(-HCA) content in different fruit morpho types of Garcinia

Indian Journal of Advances in Plant Research (IJAPR),
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Indian Journal of Advances in Plant Research (IJAPR)
Research Article
Estimation of hydroxy citric acid (-HCA) content in different fruit morpho
types of Garcinia indica Choisy
*
Krishna, A., Niveditha, M. and Vasudeva, R.
Department of Forest Biology and Tree Improvement, College of Forestry, Sirsi-581 401, University of Agricultural Sciences,
Dharwad, Karnataka, India..
*
Corresponding Author: A. Krishna; Email: [email protected]
ABSTRACT
In recent years, Garcinia indica has gained popularity because of its newly identified organic acid. The fruit rind is rich in Hydroxycitric acid (-HCA), an important biologically active plant metabolite used as an anti-obesity drug. It inhibits the
conversion of carbohydrates into fats by inhibition of ATP citrate lyase, an important enzyme in Kreb‟s cycle. Therefore it is
important to evaluate species and understand its character which has greater impact on the productivity. With this background the
present investigation was taken up in the College of Forestry, Sirsi. The trees were grouped based on the fruit colour into four
major types viz. red; green; orange and yellow morpho-types. One of the important and major acids present in all the four morpho
types of Garcinia indica is Hydroxycitric acid (-HCA). Highest HCA percent was encountered in green morpho (20.35 g / 100 g in
dry rind; whereas least HCA percent was in orange morpho (5.7 g / 100 g in dry rind in dry rind) and the other two morpho are
intermediates. Maximum HCAL content was in yellow (0.71 g / 100 g in dry rind) and the least in orange morpho (0.28 g / 100 g
in dry rind. Of all the morpho types, green and red morpho type fruit contain high amount of HCA than other two morpho -types
(20.35 g per 100 g dry weight and 19.5 g per100 g dry weight respectively).This study clearly shows that green and red morpho
trees can be recommended for cultivation for high HCA yield.
Key words: Garcinia indica, Morphotypes, HCA content and productivity
INTRODUCTION
India is endowed with rich diversity of tropical fruits. One
among them, Garcinia indica (kokam or murugalu), is an
evergreen, dioecious tree endemic to the Western Ghats. Due to
the increased demand owing to its medicinal, nutritive and
industrial value is fast becoming one of the most important non timber forest product (NTFP) species of the coastal districts of
Karnataka. Generally fruit colour is red and occasionally trees
bearing pale yellowish fruit, greenish as well as orange
colouration are also found. Traditional healers believe that
yellow morpho fruits have better healing properties when used
for stomach ailments. However the physical and chemical
characterizations of different morpho-types have been very
scantily attempted. With this background the present study was
undertaken to identify and characterize different morpho-types
morphologically and chemically for various fruit traits.
In recent years the species has gained popularity because of
its newly identified organic acid. The fruit rind is rich in Hydroxycitric acid (-HCA), an important biologically active
plant metabolite used as an anti-obesity drug (Heymsfield et al.,
1998). It inhibits the conversion of carbohydrates into fats by
inhibition of ATP citrate lyase, an important enzyme in Kreb‟s
cycle. The seeds yield a valuable edible fat known in commerce
as „Kokam Butter‟. Several value added products are being
prepared from kokam and are popular in Indian and International
market. Products such as jam, pickles, chutney, squash, dried
fruit rind, pulp, kokam syrup, kokam agal, kokam oil, stearic acid
from kokam fat, kokam rind acid, kokam colour, ointment etc are
also being exported. Kokam oil can be directly used for lighting
and frying. It is also used in chocolate, confectionary, cosmetic
industry in lipsticks and crack creams and locally used for
healing of cracks on leg. The species has been declared as
“threatened” as per the IUCN guidelines.
G. indica has multifarious uses and health advantages such
as anti-obesity property, rich vitamin C, anti-oxidant property
and rich fatty acids. This undomesticated species shows
tremendous variability in fruit morphology and uses. However,
till date no serious efforts have been made to characterize for
chemical properties, except for few sporadic reports.
Characterization is most significant step and a first s tep in
assessing variability. Systematic characterization of chemical
characters of available germplasm would provide knowledge on
the extent of genetic diversity in the fruits species and facilitate
in identifying the superior genotypes with desired character.
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Therefore, it is important to evaluate species and understand
were combined and made up to 100 ml, mixed and filtered using
different morpho types which has greater impact on Whatman‟s filter paper 1 and used for HPLC. The best fit was
Hydroxycitric acid (-HCA) productivity.
obtained with the regression equation y= 0.4146x with an R =
0.9761.The standard curve for HCA is shown in Fig.1 and 2.
MATERIALS AND METHODS
Survey was carried out to identify different morpho types of
Extraction of HCA through distilled water method
Garcinia indica Choisy. The trees were grouped based on the
Procedure for Sample extraction
fruit colour into four major types viz. Red; Green; Orange and
Each morpho type samples were subjected to oven drying.
Yellow morpho types. Global Positioning System was used to get
Dried samples were ground to a fine powder using mixer. About
the geographic positions of individual trees selected for the
1 g was weighed and subjected for extraction adopting following
study. A sample of fifteen to twenty fresh fruits was randomly
methodology.
drawn from the fruit lots of each tree and each fresh fruit was
weighed using an electronic top balance to the nearest 0.1g.
Keeping the fruits in the same order length, diameter of fruits
1 g powder was taken
were measured and recorded with the help of digital verniar
caliper in millimeter to the nearest 0.01 cm. Each fruit was cut
open and pulp weight was recorded in grams.
10 ml distilled water was added
Estimation of (-) Hydroxy citric acid (HCA) through HPLC
method
(-)Hydroxy citric acid was analyzed by reverse phase HPLC
(Make: Shimadzu, LC-8A,) on a C18 column Hibou R 250-4, 6
RP- 18 e (5 µm).The HPLC conditions were: 210 nm
wavelength, UV detector, 1 ml /min flow rate and 20μL sample
loop. The mobile phase was isocratic, 8mM sulphuric acid and
triple distillated water. A Potassium hydroxy citrate (Sigma:
tribasic monohydrate) standard sample was procured from Sigma
Chemicals. The standard was prepared using distilled water. The
retention time of standard HCA was 4.6 min. Run time for each
sample was 20 m (Jayaprakash and Sakariah, 2002).
Procedure for preparation of mobile phase
To prepare 1 lit mobile phase 0.43 ml of 8mM sulphuric
acid was added and made up to 1000 ml with triple distillated
water. Mobile phase must be prepared on the day of HPLC only,
after preparing it should be degassed with sonicating instrument.
Column was washed with Mobile phase for half an hour. Then
sample was injected into HPLC slot one by one after every 20
min. Then the retention time was noted down and comp ared with
standard solution retention time.
Preparation of pure HCA from potassium hydroxicitrate salt
Standard HCA of Garcinia species extract is not
commercially available in market. Free HCA was prepared from
Potassium hydroxy citrate tribasic monohydrate (100 mg) which
was suspended in a 50 ml beaker containing 5.0 ml distilled
water and was treated with 500 mg of Dowex 50 [H+]. The
mixture was stirred using glass rod/ magnetic stirrer for 10 min.
After 10 min resin and salt will settle at the bottom and only
supernatants was decanted. This step was repeated 6-7 times till
the resin comes to neutral pH. The washing and the supernatants
Vortexed for 10-15 min
Filtered using whatman paper 4
Add 200µl filtrate to eppendorff tubes and
Add 400 µl ethanol
Vortexed for 1 min and kept in fridge for overnight for
incubation
Centrifuge at 5000 rpm for 10 min
Take the supernatant in the another eppendorff tubes
Dry the supernatant using nitrogen gas and keep it in the
fridge (4º c)
On the day of HPLC slot add 100 µl triple distillated water
and dissolve the dried sample properly and was used for
injection to the HPLC
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6
y = 0.4146x
R2 = 0.9761
5
Area (mm2 x 100000)
4
3
2
1
0
0
2
4
6
8
10
12
14
Concentration of HCA (ug)
Concentration(µg)
Fig 1: Linear relationship between the area and concentration of standard HCA
Indian J. Adv. Plant Res.,
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4.661
200
8.555
9.045
9.419
6.997
7.413
7.851
3.637
50
5.845
6.261
100
5.216
mAU
4.213
150
0
0
2
4
6
8
10
12
14
16
18
Minutes
Fig. 2 The protocol followed for the extraction of HCA from the dried rind of Garcinia indica
RESULT AND DISCUSSION
The (–) HCA content of dry fruit rinds was estimated
adopting RP-HPLC (LC-8A: Shimadzu). The HCA was observed
at 210 nm at 4.6 minutes retention time. Highly significant
variation was observed with respect to (-) HCA and hydroxy
citric acid lactones (HCAL) content in dry rind among the four
morpho (Table No. 1; Fig-3 & 4). Highest HCA percent was
encountered in green morpho (20.35 g / 100 g in dry rind;
whereas least HCA percent was in orange morpho (5.7 g / 100 g
in dry rind in dry rind) and the other two morpho are
intermediates. Maximum HCAL content was in yellow (0.71 g /
100 g in dry rind) and the least in orange morpho (0.28 g / 100 g
in dry rind. Perhaps for the first time an association between
HCA content of the dried fruit rind and colour morpho has been
clearly established in G. indica.
Major portion of organic acids in Garcinia indica is hydroxy
citric acid [(-) HCA]. Highly significant variation was observed
with respect to (-) HCA and hydroxy citric acid lactones (HCAL)
content in dry rind among the four morpho (Table No.2). Due to
its presence in high amount in Garcinia species it is also called
garcinia acid. It has been reported that the rinds contain about 1030 per cent (-) HCA on a dry basis (Jayaprakash and Sakariah,
2002; Nayak and Rastogi, 2010; Priya Devi et al., 2012; Priya
Devi et al., 2010b). In this study, highest HCA percent was
found in green morpho (20.35 g / 100 g in dry rind) whereas least
HCA percent was in orange morpho (5.7 g / 100 g in dry rind in
dry rind) and the other two morpho are intermediates. Maximum
HCAL content was in yellow (0.71 g / 100 g in dry rind) and the
least in orange morpho (0.28 g / 100 g in dry rind; (Fig-5). In this
study perhaps for the first time an association between HCA
content of the dried fruit rind and colour morpho has been clearly
established in G. indica. Hence selection for green types would
yield a higher HCA content.
Table No. 1: Comparison for tree traits among different fruit morpho types of Garcinia indica.
The values show Mean ± S.D
Sl
Red morphoGreen morphoOrange morpho- Yellow morphoTree trait
No.
type
type
type
type
Tree height
8.5 ± 1.3
10.5 ± 4
8.6 ± 1.5
13 ± 1.7
(m)
Gbh (cm)
50.0 ± 19.3
53.8 ± 42.5
53.6 ± 38.5
78.5 ± 24.6
Table No. 2: Composition of (-) HCA and HCAL in fruit rinds
different fruit morpho-types of Garcinia Indica
HCA
Sl. No.
Different morpho types
(g/100g)
Red morpho-type
19.5±1.14
Green morpho-type
20.35±3.04
Orange morpho-type
5.7±0.35
Yellow morpho-type
14.45±0.96
of
HCAL
(g/100g)
0.40±0.03
0.33±0.43
0.28±0.03
0.71±0.07
-
4.629
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mAU
40
20
0
0
1
2
3
4
5
6
7
8
9
Minutes
Fig 3: HCA standard graph with sample
4.661
200
8.555
9.045
9.419
6.997
7.413
7.851
3.637
50
5.845
6.261
100
5.216
mAU
4.213
150
0
0
2
4
6
8
10
12
14
16
18
Minutes
Fig 4: Sample chromatograph showing peak of HCA at 4.6 min
Fig 5: Concentration of Hydroxycitric acid lactone and hydroxy citric acid among different fruit morph
types in Garcinia indica
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Nayak and Rastogi (2010) have reported that the HCA is
also present in leaves of G. indica choisy and its potential food
application. Jayaprakash and Sakariah (2002) have reported
similar results indicating that the per cent HCA ranged from 10.3
to 12.7 % in rinds and 4.1 to 4.6 % in leaves. Jayaprakasha et al.
(2003) reported that – HCA content in G.indica is tissue specific
at the level of 2.5, 0.8, 3.0 and 20.1 % in leaf, pulp, fresh fruit
and dried rind respectively. However, in a recent study from
Goa, Priya devi et al. (2010a) have reported that HCA per cent in
dried fruit rind ranged from a minimum of 19.32% to a
maximum of 37.39 %. Among them, 53 accessions recorded
more than 30 per cent (-) HCA in rinds. Hegde et al., (2010) have
compared Garcinia indica and G. gummi-gutta for
morphological and chemical traits and reported that the (-) HCA
content in G. indica ranged from 7.0 to 13.0% among higher fruit
yielding trees. The lactone content was 4.9 to 10.5 %, In case of
yellow morpho type HCA content ranged from 9.0 to 11.9%.
-HCA is susceptible to lactonization especially during
evaporation and concentration. The lactone has very low
biological activity compared with free – HCA (Jayaprakash and
Sakariah, 2002). In the current study the levels of lactones were
lower, suggesting that the estimates of the – HCA is nearer to the
real value present in different morpho types. Utpala et al., (2010)
reported that the percentage of (-) HCA among north eastern
species of Garcinia ranged from 0.09 % to 5.81 % while that
among the Western Ghats was higher and ranged from 8.5 % to
The local healers generally believe that the „yellow‟ morpho
fruits have better healing properties than the „red‟ morpho hence
it is priced higher. However the results of this study do not
support this notion as on an average the „red‟ morpho has higher
(-) HCA content than the „yellow‟ morpho. Alternatively healing
property might not be related to the (-) HCA content. Among the
major active compounds in Garcinia species, (-) HCA is
predominant and garcinol is the next. Both of these compounds
play beneficial role in human health since they have anti cancer
and anti-obesity properties. Garcinol is a yellow colored, fat
soluble pigment found in the rinds of Garcinia indica at level of
2-3 %. In fact all Garcinia species have some amount of garcinol
(Yamaguchi et al., 2000; Bakana et al., 1987; Sahu et al., 1989).
Garcinol can be separated from fruit rinds by ethanol or hexane
extraction (Yamaguchi et al., 2000; Padhye et al., 2009). It would
be interesting to know if the „white‟ morpho has higher garcinol
content and hence preferred by the local healers.
One of the important and major acids present in all the four
morpho types of Garcinia indica is (-) HCA. Of all the morpho
types, green and red morpho type fruit contain high amount of
HCA than other two morpho-types (20.35 g per 100 g dry weight
and 19.5 g per100 g dry weight respectively).This study clearly
shows that green and red morpho trees can be recommended for
cultivation for high HCA yield.
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