of GnRH isolated from the sea
525–534, 2012.
lamprey (Petromyzon marinus) with negligible endocrine
Keywords: Lamprey GnRH-III; lGnRH-III analogs;
activity in mammalian systems. Data concerning the
solution structure GnRH-I; solution structure lGnRH-III;
superior direct anticancer activity of lGnRH-III have
antiproliferative activity
been published, raising questions on the structure–
activity relationship. We synthesized 21 lGnRH-III
analogs with rational amino acid substitutions and
studied their effect on PC3 and LNCaP prostate cancer
cell proliferation. Our results question the importance of
This article was originally published online as an accepted
preprint. The ‘‘Published Online’’ date corresponds to the
preprint version. You can request a copy of the preprint by
emailing the Biopolymers editorial office at biopolymers@wiley.
com
the acidic charge of Asp6 for the antiproliferative activity
and indicate the significance of the stereochemistry of Trp
INTRODUCTION
in positions 3 and 7. Furthermore, conjugation of an
he decapeptide gonadotropin-releasing hormone-I
(GnRH-I; pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-ProGly-NH2) has a pivotal role in orchestrating and
maintaining normal reproductive events by regulating the secretion of pituitary gonadotropin hormones.1 In addition, GnRH-I and many of its analogs exhibit
an antiproliferative effect on various cancer cells, which can
be exerted by indirect or direct ways.2–4 Chronic administration of GnRH analogs desensitizes the pituitary gonadotroph
cells, results in arrest of gonadotropin secretion, and thereby
suppresses ovarian and testicular function (chemical castration). Thus, chemical castration is a therapeutic approach of
hormone-dependent tumors such as prostate and breast cancer. Thousands of synthetic peptide analogs of GnRH have
been synthesized since its discovery, and several of them
(agonists or antagonists) have been used in breast and/or
prostate cancer therapy.4 In addition, GnRH analogs also
exert anticancer activity by directly affecting the hormonedependent and -independent cancer cells, an effect which
acetyl-group to the side chain of Lys8 or side chain
cyclization of amino acids 1-8 increased the
antiproliferative activity of lGnRH-III demonstrating
6
8
that the proposed salt bridge between Asp and Lys is not
crucial. Conformational studies of lGnRH-III were
performed through NMR spectroscopy, and the solution
structure of GnRH-I was solved. In solution, lGnRH-III
adopts an extended backbone conformation in contrast to
Additional Supporting Information may be found in the online version of this
article.
Correspondence to: Paul Cordopatis, Laboratory of Pharmacognosy and Chemistry
of Natural Products, Department of Pharmacy, University of Patras, GR-265 04
Patras, Greece; e-mail: [email protected] or Eleni V. Pappa, Laboratory of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy,
University of Patras, GR-265 04 Patras, Greece; e-mail: [email protected]
C 2012
V
Wiley Periodicals, Inc.
PeptideScience Volume 98 / Number 6
T
525
526
Pappa et al.
Table I
Yield of Synthesis and Physicochemical Properties of lGnRH-III Analogs
Peptide Code
lGnRH-III
Group A
Group B
Group C
a
Sequence
Yield (%)
pGlu1-His2-Trp3-Ser4-His5-Asp6-Trp7-Lys8-Pro9-Gly10-NH2
I
[Asn6]-lGnRH-III
II
[Asp6(OMe)]-lGnRH-III
III
[Glu6]-lGnRH-III
IV
[Gln6]-lGnRH-III
V
[DTrp3]-lGnRH-III
VI
[DTrp7]-lGnRH-III
VII
[DTrp3, DTrp7]-lGnRH-III
VIII
[DTrp3, DTrp7]-lGnRH-III-NHEt
IX
[Tic3, Tic7]-lGnRH-III
X
[DTic3, DTic7]-lGnRH-III
XI
[DTrp3, DTic7]-lGnRH-III
XII
[DTic3, DTrp7]-lGnRH-III
XIII
[NMeGlu1]-lGnRH-III
XIV
[cyclo(Glu1, Lys8)]-lGnRH-III
XV
[cyclo(Ac-Glu1, Lys8)]-lGnRH-III
XVI
[e-N-Ac-Lys8]-lGnRH-III
XVII
[Glu1, e-N-Ac-Lys8]-lGnRH-III
XVIII
[Ac-Glu1, e-N-Ac-Lys8]-lGnRH-III
XIX
[Lys5, His8]-lGnRH-III
XX
[cyclo(Glu1, Lys5), His8]-lGnRH-III
XXI
[cyclo(Ac-Glu1, Lys5), His8]-lGnRH-III
76.1
69.8
54.8
71.1
73.1
70.7
58.6
67.9
78.3
73.2
77.8
74.4
78.5
81.3
45.2
53.1
80.8
67.1
80.4
81.0
39.1
43.7
HPLCb
tR (min)
7.09
7.12
7.58
7.23
7.18
7.27
7.32
7.20
7.19
7.14
7.23
6.93
6.83
7.73
7.86
9.61
7.96
7.21
8.11
7.31
7.43
8.60
MH+obsd/(calcd)c
1260.33/1259.33
1258.92/1258.35
1274.02/1273.36
1273.66/1273.36
1272.10/1272.37
1259.70/1259.33
1260.23/1259.33
1259.83/1259.33
1287.92/1287.38
1205.55/1205.28
1205.58/1205.28
1231.97/1232.31
1231.94/1232.31
1292.11/1291.37
1276.17/1275.33
1318.14/1317.37
1302.23/1301.37
1320.19/1319.38
1362.33/1361.42
1259.71/1259.33
1276.08/1275.33
1318.21/1317.37
a
Yields were calculated on the basis of the amino acid content of the resin. All peptides were at least 98% pure.
For elution conditions, see Materials and Methods section.
c
Data obtained by ESI-MS, observed (obsd) and calculated (calcd) m/z values of MH+.
b
could be mediated through the GnRH receptors (GnRHRs)
highly expressed in those cells.
The discovery of lGnRH-III (pGlu-His-Trp-Ser-His-AspTrp-Lys-Pro-Gly-NH2), a native GnRH analog isolated from
sea lamprey (Petromyzon marinus)5 that stimulates the
release of estradiol and progesterone in the adult female sea
lamprey,6,7 has stimulated further research on their potential
medicinal uses. Functional activity of lGnRH-III has been
found in the hypothalamus of several (mammalian) species,
including rats, cows, and sheep, and it was originally suggested
that lGnRH-III might act as a mammalian Follicle-Stimulating
Hormone (FSH) releasing factor.8–11 However, the data concerning the effect of lGnRH-III on the selective secretion of
FSH are inconsistent.12–14 While questions concerning the endocrine activity of lGnRH-III are yet not answered, studies
concerning the direct anticancer activity of lGnRH-III have
been published in the last decade.15–18 lGnRH-III differs in the
sequence 5-8 from the mammalian GnRH (GnRH-I)5 and is a
weak agonist of the mammalian GnRHR. The ability of
lGnRH-III to inhibit proliferation of cancer cells, combined
with the weak GnRH-I agonistic activity, makes it an excellent
starting compound for the development of peptide analogs
with high and selective anticancer activity. Currently, new
GnRH analogs have been synthesized to enhance the anticancer potency of native lGnRH-III.5,13,16,17 Those studies have
shown that modifications in positions 5-8 decrease the anticancer activity of lGnRH-III. Systematic replacement of residues 5-8 of lGnRH-III by Ala19 showed that mutation of Asp6
or Trp7 resulted in the loss of antiproliferative effect probably
due to an ionic interaction between these residues, which
might stabilize the biologically active conformation of lGnRHIII. In addition, the importance of indole rings of Trp3,7 was
also proposed.16 However, further information on the structure–activity relationships of lGnRH-III is still lacking.
To further investigate the structure–activity relationship
of lGnRH-III, we synthesized 21 new lGnRH-III analogs and
studied their direct antiproliferative effect on prostate cancer
cells. The analogs were arranged into three groups: (A) analogs with single amino acid changes in position 6, (B) analogs with modifications in positions 3 and/or 7, (C) analogs
with amino acid changes in positions 1, 5, 8 and cyclic analogs (Table I). In particular, we studied the importance of the
Asp in position 6 by incorporation of several amino acids
[Asn, Asp(OMe), Glu, Gln]. DTrp and L/D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) were inserted in
positions 3 and/or 7 to further investigate the role of the
Biopolymers (Peptide Science)
Structure–Activity Studies of lGnRH-III
indole ring of Trp and its stereochemistry. In the last group,
the importance of the amino acids in positions 1 and 8 are
mainly studied. The utility of backbone cyclization has been
well established in peptides, and it has been demonstrated to
increase biological activity and selectivity20,21 since they are
usually more stable in metabolism than the parent linear
molecules.22 Therefore, cyclopeptides are of great importance
and interest both in pharmaceutical and chemical respect.
According to these considerations, we also synthesized 1-8
and 1-5 side chain cyclic peptides and studied their antiproliferative activity on prostate cancer cells.
Moreover, until today, some sporadic attempts have been
done to study the solution conformation of lGnRH-III, but
there is still no NMR 3D model available. According to the
results of a previous molecular dynamics simulation study by
Lovas and coworkers,23 lGnRH-III has an extended helical conformation from residues 2 through 7, which is stabilized by
intramolecular hydrogen bonds and slightly polar interactions.
However, that conformational model has not been confirmed by
other studies.17 The determination of the conformational characteristics of lGnRH-III is a crucial step for in depth study of
structure–activity relationship. In that direction, we studied the
solution structure of lGnRH-III through NMR spectroscopy,
and a conformational model of this decapeptide is presented.
Moreover, the solution structure of GnRH-I (PDB 1YY1) was
studied under the same experimental conditions to elucidate the
conformational differences between GnRH-I and lGnRH-III.
Peptide Synthesis
The linear and the cyclic peptides (Table I) were rationally designed
and synthesized manually. 9-Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids and peptide reagents were obtained from CBL
(Patras, Greece), Bachem (Bubendorf, Switzerland), and Novabiochem (Läufelfingen, Switzerland). All solvents and reagents used for
solid-phase synthesis were of analytical quality. Purification of crude
peptides was achieved by semipreparative high-performance liquid
chromatography (HPLC) (Mod.10 ÄKTA, Amersham Biosciences,
Piscataway, NJ) coupled to a UV/Vis detector from Amersham Pharmacia Biotech on a Supelcosil C18 (5lm particle size, 8 mm 3 250
mm, Sigma-Aldrich, St Louis, MO). Analytical HPLC (Waters, Malva,
Milford, CT) equipped with a Waters C18 column (symmetry, 3.5
lm, 4.6 mm 3 75 mm) produced single peaks with at least 98% of
the total peak integrals (integrated with Empower software). Electrospray ionization-mass spectrometry (ESI-MS, Micromass-Platform LC
instrument, Waters Micromass Technologies, Milford, MA) was in
agreement with the expected mass. The physicochemical properties of
the new analogs are summarized in Table I.
Linear Peptides. Sieber amide resin (0.45 mmol/g capacity) was
used as a solid support for the linear peptides. All peptides were
synthesized manually following a Fmoc strategy. First, the Fmoc
527
protecting group on the resin was removed by treatment with 20%
piperidine/N,N-dimethylformamide (DMF) and then, the coupling
reaction was performed by using threefold molar excesses of activated Fmoc-amino acids throughout the synthesis. The amino acids
were activated essentially either in situ using diisopropylcarbodiimide/1-hydroxy-benzotriazol (HOBt) in DMF for 2.5 h or with
O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate,
HOBt, and N,N-diisopropylethylamine (DIEA) (2.45:3:6). The
Fmoc deprotection step was accomplished by treatment with 20%
piperidine in DMF or by 2% 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) in DMF containing 2% piperidine for 2–3 min24 to prevent
the potential migration of Dde and DMab groups to other functional groups after the incorporation of Fmoc-DLys(Dde)-OH or/
and Fmoc-Glu(DMab)-OH. Regarding analogs XVI, XVII, and
XVIII after the removal of the DMab and/or Dde protecting groups
by 2% hydrazine in DMF (3 3 2 min), on-resin acetylation of the
Na-amino group of Glu1 (peptide XVIII) and of Ne-amino group of
Lys8 (peptides XVI, XVII and XVIII) was performed by 0.5M Ac2O
in DMF for 1 h. The resin was washed manually with DMF,
dichloromethane (DCM), and diethyl ether successively to remove
the solvents excess. The peptidyl resin was treated with the splitting
mixture trifluoroacetic acid (TFA)/DCM/anisole/1,2-ethanedithiol
(EDT)/H2O (92:3:2:1:2, v/v/v/v/v), for 3 h to remove the protecting
groups and the peptide from the resin. The resin was filtered off,
and the solution was concentrated. Peptides were isolated by precipitation with cold diethyl ether, centrifuged, dissolved in water with a
few drops of acetic acid, and lyophilized. Purification of crude peptides was achieved by semipreparative HPLC (Mod.10 ÄKTA, Amersham Biosciences) on Supelcosil C18 (5 lm particle size, 8 mm 3
250 mm, Sigma-Aldrich) Analytical HPLC (Waters, Malva)
equipped with a Waters C18 column (symmetry, 3.5 lm, 4.6 mm 3
75 mm) produced single peaks with at least 98% of the total peak
integrals (integrated with Empower software). ESI-MS (MicromassPlatform LC instrument, Waters Micromass Technologies) was in
agreement with the expected mass. The physicochemical properties
of the new analogs are summarized in Table I.
Cyclic Peptides. The precyclic peptides (analogs XIV, XV, XX,
XXI) were synthesized on a Sieber amide resin (0.45 mmol/g
capacity) utilizing a standard Fmoc solid-phase synthesis protocol
described above. Fmoc-protected peptidyl resins were treated with
2% DBU in DMF containing 2% piperidine for 2–3 min. Bocprotecting group was inserted on the free terminal amino group of
the XIV and XX after treatment with (Boc)2O (3 equiv.)/DIEA (3
equiv.) in DMF (1 h, RT) while, on-resin N-terminal acetylation of
the peptides XV and XXI was performed by using 0.5M Ac2O in
DMF for 1 h. After that step, Dde and DMab protecting groups
were removed by using 2% hydrazine in DMF (3 3 2 min). Then,
the amide bond between c-carboxyl group of Glu1 and the e-amino
group of Lys in position 8 (analogs XIV, XV) or in position 5 (analogs XX, XXI) was carried out on the resin, using a mixture of
PyBoP/HOBt/DIEA (1.5 equiv./1.5 equiv./2.0 equiv, 3 3 4 h, RT608C), monitoring the formation of the lactam bridge by Kaiser
test. Cleavage from the resin and deprotection of the amino acid
side chains of the cyclopeptides were performed as reported above.
The resin was filtered off, and the solution was concentrated. The
crude product was precipitated with cold diethyl ether, centrifuged,
and lyophilized. Cyclopeptides were analyzed by analytical RPHPLC and characterized using the procedure described above. The
528
Pappa et al.
physicochemical properties of the new analogs are summarized in
Table I.
All peptides were purified by semipreparative HPLC on a RP C18 support using a linear gradient from 20% to 35% of solvent A
(0.1% TFA/H2O) and solvent B (0.1% TFA/acetonitrile) for 15 min
at a 2 mL/min flow rate and UV detection at 215 and 280 nm.
Eluted peptides were lyophilized immediately. The purity of each
peptide was verified by an analytical using a reversed-phase C18 column at a 1 mL/min flow rate with the same solvent system as in the
preparative HPLC. The molecular weight of the peptides was confirmed by using ESI-MS. The HPLC chromatogram showed that the
purity of the peptides was >98%, while ESI-MS showed the correct
molecular ion for the peptide.
Cell Culture and Proliferation Assay
The human prostate cancer epithelial cell lines PC3 and LNCaP
(ATCC, American Type Culture Collection) were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS),
100 U/mL penicillin, and 100 lg/mL streptomycin. Cultures were
maintained in 5% CO2 and 100% humidity at 378C. Cell culture
reagents were from BiochromKG (Seromed, Germany).
Slow-growing LNCaP and fast-growing PC3 cells were seeded
into 48-well culture plates at 1 3 104 cells/well. After a 24-h incubation period, adherent cells were treated with the peptides in 2.5%
(v/v) FBS-RPMI-1640. At the end of the incubation period, cells
were fixed with methanol and stained with 0.5% crystal violet in
20% methanol for 20 min.25 After gentle rinsing with water, the
retained dye was extracted with 30% acetic acid, and the absorbance
was measured at 590 nm.
Comparison of mean values among groups was done using
ANOVA and the unpaired Student t-test. Homogeneity of variance
was tested by Levene’s test. Each experiment included at least triplicate measurements for each condition tested. All results are
expressed as the mean 6 SD of at least three independent experiments. Values of P less than 0.05 were taken to be significant (*P <
0.05, **P < 0.01, ***P < 0.001).
NMR Spectroscopy
Samples of synthetic GnRH-I and 1GnRH-III in deuterated dimethyl sulfoxide (DMSO-d6) were used for NMR studies. Data were
acquired in a wide range of temperatures from 298 K up to 343 K on
a Bruker Avance 400 MHz spectrometer. 1H 1D NMR spectra were
recorded using spectral width of 12–17 ppm with or without presaturation of the H2O signal. 1H-1H 2D Total Correlation Spectroscopy
(TOCSY)26,27 were recorded using the MLEV-17 spin lock sequence
using sm ¼ 80–100 ms, and 1H-13C HSQC28,29 spectra with 200.791
ppm spectral width in F1. 1H-1H TPPI Nuclear Overhauser Effect
Spectroscopy (NOESY)30,31 spectra were acquired using mixing time
sm ¼ 400 ms applying water suppression during the relaxation delay
and mixing time. All 2D spectra were acquired with 10.014 ppm spectral width, consisting of 2K data points in the F2 dimension, 16–32
transients, and 512–1024 complex increments in the F1 dimension.
Raw data were multiplied in both dimensions by a pure cosinesquared bell window function and Fourier-transformed to obtain
2048 3 2048 real data points. A polynomial baseline correction was
applied in both directions. For data processing and spectral analysis,
the standard Bruker software (XWinNMR 3.5) and XEASY program32 (ETH, Zurich) were used.
NOE Constraints
Intensities of dipolar connectivities were mainly extracted by
NOESY maps acquired at 298 K and 400 ms of mixing time. 409
and 299 NOESY cross-peaks were assigned in both dimensions for
GnRH-I and 1GnRH-III peptides, respectively, in DMSO-d6. The
number of unique cross-peaks was 198 and 150 (20 and 15 NOEs
per residue for GnRH-I and 1GnRH-III, respectively). Their intensities were converted into upper limit distances through CALIBA.33
Sequential constraints, number and range of NOEs are illustrated in
Supporting Information Figures S1 and S2.
Structure Calculations and Refinement
The NOE-derived structural information extracted from the analysis
of NOESY spectra acquired in DMSO-d6 solutions under identical
experimental conditions for both peptides was introduced to
DYANA34,35 software for structure calculation. 113 and 87 NOE
constraints for GnRH-I and 1GnRH-III, respectively, were found
meaningful and used in DYANA calculations. A force constant of
133.76 kJ mol1 Å2 is applied for the distance constraints. The 20
models’ ensembles of both GnRH peptides are illustrated in Figures
4 and 5 (Figures are generated with MOLMOL36). Structural calculations have been performed on IBM RISC6000 and xw4100/
xw4200 HP Linux workstations.
RESULTS
Synthesis of Peptides
The application of Fmoc/tBu solid phase strategy combined
with the appropriate side chain protecting groups resulted in
the appropriate peptides with good yields of 40–81% (Table
I). The preparation of side chain to side chain-cyclized peptides through lactam bridge formation requires orthogonal
protecting groups for side chain amino and carboxylate functionalities. Our approach to acetylate or protect by the Boc
protecting group the N-terminal amino group of the protected precyclic peptide was effective to prevent the potential
formation of Na-pyroglutamyl chain-terminated peptides,
often favored during the deprotection step of Dmab group.37
Effect on Prostate Cancer Cell Proliferation
To study the anticancer activity of lGnRH-III and lGnRH-III
analogs, we tested their effect on the anchorage-dependent
proliferation of the well-established prostate carcinoma cell
lines, LNCaP and PC3, that mimic different stages on the
progression of prostate cancer. LNCaP cells are androgensensitive and express a large number of GnRHRs,16,17 and
PC3 cells are highly malignant, androgen-insensitive, and
express lower number of GnRHrs.6,38,39 lGnRH-III inhibited
the proliferation of LNCaP cells in a concentration-dependent manner having a maximal effect (21.5% reduction) at the
529
of lGnRH-III and significant (P < 0.01) effect on PC3 proliferation, incorporation of DTic in positions 3 and 7 (analog
X) led to increased antiproliferative effect on both cancer cell
lines (Figure 2). The antiproliferative effect of [DTrp3,
DTic7]-lGnRH-III (analog XI) and [DTic3, DTrp7]-lGnRHIII (analog XII) was higher than that of lGnRH-III. In detail,
analog XI inhibited the proliferation of LNCaP and PC3 cells
by 36.5 6 0.9% (P < 0.001) and 32.4 6 4.0% (P < 0.001),
respectively, and peptide XII, by 36.7 6 0.7% (P < 0.001)
and 31.6 6 2.7% (P < 0.001), respectively.
FIGURE 1 Effect of lGnRH-III and analogs I–IV on the proliferation of LNCaP and PC3 cells at a final concentration of 20 lM, after
144 h incubation for LNCaP cells and 96 h for PC3 cells. The cell
number was estimated as described under Materials and Methods
section. Results are presented as % inhibition relative to the control
and are the mean 6 SEM of three independent experiments. ***P <
0.001.
concentration of 20 lM. All analogs of lGnRH-III were tested
at 20 lM concentration.
Analogs Modified in Position 6 (Group A)
[Asn6]-lGnRH-III (analog I) and [Glu6]-lGnRH-III (analog
III) did not have significant inhibitory effect on LNCaP and
PC3 cell proliferation whereas, the antiproliferative effect of
analog [Gln6]-lGnRH-III (IV) was similar to that of lGnRHIII. Substitution of Asp in position 6 by Asp(OMe) (peptide
II) resulted in the most effective analog of that group (Figure
1); the inhibition effect on LNCaP cell proliferation, was
higher compared to lGnRH-III (32.1 6 0.9%, P < 0.001).
Furthermore, while lGnRH-III was ineffective on PC3 cell
proliferation, analog [Asp6(OMe)]-lGnRH-III had significant
(27.7 6 2.1%, P < 0.001) antiproliferative effect.
Analogs with Amino Acid Changes in Positions 1, 5, 8
and Cyclic Compounds (Group C)
The antiproliferative effect of [NMeGlu1]-lGnRH-III (analog
XIII) on LNCaP cells was 30% lower than that of lGnRH-III
whereas it had no effect on PC3 cell proliferation. Cyclic analogs [cyclo(Glu1, Lys8)]-lGnRH-III (XIV) and [cyclo(AcGlu1, Lys8)]-lGnRH-III (XV) had increased inhibitory effect
on cell proliferation compared to lGnRH-III; their effect on
LNCaP cells was 36.0 6 2.0 (P < 0.001) and 35.861.0 (P <
0.001), respectively, while on PC3 cells the observed effect
was 33.8 6 2.0 (P < 0.001) and 30.6 6 3.0 (P < 0.001),
respectively (Figure 3). Acetylation of Ne amino group of
Lys8 (analog XVI) led to significant increase in antiproliferative effect on both cell lines compared to lGnRH-III. Interestingly, further modifications on that analog in position 1
(analogs XVII and XVIII) decreased the effect on LNCaP cell
proliferation compared to [e-N-Ac-Lys8]-lGnRH-III (Figure
3). On PC3 cell line, analog XVII had similar and XVIII had
higher effect than analog with simple acetylation of Ne amino
group of Lys8. Analog XIX {[Lys5, His8]-lGnRH-III} resulting
Analogs with Modifications in Position 3 and/or 7
(Group B)
Analog [DTrp3]-lGnRH-III (V) had increased antiproliferative activity on LNCaP cells and significant (P < 0.001) effect
on PC3 cell proliferation compared to lGnRH-III, while substitution of Trp7 by DTrp (analog VI) dramatically decreased
the effect (no significant effect on both cell lines). Simultaneous incorporation of DTrp in positions 3 and 7 (analog VII)
decreased the antiproliferative effect induced by analog V
whereas, further modification of the C-terminal amide in
analog [DTrp3, DTrp7]-lGnRH-III-NHEt (peptide VIII)
increased it. Although analog [Tic3, Tic7]-lGnRH-III (IX)
caused lower inhibition of LNCaP cell proliferation than that
FIGURE 2 Effect of lGnRH-III and analogs V–XII on the proliferation of LNCaP and PC3 cells at a final concentration of 20 lM,
after 144 h incubation for LNCaP cells and 96 h for PC3 cells. The
cell number was estimated as described under Materials and Methods section. Results are presented as % inhibition relative to the
control and are the mean 6 SEM of three independent experiments.
**P < 0.01; ***P < 0.001.
530
Pappa et al.
FIGURE 3 Effect of lGnRH-III and analogs XIII–XXI on the proliferation of LNCaP and PC3 cells at a final concentration of 20 lM, after 144 h incubation for LNCaP cells and 96 h for PC3 cells. The cell
number was estimated as described under Materials and Methods section. Results are presented as % inhibition relative to the control and
are the mean 6 SEM of three independent experiments. ***P < 0.001.
after an exchange of positions between the two basic amino
acids, His and Lys, had similar effect to lGnRH-III on LNCaP
and PC3 proliferation. The antiproliferative effect on LNCaP
cells of 1-5 cyclic analogs XX and XXI was lower than that of
lGnRH-III and evidently decreased compared to counterparts 1-8 cyclic analogs (XIV and XV, Figure 3).
NMR Studies
Resonance Assignment. TOCSY maps of GnRH-I and
1GnRH-III peptides were first analyzed to assign the individ-
ual spin patterns of amino acids through scalar connectivities
(Figure 4). Sequential, medium-, and long-range connectivities were identified from NOESY maps acquired with
sm ¼ 400 ms. Proton chemical shift peptides are reported in
Supporting Information Tables S1 and S2.
As far as the GnRH-I peptide is concerned, NHNH connectivities of the type (i,i + 1) are detected for the region spanning the residues His2-Leu7 while NHNH (i,i + 2) NOE
cross-peaks are detected for the residue pairs His2-Ser4, Trp3Tyr5, Tyr5-Leu7, and Arg8-Gly10. On the other hand, Ha
NH
connectivities of the type (i,i + 1) are observed for the fragments pGlu1-Tyr5 and between the residues Leu7-Arg8, while
HbNH type (i,i + 1) connectivities have been observed for
the N-terminal tetra-peptide segment (pGlu1-Ser4) and for the
Tyr5-Gly6 pair. Moreover, Ha
NH (i,i + 2) NOEs are identified among amino acids almost all over the peptide sequence.
More than 30 medium-range NOEs were observed in the
NOESY maps acquired with sm ¼ 400 ms. The most characteristics among them are Hb
NH type connectivities between
amino acids Ser4-Gly6 and protons of the aromatic ring of His2
with all backbone protons of Ser4 as well as NOEs between
Leu7-Gly10 and Tyr5-Arg8. Long-range NOE signals involve the
amide proton of N-terminal Serine at position 4 of the peptide
sequence and the C-terminal Proline at the position 9 (four
NOEs) as well as the amine protons of the CO-NH2 terminal
group (one NOE). These data provide a solid experimental evidence for the spatial proximity of the peptide’s termini.
FIGURE 4 Characteristics 1H-1H TOCSY fingerprint regions of GnRH-I and 1GnRH-III peptides recorded at 400 MHz NMR and in DMSO-d6 at 298 K.
FIGURE 5 Backbone representation of the family of 20 energy
minimized DYANA models calculated for the GnRH-I peptide illustrated in blue. Side chains are illustrated in yellow. Figures are generated with MOLMOL.
Although the 1GnRH-III exhibits a lower number of NOE
cross-peaks compared to the GnRH-I, the number of identified NOEs is satisfactory, and 1GnRH-III can be defined by a
single well-determined structure. The NOE pattern involving
NHNH connectivities, spanning the residues His2-Lys8, for
this peptide is rather similar to these of the GnRH-I analog,
while HaNH and HbNH (i,i + 1) type connectivities have
been identified all over the peptide sequence except for Pro9.
Both long- and medium-range NOEs involving backbone protons present in GnRH-I were totally absent in the 1GnRH-III
analog. Only two HbHb (i,i + 2) type connectivities among
Trp3 and His5 as well as one NOE cross-peak between the
eNH of Lys8 and the Hb of Asp6 have been assigned. Moreover,
the interaction among the aromatic rings of His5 and Trp7 is
manifested by the existence of two medium-range NOEs
between the He proton of His5 and the Hd proton of Trp7.
Structure Calculation and Conformational Analysis
The NOE-derived structural information extracted from the
analysis of NOESY spectra acquired in DMSO under identical
experimental conditions (sm ¼ 400 ms) was introduced to
DYANA for structure calculations. The average target function
for the DYANA family of 20 calculated models was found 9.2
3 102 6 1.7 3 102 Å2 for GnRH-I and 0.36 6 1.64 3 103
Å2 for 1GnRH-III, respectively. No consistent violations
existed at the final DYANA run, and no constraint violation
was found larger than 0.20 Å. The RMSD values for the
GnRH-I 20 models’ ensembles were calculated as 0.52 6 0.24
Å (BB) and 0.96 6 0.19 Å (HA). Similarly, the 1GnRH-III
DYANA 20 models’ ensembles exhibit pairwise RMSD values
for all residues 0.70 6 0.18 Å (BB) and 1.52 6 0.60 Å (HA).
3D Solution Models
Despite the fact that a typical HaNH (i,i + 3) connectivity
has not been observed for the fragment Ser4-Arg8, the presBiopolymers (Peptide Science)
531
ence of the HaNH and NHNH (i,i + 2) connectivities—
together with the absence of HaNH (i,i + 1) type NOEs
for Gly6—suggest the existence of a b-turn structure in this
region. In this conformation, the N-terminal region is close
to the C-terminal and the spatial proximity of the two termini is manifested by long-range NOEs between: (i) the NH
proton of Ser4 and various Pro9 protons, and (ii) the NH
proton of the C-terminal amide. Since these long-range constraints might be fundamental for the b-turn formation and
the convergence of the two peptides’ termini, structure calculations were also performed without the above-mentioned
long-range NOEs. The resulted ensemble of 20 DYANA models (PDB 1YY1) is also characterized by the formation of the
b-turn and the highly flexible peptide termini. This conformation led us to conclude that the peptide preserves the hairpin-like conformation, and the NOE contacts that determine
this conformation are all among the residues of the Ser4-Arg8
loop.
In the GnRH-I solution structure presented here, Gly6 is
found in the tip of the loop formed by residues Ser4-Arg8
(Figure 5). NOEs of (i,i + 2) between Ha Ser4 and HN Gly6
as well as Gly6 HaHN Arg8 indicate that achiral Glycine
residue in position 6 allows the turn formation and the Ushape fold of the peptide. Additionally, the side chains of
Tyr5 and Leu7 seem to be in a short distance, while the
observed NOEs between the Tyr5 aryl protons and the Leu7
methyl protons support the parallel orientation of their side
chains. Moreover, the side chains of His2 and Trp3 present
different orientation, while Trp3 orients its indole ring to the
same direction with the Tyr5 aryl ring.
The substitution of the GnRH-I tetra-peptide segments
Tyr5-Gly6-Leu7-Arg8, which is located in the middle of the
GnRH-I sequence with the His5-Asp6-Trp7-Lys8 segment in
1GnRH-III, seems to impose great structural changes in the
stereochemistry of the latter peptide. The spatial arrangement of both N- and C-terminal fragments is totally different
with respect to the peptide representing the native hormone,
since no long-range NOEs involving the terminal residues
have been identified (Figure 6). Furthermore, there is no experimental evidence for a b-turn formation in the peptide
segment 4-8, and consequently 1GnRH-III does not maintain
the hairpin structure obtained in many GnRH analogs.40,41
Despite the lower number of NOE cross-peaks identified in
the NOESY spectra of 1GnRH-III, the peptide seems to
adopt a rather defined backbone conformation. Our NMR
data suggest that the molecule adopts extended backbone geometry with three irregular turns/bends around amino acids
Trp3, Ser4, and Pro9. The side chains of His2 and Trp3 still
occupy different region in space, with the indole ring of Trp3
found on the opposite site of the peptide backbone level in
532
Pappa et al.
FIGURE 6 Backbone representation of the family of 20 energy
minimized DYANA models calculated for the 1GnRH-III peptide
illustrated in blue. Side chains are illustrated in cyan. Figures are
generated with MOLMOL.
respect to the His2 side chain and pointing towards the
indole ring of Trp7. Moreover, the aromatic ring of His5 is
found within NOE distance with Trp7 indole ring supporting
the parallel orientation of their side chains.
DISCUSSION
The ability of lGnRH-III to inhibit proliferation of cancer
cells, combined with the weak GnRH-I agonistic activity,
makes it an excellent starting compound for the development
of peptide analogs with enhanced selectivity and anticancer
activity. In this study, we report the synthesis of 21 new
lGnRH-III analogs and their impact on the proliferation of
two different prostate cancer cell lines. Moreover, to investigate the conformational resemblance or diversity in GnRH
family, we studied the conformational properties of lGnRHIII and GnRH-I through NMR spectroscopy and their solution models are presented herein.
In our previous studies,41 we have shown that GnRH-I
does not exhibit significant antiproliferative activity on
LNCaP and PC3 cells even at the concentration of 100 lM.
Results of this study provide experimental evidence that the
inhibitory effect of lGnRH-III, at the concentration of 20
lM, on LNCaP cell proliferation was 21.5 6 0.9% while, on
PC3 cell it was negligible. The absence of high affinity binding sites on PC3 cells42 and the lower number of GnRHRs
expressed on them39 compared to LNCaP cells, may be
related to the lack of responsiveness of PC3 cells following
lGnRH-III treatment. However, many of the new analogs of
lGnRH-III presented on this study had significant antiproliferative effect on both cancer cell lines. Our observations
confirm the results of other studies that additional mechanisms may mediate the biological effects of GnRH analogs
on cancer cells which have not yet been clarified.42
In lGnRH-III, the position 6 is occupied by Asp, whereas
mammalian GnRH (GnRH-I) bears Gly at this position.
Although Gly6 is often replaced by D-amino acids to enhance
the biological activity of GnRH-I analogs, previous studies
report that removal of the Asp6 in lGnRH-III resulted in
complete loss of biological activity.19,43 To further investigate
the significance of this position, we substituted Asp6 by Asn,
Asp(OMe), Glu, and Gln. Incorporation of Asp(OMe)
instead of Asp6 significantly increased the antiproliferative
activity of lGnRH-III on LNCaP cell line. Furthermore,
Asp(OMe)-analog was the only analog of that group, with
significant effect on PC3 proliferation. Insertion of Gln was
well tolerated, while no antiproliferative activity on LNCaP
cells was observed when Glu or Asn substitutes Asp6. These
findings suggest that neither the acidic function of the side
chain of Asp6 nor the putative salt bridge (Asp6-Lys8) are
crucial for the antiproliferative activity of lGnRH-III, and we
presume that different intramolecular interactions, that
deserve further investigation, may occur and preserve the
bioactive conformation.
According to Herédi-Szabó et al.,19 indole rings of Trp3,7
are crucial for the anticancer activity, probably due to interactions of these residues with the N-terminal which stabilizes
the biological active conformation of lGnRH-III. In our
study, the antiproliferative effect of the analog with DTrp in
position 3 was significantly higher than lGnRH-III whereas
DTrp7 analog was ineffective. Introduction of DTrp instead
of Trp3,7 decreased the anticancer activity while, combined
modification on the C-terminal resulted in a significant
increase of the anticancer potency in both cancer cell lines.
Tic is a conformationally restrained imino acid that has been
incorporated to GnRH antagonists as local structure determinant.44 In addition, we have previously shown that substitution of Trp3 in GnRH-I by L- or D-Tic increased the antiproliferative effect of the analogs.45 In this study, results
show that substitution of Trp3,7 by Tic decreases the antiproliferative effect of lGnRH-III on LNCaP cells and slightly
improves the effect on PC3 cancer cells. In contrast, the inhibitory activity of the analogs [DTic3,7]-lGnRH-III (X),
[DTrp3, DTic7]-lGnRH-III (XI), and [DTic3, DTrp7]lGnRH-III (XII) was significantly higher. Based on these
findings, we presume that introduction of DTic, although
lacks the indole ring, preserved the biologically active conformation of lGnRH-III. Furthermore, the importance of the
stereochemistry of the residues in positions 3 and 7 is also
indicated.
Conjugation of an acetyl-group via the side chain of Lys8
significantly increases the antiproliferative activity of lGnRHIII suggesting that the putative salt bridge between Asp6 and
Lys8 in lGnRH-III, proposed in an earlier structure activity
study16 and demonstrated by our findings, is not important
for the anticancer activity. Similar elevation of the antiproliferative effect was observed by analogs with side chain
FIGURE 7 Superimposition of the backbone of the two hormones, lGnRH-III is illustrated in yellow while GnRH-I in blue. The
calculated RMSD of both peptides for the segment 1-6 found to be
0.815Å.
cyclization of amino acids 1-8 (peptides XIV, XV) probably
due to a more constrained conformation. On the other hand,
although exchanging places between the weakly basic His5
and the more basic Lys8 did not alter the anticancer activity
of the parent hormone, simultaneous 1-5 side chain cyclization in analogs XX and XXI resulted in lower antiproliferative effect.
In our attempt to elucidate conformational characteristics
of lGnRH-III, the solution model was determined through
NMR spectroscopy in DMSO, a solvent widely used for solubility reasons and is an acceptable medium of mimicking a
prototypical hydrophobic environment. Furthermore, the 3D
solution model of GnRH-I was also determined to shed light
on the conformational differences between the two peptides.
The obtained results show substantially different conformational features between the 1GnRH-III and the GnRH-I
(Figure 7). According to our NMR study, the network of sequential NOE connectivities present in GnRH-I spectra suggest the existence of b-turn structure for the Ser4-Arg8 segment. Long-range NOE signals between the N- and C-termini provide solid experimental evidence for the spatial
proximity of the two termini. On the other hand, 1GnRH-III
does not exhibit the GnRH-I conformation (U-shape). In
spite of earlier publications suggesting the existence of some
helical properties,23 such backbone conformation cannot be
supported by our results. In contrast, the 3D models of
1GnRH-III can be characterized by the extended backbone
conformation forming a kink around Asp6 and two more
turns/bends around Trp3 and Pro9. The present lGnRH-III
models are in agreement with Mezo et al.,17 where a relatively
ordered extended like backbone conformation is proposed
for the central region of the 1GnRH-III peptide.
The substitution of the small and flexible residue Gly in
position 6 of the GnRH-I sequence with Asp6 in 1GnRH-III
does not favor the formation of the b-type conformation in
the 4-8 segment of the peptide sequence. Moreover, the subBiopolymers (Peptide Science)
533
stitution of Leu7 with the bulky Trp7 differentiates remarkably the conformation of the peptide. This also becomes evident by the observed NOE interactions between His5 and
Trp7, which do not favor the spatial proximity of the two termini. As a consequence, the hydrophobic core present in
GnRH-I, is lost in 1GnRH-III.
Furthermore, the proposed ionic interaction between
Asp6-Lys8 residues16 is in agreement with our findings. Trp7
changes its orientation pointing away from Lys8 and consequently no NOE interaction is found between their side
chains (as in the Leu7-Arg8 case in GnRH-I), and Asp6 is
found to be within NOE distance with Lys8 giving rise to one
interaction between eNH of Lys8 and Hb of Asp6.
In conclusion, we synthesized 21 new lGnRH-III analogs
and the structure–activity studies show that several of
them had similar or higher activity than that of the parent
hormone. Analogs II, V, VIII, X, XI, XII, XIV, XV, and XVI
had higher antiproliferative activity than lGnRH-III on
LNCaP cells and significant activity on PC3 cell. The modifications of those vary significantly but their inhibitory
effect is almost the same, hampering thus the identification
of one particular ‘‘lead compound.’’ However, we have
studied ‘‘key’’ positions of lGnRH-III, providing new information regarding the structure–activity relationship of
lGnRH-III. Our observations are illustrating that lGnRHIII is a promising molecule for the development of peptide
analogs with increased and potentially selective anticancer
activity.
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Publication: Molecular Vision; Type: Research Article
Article ID: 999; Volume: 19; EJP Article Number: 2013MOLVIS0007
Formatted: Numbering: Continuous
2
Saffron administration prevents selenite-induced
cataractogenesis
3
4
Olga E. Makri,1 Anastasia-Varvara Ferlemi,2 Fotini N. Lamari,2 Constantine D.
Georgakopoulos1
5
1
6
7
2
1
Department of Ophthalmology, Medical School, University of Patras, Patras, Greece
Laboratory of Pharmacognosy & Chemistry of Natural Products, Department of Pharmacy,
University of Patras, Greece
8
9
10
Correspondence to: Constantine D. Georgakopoulos, Ophthalmology, Morfou 1 Street,
26441 Patras, Greece; Phone: +30 2610 999904, FAX: +30 2610 434914; email:
[email protected]
11
Abstract
12
13
14
Purpose: The present study sought to investigate whether Crocus. sativus stigmas (saffron)
extract prevents selenium-induced cataractogenesis in vivo, and to study the its possible
protective mechanism.
15
16
17
18
19
20
21
22
23
24
25
26
27
Methods: Wistar rat pups were randomized into 3 three groups. Group I (control) received
subcutaneous injection of normal saline on postnatal day 10. Groups II (selenite-treated) and
III (selenite+saffron-treated) received subcutaneous injection of sodium selenite (20 µmol/kg
bodyweight) on postnatal day 10. Group III also received intraperitoneal injections of saffron
extract (60 mg/kg bodyweight) on postnatal days 9 and 12. On postpartum day 21, rats were
sacrificed and the lenses were isolated and examined for cataract formation. Activities of
superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, (CAT) and
glutathione levels, as markers of antioxidant defense, were measured in the isolated lenses.
Levels of the indicator of lipid peroxidation, malondialdehyde (MDA), and protein oxidation
(sulfhydryl content) in the lens were also determined. The Effect effect of the different
treatments on lens protein profile was evaluated with the through an estimation of the soluble
to insoluble protein ratio and SDS–PAGE sodium dodecyl sulfate polyacrylamide gel
electrophoresis analysis of the water-soluble fraction (WSF) of lens proteins.
28
29
30
31
32
33
Results: Saffron demonstrated significant protection against selenite-induced
cataractogenesis in vivo. The mean activities of superoxide dismutaseSOD, glutathione
peroxidaseGPx, catalase, CAT and glutathione levels were significantly increased in group
III compared to the selenite-treated group. Saffron significantly prevented selenite-induced
lipid peroxidation, protein oxidation, as well as and proteolysis and insolubilization of the
lens WSF.
34
35
36
37
38
Conclusions: Saffron extract prevented selenite-induced cataract formation in Wistar rats,
possibly by through the reinforcement of antioxidant status, reduction of the intensity of lipid
peroxidation, protection of the sulfhydryl groups, and inhibition of proteolysis of the lens
WSF. These findings highlight the anti-cataractogenic potential of saffron by virtue of its
antioxidant property.
39
Introduction
40
Cataract is a degenerative condition and is the consequence of results from the loss of the
41
crystalline lens transparency. According to data provided by the World Health Organization,
42
cataract is one of the major causes of visual impairment globally and the first cause of
Page 1 of 20
Comment [Au1]: Do you mean: postnatal?
Comment [Au2]: Do you mean: or?
43
blindness, accounting for 51% of cases worldwide [1]. It is estimated that about 40 million
44
people will eventually have severely reduced vision due to cataract by the year 2020 [2],
45
especially in the developing countries of Asia and Africa [3]. The only available treatment
46
for cataract is the surgical removal of the opaque lens and its replacement with an artificial
47
one to restore visual acuity. The aforementioned procedure has been optimized over the last
48
decades so as to become an everyday surgery in the developed countries. Nevertheless,
49
cataract cataract-induced blindness remains a severe health problem in the developing
50
countries, due to the low socioeconomic status of the patients and limitations in the
51
acceptability, accessibility, and affordability of cataract surgical services [4].
52
Since surgical treatment is not widely available, many researchers seek for have investigated
53
pharmaceutical agents to prevent this disease. It has been estimated that a delay of 10 years
54
in the onset of cataract, by any means, would halve the number of patients needing surgery
55
[2]. Actually, over the last few years, great emphasis has been laid placed on exploring the
56
possible protective effects of several natural products against cataract formation. Natural
57
products are privileged candidates for drug discovery, since they are often endowed with
58
multiple functions. Cataractogenesis is considered to be a multi-factorial disease, correlated
59
with various pathogenetic mechanisms, that have not been completely clarified. There is a
60
large body of evidence demonstrating that oxidative stress (i.e., measurably increased levels
61
of reactive oxygen species ROS and oxidized substrate molecules - —lipids,
62
sulphydrylssulfhydryls, nucleic acids) is compulsory to cataract development [5-9].
63
Consequently, enhancement of the antioxidant defenses of the lens could prevent or delay the
64
onset of cataract. Based on that premise, numerous natural products, with known antioxidant
65
properties, have been evaluated during in the last decades.
66
Saffron consists of the dried stigmas of Crocus sativus L., which is a bulbous perennial plant
67
of the family Iridaceae. Saffron is has been used and consumed since antiquity, and is one of
68
the most expensive spices; it is used for flavoring and coloring food preparations, as a
69
perfume, and also as a dye. It is cultivated in many Mediterranean countries, like including
70
Spain, Greece, and Turkey, as well as in India and Iran (its major producer) [10]. Saffron’s
71
The value of saffron is attributed to its characteristic phytochemical components; ,
72
specifically the unique hydrophilic carotenoids, i.e., crocins (glycosidic esters of crocetin),
73
picrocrocin (terpenoid glycoside responsible for the exquisite taste), and safranal (the main
74
essential oil component) [11-13]. Hippocrates and Dioscurides mention the use of saffron for
75
the treatment of ophthalmic disorders, among other uses. Since then, saffron has been
Page 2 of 20
76
constantly used in traditional medicine, as therapy of for several conditions such as insomnia,
77
depression, bronchospasm, cardiovascular diseases, gastrointestinal disorders, menstrual
78
pain, menopausal problems, as analgesic, and even against cancer [10,14]. Some of the
79
medicinal-biologic properties of saffron, or its components, are attributed to its antioxidant
80
features, which have been highlighted in several studies [15-21]. The objective of the present
81
study is to investigate, for the first time in the literature, the effect of saffron extract against
82
selenite-induced experimental cataract in vivo.
83
Methods
84
Chemicals
85
Commercially available saffron was kindly provided from by the Cooperative de Safran
86
(Krokos Kozanis), West Macedonia, Greece. Sodium selenite (44%–46%), catalase (CAT)
87
from bovine liver (4966 U/mg), Purpald, N-ethylmaleimide, L-glutathione reduced,
88
glutathione oxidized, glutathione peroxidase (GPx), and N-acetyl-cysteine were purchased
89
from Sigma-Aldrich (St. Louis, MO 63,103, USA). A Superoxide Dismutase Assay Kit from
90
Cayman Chemical Company (Ann Arbor, USA) was used for the determination of
91
superoxide dismutase (SOD) activity.
92
Preparation of saffron extract
93
Saffron stigmas powder was extracted with methanol:water (50%v/v; 18 mlL/ 250 mg) for 4
94
h at room temperature, in the absence of light and with continuous stirring. The extract was
95
centrifuged, filtered, and evaporated to dryness in using a Speed Vac System (Labconco
96
Corp., Kansas City, MO). The composition was screened with HPLC on a Supelcosil C-18
97
column as previously described [13,20].The residues were stored at −20 °C until further use.
98
Samples were redissolved in normal saline and sterilized though membrane filtration (0.2 μm
99
i.d.) for the injections.
100
Animals
101
New born suckling Wistar rat pups were used in our experiment. The pups were housed
102
along with their mothers in stainless-steel cages in well well-ventilated rooms with controlled
103
temperature (23 °C) and humidity conditions with and 12 h:12 h light-dark cycles12 h
104
dark/light cycles. The mothers were maintained on a standard laboratory animal diet in the
105
form of dry pellets and provided tap water ad libitum throughout the experimental period.
106
Animals were randomly assigned to 3 three groups:
Page 3 of 20
Comment [Au3]: Please provide the
manufacturer’s city.
Comment [Au4]: Molecular Vision style requires
a materials source and source’s location for ALL
materials used. Please carefully check your
manuscript and complete all missing materials
source information, and complete areas where
materials source information is missing.
Comment [Au5]: Can you say: reduced Lglutathione, oxidized glutathione?
Comment [Au6]: Molecular Vision style requires
that any abbreviated term be used at least three
times. For two or fewer times, the term should be
spelled out in full.
Comment [Au7]: Please provide sufficient
details of the protocols used so that readers
understand what was done.
Formatted: Font: Not Italic
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
108
109
Formatted: Bulleted + Level: 1 + Aligned at:
0.25" + Indent at: 0.5"
normal saline.;

110
111
Group I (control, n=9 animals), which received only subcutaneous injection of
Group II (Seleniteselenite-treated, n=8 animals), which received a subcutaneous
injection of sodium selenite (20 µmol/kg bodyweight) on postnatal day 10.; and

Group III (Seleniteselenite+Saffronsaffron-treated, n=9 animals), which received a
112
subcutaneous injection of sodium selenite (20 µmol/kg bodyweight) on postnatal day
113
10 and intraperitoneal injections of saffron extract (60 mg/kg bodyweight) on
114
postnatal days 9 and 12.
115
Cataract formation could be evaluated from the 16th day, when the pups opened their eyes,
116
with the help of an ophthalmoscope and later on with the naked eye. Bilateral cataracts were
117
observed in the animals used for this study. On postpartum day 21, rats were anaesthetized
Comment [Au8]: Do you mean: postnatal?
118
with diethyl ether and then sacrificed by cervical dislocation. The eyes were enucleated and
119
lenses were at once excised intracapsularly through an incision 2 mm posterior to the limbus
Comment [Au9]: According to Molecular Vision
style, methods of animal euthanasia must be clearly
spelled out: dosage, type, route, etc.
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under surgical microscope magnification. Both lenses were obtained from each rat and
121
morphological examination for cataract formation was performed by gross examination of
122
lenses under the magnification of the dissecting microscope against a background of black
123
grid lines.
124
Staging of the cataract formation was conducted by an examiner, blinded regarding the
125
studied study groups, based on a scale 0 to 3 according to from Geraldine et al. [22]. The
126
degree of opacification was graded as follows:
127

Grade 0: absence of opacification (gridlines clearly visible; Figure 1A);
128

Grade 1: a slight degree of opacification (minimal clouding of gridlines, with
129
130
gridlines still visible; Figure 1B);

131
132
133
Formatted: Bulleted + Level: 1 + Aligned at:
0.25" + Indent at: 0.5"
Grade 2: presence of diffuse opacification involving almost the entire lens (moderate
clouding of gridlines, with gridlines faintly visible; Figure 1C);

Grade 3: presence of extensive thick opacification involving the entire lens (total
clouding of gridlines, with gridlines not seen at all; Figure 1D)
134
The experiments were conducted in strict compliance with the ARVO Association for
135
Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and
136
Vision Research and Guiding Principles in the Care and Use of Animals [23]. The study has
137
been approved by the University Hospital Bioethics Committee.
Page 4 of 20
Comment [Au10]: I would recommend stating
this at the beginning of the section.
138
Tissue treatment
139
Prior to biochemical analysis, the lenses were washed in ice-cold saline to remove blood and
140
then weighed carefully. The lenses were homogenized in 30 mM phosphate buffer, pH 7.6,
141
(10% w/v), and centrifuged at 15,000 rpm for 20 min at 4 °C. The supernatant obtained was
142
stored at −30 °C, pending further analysis.
143
Analytical methods
144
Assay of superoxide dismutaseSOD activity
145
Superoxide dismutase SOD activity was determined according to the Superoxide Dismutase
146
Assay Kit, Cayman Chemical Company, which is based on the formation of a tetrazolium
147
salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine.
148
The absorbance was read at 450 nm. The results are expressed as units/mLml, where one unit
149
of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the
150
superoxide anion.
151
Assay of catalase activity
152
The peroxidative activity of catalase CAT was determined according to the modified method
153
of Sinha [24]. The method is based on the reaction of the enzyme with methanol in the
154
presence of an optimal concentration of H2O2. The formaldehyde produced is measured
155
colorimetrically at 540 nm with reaction with Purpald (4-amino-3-hydrozino-5-mercapto-
156
1,2,4-triazole) and oxidation with KIO4. Standard concentrations of formaldehyde (0–75 μΜ)
157
were used for the construction of a calibration curve. All determinations were performed in
158
triplicate. The results are expressed as nmol/min/mLml.
159
Glutathione Peroxidase peroxidase (GPx) activity
160
GPx activity was determined colorimetrically, according to following Rotruck et al. [25], to
161
estimate the rate of the glutathione oxidation by H2O2. Reduced glutathione was used as
162
substrate and DTNB [5,5′-dithio-bis(2-nitrobenzoic acid)] as a chromogen. Suitable aliquots
163
of standard concentrations of GPx (0.025–1 U/mLml) were, also, used for the construction of
164
the calibration curve. The color that was developed was read against a reagent blank at 412
165
nm. Results were expressed as units/mg tissue (one unit was the amount of enzyme that
166
converted 1 μmol of reduced to the oxidized form of glutathione in the presence of H2O2/
167
min).
Page 5 of 20
Comment [eXtyles11]: If rpm is used in the
context of centrifugation, then it needs to be replaced
with “g” gravity. The formula to convert rpm to g is:
RCF = (1.119 × 10−5) × (rpm)2 × r, where r is the
mean radius of rotation in cm, and rpm is the
centrifuge speed in revolutions per minute.
168
Estimation of reduced and total glutathione content
169
A modified modification method of Hissin and Hilf’s method was used for the determination
170
of reduced (GSH) and oxidized glutathione (GSSG) content in the lens [26]. The two forms
171
of glutathione were estimated fluorometrically after reaction with o-phthalaldehyde (OPT).
172
The samples were treated with 50% TCA and centrifuged to eliminate the protein content.
173
The supernatant was extracted three times with diethyl ether. The aqueous fraction was used
174
to determine the GSH and GSSG content; GSH selectively reacts reacted with OPT
175
(10 mg/mL ml in pure methanol) at pH 8.0 (500 mM Na2HPO4 buffer), whereas after the
176
addition of 5 mM NEM in the samples, only GSSG reacted with OPT at pH 12.0 (0.2 N
177
NaOH). Standard concentrations of GSH and GSSG (0.75–15 μΜ) were used. The excitation
178
wavelength was at 340 nm and the fluorecencefluorescence intensity was determined at 420
179
nm. The respective concentrations in nmol/mg wet tissue are were calculated from the
180
respective calibration curves. The concentration of total glutathione was calculated by adding
181
GSH and GSSG.
182
Estimation of protein sulfhydryl content
183
The protein sulfhydryl content was estimated using the Ellman’s procedure as slightly
184
modified by Sedlak and Lindsay [27]. Briefly, the pellets from the samples treated with 50%
185
TCA for the determination of glutathione were redissolved with a Tris-EDTA-guanidine HCl
186
(3 mM: 3 mM: 3M) buffer, pH 8.9. In a 96-well microplate, 250 μL μl of the sample were
187
was mixed with 20 μL μl DTNB [5,5′-dithio-bis(2-nitrobenzoic acid)]. After incubation for
188
15 min at room temperature, the absorbance was read at 412 nm. The content of protein
189
sulfhydryls was calculated using a calibration curve prepared with N-acetyl-cysteine
190
(0.06 mM-–1 mM). The protein content of each sample was evaluated using the Bradford
191
assay [28]. Since NAC contains only one sulfhydryl group, the results were expressed as
192
nmol -–SH/mg protein.
193
Determination of Lipid lipid Peroxidationperoxidation
194
Lipid peroxidation was determined by the evaluation of malondialdehyde (MDA) levels in
195
the rat lens. Due to the small volume of tissue sample, the fluorimetric method of Grotto et
196
al. [29] and Jentzsch et al. [30] was used with slight modifications to measure MDA levels
197
using a 96-well microplate. MDA was determined after the reaction with thiobarbituric acid
198
and extraction with n-butanol. The excitation wavelength was at 515 nm and the fluorescence
199
intensity was determined at 553 nm. The results were expressed as nmol MDA/g tissue
200
against a standard curve (0.05–10 μΜ MDA).
Comment [Au12]: Molecular Vision style
requires that any abbreviated term be used at least
three times. For two or fewer times, the term should
be spelled out in full.
Comment [Au15]: Should this be defined?
Page 6 of 20
201
202
Sodium- dodecyl- sulfate polyacrylamide gel (SDS–PAGE) electrophoresis of
water soluble proteins
203
Samples (30 μg protein) were loaded on a 12% polyacrylamide gel (1 mm thickness)
204
according to a modified modification of method of Laemmli’s method [31]. Before loading,
205
lens homogenate was mixed with sample buffer (35% v/v glycerol, 18% v/v 2-
206
mercaptoethanol, 230 mM Tris/HCl pH=6.8, 10% w/v sodium dodecyl sulfate [SDS], 0.3%
207
w/v bromophenol blue). Each mixture was heated at 100 °C for 5 min and then it was
208
centrifuged for 3 min. The protein mix was left to cool down at room temperature.
209
Electrophoresis was performed in using a Cleaver Scientific Ltb (Warwickshire, UK)
Comment [Au16]: Do you mean: Ltd.?
210
apparatus, at 100V for approximately 2 h. For the visualization of protein lanes, gels were
Comment [Au17]: Please provide the
manufacturer’s city.
211
stained with 0.5% w/v Coomassie brilliant blue in methanol: acetic acid: water (3:1:6 v/v/v)
212
and destained. Image J Launcher was used for the densitometric analysis of gels (at least four
213
different experiments).
214
Determination of lens proteins
215
The determination of insoluble and soluble protein content in the rat lenses of each group
216
was estimated by using the Bradford assay, using with BSA bovine serum albumin as a
217
standard [28]; and the results were expressed as mg protein/ mg wet tissue. The ratio of
218
soluble to insoluble proteins per sample was calculated.
219
220
All variables were tested for normality with the Kolmogorov–Smirnov test. Differences in
221
clinical score between the groups were estimated with the Mann–Whitney U test. One-way
222
ANOVA analysis of variance was used to detect differences in continuous variables among
223
the three groups. The Bonferroni post hoc test was used for pairwise comparisons. A p value
224
less than 0.05 was considered statistically significant. The Analysis analysis was performed
225
using SPSS 15.0 software (SPSS, Inc., Chicago, IL).
226
Results
227
Morphological assessment of cataract formation
228
In group I, all the eyes were clear. In group II, 100% of the eyes developed moderate to
229
severe cataract, indicating success in establishing the selenite-induced cataract model in our
230
experiment. Stage 2 was the common endpoint of the cataractogenic process, accounting for
231
62.5% of the eyes; (median 2, (range 2–3). On the contraryIn contrast, saffron
232
coadministration (group III) significantly retarded selenite-induced cataractogenesis in vivo.
Page 7 of 20
233
In this group, 38.9% of the lenses exhibited stage 1 cataract, 44.4% of the eyes developed
234
stage 2 cataract, and only 16.7% developed dense cataract; (median 2, (range 1–3; Table 1,
235
Figure 1).The difference in exhibited cataract exhibited between in the control and selenite-
236
treated group was significant (p<0.0001, Mann–Whitney U test). The difference of in
237
cataract frequency and severity between groups II and III was statistically significant
238
(p<0.05, Mann–Whitney U test).
239
Antioxidant status in the lens
240
Superoxide dismutaseSOD activity
241
The activity of SOD (mean±standard deviation [SD]) was significantly lower in the lenses of
242
the group II compared to that of the normal lenses (p<0.001; Table 2). However, saffron co-
243
administration (group III) resulted in significantly higher SOD activity levels compared to
244
the group II, that which received only sodium selenite (p<0.05), although they are were
245
lower than those of the group I (control; p<0.05).
246
Catalase activity
247
The mean activity of CAT (mean±SD) in the lenses of group II rats was significantly lower
248
than that of the lenses in control group (p<0.01; Table 2). In the group III, CAT activity was
249
significantly higher than in group II (p<0.05). In fact, saffron coadministration resulted in the
250
prevention of the loss of CAT activity, which was measured at levels similar to those of the
251
control group (p>0.05).
252
Glutathione Peroxidase peroxidase (GPx) activity
253
The mean activity of GPx (mean±SD) in lenses of group II was significantly lower than that
254
of the lenses in the control group (p<0.001; Table 2). In the selenite+saffron treated group
255
(III), GPx activity was significantly higher than in the selenite-treated group (p<0.001).
256
Reduced and total glutathione
257
For the further evaluation of the antioxidant status of the lenses, the reduced and total
258
glutathione levels (mean±SD) were evaluated (Table 3). Selenite administration resulted in a
259
significant reduction of reduced glutathione in comparison with normal lenses (p<0.05).
260
Cotreatment with saffron (group III) restored the reduced glutathione concentration, which
261
reached to a level that was not statistically significantly different from the control group
262
(p>0.05). The Lens lens total glutathione pool was depleted after selenite administration,
263
while saffron showed a sparing effect on total glutathione content.
Page 8 of 20
264
Levels of lens protein sulfhydryls
265
The level of protein sulfhydryl groups is an important indicator of tissue protein oxidation. In
266
group II, protein sulfhydryl content (mean±SD) was significantly lower compared to control
267
(p<0.001; Table 4). However, in group III, lens protein sulfhydryl content was significantly
268
higher than the concentration observed in the selenite-treated group (p<0.05), demonstrating
269
its protective effect against protein oxidative damage. Sulfhydryl content in the group III
270
reached a level that was not statistically significant lower from than group I (p>0.05).
271
Levels of the indicator of lipid peroxidation in lens (malondialdehyde)
272
Malondialdehyde The MDA level (MDA; mean±SD) reflects the overall tissue lipid
273
peroxidation [32]. MDA in In group II, this was 91.6% higher than in group I (p<0.0001;
274
Table 4). In group III, MDA it was 54.8% lower compared to the selenite-treated group
275
(p<0.0001) and similar to those that of the control group (p>0.05).
276
Effect of Saffron saffron on Lens lens Proteinsproteins
277
Calculation of the soluble to insoluble ratio (mean±SD) revealed a significantly lower ratio
278
in the selenite-treated group when compared to the control group (p<0.0001; Table 4). In
279
group III, saffron resulted in a significantly higher ratio in comparison to the group II
280
(p<0.05). The ratio in group III was still significantly lower from than the that of normal
281
lenses (p<0.001).
282
The relative changes in the water-soluble fraction (WSF) of lens proteins were detected by
283
SDS–polyacrylamide gel electrophoresis (PAGE) analysis. Figure 2 shows the a significant
284
decrease of in low molecular weight proteins in the selenite-treated lens; proteins between 10
285
and 30 kDa in group II were lower (~35%) than those in group I. Saffron coadministration
286
seems to diminish the observed proteolysis of those these proteins, since the densitometric
287
analysis revealed a significant increase of protein (25–30 kDa) bands intensity (13%–27%)
288
compared to group II. Particularly, the intensity of the 19–21 kDa protein band in group III
289
was similar to that of the control (group I).
290
Discussion
291
Over the last In recent years, studies have been conducted to evaluate the possible beneficial
292
effects of saffron or its components in ophthalmology. All the published data are with make
293
reference to the pathology of the retina and its possible beneficial effects in conditions such
294
as ischemic retinopathy, age age-related macular degeneration, or other neurodegenerative
295
conditions [33-38]. However, despite the continuously increasing literature on the
Page 9 of 20
296
neuroprotective effects of saffron or its components in the retina, to date, no study has been
297
conducted to evaluate saffron’s its possible effects against cataract formation.
298
The purpose of our study was to examine in vivo the potential anti-cataract effects of saffron
299
extract in selenite-induced cataract. Our clinical observations of the morphological
300
examination of the lenses showed that cataract formation caused by selenite administration
301
was attenuated by the administration of saffron, suggesting its anti-cataract potential.
302
Furthermore, biochemical analyses were conducted to support the clinical observation.
303
The selenite cataract model was selected because of the for its rapid, effective, and
304
reproducible cataract formation [39,40]. Although the rate of lens opacification is much more
305
rapid than in human cataract, the selenite model shows an amount of general similarities to
306
human senile nuclear cataract, such as the formation of vesicles, increased levels of calcium,
307
proteolysis, a decrease of the in water-soluble proteins, the presence of insoluble proteins,
308
and diminished amounts of GSH. The mode of action of sodium selenite in cataractogenesis
309
has not been completely defined, but it is indicated that the main biochemical events, —
310
induced by selenite overdose, —are triggered by the selenite selenite-induced oxidative stress
311
and the generation of reactive oxygen species (ROS) in the aqueous humor in combination
312
with a decrease in the activity of antioxidant enzymes such as SOD, CAT, GPx, glutathione
313
transferase, and glutathione reductase, and of as well as GSH content in the lens [40,41].
314
More precisely, it is assumed that oxidative damage occurs to critical sulfhydryl groups of
315
proteins of the lens epithelium membranes. The aforementionedThis, in combination with
316
oxidative stress-–induced lipid peroxidative damage of lenticular membranes, leads to the
317
inactivation of membrane proteins, such as Ca2+-ATPase. Inhibition of the function of Ca2+-
318
ATPase results in loss of calcium homeostasis and calcium accumulation in the lens. As a
319
consequence of calcium influx in rodent lenses, calpains, a family of well well-characterized
320
calcium dependent proteases, are activated. Calpain activation, mainly m-Calpain and Lp82
321
in rodent lenses, induces rapid proteolysis of the water soluble proteins, that is, lens
322
crystallins, and cytoskeletal proteins. Proteolysis exposes the hydrophobic regions of lens
323
proteins, which then interact to form insoluble aggregates [39]. Insolubilization results in
324
precipitation and aggregation of lens fragmented proteins, and finally, in loss of lens
325
transparency [40].
326
SOD and CAT make up a primary line of defense against superoxide anion (O2−) and
327
hydrogen peroxide (H2O2), respectively. More precisely, SOD is a specific scavenger of
328
superoxide anion. It converts harmful superoxide radicals to H2O2, which is detoxified by
Page 10 of 20
Comment [Au18]: Can this be defined?
Formatted: Not Superscript/ Subscript
329
CAT to harmless by-products. GPx, which belongs to the family of selenoproteins, is present
330
in relatively large amounts in the epithelium of the lens [42]. It catalyzes the reduction of a
331
variety of hydroperoxides with the help of its reducing substrate, GSH. The enzyme GPx
332
maintains the integrity of the phospholipid bilayer of membranes by putting a brake on the
333
lipid peroxidation initiated by superoxide. In our experiment, administration of sodium
334
selenite resulted in significant reduction of the activities of the antioxidant enzymes SOD,
335
CAT, and GPx, whereas such a decline was prevented in the lenses of animals co-treated
336
with saffron. It could be postulated that saffron, improves the antioxidant defense
337
mechanisms of the lens, leading to restoration of the levels of SOD, CAT, and GPx activities,
338
in spite of exposure to sodium-selenite.
339
GSH, a tripeptide of glycine, glutamic acid, and cysteine, is found in unusually high levels in
340
the crystalline lens and is believed to play a significant role in the maintenance of the
341
reduced state in the lens. GSH (via its side sulfhydryl group) is involved in the detoxification
342
of potentially damaging oxidants such as H2O2 and dehydroascorbic acid, and in the
343
protection against oxidation of membrane –SH groups, that which are important for cation
344
transport regulation. Finally, it protects cytoskeletal proteins and prevents the cross linking of
345
soluble crystallins, and, therefore, maintenance of its levels is vital for the preservation of
346
lenticular transparency [43-45].GSH levels decrease with age and are found reduced in most
347
types of cataract [45,46]. Furthermore, selenite overdose has been proved to cause a
348
significant depletion of lens GSH levels [47,48]. Restoration of GSH and total glutathione
349
levels could be attributed to the beneficial effects of saffron treatment on the enzymes
350
involved in glutathione synthesis [49,50].
351
The crystallins are subject to oxidative changes, which include including the formation of
352
disulfide and other inter- and intramolecular cross-links, resulting to in their aggregation
353
[51]. Furthermore, one of the primary events in selenite selenite-induced cataractogenesis
354
theory is the oxidative damage that occurs to proteins of the lens epithelium membranes [39].
355
Free sulfhydryl (–SH) groups of proteins are mainly responsible for their antioxidant
356
response, and they can serve as a sensitive indicator of oxidative stress [52]. Consequently,
357
the reduced level of protein sulfhydryl content is an indication of tissue protein oxidation. In
358
the present study, in contrast to the lower level of lens protein sulfhydryl content of the
359
selenite-treated group, saffron coadministration kept the protein sulfhydryl content near to
360
control levels, indicating its protective effect against oxidative damage.
Page 11 of 20
361
Free radical radical–induced lipid peroxidation is a highly destructive process and has been
362
implicated as one of the main components of the pathogenetic mechanism of selenite
363
selenite-induced cataractogenesis [47]. The accumulated peroxidation products damage vital
364
membrane structures [53]. MDA is a product of the breakdown of mainly unsaturated fatty
365
acids through the oxidation mechanism [54]. In the present study, such a disturbance of
366
membrane lipids from selenite probably resulted in the observed increase in MDA lens
367
levels, when compared to normal lenses. The observed almost twofold reduction in the MDA
368
level in the selenite+saffron-treated group suggests that saffron possibly preserved the
369
structural integrity of the lens, thereby preventing the its opacification of the lens.
370
Selenite cataract is characterized by a marked decline in water water-soluble proteins through
371
protein insolubilization either by excessive proteolysis by calpains or through structural
372
alterations resulting from sulfhydryl oxidation [40]. The selenite-induced alteration in the
373
lens protein profile has been depicted through the calculation of the soluble/insoluble
374
proteins ratio in the selenite group in our experiment. Saffron supplementation prevented
375
protein degradation and aggregation, resulting in less cataract formation. Previous studies
376
showed that many of the soluble proteins in the approximate molecular weight ranges of 23–
377
31 kDa are from β-crystallin, 19–20 kDa are from α-crystallin, and the smear of polypeptides
378
from 21 to 23 kDa are γ-crystallins [55]. Saffron coadministration resulted in considerable
379
preservation of the pattern of lens proteins with molecular size 19 ~–21 kDa, and 25–30 kDa,
380
which probably denotes inhibition of crystalline proteolysis.
381
To conclude, in the present in vivo study, we showed, for the first time in the literature, that
382
intraperitoneal administration of saffron extract could significantly protect against nuclear
383
opacity formation in selenite selenite-treated pups. This fact indicates that saffron
384
components/metabolites can penetrate the blood-aqueous barrier and reach the aqueous
385
humor. It has been recently shown that crocetin, the main crocin metabolite, is determined in
386
cerebral tissue after intraperitoneal administration of saffron extract [56]. The extent of tissue
387
damage caused by selenite and the protective effect of saffron were evaluated clinically and
388
biochemically. Saffron’s The modulatory effect of saffron seems to be associated with a
389
reduction of the intensity of lipid peroxidation and protein oxidation, maintenance of
390
antioxidant status, and inhibition of proteolysis of the lens WSF. This study has focused on
391
the scientific validation of saffron as an anticataract agent, suggesting that saffron, (or its
392
components, ) exhibits a promising anti-cataractogenic effect. However, it is important to
393
emphasize that certain differences exist between human and selenite experimental cataract.
Page 12 of 20
Comment [Au19]: Please check the change.
394
This preliminary study is encouraging, but further studies are research is required to find out
395
determine whether a similar anti-cataractogenic potential can be demonstrated in humans.
396
Acknowledgments
397
398
The study was supported by ‘K. Karatheodoris’ Grant No C913 from the Research
Committee, University of Patras, Greece.
399
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400
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<jrn>52. Radi R, Bush KM, Cosgrove TP, Freeman BA. Reaction of xanthine oxidasederived oxidants with lipid and protein of human plasma. Arch Biochem Biophys 1991;
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<jrn>54. Qi HP, Wei SQ, Gao XC, Yu NN, Hu WZ, Bi S, Cui H. Ursodeoxycholic acid
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<jrn>55. David LL, Shearer TR. Calcium-activated proteolysis in the lens nucleus during
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<jrn>56. Linardaki ZI, Orkoula MG, Kokkosis AG, Lamari FN, Margarity M. Investigation
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559
Page 16 of 20
560
requires that all figure legends be written in
complete sentences. Please rewrite all sentence
fragments in this legend and review this point for all
figures.
561
Figure 1. Transillumination pictures of lenses representative of each grade. Lens
562
opacification of experimental animals corresponding to each grade. A: Grade 0: absence of
563
opacification (gridlines clearly visible; A); B: Grade 1: a slight degree of opacification, with
564
minimal clouding of gridlines (B); C: Grade 2: diffuse opacification involving almost the
565
entire lens, with gridlines faintly visible, (C); D: Grade 3: extensive dense opacification
Formatted: Font: Bold
566
involving the entire lens (gridlines not visible; D).
Comment [Au22]: Can these sentences be
combined into one title?
Page 17 of 20
567
568
Figure 2. Sodium- dodecyl- sulfate PAGE polyacrylamide gel electrophoresis of the lens
569
water soluble protein fraction. An equal amount of protein (30 μg) was loaded on 12%
570
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). The first right lane
571
indicates the molecular weight marker; Group I lane represents the protein profile of the
572
normal untreated lens; Group II, the selenite-treated group; Group III, the selenite+saffron-
573
treated group. In the normal untreated lens water-soluble fraction (WSF), the prevalence of
Page 18 of 20
574
proteins with a molecular mass of 19–30 kDa (characteristic of crystallins) is shown. Selenite
575
treatment led to a significant reduction (~35%) of in those these proteins, which was
576
prevented by saffron coadministration.
577
Table 1. Morphological assessment of cataract formation of each group’s isolated lenses
No of lenses with different degree of
opacification
Group
Grade Grade Grade Grade Median
0
1
2
3
(range)
I (n=18)
18
0
0
0 0 (0)
II (n=16)
0
0
10
6 2 (2–3) *
III (n=18)
0
7
8
3 2 (1–3) *, ‡
578
Incidence and staging of cataract formation of the isolated lenses in each experimental group.
579
Group I: Control, Group II: Selenite-treated, Group III: Selenite+Saffron-treated. The Mann–
580
Whitney U test was used for pairwise comparisons. Different symbols indicate statistically
581
significant difference between groups. * p<0.0001 compared with group I. ‡ p<0.05
582
compared with group II.
583
Table 2. Activities of antioxidant enzymes of the lenses of each group.
Enzyme
Group I
Group II
Group III
Superoxide dismutase (unit/ml)
0.37±0.07
0.18±0.05**
0.26±0.03#, ‡
Catalase (nmol/min/ml)
0.07±0.01
0.04±0.005*
0.067±0.02‡
Glutathione peroxidase (μmol
glutathione oxidized/min/mg tissue)
14.71±1.39
7.39±1.30**
12.34±1.93†
584
Activities of the enzymes superoxide dismutase, catalase and glutathione peroxidase of the
585
lenses of each experimental group. Group I: Control, Group II: Selenite-treated, Group III:
586
Selenite+Saffron-treated. Each value represents the mean ± SD of six determinations.
587
Statistical analysis was performed by one-way ANOVA with Bonferroni’s adjustment .
588
Different symbols indicate statistically significant difference between groups of each enzyme
589
activity. *p<0.01; **p<0.001; #p<0.05 compared with group I. ‡p<0.05; †p<0.001 compared
590
with group II.
591
Table 3. Total and reduced glutathione levels.
Parameters
Group I
Group II
Group III
Reduced
glutathione
(nmol/mg
wet tissue)
0.33±0.1
0.17±0.02* 0.31±0.13
Page 19 of 20
Total
glutathione
(nmol/mg
wet tissue)
0.50±0.17 0.36±0.07
0.52±0.18
592
Reduced and total glutathione levels in the lens of Wistar rat pups of each group. Group I:
593
Control, Group II: Selenite-treated, Group III: Selenite+Saffron-treated. Values represent the
594
mean ± SD of six determinations. Statistical analysis was performed by one-way ANOVA
595
with Bonferroni’s adjustment. Symbol indicates statistically significant difference between
596
groups II and I (p<0.05)
597
598
Table 4. Levels of the indicators of protein (sulfhydryl content) and lipid (malondialdehyde)
oxidation and ratio of soluble/insoluble proteins in lens of each group.
Component analyzed
Group I
Group II
Group III
Protein sulfhydryl (nmol
-SH/mg protein)
0.93±0.1
0.67±0.08**
0.82±0.1‡
Malondialdehyde
(nmol/g tissue)
56.64±12.38 108.51±13.63* 49.04±18.22†
Soluble/insoluble
proteins
2.28±0.27**,
2.91±0.09
1.88±0.27*
‡
599
Quantitative analysis of protein sulfhydryl groups and malondialdehyde levels in the lenses
600
of each experimental group. Calculated soluble to insoluble proteins ratio in each group
601
lenses. Group I: Control, Group II: Selenite-treated, Group III: Selenite+Saffron-treated.
602
Data represent the mean ± SD of six determinations. Statistical analysis was performed by
603
one-way ANOVA with Bonferroni’s adjustment. Different symbols indicate statistically
604
significant difference between groups. *p<0.0001; **p<0.001 compared with group I.
605
†
p<0.0001; ‡p<0.05 compared with group II.
606
Page 20 of 20
DOI 10.1515/jpem-2012-0183 J Pediatr Endocr Met 2012; aop
Nikolitsa Koutroumani, Ioanna Partsalaki, Fotini Lamari, Athina Dettoraki,
Andrea Paola Rojas Gil, Alexia Karvela, Eirini Kostopoulou and Bessie E. Spiliotis*
Protective mechanisms against oxidative stress
and angiopathy in young patients with diabetes
type 1 (DM1)
Abstract
Objective: Advanced glycation end-products (AGEs) via
their receptor, RAGE, are involved in diabetic angiopathy.
Soluble RAGE, an inhibitor of this axis, is formed by enzymatic catalysis (sRAGE) or alternative splicing (esRAGE).
Malondialdehyde (MDA) is an oxidative stress marker, and
ferric reducing ability of plasma (FRAP) is an anti-oxidant
capacity marker.
Methods: In isolated mononuclear blood cells from 110
DM1-patients (P) and 124 controls (C) (4–29 years) RAGE
mRNA (g) and protein expression (pe) were measured
by RT-PCR and Western immunoblotting, respectively.
Plasma levels of CML (AGEs) and sRAGE were measured
by ELISA, MDA by flurometry and FRAP according to
‘Benzie and Strain’.
Results: P showed: (i) higher g of RAGE, especially in p > 13
years of age and > 5 years DM1, (ii) increased pe of esRAGE
in DM1 > 5 years and (iii) increased FRAP and MDA.
Conclusions: The increased esRAGE and FRAP with
increased levels of CML and MDA possibly reflects a protective response against the formation of diabetic complications in these young diabetic patients.
Keywords: advanced glycation end-products (AGEs);
diabetes mellitus type 1; oxidative stress; RAGE; soluble
RAGE.
*Corresponding author: Bessie E. Spiliotis, University of Patras
School of Medicine, Research Laboratory of the Division of Pediatric
Endocrinology and Diabetes, Department of Pediatrics, Patras,
Achaia, Greece, E-mail: [email protected]
Nikolitsa Koutroumani: University of Patras School of MedicineResearch Laboratory of the Division of Pediatric Endocrinology and
Diabetes, Department of Pediatrics, Patras, Achaia, Greece
Ioanna Partsalaki: University of Patras School of MedicineResearch Laboratory of the Division of Pediatric Endocrinology and
Diabetes, Department of Pediatrics, Patras, Achaia, Greece
Fotini Lamari: University of Patras, Laboratory of Pharmacognosy
and Chemistry of Natural Products, Department of Pharmacy, Patras,
Achaia, Greece
Athina Dettoraki: University of Patras School of Medicine-Research
Laboratory of the Division of Pediatric Endocrinology and Diabetes,
Department of Pediatrics, Patras, Achaia, Greece
Andrea Paola Rojas Gil: University of Patras School of MedicineResearch Laboratory of the Division of Pediatric Endocrinology and
Diabetes, Department of Pediatrics, Patras, Achaia, Greece; University of Peloponnese, Faculty of Human Movement and Quality of Life
Sciences, Department of Nursing, Orthias Artemidos and Plateon
Sparta, Sparta, Lakonias, Greece
Alexia Karvela: University of Patras School of Medicine-Research
Laboratory of the Division of Pediatric Endocrinology and Diabetes,
Department of Pediatrics, Patras, Achaia, Greece
Eirini Kostopoulou: University of Patras School of MedicineResearch Laboratory of the Division of Pediatric Endocrinology
and Diabetes, Department of Pediatrics, Patras, Achaia,
Greece
Introduction
Diabetes mellitus type 1 (DM1) affects up to 35 per 100,00
children, adolescents and young adults every year,
with a rising prevalence within the last decade (1). In
adults it is commonly associated with microvascular
complications (diabetic nephropathy, neuropathy and
retinopathy) although these may rarely also develop
in adolescents (2). The development of adult diabetic
complications is strongly correlated to hyperglycemia
and the duration of diabetes and several pathological mechanisms have been proposed that contribute to
these complications.
One of these mechanisms is the production of
advanced glycation endproducts (AGE), which are products derived from the non-enzymatic glycation and oxidation of plural substrates, such as proteins, lipids and
nucleotides. Intermediate products of these processes are
the amadori products, the best known of which is glycosylated hemoglobin A1C (HbA1c) (3). The accumulation of
AGEs can directly modify the extracellular matrix (ECM)
resulting in increased stiffness of the vasculature. They
can also modify hormones, cytokines and free radicals
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2 Koutroumani et al.: Protective mechanisms against oxidative stress and angiopathy
that elicit oxidative stress in several tissues and subsequently evoke thrombotic and inflammatory processes
intracellularly, via their receptor, RAGE. A common AGE
that accumulates in diabetes is Nε-(carboxymethyl)Lysine (CML), which is believed to be lipid and protein
adducts that have been found to impair nitric oxide (NO)
production and activate the ‘nuclear factor kappa-lightchain-enhancer of activated B cells’ (NF-κB), thereby
increasing atherogenesis, oxidative stress and inflammation (4).
Lipid peroxidation in diabetes has been shown
to impair LDL uptake by the endothelial cells and it is
one of the proposed mechanisms involved in diabetic
microangiopathy and retinopathy (5). Malondialdehyde
(MDA) has been found to be an important marker of
lipid peroxidation responsible for the milieu of oxidative stress and its levels have been found increased in
adult DM1 patients (6, 7). Furthermore, in DM1 adult
patients with retinopathy the increased levels of MDA
coexist with a reduction of their anti-oxidant power, as
indicated by a reduction of the ferric reducing ability of
plasma (FRAP) (8).
The most widely studied receptor for AGEs is RAGE.
RAGE is a cell surface receptor that belongs to the immunoglobin superfamily of receptors. Full-length RAGE receptor is comprised of one cytoplasmic domain, responsible
for intracellular signaling, one transmembrane domain
and two extracellular C-type domains preceded by one
V-type immunoglobulin type domain that plays a critical
role in ligand binding. Other forms of RAGE that appear
to lack the cytosolic and transmembrane domains are
known as soluble receptors for AGEs and can be detected
in the circulating blood. These can be produced by alternative splicing of RAGE pre-mRNA transcripts forming
the endogenous secretory RAGE (esRAGE) and by enzymatic cleavage of the full length RAGE, forming circulating soluble RAGE (sRAGE). Both esRAGE and sRAGE can
bind AGEs in the circulation, thus preventing the adverse
effects of the activated AGEs-RAGE axis intracellularly
(9).
Limited reports have investigated the physiological
mechanisms of AGEs accumulation in relation to oxidative stress in a milieu of type 1 diabetic children, adolescents and young adults. Therefore, the aim of our study
was to investigate oxidative stress (MDA) and the anti-oxidant capacity of the body (FRAP) in children, adolescents
and young adults with DM1 in relation to the activation
of the AGEs (CML)-RAGE axis and its soluble isoforms,
sRAGE and esRAGE, in order to investigate their possible role in the predisposition for, or the protection from,
diabetic complications.
Patients and methods
Patient population
The study population consisted of 110 patients with DM1 (P) [90
children and adolescents (4–18 years) and 20 young adults (18–29
years)] and 124 age- and sex-matched (92 children and adolescents
and 32 young adults) normal non-diabetic control subjects (C)
(Table 1). In all the subjects, BMI (Body Mass Index), BMI percentile
(BMI%), Tanner stage and waist circumference (WC) were obtained,
together with measurements of glycosylated hemoglobin (HbA1C), total cholesterol, HDL and LDL cholesterol, triglyceride, liver enzymes
(SGOT, SGPT), and urea and creatinine plasma levels (Table 1). In all
the P, the duration of diabetes was also noted.
DM1 patients were followed-up on a regular basis in the Outpatient Clinic of the Division of Pediatric Endocrinology and Diabetes
of the University Hospital of Patras, Greece. The inclusion criteria
for the controls were: (a) absence of diabetes and any other related
autoimmune disease and (b) absence of obesity. Informed parental
consent and children’s assent were obtained in all cases. The study
was approved by the Ethical Committee of the University Hospital of
Patras (Patras, Greece).
Isolation of human peripheral blood
mononuclear cells
The isolation of the human peripheral blood mononuclear cells (PBMCs) was performed as previously reported by our research group
(10). Specifically, 20 mL of venous blood were obtained from all the
subjects and this was layered over a Ficoll-Paque (Histopaque 1077;
Sigma Aldrich, Dorset, UK) layer at a 2:1 ratio and centrifuged at
805 × g for 35 min at 20oC. PBMCs were collected from the interface
and washed with RPMI (Roswell Park Memorial Institute) media 1640
(GIBCO Invitrogen Corporation, Carlsbad, CA, USA) in order to remove the plasma and ficoll, according to the manufacturer’s instructions. The isolated pellets of PBMCs were stored at –80°C in order to
isolate total RNA and proteins subsequently.
Total RNA extraction and reverse
transcription PCR
Total RNA extraction from the PBMCs was performed according to
the manufacturer’s instructions with the mammalian total RNA,
miniprep kit (Sigma, Aldrich). DNaseI (Thermoscrip RT, Invitrogen
Corporation, Carlsbad, CA, USA) treatment of all the samples was
included. Preparation of cDNA from total RNA was performed using a reverse transcription system (Thermoscrip RT, Invitrogen Corporation). From the cDNA samples RAGE and β-actin mRNA were
amplified with: RAGE primers: forward = 5′AAC-TGC-AGG-CTC-TGTGGG-AGG-AT 3′, reverse = 5′CCT-TGC-CAA-GCT-TTC-TTC-TAC-AAC-C 3′
and β-actin primers: forward = 5′AAG-GCC-AAC-CGT-GAA-AAG-ATGACC 3′, reverse = 5′-ACC-GCT-CGT-TGC-CAA-TAG-TGA-TGA 3′ under the
following conditions: denaturation at 95oC for 1 min, annealing at
55oC for 30 s and extension at 72oC for 1 min, for 35 cycles. PCR bands
were quantified by densitometry with scion image (version 4.0.3.2,
Scion Corporation, Frederick, MD, USA).
Koutroumani et al.: Protective mechanisms against oxidative stress and angiopathy Subjects
Sex, male/female
Age, years
Height, cm
Height SDS
Tanner stage
Weight, kg
Body mass index, cm/kg2
Body mass index, cm/kg2%
Body mass index SDS
Waist circumference, cm
DM1 duration, years
HbAlc, %
Cholesterol, mg/dL
LDL cholesterol, mg/dL
HDL cholesterol, mg/dL
Triglycerides, mg/dL
SCOT, U/L
SGPT, U/L
Urea, mg/dL
Creatinine, mg/dL
Micro-angiopathy
Diabetic patients
Controls
110
59/51
14.23 ± 5.19
154.39 ± 19.02
0.11 ± 1
I: 19
II: 17
III: 4
IV: 18
V: 46
51.15 ± 17.8
20.71 ± 3.90
65.60 ± 22.88%
0.29 ± 1.08
70.78 ± 9.6
5.65 ± 4.29
8.06 ± 1.62
164.81 ± 27.13
94.05 ± 29.1
56.69 ± 12.1
66.98 ± 30.71
18.92 ± 5.69
17.8 ± 9.3
31.3 ± 6.83
0.72 ± 0.18
–
124
61/63
14.05 ± 4.97
150.57 ± 16.50
–0.38 ± 1.18
I: 11
II: 11
III: 7
IV: 22
V: 32
44.44 ± 14.33
18.99 ± 3
47.51 ± 28.10%
–0.13 ± 0.93
66.93 ± 8.9
N/A
5.11 ± 0.42
164.16 ± 29.16
95.18 ± 25.76
55.83 ± 12.35
66.83 ± 22.10
17.57 ± 3.82
16.5 ± 5.5
23.57 ± 6.02
0.8 ± 0.12
–
3
p-Value
NS
NS
NS
NS
0.002
NS
0.003
0.001
0.001
0.003
0.019
0.001
NS
NS
NS
NS
NS
NS
NS
NS
Table 1 Clinical and biochemical characteristics of the DM1 patients and the controls.
Data are means ± SD. Statistical significance, p ≤ 0.05. NS, not significant.
Western immunoblotting
Plasma assays
Protein samples of isolated PBMCs were lysed with laemnli sample
buffer (2 × SDS sample buffer) prepared in the laboratory. Protein
content in the samples was approximately estimated by SDS-PAGE
electrophoresis and staining with Coomassie Blue. Whole cell lysates
were separated by SDS-PAGE and proteins were transferred to nitrocellulose membranes (Amersham Biosciences plc, Buckinghamshire, UK). The membranes were blocked for 1 h at room temperature
(RT) and then incubated with IgG: anti-RAGE (Biotechnology, Santa
Cruz, CA, USA) diluted 1:150, overnight at RT. Furthermore, the protein measurements were corrected with the expression of β-tubulin,
which was detected with an anti-β-tubulin antibody (Cell signaling
Technology, Inc., Danvers, MA, USA) diluted 1:500, overnight at RT.
Band detection was performed with chemiluminescence reagent ECL
(GE Healthcare Bio-Sciences AB, Sweden). The Western blot signals
(complexed protein bands) were quantified by densitometry with
scion image (version 4.0.3.2, Scion Corporation).
The plasma levels of the total soluble RAGE (esRAGE and sRAGE)
(11) were measured by a sandwich ELISA kit (Human Immunoassay
RAGE, Quantikine, R & D Systems, Minneapolis, MN, USA). Measurements were performed according to the manufacturer’s instructions
and the intra-assay coefficient of variation for repeated measurements was 7.5%.
The plasma levels of CML were measured by a sandwich ELISA
kit (CirculexTM Anti-CML Human autoantibody, Qzzuantikine, Cyclex
Co, Ohara, Japan). Measurements were performed according to the
manufacturer’s instructions and the intra-assay precision was 5.6%,
whereas the inter-assay precision was 8.7%.
Malondialdehyde (MDA) was determined fluorometrically according to the protocol by Papandreou et al. (12). In brief, 25 μL of 90
mM butylated hydroxytoluene (BHT) was added to 250 μL of plasma
samples or MDA standards (0.05–5 μM), to prevent further lipid peroxidation. Samples were hydrolysed in mild alkaline conditions at 60°C
for 30 min in order to release bound MDA and the proteins were precipitated with 10% trichloroacetic acid (TCA). After being kept on ice
for 10 min, mixtures were centrifuged and the supernatants obtained were incubated with 0.8% thiobarbituric acid (TBA) at 95°C for 90
min. The supernatants were then extracted with n-butanol and fluorescence was measured at 535 nm (emission wavelength) after excitation at 515 nm on a Perkin-Elmer LS55 fluorescence spectrometer. All
determinations were carried out at least two times and in duplicates.
Blood samples
Venous blood samples were obtained with heparinised syringes. The
plasma was collected during the procedure of the PBMCs isolation
and then stored at –80oC. The plasma samples were used to measure
the levels of sRAGE, CML, FRAP and MDA.
The ferric reducing ability of plasma (FRAP) was determined by
a modified method of the Benzie and Strain protocol (13, 14). In particular, the working FRAP reagent was produced by mixing 300 mM
acetate buffer (pH = 3.6), 10 mM 2,4,6-tripyridil-s-triazine (TPTZ) solution and 20 mM FeCl 6H2O in a 10:1:1 ratio just before use. This reagent
was then loaded (150 μL) to each well of a 96-well micro titer plate
together with a total of 5 μL of sample and 15 μL of ddH2O. A reading
at 595 nm was taken and also a standard linear curve of Ferrous sulfate (FeSO4 7H2O) was produced by known concentrations (10 3–10 5
M). FRAP values were determined according to the standard curve.
exhibited significantly increased height SDS, weight, body
mass index (BMI), BMI %, BMI SDS and waist circumference when compared to their controls, but these values were
within the normal range, therefore they were not considered
as risk factors for diabetic complications. Furthermore, the
diabetic patients had not developed any micro- or macroangiopathy and their glycemic profile showed that their
diabetes was moderately controlled (HbA1c = 8.0% ± 1.8%).
RAGE mRNA and protein expression
in PBMCs
Our subjects were grouped according to age, DM1 duration and
HbA1c levels (for DM1 patients), LDL cholesterol levels, pubertal
stage, waist circumference, body mass index (BMI) and BMI%tiles.
Expression levels of mean values with ± standard deviation (SD) and
correlations were performed using the SPSS computer program (17.0
version for Windows, Mann-Whitney or Kolmogorov 2-tailed t-test,
Pearson’s or Spearman’s correlation coefficients). The threshold of
statistical significance was defined as p ≤ 0.05.
Results
Clinical and biochemical characteristics
of the study subjects
All the clinical and biochemical characteristics of the
subjects are presented in Table 1. The diabetic patients
Controls
Diabetic patients
Relative gene expression, %
A
B
The gene expression (mRNA) of RAGE was increased in
the overall sample of DM1 patients in comparison to the
controls, (p = 0.007) (Figure 1A). More specifically, RAGE
mRNA expression was increased in the DM1 patients > 13
years of age in comparison to their respective controls
(p = 0.039) (Figure 1B), and in the DM1 patients with > 5
years duration of diabetes in comparison to the controls
(p = 0.002) (Figure 1C).
The protein expression of the non-glycosylated esRAGE
isoform is detected at 46 kDa (15). The esRAGE protein
expression tended to increase in the DM1 patients in comparison to their controls in the overall sample (Figure 2A)
and this tendency remained even after the samples were
grouped according to their age ( ≤ or > 13 years of age)
(Figure 2B). esRAGE, however, was significantly increased
Controls ≤13 years
Diabetic patients ≤13 years
Controls >13 years
Diabetic patients >13 years
100
90
80
70
60
50
40
30
20
10
0
RAGE mRNA
C1
RAGE
β-Actin
C2
P1
DM1 >5 years
C
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
DM1 ≤5 years
Controls
RAGE mRNA
P2
C1
C2
P1
RAGE mRNA
P2
RAGE
β-Actin
C1
C2
P1
P2
RAGE
β-Actin
Figure 1 RAGE mRNA expression in DM1 patients and their controls in (A) the overall sample, (B) grouped according to age ( ≤ or > 13 years)
and (C) grouped according to DM1 duration ( ≤ or > 5 years).
(A) The DM1 patients had an increased expression of RAGE in comparison to the controls (*p = 0.007) in the overall group. (B) The DM1
patients > 13 years of age had an increased RAGE mRNA expression in comparison to their respective controls (#p = 0.039), (C) the DM1
patients with > 5 years duration of diabetes had an increased RAGE mRNA expression in comparison to the controls (**p = 0.002). (Data are
plotted as mean ±SE.)
Koutroumani et al.: Protective mechanisms against oxidative stress and angiopathy Relative protein expression, %
Controls
100
90
80
70
60
50
40
30
20
10
0
Controls > years
Diabetic patients
100
90
80
70
60
50
40
30
20
10
0
A
100
90
80
70
60
50
40
30
20
10
0
B
esRAGE protein (46 kDa)
C1
C2
P1
RAGE
β-Tubulin
Controls
C1
C2
P1 P2
C3 C4
DM1 >5 years
C
P2
DM1 ≤5 years
5
P3 P4
C1
C2
P1 P2
P3
P4
RAGE
β-Actin
RAGE
β-Tubulin
Figure 2 Protein expression of 46 kDa isoform of RAGE (esRAGE) in DM1 patients and their controls in (A) the overall sample, (B) grouped
according to age ( ≤ or > 13 years) and (C) grouped according to DM1 duration ( ≤ or > 5 years).
(C) The DM1 patients with > 5 years of diabetes had an increased esRAGE protein expression in comparison to the DM1 patients with ≤ 5
years of diabetes (*p = 0.008) and the controls (**p = 0.041). (Data are plotted as mean ±SE.)
in the DM1 patients with > 5 years duration of diabetes
when these were compared with the DM1 patients with ≤ 5
years of diabetes (p = 0.008) and also with the controls
(p = 0.041) (Figure 2C).
sRAGE plasma levels
sRAGE plasma levels showed no significant differences
between the DM1 patients and the controls (data not
shown), but they had a tendency to increase in DM1
patients with the increasing levels of their HbA1c and
especially with HbA1c > 9% (Figure 3).
CML plasma levels
CML plasma levels tended to increase in the DM1 patients
when compared to the controls in the overall sample
(Figure 4A) and this increase was significant in the DM1
patients that were ≤ 13 years of age (Figure 4B) and the DM1
patients with ≤ 5 years duration of diabetes (Figure 4C).
MDA plasma levels
MDA plasma levels were increased in DM1 patients in comparison to the controls in the overall sample (p = 0.001)
Controls
HbA1c <7%
HbA1c 7.1-8%
HbA1c 8.1-9%
HbA1c 9.1-10%
HbA1c>10.1%
2.0
Plasma levels, pg/mL
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
sRAGE plasma levels
Figure 3 sRAGE plasma levels in DM1 patients and their controls, in relation to their Hemoglobin A1c (HbA1c) levels.
In DM1 patients, sRAGE plasma levels tended to increase with HbA1c > 9%. (Data are plotted as mean ± SE.)
Controls
Plasma levels, ng/mL
9
8
Diabetic patients
A
Controls >13 years
9
7
6
5
4
3
2
1
0
9
B
8
Controls
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
≤5 years DM1
>5 years DM1
C
0
0
CML plasma levels
CML plasma levels
CML plasma levels
Figure 4 CML plasma levels in DM1 patients and their controls in (A) the overall sample, (B) grouped according to age ( ≤ or > 13 years) and
(C) grouped according to DM1 duration ( ≤ or > 5 years).
(A) The DM1 patients had a tendency to increase CML in comparison to their controls. (B) The DM1 patients ≤ 13 years of age had increased
CML plasma levels in comparison to their age-matched controls (*p = 0.034). (C) The DM1 patients with ≤ 5 years of diabetes had increased
CML plasma levels in comparison to the controls (#p = 0.027). (Data are plotted as mean ± SE.)
(Figure 5A). Also, MDA plasma levels were increased
in DM1 patients regardless of age [ ≤ 13 years (p = 0.001)
and > 13 years (p = 0.012), of age] in comparison to their
controls (Figure 5B) and regardless of the duration of diabetes [ ≤ 5 years (p = 0.01) and > 5 years (p = 0.01)] in comparison to the controls (Figure 5C).
the FRAP plasma levels tended to increase in the DM1
patients regardless of age in comparison to their age
matched controls (Figure 6B) but they were significantly
increased in the DM1 patients regardless of the duration
of diabetes [ ≤ 5 years (p = 0.052) and > 5 years (p = 0.012)]
in comparison to the controls (Figure 6C).
FRAP plasma levels
Correlations
The FRAP plasma levels were significantly increased in
the DM1 patients in comparison to the controls in the
overall sample (p = 0.005) (Figure 6A). More specifically,
In the overall sample of the diabetic patients: (a) the
sRAGE and MDA plasma levels were negatively correlated
with the duration of the diabetes (r = –0.247, p = 0.027 and
Controls
Plasma levels, μM
2.5
Diabetic patients
2.5
A
Controls >13 years
B
Controls
2.5
2.0
2.0
2.0
1.5
1.5
1.5
1.0
1.0
1.0
0.5
0.5
0.5
0
0
MDA plasma levels
≤5 years DM1
>5 years DM1
C
0
MDA plasma levels
MDA plasma levels
Figure 5 MDA plasma levels in DM1 patients and their controls in (A) the overall sample, (B) grouped according to age ( ≤ or > 13 years) and
(A) The DM1 patients had increased MDA plasma levels in comparison to controls (*p = 0.001) in the overall group. (B) The DMI patients with
≤ and > 13 years of age had increased MDA plasma levels in comparison to their controls (**p = 0.001, ***p = 0.012). (C) The DMI patients with ≤ and > 5 years duration of diabetes had increased MDA plasma levels in comparison to the controls (#p = 0.01, ∞p = 0.01). (Data are plotted
as mean ± SE.)
Koutroumani et al.: Protective mechanisms against oxidative stress and angiopathy Controls
450
Plasma levels, mM
400
Diabetic patients
A
Controls >13 years
450
400
Controls
450
B
400
350
350
350
300
300
300
250
250
250
200
200
200
150
150
150
100
100
100
50
50
50
0
0
FRAP plasma levels
≤5 years DM1
7
>5 years DM1
C
0
FRAP plasma levels
FRAP plasma levels
Figure 6 FRAP plasma levels in DM1 patients and their controls in (A) the overall sample, (B) grouped according to age ( ≤ or > 13 years) and
(A) The DM1 patients had increased FRAP plasma levels in comparison to the controls (*p = 0.005) in the overall group. (B) The DM1 patients
with ≤ and > 13 years of age had a significant tendency to increase the FRAP plasma levels in comparison to their controls. (C) The DM1
patients with ≤ and > 5 years of diabetes had increased FRAP plasma levels in comparison to the controls (**p = 0.052, #p = 0.012). (Data are
plotted as mean ± SE.)
r = –0.349, p = 0.020, respectively), (b) the protein expression of the 46 kDa isoform of RAGE (esRAGE) was positively correlated with the duration of the diabetes (r = 0.530,
p = 0.005) and negatively correlated with the MDA plasma
levels (r = –0.661, p = 0.038). (c) The gene expression of
RAGE was negatively correlated with the FRAP plasma
levels (r = –0.474, p = 0.015) and (d) the CML plasma levels
were positively correlated with the FRAP plasma levels
(r = 0.306, p = 0.021) (data not shown).
Discussion
Activation of the AGEs-RAGE axis plays a central role in
the development of diabetic complications. This activated axis leads to diabetic angiopathy in adults (16). It
has always been of clinical interest that the younger DM1
patients do not develop microangiopathy frequently, even
with a duration of DM1 > 5 years which is a crucial time
period after which adults with DM1 present with these
complications.
In our DM1 patients, we found a tendency of the
plasma levels of CML (AGE) to increase, a tendency that
was still evident when the subjects were grouped according to age and duration of diabetes. In particular though,
the younger DM1 patients ( ≤ 13 years of age) and those
with less DM1 duration ( ≤ 5 years) increased CML significantly. Previous reports have also shown significantly
increased CML levels in DM1 children and adolescents
that were not associated, though, with age and duration
of diabetes, but were mostly associated with DM1 complications (17, 18). However, this significant increase
was not associated with an increase in the RAGE mRNA
expression in these young patients, possibly indicating
that these increased circulating CML levels had not fully
activated the AGEs-RAGE axis. Furthermore, RAGE mRNA
expression was significantly increased in the older DM1
patients ( > 13 years of age) and in those with a longer
duration of diabetes ( > 5 years). Previous studies have
also reported increased mRNA expression of RAGE with
increased duration of diabetes (19) but our study has
shown, for the first time, that the age cut-off of 13 years is
of great significance in childhood DM1.
Conversely, the protein expression of the endogenous
inhibitor of AGEs, esRAGE (RAGE 46 kDa), increased with
the duration of diabetes and the levels of total soluble
RAGE (sRAGE and esRGE) tended to increase with the
increased levels of HbA1C ( > 9%) and negatively correlated
with the duration of the patients’ diabetes. The increase in
intracellular esRAGE in the patients with increased duration of diabetes ( > 5 years) could be part of a protective
mechanism that is activated in these young DM1 patients
in an attempt to keep the circulating AGEs within ‘normal’
levels, as the CML levels did not significantly differ in this
group from their respective controls. It is, however, of
great interest that the sRAGE levels did not significantly
change, but do show a negative correlation with the duration of diabetes. This negative correlation may possibly
indicate that some of the AGEs’ homeostatic mechanisms
are negatively affected by the duration of the DM1, even
in these young patients. A clinical report (EURODIAB) in
adults with DM1 has shown that sRAGE increases in DM1
patients that already show macro- and micro-angiopathy
when these were compared with diabetic patients with
no angiopathy (20). Therefore, the tendency of sRAGE to
increase especially with increased HbA1c may also reflect
a counteractive mechanism in these children attempting
to decrease their risk for diabetic complications.
In addition, we found increased MDA and FRAP
plasma levels in the DM1 subjects. Previous reports have
shown increased MDA plasma levels in children and
adults with DM1 (6, 7, 21, 22). It has also been shown that
MDA plasma levels progressively increase in the older
patients with DM1 (23) in comparison to the younger ones.
This increase in MDA plasma levels has not been shown
by other studies to be accompanied by an increase in
FRAP. Nowak et al. (8) showed in 2010 that DM1 patients
who developed nephropathy have decreased antioxidant
capacity as evaluated by their decreased FRAP plasma
levels. Also, Firoozrai et al. in 2007 (7) correlated the
decreased antioxidant defense (decreased FRAP) of their
adult DM1 patients with their poor glycemic control.
Therefore, it is possible that the increased FRAP in
our young DM1 patients is in response to the increased
MDA, showing that our young DM1 patients seem to
still possess the ability to combat the oxidative stress
mediators, which could put them in danger for vascular
complications. This hypothesis is also supported by
Astaneie et al. in 2005 (24), who showed the existence
of increased anti-oxidant capacity in adult DM1 patients,
through markers like FRAP and EGF, which also did not
exhibit changes in oxidative stress markers like the lipid
perodixation marker of the thiobarburitic reactive substances (TBARS).
In conclusion, the increased protein expression of
the endogenous inhibitor of AGEs, esRAGE, in our young
patients with a duration of diabetes > 5 years together
with the tendency of sRAGE to increase with HbA1c > 9%,
seem to indicate that these protective factors are possibly
recruited in the face of increased AGEs (CML), in these
young DM1 individuals, in order to decrease the risk of
AGE-induced complications. The counteracting effect
of the increased FRAP levels in the face of the increased
MDA levels, may also indicate that these young diabetic
patients have possibly preserved their body’s ability to
activate the physiological mechanisms recruited normally to reduce oxidative stress. It therefore seems that
the younger patients with DM1 may recruit protective
mechanisms against glycation and oxidative stress that
may deem them capable of reducing their risk for diabetic
complications.
Received June 11, 2012; accepted August 4, 2012
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In Search of a “Natural” Treatment of Cataract
Anastasia-Varvara Ferlemi1, Olga E Makri2, Constantine D Georgakopoulos2 and Fotini N Lamari1*
1
2
Department of Pharmacy, School of Medicine, University of Patras, 26504 Patras, Greece
Department of Ophthalmology, School of Medicine, University of Patras, 26504 Patras, Greece
In cataract, opacification of the lens reduces visual acuity, finally
leading to blindness if untreated. Cataract is the leading cause of
blindness worldwide – there are about 50 million blind people in the
world and cataract formation is the cause in half of these cases [1].
Today, cataract is treated by surgery. The cost of the surgery places
it beyond the reach of individuals in many parts of the world and there
is an urgent need for less expensive, non surgical approaches to cataract
treatment [2]. It was estimated that extra-capsular surgery used with
95% coverage of the population would avert more than 3.5 millions
disability-adjusted life years (DAILY) per year globally [3]. In 2008, a
study showed that the mean total cost of the operation per patient was
€714 (SD = €57) and ranged from €437 in Denmark to €1087 in Italy
[4]. The financial encumbrance is enormous considering that 28,000
new cases are reported daily [5]. Thus, pharmaceutical interventions
that will maintain the transparency of the lens are intensively sought
after.
Clearly, to develop such treatments, a precise understanding of
the underlying pathophysiological mechanism of cataract formation
is required. There are three discrete categories of cataract: the “agerelated”, that is associated with aging, “congenital cataract” that is
present at birth indicating pathological changes during embryonic
development of the lens, and “sugar cataract” which is associated either
with diabetes mellitus [6-8] or galactosemia [9].
Sun light and oxygen, that the lens is exposed to, are associated
with extensive damage to proteins and other constituents of the
tissue. Other risk factors include environmental stress factors such as
smoking, excessive UV-light exposure and electromagnetic radiation,
life threatening diseases like coronary heart diseases [8], renal failure
and many drugs [7]. These factors contribute to the depletion of
antioxidants [e.g. vitamins C and E, carotenoids and glutathione
(GSH)] and reduction of antioxidant enzymes [e.g. superoxide
dismutase (SOD), catalase (CAT) and glutathione reductase/peroxidase
(GSR/GPx)]. Furthermore, they may diminish protease (i.e. caspase,
nuclease) activity, whose role is the second level of defense (e.g. removal
of obsolete proteins).
Diabetes mellitus is another important risk factor. Hyperglycemia
induces oxidative stress through a complicated pathway. Evidently,
during hyperglycemia the accelerated flux of sorbitol through
the polyol pathway and enhanced oxidative stress are implicated
in the pathogenesis of secondary diabetic complications, such as
cataractogenesis [10]. Aldose reductase (AKR1B1) catalyzes the first
and rate-limiting step of the polyol pathway of glucose metabolism.
Several biochemical mechanisms are involved in the opacification
of the lens and they have been thoroughly described: i) non enzymaticglycation, ii) oxidative stress, iii) polyol pathway and iv) activation
of calpain proteases. The established feature of cataract is the major
structural modifications of the water soluble crystallins [11]. Ageing
process, diabetes and oxidants lead to impaired membrane function,
which results to pathologically elevated levels of intracellular Ca2+
[12,13]. Under these conditions, calpains (calpain 1 or μ-calpain, Lp85,
calpain 2 or m-calpain, calpain 10 and Lp82) are over activated and
the resulting deregulated proteolysis of soluble crystallins leads to
Med Aromat Plants
ISSN: 2167-0412 MAP, an open access journal
their insolubilization and aggregation. Over activated calpains also
degrade the cytoskeleton proteins, which can further elevate Ca2+ [14].
The above mechanisms compromise lens transparency and induce
cataractogenesis. There are numerous models for in vitro and in vivo
study of cataractogenesis. At in vitro experiments, cataractogenic
agents such as galactose, glucose, naphthalene, selenite, transforming
growth factor-β, methylglyoxal are added to the lens culture. The in
vivo models include induction of cataractogenesis especially in rodents
through streptozotocin-induced diabetes, galactose feeding, ionizing
radiation, inhibition of cholesterol synthesis, steroid treatment, and
administration of selenite overdose inducing oxidative stress [5].
Over the last decade, an increasing number of studies indicates the
possible role of some plant extracts or isolated agents against cataract
formation. Plant extracts, like extract of Ocinum sanctum [15], aqueous
garlic extract [16], onion juice [17], as well as fraction of flavonoids
extracted from Emilia sonchifolia [18], polyphenolic compounds of
Camelia sinensis [19] and natural products such as curcumin [20],
ellagic acid [21], luteolin [22], acetyl-L-carnitine [23,24], lycopene
[25], resveratrol [26] have been proved to ameliorate selenite-induced
cataract formation by enhancing antioxidant enzyme activity and
inhibiting radical formation and lipid peroxidation. Additionally to
their antioxidant potential, some plants or their compounds exhibit
anticataract action via maintaining Ca2+-ATPase, prevention of Ca2+
accumulation, thus inhibiting calpain activation. Such compounds are
drevogenin D, an aglycone triterpene [27] and the flavonoid fraction
extracted from Vitex negundo [28]. Furthermore, it has been found that
the carotenoid, astaxanthin, delays lens crystalin precipitation [29],
whereas ellagic acid prevents the alteration of lenticular, non-crystalin
proteins [30].
Diabetic cataract and the possible preventing properties of some
plants extracts or compounds have, also, been studied, but not as
thoroughly as the selenite model. Zingiber officinalis (ginger) extract
[31], Allium sativum methanolic extract [32], turmeric and curcumin
[33] inhibited the polyol pathway and the advanced glycation of
proteins, and protected lens antioxidant enzymes in streptozotocin–
induced cataract model. Hydroxytyrosol and oleuropein from olive
leaves, as well as caffeine protected lenses from oxidative stress in an
alloxan- and a galactose-induced cataractogenesis model, respectively
[34].
*Corresponding author: Fotini N Lamari, Department of Pharmacy, School of
Medicine, University of Patras, 26504 Patras, Greece, Tel. 302610969335; E-mail:
[email protected]
Received December 20, 2012; Accepted December 22, 2012; Published
December 24, 2012
Citation: Ferlemi AV, Makri OE, Georgakopoulos CD, Lamari FN (2013) In
Search of a “Natural” Treatment of Cataract . Med Aromat Plants 1: e144.
doi:10.4172/2167-0412.1000e144
Copyright: © 2013 Ferlemi AV, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Volume 2 • Issue 2 • 1000e144
Citation: Ferlemi AV, Makri OE, Georgakopoulos CD, Lamari FN (2013) In Search of a “Natural” Treatment of Cataract . Med Aromat Plants 1: e144.
doi:10.4172/2167-0412.1000e144
Page 2 of 3
An increasing number of herbal extracts is documented to have
anticataractogenic potential; whether the tested doses and dosage
schemes can be translated in effective agents that will prevent cataract
formation in humans is widely discussed. Most studies focus on the
antioxidant properties of the natural products. Thus, members of the
polyphenol family, i.e. flavonoids, galloyl glycosides, caffeoylquinic
acids, known for their antioxidant properties are privileged candidates
for development of efficacious preventive agents. Beyond this approach,
Babizhayef et al. [35-37] have shown that a natural histidine dipeptide
can prevent and reverse cataract. N-acetylcarnosine has been used
in several clinical studies as eye drops, in order to assess its ability
to treat senile cataract. First results were encouraging, so Innovative
Vision Products Inc. (INV) scientists developed the lubricant eye
drops Can-C, designed as 1% N-acetylcarsosine (NAC) prodrug of
L-carnosine and tested in 75 symptomatic patients 54-78 years old for 9
months. That small-scale study showed that NAC is safe and efficacious
for prevention and treatment of senile cataract for long-term use.
The use of “omics” technologies might reveal new molecular targets
and, thus, specificity in pharmacological targeting might reveal more
effective therapeutics. Inhibition of aldose reductase is the commonest
target in order to combat diabetic cataract. Previous studies using
normal rats, dogs and mice have identified that AKR1B1 inhibitors
are potential drugs to prevent high glucose- and galactose-induced
cataracts. Nonetheless, the clinical utility of AKR1B1 inhibitors
remains uncertain [38]. Another emerging molecular target is the lens
calpains because calpain over activation is the convergence point of all
implicated factors, i.e. ageing, diabetes, and oxidation. Among natural
products, chalcones were indicated as calpain inhibitors in a recent in
vitro study [39]. Besides that, the topical application as eye drops of
those agents remains an attractive drug delivery means [35,40].
The screening of natural compounds for the identification of potent
anticataract agents is a difficult and long-term goal. However, as far
as cataract remains a significant public health problem and the only
way of treatment is, the unprofitable for the society and for individuals,
surgery operation, researchers have to keep seeking an agent that could
prevent cataract formation. Nature has certainly arranged it for us.
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doi:10.4172/2167-0412.1000e144
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Citation: Ferlemi AV, Makri OE, Georgakopoulos CD, Lamari FN (2013) In
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