Journal
Journal of Applied Horticulture, 14(2): 98-101, 2012
Appl
Isolation of biomolecules of pharmacological importance
from Garcinia indica fruit and evaluation of total antioxidant
activity
P. Gayathri and P. Govindaraju*
Department of Biochemistry, Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University,
Coimbatore – 641 003, Tamil Nadu, India. *E-mail: govindtnau@yahoo.co.in
Abstract
An investigation was undertaken to study the antioxidant activity of various solvent extracts of the fruit of Garcinia indica using FRAP
assay and to separate the compounds in the potential extract through TLC, HPLC and analyse using GC-MS. The study revealed that
methanol and ethyl acetate extracts showed a higher antioxidant value than the other extracts. The compounds present in the methanol
extracts were separated by TLC, HPLC and analysed using GC-MS. The results of TLC revealed the separation of two different spots
in case of phenols and a single spot in case of alkaloids. The eluted compounds, subjected to HPLC, separated into 8 peaks in case
of phenolics and 8 peaks in case of alkaloids with varying retention time. The HPLC fractions were subjected to GC-MS to identify
the compounds in comparison with the Wilcon-NIST library. The study is useful in identifying the bioactive compound for anticancer
activity using cell lines.
Key words: Garcinia indica, methanol, ethylacetate, chloroform, hexane, FRAP, antioxidant
Garcinia indica Linn belongs to family Clusiaceae commonly
called as ‘Kokum’ in Maharashtra and cultivated in Konkan,
Goa and the western region of India. Fruits of G. indica have
been suggested in the Indian system of medicine for a number of
diseases. These include its usefulness as an infusion, in skin rashes
caused by allergies, to relieve sunstroke, remedy for dysentery,
an appetizer, liver tonic, to allay thirst and as a cardiotonic
(Khare, 2007). The fruit rind contains polyisoprenylated
benzophenones, garcinol, its isomer isogarcinol, xanthochymol,
and isoxanthochymol (Kirtikar and Basu, 1991). Garcinol has
antioxidative, chelating, free radical scavenging, antiglycation,
anticancer, anti-inflammatory and antiulcer activities (Sheth et
al., 2006; Chattopadhyay et al., 2006). Kokum contains other
compounds with potential antioxidant properties which include
citric acid, malic acid, polyphenols, carbohydrates, anthocyanin
flavonoids and ascorbic acid (Ho et al., 2002; Yamaguchi et al.,
2000).
Free radicals include hydroxyl, superoxide, nitric oxide, nitrogen
dioxide, peroxyl, lipid peroxyl and hydrogen peroxide, which are
generated by products of normal cellular metabolism (Nordberg
and Arner, 2001). The mitochondrial leakage of these reactive
oxygen species leads to oxidative damage of cell components such
as proteins, lipids and nucleic acids (Block et al., 1992). Increased
oxidative stress has been proposed to be one of the major causes
of the pathogenesis of cancer, cardiovascular disease, diabetes
mellitus, neurodegenerative diseases (Alzheimer’s disease
and Parkinson’s disease), autoimmune disorders, rheumatoid
arthritis and ageing (Leifert and Abeywardena, 2008; Leja et
al., 2003; Li et al., 2007). Natural antioxidants such as vitamins
and polyphenols, in vegetables and fruits, are considered to be
responsible for health benefits (Li et al., 2008; Stangeland et al.,
2009). As an important category of phytochemicals, phenolic
compounds universally exist in plants, and have been considered
to have high antioxidant ability and free radical scavenging
capacity, with the mechanism of inhibiting the enzymes
responsible for ROS production and reducing highly oxidised
ROS. In recent years, considerable interest has been shown in
determining total phenolic contents and antioxidant activity of
vegetables, fruits, spices, medicinal plants and algae (Gan et al.,
2010; Ghasemzadeh et al., 2010; Cai et al., 2004; Devasagayam
et al., 2006; Rezaeizadeh et al., 2011).
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Introduction
The aim of this study was to evaluate the total antioxidant activity
of the various extracts of G. indica fruits and to separate the
components of the extract with high activity through TLC, HPLC
and analysed by GC-MS.
Materials and methods
Chemicals and reagents: 2,4,6-tripyridyl-S-triazine (TPTZ)
from M/s. Sigma-Aldrich Chemicals, Bangalore; HPLC grade
methanol, ascorbic acid, bismuth nitrate, sodium acetate, glacial
acetic acid, ferric chloride, potassium iodide, sodium carbonate,
folin ciocalteau reagent, hexane, chloroform, methanol and
ethyl acetate from M/s Merck, Mumbai were used in the study.
Deionized water was used for HPLC analysis. Ready made plates
coated with alumina from M/s Merck, Mumbai was used for the
separation of active components.
Plant material: G. indica fruits were collected from the local
market in Coimbatore.
Preparation of various solvent extracts of G. indica fruit:
Fruits were cut open and the pulp was separated from the rind.
The fruit rinds and pulp were allowed to dry in the shade. The
dried fruit (rind and pulp) was subjected to size reduction to a
Isolation of biomolecules of pharmacological importance from Garcinia indica fruit
Total antioxidant activity: The total antioxidant activity of
the extract of G. indica was assayed by FRAP method (Ferric
Reduction Antioxidant Power) (Benzie and Strain, 1996). One
hundred microlitre of the diluted sample was added to 3 mL of the
FRAP reagent. The absorbance of the mixture was measured at
593 nm in 1cm light path at 37º C using ELICO SL159 UV–Vis
spectrophotometer after 4 min. Briefly, the FRAP reagent was
prepared from sodium acetate buffer (300 mM, pH 3.6), 10 mM
TPTZ solution (40 mM HCl as solvent) and 20 mM iron (III)
chloride solution in a volume ratio of 10:1:1, respectively. The
FRAP reagent was prepared fresh every time and warmed to 37° C
in a water bath before use. A standard solution of 1 mM ascorbic
acid was tested in parallel and the results were expressed as mg
ascorbic acid equivalents/g dry weight of fruit.
Thin layer chromatography: TLC of various extracts of
G. indica whole fruit was performed using readymade plates
coated with alumina. In brief, the plates were cut to the required
dimension (8 x 4 cm) and the extract was spotted using a
capillary tube leaving a distance of 1 cm from the bottom, for
10-15 times to get a concentrated spot. The spotted plates were
placed in a solvent system of Chloroform: acetic acid (9:1) in a
closed chamber and allowed for the run till the solvent mixture
reached three-fourth of the plate. The plate was air dried and
sprayed using the spray reagent Folin’s Ciocalteau (1:1 dilution)
reagent followed by 20% Sodium carbonate, air dried followed by
subsequent drying using drier to identify the presence of phenols.
The solvent system chloroform : methanol (9:1) was used for the
run and the Dragendorff’s reagent was used for spray to identify
the presence of alkaloids. Dragendorff’s reagent was prepared
as under (Sarkozi et al., 2006): Dragendorffs reagent: Solution
A - 1.7 g bismuth nitrate in 100 mL distilled water: acetic acid
(8:2); Solution B - 40 g KI in 100 mL distilled water; Solution
C- 20 mL glacial acetic acid + 70 mL distilled water; Solution D
- Mix 5 mL of solution A, 5 mL of olution B and 90 ml of solution
C just before use.
High performance liquid chromatography (HPLC) analysis
conditions: Methanolic G. indica extract fractions separated by
TLC were filtered using a 0.45 μm nylon filter. 15 μL of the extract
was separated on a Waters dual-pump high-performance liquid
chromatograph (HPLC) using a Waters C-18 Symmetry column
(Milford, MA) and Diode Array Detector. The method used HPLC
grade methanol (mobile phase A) and HPLC grade water (mobile
phase B) with the following linear gradient programme: 85% A,
0 min.; 75% A, 15 min.; 70% A, 20 min.; 45% A, 24 min.; 10%
A, 28 min.; 0% A, 30 min.; and 85% A, 35-40 min. Analytes
were detected at 280 nm and the mobile phase flow rate was 1
mL/min.
GC-MS conditions: The HPLC fractions of phenolics and
alkaloids of G. indica fruit extract were subjected to GC-MS on
a Perkin-Elmer Clarus 500 of mass range between 50-450 m/z
with a flow rate of 1 mL/min. The injection volume was 1 μL
and the Library used was Willey & NIST.
Results and discussion
The results of the total antioxidant activity determined by the
FRAP assay revealed that the methanol extracts of G. indica fruit
had the highest antioxidant activity in terms of mg ascorbic acid
equivalents compared to the other solvent extracts. This may
be due to the efficient extraction of various active components
of antioxidant property especially the phenolics in the methanol
extract.
Table 1. Total antioxidant activity of G. indica fruit
S.No Extract (1g dried sample)
Total antioxidant activity
1. Aqueous
1.32 ± 0.13
2. Chloroform
0.63 ± 0.08
3. Ethyl acetate
6.53 ± 0.62
4. Methanol
7.60 ± 0.51
5. Petroleum ether
0.79 ± 0.25
6. Hexane
2.87 ± 0.39
Values are mean ± SD of triplicates and expressed as equivalents of mg
ascorbic acid
Phenolic compounds have specific health effects even though
they are nonnutritive compounds. Antioxidant properties of
phenolic compounds play a vital role in the antioxidative defence
mechanisms of biological systems (Wright et al., 2001). The
antioxidative effect of phenolics is due to a direct free radical
scavenging activity (Halliwell et al., 2000; Halliwell, 1996),
reducing activity and an indirect effect arising from chelation
of prooxidant metal ions. The chelation of metal ions generally
requires ortho-dihydroxylation on the phenyl ring in phenolic
acids and flavonoids or the presence of a 3- or 5-hydroxyl group in
flavonoids (Shahidi, 2000; Shahidi, 2007). Natural antioxidants
can protect the human body against free radicals, and have also
been shown to retard the progress of a variety of chronic diseases
(cancer, heart disease, diabetes, etc.), as well as ameliorating or
retarding lipid rancidity in foods (Wettasinghe and Shahidi, 1999;
Kinsella et al., 1993).
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coarse powder. The coarsely powdered form of shade dried whole
fruit was extracted using various solvents viz., hexane, chloroform,
methanol, ethyl acetate using mechanical grinder. Then the
extracts were centrifuged and the supernatant was collected and
the solvent was evaporated at room temperature. The semisolid
extract obtained was stored in an airtight container in refrigerator
for further use. The solution of aqueous extract was prepared by
using normal saline as solvent for experiment. The suspension of
hexane, chloroform, methanol and ethyl acetate extract of G. indica
fruit was prepared in distilled water.
99
Thin Layer Chromatography of methanol extract of G. indica whole
fruit was performed using various solvent systems to identify the
presence of secondary metabolites viz., phenols and alkaloids. The
results revealed the presence of phenolic compounds and alkaloids
which was confirmed by the blue and orange colour spots obtained
after spraying with the spray reagent.
HPLC analysis of TLC phenolics fraction of G. indica whole fruit
revealed the separation of the TLC phenol fraction into 4 major
peaks and 5 minor peaks (Fig. 1) with retention time as mentioned
in the Table 2. HPLC analysis of TLC alkaloid fraction of G.
indica revealed the separation of the TLC alkaloid fraction into
3 major peaks and 5 minor peaks (Fig. 2) with retention time as
mentioned in the Table 3.
The phenolic HPLC fraction of G. indica extract was subjected
to GC-MS and the compounds identified in the fraction upon
comparison with the library are shown in Table 4. The alkaloid
HPLC fraction of G. indica extract was subjected to GC-MS and
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Isolation of biomolecules of pharmacological importance from Garcinia indica fruit
Table 2. HPLC peaks of TLC phenolics fraction with retention
time
Peak No
1
2
3
4
5
6
7
8
9
Retention time
2.635
3.147
5.005
5.621
6.208
7.125
9.248
18.400
20.970
Table 3. HPLC peaks of TLC alkaloid fraction with retention
time
Fig. 1. HPLC of TLC phenolics fraction- G. indica
Peak No
1
2
3
4
5
6
7
8
Retention time
3.135
4.133
5.312
6.144
9.621
18.325
20.853
34.630
Fig. 2. HPLC of TLC alkaloid fraction- G. indica
the compounds identified in the fraction upon comparison with
the library are shown in Table 5.
Based on the study, it is concluded that G. indica fruit is a potent
source of antioxidants. The compounds identified through GCMS can be tested for antioxidant and anticancer activity using
different cell-lines. Further identification of active principles
present in the extract and structural elucidation can be done using
LC-MS and NMR.
Compound
Trans, Trans-3,12-Dimethoxy-1,6,10dodecatriene
Tridecane,2,2,4,10,12,12-Hexamethyl7(3,5,5-trimethylhexyl
Tetradecanoic Acid, 10,13-Dimethyl,Methyl Ester
Octadecane
M.W Formula
224 C14H24O2
1,7-Dimethoxy-1,6-Heptadiene
394
C28H58
270
C17H34O2
254
C18H38
156
C9H16O2
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Table 4. GC-MS of G. indica extract - Phenolics -HPLC
fraction
Table 5. GC-MS of G. indica extract-Alkaloid-HPLC fraction
Compound
M.W Formula
6-Methyl-7-(Trimethylsilyl)-5-Hepten-1-ol
200 C11H24OSi
2R,3R,4R,5S,6R,8S,10S)-5-Acetoxy-10Benzyloxy-8-Benzyloxymethyl
498
C29H38O7
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Tetradecanoic Acid, 10,13-Dimethyl, Methyl ester
270
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