Chapter 3

PRELIMINARY PHYTOCHEMICAL SCREENING

AND IN VITRO ANTIOXIDANT EVALUATION

OF GARDENIA GUMMIFERA LINN. F. ROOT

113

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.

134

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

136

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.