contribution to the study of the pharmaceutical

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CONTRIBUTION TO THE STUDY OF THE
PHARMACEUTICAL QUALITY OF SOME
CHAMOMILE COMERCIAL SAMPLES. NOTE I.
THE ANALYSIS OF THE VOLATILE OIL
OANA CIOANCĂ*, ANA CLARA APROTOSOAIE, ADRIAN ŞPAC,
MONICA HĂNCIANU, URSULA HELENA STĂNESCU
University of Medicine and Pharmacy “Gr. T. Popa” Iasi, Faculty of
Pharmacy, 16 University Str., Iasi, 700117
*corresponding author: [email protected]
Abstract
Chamomile is well known and widely used for its therapeutic purposes. It is
generally approved that natural products not necessarily mean safe or pure products, thus
the aim of our research was to assess the degree to which chamomile tea manufacturers
comply with the regulations imposed by the European pharmaceutical distribution market
(Romania being part of the European Union) of such a tea. In the present study we present
the results obtained in the phytochemical analysis of the volatile oils extracted from 10
commercial samples of chamomile tea. Important qualitative and quantitative differences
were noted for the separated volatile fractions.
Rezumat
Muşeţelul este o plantă medicinală foarte cunoscută şi extrem de utilizată în scop
terapeutic. Se ştie că produse naturale nu înseamnă produse sigure sau neimpurificate, de
aceea scopul cercetării noastre a fost acela de a urmări gradul în care producătorii de ceaiuri
medicinale respectă regulile impuse de comercializarea prin reţeaua farmaceutică
europeană (România fiind membră a Uniunii Europene) a acestor tipuri de produse. În
cadrul studiului de faţă vom prezenta rezultatele obţinute în urma studiului fitochimic a
uleiurilor volatile extrase din 10 mostre de ceai de muşeţel comercializat. Au fost observate
diferenţe calitative şi cantitative importante între fracţiunile volatile separate.
Keywords: chamomile, volatile oil, differences, quality
Introduction
Chamomillae flos represents one of the most common medicinal
teas used both for it’s pleasant taste as well as for therapeutic purposes, but
most of the vegetal material commercialized by pharmacies has poor
quality. Thus, the aim of our study was to investigate this aspect, pursuing
the requirements the chamomile flowers should comply with the European
Pharmacopoeia (PhEu) [10].
We used nine commercial samples of chamomile bought from
pharmacies before Romania became an European Union (EU) member, and
FARMACIA, 2010, Vol.58, 3
309
a German producer sample was added.
Our research was centered on: the pharmacognostic macroscopic
study of the vegetal material (identification of the vegetal and mineral
impurities, the congruence of the product to what the chamomile
inflorescences should be); the phytochemical analysis (the composition of
the volatile oil, flavones and polyphenolic acids); and the microbiological
contamination. In this paper, we are going to present the results obtained for
the study of the volatile fraction extracted from the 10 chamomile samples.
Materials and methods
The material was represented by 9 commercial chamomile samples
(bought from pharmacies in April-May 2005) from different inland
producers. The samples were codified C1-C9 bearing in mind the
packaging, tea-bags and 50-100g sacks (for the last type of samples using
the term “bulk”). The sample C0 (tea-bags) made by Sidroga, found only in
pharmacies within EU countries, was included in the study. For this last
sample, the flowers are of cultivated chamomile and comply with the PhEu
requirements [2,3,12].
The volatile oils were obtained by hydro-distillation in a NeoClevenger type apparatus from the 10 samples of Chamomillae flos,
according to the method described in the Romanian Pharmacopoeia Xth
Edition [11].
Methods:
We established the extraction yield, the colour and aspect
characteristics for each oil sample.
The chemical analysis pursued the identification of the terpenoidic
compounds from the volatile fractions, using thin layer chromatography
(TLC) followed by gas chromatography with mass spectrometry detection
(GC-MS) [1].
GC analysis of the samples was performed on a Gas Chromatograph
type Agilent 6890 with 5975 Mass Selectiv Detector, HP 5 MS capillary
column (30 m x 0.25 mm x 0.25 µm) with helium as mobile phase at a
constant flow of 1 mL/min (average velocity of 36 cm/sec). Sample
solutions were injected (0.2 µL) in split mode (1:10) at 260°C. The column
temperature was lineary programmed from 35 - 260°C at 10°C/min.
Transfer line was heated at 260°C. Evaluation of the results was performed
according to the software (ChemStation) including a mass spectral library
(Wiley), which was used for identifying organic compounds extracted from
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the oils. The identification of the components was based on the comparison
of their mass spectra in the apex of each peak with those of the analytical
standards from Willey Mass Spectral Library. For quantification purposes
the percent area was reported.
Reagents:
All chemical and reagents were of analytical grade or of
chromatographic quality and were purchased from Merck (Darmstadt,
Germany), Sigma Aldrich (Seelze, Germany) or Fluka (Buchs,
Switzerland).
Results and discussion
Calculating the extraction yield for the volatile oils separated by
hydro-distillation from the vegetal material we obtained the results
presented in table I and figure 1.
Table I
The volatile oil content and aspect for the chamomile samples
C0
Sidroga
s
Volatile oil
content (%)
1.15
slightly viscous liquid
indigo-blue
C1
Belin
s
0.58
slightly viscous liquid
blue-indigo
C2
C3
C4
Celmar
Fares
Ciprod
s
s
s
0.43
0.43
0.29
viscous liquid
viscous liquid
slightly viscous liquid
blue-indigo
blue-indigo
green-blue
C5
C6
Hofigal
Digitalis
s
v
0.36
0.72
slightly viscous liquid
extremely viscous liquid
blue-green
dark blue-indigo
C7
C8
Cyani
StefMar
v
v
2.15
0.43
slightly viscous liquid
slightly viscous liquid
blue-green
blue-green
C9
Plafar
Botosani
v
0.72
slightly viscous liquid
light blue
Code
Producer
Type
Aspect
Colour
where: s - tea-bag; v - „bulk” package
Figure 1
Graphic representation of the extraction yield for the volatile oil
from the chamomile samples
FARMACIA, 2010, Vol.58, 3
311
Bearing in mind the obtained values we can state that two of the
samples, C4 and C5, did not comply with the pharmacopoeial (both Ph.Eu
and FR X) standards [10,11] that require a minimum amount of 0.4 mL of
volatile oil for 100g of dried vegetal material. Overall, the volatile oil
content of the chamomile samples varies between 0.29 – 2.15%. Also,
comparing the volatile oil content of the tea-bagged samples we notice that
this is lower than the amount registered for the “bulk” samples. Regardless
of the Sidroga sample (from EU) the first category had a content of 0.29%
to 0.58%, whereas the “bulk” samples provided quantities varying between
0.72% and 2.15% of volatile oil. This fact is in accordance with what we
already know about the volatile fractions: for a vegetal material that is
grounded, the higher the powdering degree, the higher loss of volatile
compounds, especially when the tea-bag material is permeable and there is
no other protection (tea bags wrapped in individual waxed envelopes,
included in a box with a plastic wrapper, as for the C0 sample).
Assessing the physical aspects of the 10 volatile oils, the one
obtained from the Sidroga sample had the organoleptic characteristics that
best comply with the standards for the chamomile volatile oil (table I). The
color of the oil samples varies from blue-indigo (C1-C3) to light-blue (C9)
and blue-green (C5- macroscopic analysis revealed the presence of the
leaves and stalk), with important differences regarding the mobility – from
slightly viscous (C1, C4, C5) to extremely viscous for the C6 sample.
The TLC for the volatile oils on Kieselgel G60 (mobile phase
toluene: ethyl acetate 95:5) revealed the images presented below, in figure
2.
Figure 2
TLC for chamomile volatile oil samples; C0-C9 = samples; standards: standard
chamomile volatile oil, bisabolol and bornyl acetate
We noticed the lack of uniformity regarding the chromatographic
aspects, even if the samples C1-C3 might have a similar compound spectra.
The presence of azulene (Rf=0.94) in all samples (excepting sample C5) is
important not only for the colour of the oil, but it has a certain importance in
the antiinflammatory action of the volatile fraction. C0 sample lacks
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bisabolol (Rf=0.41), but for most of the samples is present. At Rf=0.5 there
is a brown spot which, probably belongs to the spiroethers, that for C4-C6
and C8 samples fades out.
The GC-MS allowed us to identify a large number of compounds;
the main components are presented in table II.
Table II
Main compounds identified in the chamomile volatile oils–selective table
RT*
(min.)
Area (%)
Compound
C0
C1
C2
C3
C4
C5
C6
C7
C8
C9
-
-
-
0.01
-
0.04
0.01
5.692
α-pinene
0.03
-
0.01
6.480
6-methyl-5-hepten-2-one
0.05
-
0.01
-
-
-
0.01
-
-
-
7.112
p-cymene
0.02
-
0.05
0.30
-
0.05
0.04
-
0.05
0.04
7.173
limonene
0.04
0.07
0.03
0.12
0.04
-
0.04
-
0.04
0.03
7.233
1,8-cineol
0.02
-
-
-
-
-
0.02
-
0.12
-
7.649
artemisia ketone
7.995
artemisia-alcohol
0.03
0.15
0.07
0.09
0.04
0.07
-
0.08
-
0.03
-
0.31
0.15
0.01
8.393
α-thujone
-
0.10
-
-
-
-
0.07
-
-
0.13
9.129
L-menthone
0.10
0.41
0.29
-
-
-
0.14
-
-
-
9.285
isomenthone
0.12
0.51
0.30
-
-
-
-
-
-
-
9.319
l-borneol
-
-
-
-
0.05
0.12
-
-
0.33
0.23
11.016
trans-anethole
1.29
-
-
0.32
0.10
0.25
0.07
-
-
0.04
12.496
berkheyaradulene
0.28
0.26
0.21
-
0.24
-
0.16
-
-
0.20
13.137
pirethryn I
-
-
0.07
-
-
-
-
-
-
13.232
trans-β-farnesene
14.07
1.13
1.12
3.93
2.49
2.71
4.32
11.34
3.24
11.13
13.448
alloaromadendrene
-
0.45
0.76
-
-
-
0.09
-
-
0.17
13.786
β-selinene
0.59
-
0.47
-
-
-
0.18
-
-
0.62
13.630
α-curcumene
0.36
-
0.18
-
-
-
0.13
-
-
0.20
13.682
germacrene D
1.11
0.41
0.21
0.66
0.17
0.17
0.28
-
0.33
0.89
13.561
(E,Z)-α-farnesene
0.40
0.24
-
-
-
-
-
-
-
0.51
13.760
eremophyllene
-
-
-
-
-
-
-
-
0.47
0.42
13.881
biciclogermacrene
-
-
0.16
0.48
-
0.20
0.30
-
0.54
0.87
14.340
cis-α-bisabolene
0.12
-
-
-
-
-
-
-
-
-
14.348
α-muurolene
-
-
-
-
-
-
-
-
-
0.06
14.868
(-)-spathulenol
2.50
5.01
5.27
10.75
6.53
2.56
2.90
3.61
3.00
2.80
15.733
16.002
α-bisabololoxide B
α-bisabolol
8.34
-
21.38
0.95
14.42
-
20.49
1.57
3.43
0.85
4.29
0.99
13.58
-
3.47
1.87
3.63
1.00
2.69
-
16.062
bisabolonoxide
-
10.64
8.75
11.14
10.28
13.46
-
31.53
13.25
26.20
16.106
β-bisabolene
21.92
-
0.11
-
-
-
0.07
-
-
0.09
16.616
chamazulene
8.25
2.92
4.97
5.71
0.26
1.84
11.48
3.64
1.76
0.38
16.772
bisabololoxide A
10.76
7.77
9.08
9.60
7.72
10.66
10.29
19.76
10.24
11.59
18.131
en-yne-dicycloether
3.13
1.19
0.71
3.29
1.61
1.23
0.84
1.61
1.65
1.53
20.719
ambretolide
-
-
-
1.36
-
-
-
-
-
-
24.286
octadecane
-
-
-
-
-
-
0.18
-
0.87
0.02
24.987
tetracosane
0.14
0.31
0.28
-
1.36
12.51
0.65
-
2.01
0.46
25.316
eicosane
0.07
-
0.02
-
0.54
6.13
0.08
1.73
2.18
0.04
* RT – Retention time (minutes).
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313
As shown in the table, the volatile oils had a complex chemical
composition, being mainly constituted of sesquiterpenes, aliphatic
compounds, spiroethers and, excepting C7, monoterpenes. The compound
spectra as well as the quantity of each component varies a lot from one
sample to another. As a matter of fact, the only compounds found in all
samples were: chamazulene, en-in-dicycloether, α-bisabololoxide A, αbisabololoxide B, t-β-farnesene and spathulenol - a compound important for
its antifungal properties, the first 5 being considered the major specific
components for the chamomile volatile oil.
Excepting the samples C4 and C5, sesquiterpenes were the major
fraction (38.45% - 77.16%) of the volatile oil (the literature mentions values
of 75-90%). The noted differences are not only quantitative, but qualitative
too. For example, alpha-bisabolol and its oxides A and B represent the most
important oxygenated sesquiterpenes with pharmacologic activity; but as the
internal standard Sidroga (C0) the samples C2, C6 and C9 lack α-bisabolol.
Another major sesquiterpene-oxide identified in samples C1-C5, C7-C9 is
bisabolonoxide. Regarding the oxygenated sesquiterpenes distribution, a
recent study showed that: α-bisabolol, bisabololoxide B and bisabolonoxide
are specific for the ligulate and tubular florets, whereas spathulenol and
bisabololoxide A are found in the receptacle and tubular florets [7].
All volatile oils contained t-β-farnesene as a major hydrocarbonated
sesquiterpene, distinguished in high concentrations in the leaves and roots of
Chamomilla recutita [5]. For C0, C1, C2, C4, C6 and C9 samples we
identified another hydrocarbonated sesquiterpene – berkheyaradulene, that
is specific for the roots of Berkheya radula (Harv.) de Willd. [4] and the
Silphium perfoliatum rhizome [9], a common Asteraceae species for our
country.
En-yne-dicycloether, with important spasmodic and antimicrobial
activity, was present in all samples, the tea-bagged samples containing
similar amounts (1.61%-C7, 1.65%-C8, 1.53%-C9) excepting C6 (0.86%),
whereas the “bulk” samples the differences were significant: 0.71%-C2
comparing to 3.29%-C3 or 3.13%-C0.
The main identified monoterpenes, even if poorly represented, were:
artemisia-alcohol, artemisia-ketone, l-borneol and limonene. Limonene
found in all samples excepting C5 and C7 is characteristic rather to the
volatile oil from the stems, wheres linalool (C0, C2) belongs to root oil [5].
The thujone from C1, C6, C9 is a compound found in the ligulate florets,
where is highly concentrated [7].
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FARMACIA, 2010, Vol.58, 3
Conclusions
The qualitative and quantitative chemical study of the chamomile
samples revealed that the extracted volatile oils differ a lot from one sample
to the other and the composition is not well preserved for the samples
packed as tea-bags unless included in waxed envelopes. In the same time,
the chemical analysis showed the existence of compositional differences
more or less important, the only compounds found in all samples were:
chamazulene, en-yne-dicycloether, α-bisabololoxide A, α-bisabololoxideTB,
t-β-farnesene and spathulenol. Also, the existence of nonspecific chamomile
compounds: menthone (C0, C1, C2, C6), isomenthone (C0, C1, C2), transanethole (C0,C3-C6), pirethryn I (C2) and ambretolide (C3), suggest that
the samples have been contaminated or inadequately processed.
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Manuscript received: July 23rd 2009