PRELIMINARY PHYTOCHEMICAL SCREENING AND IN...
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Chapter 3
PRELIMINARY PHYTOCHEMICAL SCREENING
AND IN VITRO ANTIOXIDANT EVALUATION
OF GARDENIA GUMMIFERA LINN. F. ROOT
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Chapter 3(A)
Preliminary phytochemical analysis of
Gardenia gummifera Linn. f. root
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3A.1. Introduction
Phytomedicines have been used since time immemorial to treat various
ailments long before the introduction of modern medicine. It was estimated that
about 80% of all the world’s medicines are originally derived from plant sources,
especially those found in tropical regions. Medicinal plants have been used all over
the world for the treatment and prevention of various ailments, these include
atherosclerosis and stroke, myocardial infarction, certain types of cancers, diabetes
mellitus, allergy, asthma, arthritis, Crohn’s disease, multiple sclerosis, Alzheimer’s
disease, osteoporosis, psoriasis, septic shock, AIDS, menopausal symptoms, and
neurodegeneration (Aggarwal et al., 2004; Zaidan et al., 2005; Shyamala Gowri and,
Vasantha, 2010). The medicinal value of these plants lies in some chemically active
substances that produce a definite physiological action on human body. The most
important of these chemically active constituents of plants are alkaloids, tannins,
steroids, terpenoids, flavonoids and phenolic compounds (Edeoga et al., 2005;
Aiyelaagbe and Osamudiamen, 2009; Tambekar et al., 2009).
At present the demand for herbal or medicinal plant products has increased
significantly (Ajibad et al., 2005). Natural products, which come out mainly from
medicinal plants are important for pharmaceutical research and for drug
development as a sources of therapeutic agents. G. gummifera Linn. f. may have
various phytochemicals of medicinal importance as it was traditionally used for the
treatment of various ailments (Tambekar et al., 2009). Hence, the present study was
conducted to identify the different types of phytochemical constituents of G.
gummifera root extracts.
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3A.2. Materials and Methods
3A.2.1. Preparation of plant extracts
G. gummifera Linn. f. root were collected, prepared for extraction and
cleaned, chopped, shade-dried and powdered. A 50 g of dried powder was Soxhlet
extracted with 400 mL of various solvents of increasing polarity, i.e., petroleum
ether, chloroform, acetone, ethanol and methanol for 48 h. Then the extracts were
collected and the solvents evaporated under vacuum in a rotary evaporator with an
approximate yield of 0.65%, 2.6%, 4.3%, 6.8% and 10.3% (w/w) respectively. The
steps were repeated with a new set of dried powder and solvents until the required
quantity was achieved.
3A.2.2. Preliminary phytochemical screening
Preliminary phytochemical screening of petroleum ether (PEGG),
chloroform (CHGG), acetone (ACGG), ethanol (ETGG) and methanolic extracts
(MEGG) of G. gummifera Linn. f. roots were carried out for the detection of
phytoconstituents using standard conventional protocols (Khandelwal, 1995; Evans
and Trease, 2002; Kokate et al., 2008). The extracts were subjected for the
phytochemical screening assays of alkaloids, flavonoids, phenolic compounds and
tannins, glycosides, steroids, saponins, fixed oils and fats, carbohydrates, proteins
and amino acids.
(Detailed protocols are given in chapter 2, section 2.2.2. Preliminary
phytochemical screening)
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3A.3. Results
3A.3.1. Preliminary phytochemical screening
The petroleum ether (PEGG), chloroform (CHGG), acetone (ACGG),
ethanol (ETGG) and methanolic extracts (MEGG) of G. gummifera Linn. f. roots
were subjected to preliminary phytochemical screening and the results of various
phytochemical constituents are depicted in table 3.1.
Table 3.1. Phytochemical screening of Gardenia gummifera Linn. f.root.
Constituents Petroleum ether
Chloroform Acetone Ethanol Methanolic extract
Alkaloids - + + + +
Phenolic compounds
- + + + +
Flavonoids -_ + + + +
Tannins - - - - +
Glycosides - - + - +
Terpenoids + - - - +
Steroids + - - - +
Saponins - - - - +
Fixed oils and fats + - - _ -
Carbohydrates - + + + +
Proteins and amino acids - + + + +
`
“+” indicates the presence of constituents
“-” indicates the absence of constituents
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3A.4. Discussion
Plants produce primary and secondary metabolites which encompass a wide
array of functions. Primary metabolites, which include amino acids, simple sugars,
nucleic acids, and lipids are compounds that are necessary for cellular processes.
Secondary metabolites are mainly produced by the plants in response to stress.
Plants can manufacture many different types of secondary metabolites, which have
been subsequently exploited by humans owing to their beneficial role in a diverse
array of applications. Secondary metabolites are mainly responsible for the
medicinal activity of plant species (Zwenger and Basu, 2008). In the present
investigation, a qualitative phytochemical analysis of the various extracts of G.
gummifera Linn. f. namely petroleum ether, chloroform, acetone, ethanol and
methanol was conducted to detect the presence of various components of the
extracts that may be responsible for its medicinal properties. The different extracts
were found to contain flavonoids, alkaloids, tannins, phenolic compounds,
terpenoids, saponins and glycosides that may render the stipulated medicinal effects
of the plant.
Alkaloids are nitrogen-containing compounds widely distributed in different
plant groups. They are reported to possess a wide array of pharmacological
properties, and a large number of them exhibit potent physiological effects on
mammals. For example, morphine shows narcotic effects; atropine is a smooth
muscle relaxant; cocaine is a local anesthetic and a potent central nervous system
stimulant; and strychnine is a nerve stimulant. Notable alkaloids include reserpine,
an antihypertensive alkaloid from Rauwolfia serpentina, and vinblastine, one of the
antitumor alkaloids, from the Catharanthus roseus. Furthermore, the alkaloids from
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Desmodium gangeticum, Erythroxylon coca and Tinospora cordifolia are known for
its cardioprotective activity (Cseke et al., 2006; Arya and Gupta, 2011). In the
current study, the presence of alkaloids identified in all the extracts except in the
petroleum ether extract of the root of G. gummifera indicate that it is a potential
source of natural alkaloids and that may render the pharmacological effects
including the traditionally reported cardio protective properties.
The vast majority of plant-based aromatic natural products are phenols.
Phenols constitute a large class of compounds in which a hydroxyl group (–OH
group) is bound to an aromatic ring. Numerous categories of these compounds exist,
including the simple phenols, phenylpropanoids, flavonoids, tannins, and quinines
(Cseke et al., 2006). Phenolics are produced by plants mainly for their protection
against stress. The potential of phenolic compounds to protect against cardiovascular
disease are summarized by Halliwell and Gutteridge, (2007) in his review.
Flavonoids have been referred to as nature’s biological response modifiers, because
of their inherent ability to modify the body’s reaction to allergies and virus and they
showed their anti-allergic, anti-inflammatory, anti-microbial and anti-cancer
activities (Aiyelaagbe and Osamudeiamen, 2009). Flavonoids ability to scavenge
hydroxyl radicals, superoxide anion radicals and lipid peroxyl radicals highlights
many of the health-promoting functions of flavonoids in organisms which are
important for prevention of diseases associated with oxidative damage of
membranes, proteins and DNA (Abioye et al., 2013). The flavonoids
(leucopelargonin and leucocyanin derivatives and quercetin) isolated from the bark
of Ficus bengalensis Linn. exhibited antiatherogenic effect in cholesterol fed rats
(Daniel et al., 2003). Epidemiological studies suggest that higher flavonoid intake
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from fruits and vegetables are associated with decreased risk for the development of
cardiovascular disease. A large number of studies have reported the impact of
consuming flavonoid-rich foods on biomarkers of cardiovascular disease risk in
healthy volunteers or at-risk individuals. Recent evidence suggests that some
polyphenols in their purified form, including resveratrol, berberine and naringenin,
have beneficial effects on dyslipidemia in humans and/or animal models. In a mouse
model of cardiovascular disease, naringenin treatment, through correction of
dyslipidemia, hyperinsulinemia and obesity, attenuated atherosclerosis (Mulvihill
and Huff, 2010). In addition, Lisa et al. (1999) reported the antiatherogenic
properties of the citrus flavonoid naringenin. Thus, the presence of phenolic
compounds especially the flavonoids identified in the extracts of the root of
G. gummifera indicates that it would be a rich natural source of pharmacologically
active phytochemicals.
Tannins are polyphenolic secondary metabolites of higher plants. In
medicine, especially in Asian (Japanese and Chinese) natural healing, the tannin-
containing plant extracts are used as astringents, against diarrhoea, as diuretics,
against stomach and duodenal tumours, and as anti-inflammatory, antiseptic, and
haemostatic pharmaceuticals. It is also becoming clear that tannins often are the
active principles of plant-based medicines. In extensive biological tests many
representatives of this class were found to have antiviral, antibacterial, and,
especially, antitumor activity. For example, certain tannins can selectively inhibit
HIV replication (Khanbabaee and van Ree, 2001). In addition, Panchal and Brown,
(2013) reported the anti-atherogenic activities of tannins. In the present study, tannin
identified in the methanolic extract of the root of G. gummifera evidently indicate
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that, among the various extracts screened in the present study, MEGG may be the
most promising pharmacologically active extract.
Terpenes are unique group of hydrocarbon-based natural products and are
classified by the number of five-carbon units they contain: Hemiterpenes - C5;
Monoterpenes - C10; Sesquiterpenes - C15; Diterpenes - C20; Sesterterpenes - C25
(rare); Triterpenes - C30 and Carotenoids - C40. Some 30,000 terpenes were
identified thus far. The C30 terpenes are widely distributed among plant resins, cork,
and cutin. There are several important groups of triterpenes, including common
triterpenes, steroids, saponins, sterolins, and cardiac glycosides. Only a few of the
common triterpenes are widely distributed among plants. These include the amyrins
and ursolic and oleanolic acids which are common on the waxy coatings on leaves
and as a protective coating on some fruits (Cseke et al., 2006). In the present study,
among the various extracts screened for the phytochemical constituents, the presence
of steroids and terpenes were observed in PEGG and MEGG. It indicates that the
presence of these pharmacologically active phytochemicals may also contribute to
its traditionally reported medicinal properties.
Plant steroids are known to be important for their cardiotonic activities
(Micallef et al., 2009). They are also used in nutrition, herbal medicine and
cosmetics. Consumption of plant sterol esters reduces plasma LDL cholesterol
concentration by inhibiting intestinal cholesterol absorption (Carr and Jesch, 2006).
In addition, Pietri et al. (1997) reported that Ginkgo biloba extract and its terpenoid
constituents (ginkgolides A and B, bilobalide) possess significant anti-ischemic
effects.
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Saponins are high-molecular-weight triterpene glycosides, containing a sugar
group attached to either a sterol or other triterpene. They are widely distributed in
the plant kingdom (Cseke et al., 2006). Saponins are considered a key ingredient in
traditional medicine and are responsible for many pharmacological effects viz.,
antioxidant, antifungal, anticancer and anti-inflammatory. Furthermore, saponins are
used in hypercholestrolaemia and hyperglycaemia (Abioye et al., 2013; Zhang et al.,
2013). Thus, the presence of saponins observed in the methanolic extract of the root
of G. gummifera can be correlated with its traditional use for the treatment of cardiac
debility, obesity and lipolytic disorders.
The flavanols, phenols, steroids and terpenes contribute directly to
antioxidant action and hence to cardioprotection, anticancer, antidiabetic and
antiatherogenic activities. The presence of these secondary metabolites in the
different extracts supports to the medicinal properties of G. gummifera. In
conclusion, the results obtained in the current study demonstrated that among the
extracts screened for the phytochemical constituents, MEGG possess the most
classes of pharmacologically active phytoconstituents. Hence it was chosen for
further pharmacological evaluations.
Chapter 3(B)
In vitro antioxidant evaluation of
Gardenia gummifera Linn. f. root
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3B.1. Introduction
Reactive oxygen species (ROS) are generated ubiquitously in the human
body from either endogenous or exogenous sources. Excessive generation of ROS
causes oxidative stress, a deleterious process leading to the oxidation of
biomolecules such as proteins, lipids, carbohydrates and DNA. Oxidative stress is
known to play a major role in the development of several chronic ailments such as
cardiovascular diseases, different types of cancer, arthritis, diabetes, autoimmune
and neurodegenerative disorders and aging. Antioxidants play an important role in
inhibiting and scavenging radicals, thus providing protection against infections and
degenerative diseases. Antioxidants can either directly scavenge or prevent
generation of ROS. Recently, interest in finding naturally occurring antioxidants has
increased considerably to replace synthetic antioxidants. Antioxidants are substances
that delay or prevent the oxidation of cellular oxidizable substrates. They exert their
effects by scavenging free radicals, activating a battery of detoxifying proteins, or
preventing the generation of free radicals (Soares et al., 1997; Chen et al., 2009;
Erukainure et al., 2011). Various endogenous antioxidant defense mechanisms play
an important role in the elimination of ROS and lipid peroxides and therefore protect
the cells against its toxic effects (Halliwell et al., 1992). Recently, interest in finding
naturally occurring antioxidants has increased considerably to replace synthetic
antioxidants, which are being restricted due to their side effects (Patel et al., 2011;
Ravikumar and Gnanadesigan, 2011).
Plants are some of the most attractive sources of new drugs and have been
shown to produce promising results in the treatment of a number of disorders.
A great number of in vitro assays have been developed to measure the efficiency of
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natural antioxidants either as pure compounds or as plant extracts. These methods
are popular due to their high speed and sensitivity. However it is essential to use
more than one method to evaluate antioxidant capacity of plant materials because of
the complex nature of phytochemicals (Salazar et al., 2008). With this view the
in vitro antioxidant activity of various extracts of G. gummifera root was conducted.
3B.2. Materials and Methods
3B.2.1. Chemicals
Quercetin was purchased from Sisco Research Laboratories (SRL), Mumbai,
India. Ascorbic acid was obtained from Merck, Mumbai, India. 2, 2-Diphenyl-1-
picrylhydrazyl (DPPH) was purchased from Sigma Chemical Co., St. Louis, MO,
USA. All other chemicals were of analytical grade.
3B.2.2. Preparation of plant extracts
The plant material was collected, prepared for extraction and extraction
performed using different solvents as explained in section 2.2.1. The extracts were
suspended in dimethyl sulphoxide for carring out the in vitro antioxidant studies.
3B.2.3. Evaluation of in vitro antioxidant activity
The in vitro antioxidant activity of the petroleum ether (PEGG), chloroform
(CHGG), acetone (ACGG), ethanol (ETGG) and methanolic extracts (MEGG) of
G. gummifera Linn. f. roots were measured by the following assays.
3B.2.3.1. Determination of total phenolic compounds in the extracts
The amount of total phenolic compounds in the extracts was determined
using the Folin-Ciocalteu method (Yu et al., 2002). A calibration curve of gallic acid
was prepared and the results were expressed as mg GAE (gallic acid equivalents)/g
dry extract.
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3B.2.3.2. Determination of total flavonoid content in the extracts
The total flavonoid content of PEGG, CHGG, ACGG, ETGG and MEGG
were determined spectrophotometrically by the method described by Quettier-Deleu
et al. (2000). It was determined using a standard curve with quercetin and expressed
as milligrams of quercetin equivalents (QE/g of dry extract).
3B.2.3.3. Evaluation of total antioxidant capacity
The total antioxidant capacity of the extracts was determined according to
the method of Jayaprakasha et al. (2004). Ascorbic acid was used as standard and
the total antioxidant capacity was expressed as the equivalent of ascorbic acid per
gram of extract.
3B.2.3.4. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) assay
The antioxidant activity of the extracts was measured in terms of hydrogen
donating or radical scavenging ability using the stable radical DPPH. The reduction
capability of DPPH radicals is determined by the decrease in its absorbance at 517
nm (Aquino et al., 2001). Ascorbic acid was used as standard control.
3B.2.3.5. Assay of hydroxyl radical-scavenging activity
The inhibitory effect of the extracts to prevent the degradation of
deoxyribose by Fe3+ ions in presence of H2O2-EDTA-ascorbate was determined in
hydroxyl radical scavenging assay (Ohkawa et al., 1979). The reference standard
used was quercetin.
3B.2.3.6. Determination of reducing power
The antioxidant activity of the different extracts was also manifested through
their reducing power. In this assay, the Fe3+ → Fe2+ transformation was established
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as reducing capacity (Oyaizu, 1986). Ascorbic acid was used as a standard
antioxidant compound.
All the tests were performed in triplicate and the results were expressed with
the mean values.
(Detailed protocols are described in chapter 2, section 2.2.3. In vitro
antioxidant assays).
3B.2.4. Liquid chromatography-mass spectrometry (LC-MS) Analysis of MEGG
The MEGG was analyzed using LC-MS 2010A instrument (Shimadzu,
Kyoto, Japan). 10 µl of the filtered sample was injected to the manual injector using
a Microsyringe (1-20µl, Shimadzu). The mobile phase used was acetonitrile: 0.1%
OPA in methanol (80:20) in an isocratic mode. The column and pump used were
Reverse Phase C-18 (25 cm X2.5mm) (phenomenex) and SPD 10 AVP-RD
respectively. The separated compounds were then ionized using Atmospheric
pressure chemical ionisation method (APCI). The flow rate was maintained to
2 ml/min with a temperature of 25°C and spectral data were collected at 315 nm.
Mass analysis was performed in the range 50-800 m/z, under both positive and
negative ion mode. The class VP integration software was used for the data analysis.
The constituents of the extract were identified by referring the LCMS library,
Metwin 2009 (version 2.1).
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3B.3. Results
3B.3.1. In vitro antioxidant activity
(A). Phenolic contents, flavonoid contents and total antioxidant activity
The results are summarized in table 3.2. MEGG had a higher quantity of total
phenolics (52.1 ± 3.7mg GAE/g dry extract) than PEGG, CHGG, ACGG and ETGG
(2.7 ± 1.3, 22.2 ± 2.3, 28.7 ± 2.6 and 17.6 ± 2.7 mg GAE/g dry extract). MEGG,
which has a great quantity of flavonoids (39.4 ± 3.4 mg QE/g dry extract) compared
to PEGG, CHGG, ACGG and ETGG (0.64 ± 0.6, 17.8 ± 2.5, 20.4 ± 0.9 and 13.4 ±
0.6 mg QE/g dry extract). The total antioxidant activity of MEGG was 96.8 ± 3.7 mg
ascorbic acid/g dry extract. The total antioxidant activity of PEGG, CHGG, ACGG
and ETGG were 25 ± 3.2, 43.7 ± 3.1, 56.5 ± 1.4 and 32.4 ± 1.6 mg ascorbic acid /g
dry extract.
Table 3.2. In vitro antioxidant activity of the petroleum ether (PEGG), chloroform (CHGG), acetone (ACGG), ethanol (ETGG) and methanolic extracts (MEGG) of G. gummifera Linn. f. root
Gardenia gummifera extracts
Phenolic contents (mg GAE/g dry
extract)
Flavonoid contents (mg
QE/g dry extract)
Total antioxidant activity (mg Ascorbic
acid /g dry extract)
Petroleum ether extract (PEGG)
2.7 ± 1.3 0.64 ± 0.6 25 ± 3.2
Chloroform extract
(CHGG) 22.2 ± 2.3 17.8 ± 2.5 43.7 ± 3.1
Acetone extract
(ACGG) 28.7 ± 2.6 20.4 ± 0.9 56.5 ± 1.4
Ethanol extract
(ETGG) 17.6 ± 2.7 13.4 ± 0.6 32.4 ± 1.6
Methanolic extract (MEGG)
52.1 ± 3.7 39.4 ± 3.4 96.8 ± 3.7
GAE- Gallic acid, QE- Quercetin.
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(B). DPPH radical scavenging activity
The DPPH radical scavenging activity of extracts and standard exhibited a
concentration dependent reaction trend. The IC50 values of ascorbic acid, PEGG,
CHGG, ACGG, ETGG and MEGG were 4.5, 42.4, 24.7, 17.6, 28.7 and 9.3 µg/mL
respectively (Table 3.3).
(C). Hydroxyl radical scavenging activity
Extracts and quercetin, the standard antioxidant, scavenged hydroxyl radicals
in a concentration dependent manner and the estimated IC50 values of quercetin,
PEGG, CHGG, ACGG, ETGG and MEGG were 18.6, 34.7, 26.7, 25.2, 29.3 and
20.8 µg /mL respectively. MEGG has better hydroxyl radical scavenging activity
than PEGG, CHGG, ACGG and ETGG (Table 3.3).
Table 3.3. IC50 value of DPPH radical scavenging activity and hydroxyl radical scavenging activity (µg/mL) of the petroleum ether (PEGG), chloroform (CHGG), acetone (ACGG), ethanol (ETGG) and methanolic extracts (MEGG) of G. gummifera Linn. f. root
Extracts DPPH radical
scavenging activity IC50 value (µg/mL)
Hydroxyl radical scavenging activity IC50 value (µg/mL)
Standard 4.5 (Ascorbic acid) 18.6 (Quercetin)
Petroleum ether (PEGG) 42.4 34.7
Chloroform (CHGG) 24.7 26.7
Acetone (ACGG) 17.6 25.2
Ethanol (ETGG) 28.7 29.3
Methanolic extract (MEGG) 9.3 20.8
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(D). Reducing power
Ascorbic acid used as reference compound exhibited a superior reducing
power at all concentrations, compared with PEGG, CHGG, ACGG, ETGG and
MEGG (Fig. 3.1). At 0.50 mg/mL, the absorbencies of ascorbic acid, PEGG,
CHGG, ACGG, ETGG and MEGG (at 700 nm) were 0.25, 0.066, 0.092, 0.14, 0.08
and 0.18 respectively. These values reflect the following reducing capability:
ascorbic acid > MEGG > ACGG >CHGG > ETGG > PEGG.
Fig. 3.1 Reducing power of G. gummifera root extracts of petroleum ether
(PEGG), chloroform (CHGG), acetone (ACGG), ethanol (ETGG) and methanol (MEGG) compared with standard antioxidant ascorbic acid (Ascorbic acid is diluted 1: 10).
The results obtained in the present study showed that MEGG can effectively
scavenge reactive oxygen species including hydroxyl radical as well as other free
radicals under in vitro conditions. The antioxidant activity of PEGG, CHGG,
ACGG, ETGG and MEGG were compared with that of standard compounds and the
MEGG was found to be a promising antioxidant and hence it was chosen for in vivo
studies.
0
0.05
0.1
0.15
0.2
0.25
0.3
0 0.1 0.2 0.3 0.4 0.5 0.6
Abs
orba
nce
700
nm
Concentration (mg/ml)Ascorbic acid PEGG CHGG ACGG ETGG MEGG
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3B.3.2. LCMS Analysis of MEGG.
The LCMS analysis revealed the chemical composition of the extract and
constituents with potent antioxidant, anti inflammatory and anti hypercholesterolemic
effect (Table.3.5). The major compounds identified in MEGG with the potential to
improve the cardiac health and antioxidant activities includes erythrodiol, lupeol,
epicatechin, β-sitosterol, ASIATIC acid, myricetin, oleanolic aldehyde, vernolic acid,
dicaffeoylquinic acid and chlorogenic acid (Table.3.4).
Table 3.4. List of major antioxidant/cardioprotective compounds identified in MEGG by LC-MS analysis
Sl No Name of the compounds Library sequence No. Molecular Mass
1 Erythrodiol MTW/UM/2.1.1/0089/11 442.72
2 Lupeol MTW/UM/2.1.1/0322/11 426.73
3 Epicatechin MTW/UM/2.1.1/1166/11 578.54
4 β- sitosterol MTW/UM/2.1.1/0009/11 414.71
5 Asiatic acid MTW/UM/2.1.1/0008/11 488.71
6 Myricetin MTW/UM/2.1.1/7474/11 318.25
7 Oleanolic aldehyde MTW/UM/2.1.1/1655/11 440.70
8 Vernolic acid MTW/UM/2.1.1/1578/11 296.45
9 Chlorogenic acid MTW/UM/2.1.1/7816/11 354.32
10 Dicaffeoylquinic acid MTW/UM/2.1.1/1199/11 516.49
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A
B
Fig. 3.2. Mass spectrum of MEGG. (A). Mass spectrum of positive ionization. (B). Mass spectrum of negative ionization.
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Table 3.5. Compounds identified in MEGG by LCMS analysis.
Sl No Compound Name LIB;SQ;NO Molecular Mass
1 Aminobutyric Acid MTW/UM/2.1.1/2221/11 103.12
2 Hydroxyhomoargenine MTW/UM/2.1.1/0553/11 204.23
3 Betuloside MTW/UM/2.1.1/5535/11 328.36
4 Caffeic Acid Glucoside MTW/UM/2.1.1/0297/11 341.31
5 Rosmarinine MTW/UM/2.1.1/0014/11 353.42
6 Lupeol MTW/UM/2.1.1/0322/11 426.73
7 Erythrodiol MTW/UM/2.1.1/0089/11 442.72
8 Sporidesmin MTW/UM/2.1.1/6639/11 473.96
9 Asiatic Acid MTW/UM/2.1.1/0008/11 488.71
10 Dicaffeoylquinic Acid MTW/UM/2.1.1/1199/11 516.49
11 Epicatechin MTW/UM/2.1.1/1166/11 578.54
12 Oleanolic Aldehyde MTW/UM/2.1.1/1655/11 440.70
13 Beta Sitosterol MTW/UM/2.1.1/0009/11 414.71
14 3,4 Dihroxywogonin MTW/UM/2.1.1/5565/11 316.26
15 D-Mannitol MTW/UM/2.1.1/0221/11 182.17
16 Pipecolic Acid MTW/UM/2.1.1/0197/11 129.16
17 Cuminaldehyde MTW/UM/2.1.1/0005/11 148.21
18 Dimethoxy Benzoquinone MTW/UM/2.1.1/1124/11 168.15
19 Alpha Vetivone MTW/UM/2.1.1/0785/11 218.34
20 Dihydro Reservitrol MTW/UM/2.1.1/0898/11 230.27
21 Linoleic Acid MTW/UM/2.1.1/0235/11 280.45
22 Vernolic Acid MTW/UM/2.1.1/1578/11 296.45
23 Myricetin MTW/UM/2.1.1/7474/11 318.25
24 Chlorogenic Acid MTW/UM/2.1.1/7816/11 354.32
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3B.4. Discussion
Free radicals are chemical entities that can exist separately with one or more
unpaired electrons. The propagation of free radical can brings about many adverse
reactions leading to extensive tissue damage. Lipids and proteins are all susceptible
to attack by free radical. Many plant species with antioxidant activities act as
protective agents against these radicals. In the present study, the in vitro antioxidant
activity of petroleum ether (PEGG), chloroform (CHGG), acetone (ACGG), ethanol
(ETGG) and methanolic extracts (MEGG) of G. gummifera Linn. f. root was evident
from DPPH radical scavenging assay, hydroxyl radical scavenging assay and
reducing power assay.
DPPH is a stable, nitrogen-centered free radical that easily accepts an
electron or hydrogen radical to become a stable diamagnetic molecule. DPPH
produces a violet color in methanol solution and when it encounters antioxidants
(proton donors), it gets reduced to a yellow colored product, diphenylpicryl
hydrazine (Soares et al., 1997). Among the five extracts of G. gummifera root,
MEGG showed definite DPPH radical scavenging activity in comparison with
ascorbic acid and thus indicate that it possess potent proton donating ability and
could serve as free radical inhibitor or scavenger.
Hydroxyl radical is highly reactive oxygen centered radical formed from the
reaction of various hydroperoxides with transition metal ions. It attacks proteins,
DNA, polyunsaturated fatty acid in membranes and most biological molecules it
contacts (Jornot et al., 1998) and is known to be capable of abstracting hydrogen
atoms from membrane lipids (Yen and Duh 1994) and brings about peroxidic
reaction of lipids. Hydroxyl radical scavenging capacity of an extract is directly
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related to its antioxidant activity (Shukla et al., 2012). The ability of G. gummifera
root extracts to quench the hydroxyl radicals seems to be directly related to the
prevention of propagation of the process of lipid peroxidation. The potent hydroxyl
radical scavenging activity of the extracts may be due to its active hydrogen
donating ability. In the present study MEGG showed maximum hydroxyl radical
scavenging activity than the other extracts, PEGG, CHGG, ACGG and ETGG.
The total reducing power of MEGG was also comparatively higher than
PEGG, CHGG, ACGG and ETGG. The reducing capacities of the extracts were
investigated by Fe3+ → Fe2+ transformation. Presence of reductones causes the
reduction of the Fe3+/ferricyanide complex to the Fe2+ form. The reducing power of a
compound may serve as a significant indicator of its potential antioxidant activity.
The reducing properties are generally associated with the presence of reductones
which have been shown to exert antioxidant action by breaking the free radical chain
reaction donating a hydrogen atom (Meir et al., 1995; Gulçin et al., 2003).
MEGG had a higher quantity of total phenolics, flavonoids, steroids and
terpenoids when compared to other extracts PEGG, CHGG, ACGG and ETGG.
Phenolic compounds function as high-level antioxidants because they possess the
ability to absorb and neutralize free radicals as well as quench reactive oxygen
species (Rice-Evans et al 1995). Flavonoids, one of the most diverse and widespread
groups of natural compounds, are also probably the most natural phenolics capable
of exhibiting in vitro and in vivo antioxidant activities (Aiyelaagbe et al., 2009).
The LC-MS analysis for the phytochemical profiling of the methanolic
extract of G. gummifera Linn. f. root revealed the presence of ten major phytochemicals
with proven antioxidant / hypolipidemic / antiatherogenic / cardioprotective properties
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viz., erythrodiol, lupeol, epicatechin, β- sitosterol, asiatic acid, myricetin, oleanolic
aldehyde, vernolic acid, dicaffeoylquinic acid and chlorogenic acid. Among these
phytochemicals erythrodiol (Allouche et al., 2010), lupeol (Nagaraj et al., 2000),
epicatechin (Pushp et al., 2013), β- sitosterol (Gupta et al., 2011), asiatic acid
(Pittella et al., 2006), myricetin (Choi et al., 2010), oleanolic aldehyde, (Narender
et al., 2011), vernolic acid (Danish et al., 2011), dicaffeoylquinic acid (Danino et al.,
2009) and chlorogenic acid (Sato et al., 2011) are well-known for its antioxidant
activities.
In addition to the reported antioxidant activities, erythrodiol, epicatechin,
lupeol, asiatic acid, β- sitosterol and myricetin are known for its cardioprotective
effect (de Whalley et al., 1990; Sudhakar et al., 2007; Matsuoka et al., 2008; Zhang
et al., 2009; Allouche et al., 2010; Yamazaki et al., 2010).
Taken together, the result of LC-MS analysis of MEGG indicated that it is a
potential source of phytochemicals with antioxidant and cardioprotective properties.
However, further studies are required to establish its in vivo antioxidant and
cardioprotective activities. In view of this, we have evaluated the cardioprotective
efficacy of MEGG against high fat diet induced atherosclerosis and isoproterenol
induced myocardial infarction in rats and their results are described in the
subsequent chapters.
In conclusion, the results obtained in the current study demonstrated that
MEGG contained higher levels of total phenolic compounds, steroids and terpenes
which are capable of inhibiting, quenching free radicals to terminate the radical
chain reaction, and acting as reducing agents. More precisely, the in vitro
antioxidant activity of MEGG might be attributed to the synergistic effect of the
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identified antioxidant phytochemicals. The biological activities of MEGG should not
be exclusively explained based on the effects of the major compounds because it
may also include the response to other bioactive compounds present in smaller
concentrations. The antioxidant activity of PEGG, CHGG, ACGG, ETGG and
MEGG were compared with that of standard compounds and the MEGG exhibited a
promising antioxidant activity. Hence it was chosen for in vivo studies.