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Research Article CODEN: IJPRNK IMPACT FACTOR: 1.862 ISSN: 2277-8713 Vijayalakshmi K, IJPRBS, 2014; Volume 3(3): 528-538 IJPRBS

Available Online at www.ijprbs.com 528

ANTIOXIDANT PROFILE OF THE FRUIT RIND OF GARCINIA CAMBOGIA AND LEAVES OF BAUHINIA VARIEGATA – AN INVITRO INVESTIGATION

RANJANI R2, KHADIRA SA2, PRIYA N2, VIJAYALAKSHMI K1 1. Associate professor, Department of Biochemistry, Bharathi Women’s College, Chennai, Tamilnadu, India. 2. Research scholar, Department of Biochemistry, Bharathi Women’s College, Chennai, Tamilnadu, India.

Accepted Date: 23/06/2014; Published Date: 27/06/2014

Abstract: This study was carried out to evaluate the free radical scavenging activity of the

ethylacetate extract of fruit rind of Garcinia cambogia and leaves of Bauhinia variegata using

the free radical scavenging assay 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical(OH),

superoxide radical(SO), nitric oxide radical(NO), hydrogen peroxide radical(H2O2) scavenging

activity. The reducing activity (RA) was also estimated. The IC50 value of Garcinia cambogia is

42µg/ml (DPPH), 39.8µg/ml (OH), 40µg/ml (SO), 43.3µg/ml (NO), 40µg/ml (H2O2) and

28.6μg/ml (RA) and Bauhinia variegate is 43μg/ml (DPPH), 41.8μg/ml (OH), 45.1μg/ml (SO),

47μg/ml (NO), 41.3μg/ml (H2O2), and 26.2μg/ml (RA). When comparing with BHT (Butylated

hydroxy toluene), Garcinia cambogia is less efficient in scavenging NO, DPPH, SO and H2O2

whereas it is more efficient in scavenging hydroxyl radical and has high reducing activity.

Bauhinia variegata is less efficient in NO, SO, DPPH, OH radical whereas it is more efficient in

H2O2 and has high reducing activity.

Keywords: Garcinia cambogia, Bauhinia variegata, BHT, DPPH, free radical.

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Corresponding Author: DR. VIJAYALAKSHMI KRISHNAMOORTHY

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Ranjani R, Khadira Sa, Priya N, Vijayalakshmi K; IJPRBS, 2014; Volume 3(3): 528-538



INTRODUCTION

Antioxidants are chemicals produced within the body or absorbed from the diet to neutralize

the effect of free radicals. Free radicals are generated in the human body through aerobic

respiration and exist in different forms including superoxide, hydroxyl, hydroperoxyl and

peroxyl radicals. Generally, natural antioxidant enzymes in healthy individuals remove these

free radicals. Dietary antioxidants are helpful in assisting the body to neutralize free radicals

and consumption of diet rich in antioxidants reduces the harmful effects of oxidative stress. In

addition, natural antioxidants may have an important role in protecting human health.

The fruit rinds of Garcinia cambogia (GC), commonly known as kodampuli have been used in

the Indian systems of the medicine for hundreds of years for their beneficial role and

hypolipidemic effect. GC is a moderate sized, evergreen tree and flowers are unisexual, sessile

and axillary. The leaves are dark green, shiny, elliptic to obovate. The fruit may resemble a small

yellow or reddish pumpkin, or it may have a unique purple color. GC has garnered a lot of

attention of late as a popular natural weight loss aid. The reason is that the rind of this pumpkin

like fruit is rich in a substance called hydroxycitric acid (HCA) [1] shown below.

Structure of hydroxycitric acid

Bauhinia variegata Linn. (BV) (Caesalpiniaceae) is a medium-sized, deciduous tree, found

throughout India. It is commonly known as Kanchnar (Sanskrit), Mountain Ebony (English),

Mandharai (Tamil) and Raktakanchan (Hindi) [2]. The various parts of the plant viz., flower buds,

flowers, stem, stem bark, leaves, seeds and roots are practiced in various indigenous systems of

medicine and popular among the various ethnic groups in India for the cure of variety of

ailments[3][4]. The leaves of other Bauhinia species are reported to have antiophidian [5],

antidiabetic [6], antimalarial [7], antimicrobial [8] and antioxidant potential [9].

The biomechanical mechanism of liver injury includes metabolite or coenzyme effects

(depletion or stimulation), enzyme effects (inhibition or stimulation), activation to a more toxic

form and membrane disturbances. The antioxidant plays an important role in liver protection

by inhibiting the free radical formation.

Production of free radicals cause depletion of antioxidants, so consuming antioxidants may be

necessary as reported by Halliwell [10]. Currently available synthetic antioxidants like BHT, BHA

http://upload.wikimedia.org/wikipedia/commons/7/7a/Hydroxycitric_acid.png



and Gallic acid have a number of negative health effects as reported by Barlon[11] and

Branen[12]. So recently many medicinal plants are studied for their antioxidant potential. Hence,

it was planned to investigate on the antioxidant potential of Garcinia cambogia and Bauhinia

variegata.

MATERIALS AND METHODS

Collection and processing of plant samples

The fruit rinds of Garcinia cambogia and leaves of Bauhinia variegata were collected from the

local area during the month of December. The taxonomic identification of plant material was

done by Prof. P. Jayaraman, National Institute of Herbal Science (PARC), Chennai.

(Authentication No PARC/2013/468 & PARC/2013/1469).

The fruit rinds of Garcinia cambogia and leaves of Bauhinia variegata were washed with water,

shade dried at room temperature and powdered coarsely. Exactly 10g of the course powder of

fruit rinds and leaves were taken in 100ml of various solvents such as ethanol, hydro alcohol

(ethanol: water, 1:1 ratio) aqueous and ethyl acetate. The extracts were refrigerated for 72

hours and filtered through Whatmann No.1filter paper. Preliminary phytochemical screening

was done in all the extract and result has been published [13]. From the results the ethyl acetate

extract of GC and BV was found to be best and taken for the further in-vitro antioxidant study.

In-vitro antioxidant activity

DPPH free radical scavenging Activity

Free radical scavenging activity was determined by the method of Koleva et al. [14]. The ability to

scavenge the stable free radical DPPH is measured by a decrease in the absorbance at 517nm.

To an ethanolic solution of DPPH (0.05mM), an equal volume of extracts dissolved in water was

added to a final volume of 1.0ml. An equal volume of alcohol was added to the control. After

20min, absorbance was recorded at 517nm in a UV visible double beam spectrophotometer.

The scavenging activity of the plant extract was calculated using the equation 1.

Equation 1

Percentage of Inhibition = Absorbance of control – Absorbance of sample

Absorbance of control

Nitric Oxide radical scavenging Activity

Nitric oxide scavenging activity was measured spectrophotometrically by the method of

Govindarajan et al [15]. Sodium nitroprusside (5mM) in PBS (phosphate buffer solution) was

mixed with different concentrations of the extract dissolved in methanol and incubated at 25˙C

for 15 minutes. The samples were reacted with Griess reagent (1% Sulphanilamide, 2%



Phosphoric acid 0.1% napthylethylene diamine dihydrochloride) The absorbance of the

chromophore formed during diazotization of nitrite with sulphanilamide and subsequent

coupling with naphthlethylene diamine hydrochloride was measured at 546nm and compared

with that of standard solutions treated in the same way. The scavenging ability was calculated

using equation 1.

Super Oxide Anion radical scavenging Activity

Superoxide anion scavenging activity was carried out by employing NBT (nitroblue tetrazolium)

reduction assay [16]. A reaction mixture containing 0.4µl of Sodium pyrophosphate, 25µl of

Phenazine methosulphate, 25µl of NBT and 100µl of NADH was mixed with various

concentrations of extracts and incubated for 90seconds at 30˙C. Purpled colored chromogen

formed was measured spectrophotometrically at 560nm. The scavenging activity of the plant

extract was calculated using the equation 1

Hydrogen Peroxide Radical Scavenging Activity:

Hydrogen peroxide radical scavenging activity was assayed by the method of Sinha [17]. The

reaction mixture containing 500µl of phosphate buffer and 400µl of 2mM hydrogen peroxide.

The reaction mixture was incubated at room temperature for 5 minutes. Then 2ml of

dichromate reagent was added and the decrease in color intensity was measured at 570nm.

2ml of dichromate acetic acid reagent alone served as blank whereas the reaction mixture

without the compound served as control. The scavenging activity was calculated using equation

1.

Hydroxyl Radical Scavenging Activity

The scavenging of hydroxyl free radicals was measured by the method of Halliwell 10. The

reaction mixture containing 2.8mM deoxyribose, 0.05 KH2PO4 NaOH buffer at pH 7.4, 0.1mM

Ferric chloride, 0.1mM EDTA, 1mM Hydrogen peroxide, 0.1mM ascorbate with different

concentrations of extract in a final volume of 2ml. The reaction mixture was incubated for

30min at ambient temperature followed by the addition of 2ml of trichloro acetic acid and

thiobarbituric acid. The reaction mixture was kept in a boiling water bath for 30min, cooled and

the absorbance was read at 532nm in a UV-visible double beam spectrophotometer. The

scavenging activity was calculated using equation 1.

Estimation of Reducing Activity

The reducing activity (RAI) of the extract was assessed by the method of Oyaizu [18]. 2ml of

extract was added to potassium ferricyanide (2.5ml, 10g/L) and the mixture was incubated at

50oC for 20min. Trichloro acetic acid (2.5ml, 100g/L) was added to the mixture which was then

centrifuged at 650×g for 10min. 2.5ml of distilled water and 0.5ml of ferric chloride was added.



The absorbance was read at 700nm. Higher absorbance indicated greater reducing capacity

which was calculated using equation 1.

RESULTS:

The antioxidant potential of GC and BV is estimated using the free radical such as DPPH, SO

anion, NO, OH and H2O2 and the reducing capacity of GC and BV was estimated. The IC50 value

of GC and BV is shown in Table 1.

DPPH free radical scavenging activity

DPPH free radical scavenging activity of GC and BV is shown in figure 1. IC50 value for GC was

42µg/ml and for BV it was 43µg/ml. the maximum scavenging activity of GC was found to be

49% and BV was 50%.

Superoxide anion radical scavenging activity

Figure 2 shows the SO anion radical scavenging activity of GC and BV and BHT. IC50 value is

found to be 40µg/ml with GC and 45.1µg/ml for BV. The maximum scavenging activity for GC

and BV are 70% and 60% respectively.

Nitric Oxide radical scavenging activity:

Nitric Oxide radical scavenging activity was measured spectrophotometrically. Figure 3 shows

the % of inhibition and IC50 values. IC50 values of GC were 43.3µg/ml and BV was 47µg/ml.

Hydroxyl radical scavenging activity:

Figure 4 shows the hydroxyl radical scavenging activity of GC and BV. BHT was used as the

standard. IC50 values for GC was 39.8µg/ml and BV was 41.8µg/ml. and the maximum

scavenging activity of GC was 69% and BV was 60%.

Hydrogen Peroxide radical scavenging activity:

Figure 5 shows the hydrogen peroxide radical scavenging activity of GC and BV. BHT was used

as the standard. The IC50 value was 40µg/ml for GC and 41.3µg/ml for BV. The maximum

scavenging activity of GC was 60% and BV was 60%.

Reducing activity:

Figure 6 shows the reducing activity of GC and BV. GC possessed a reducing power of28.6µg/ml

while BV showed 26.2µg/ml respectively. The maximum scavenging activity of GC was 69% and

BV was 60%.

DISCUSSION:

This study was carried out to evaluate the antioxidant potential of Garcinia cambogia and

Bauhinia variegata comparing it with the standard antioxidant, BHT. Antioxidants exert their



mode of action by suppressing the formation of reactive oxygen species either by inhibition of

enzymes or by chelating trace elements. DPPH is widely used to evaluate the free radical

scavenging effect of natural antioxidant and it is reduced to its corresponding hydrazine. Hence

change in colour depends on number of electrons taken up[19]. This assay was used to

determine the ability of Garcinia cambogia and Bauhinia variegata to reduce DPPH radical to its

corresponding hydrazine. Nitric oxide is an important chemical mediator generated by

endothelial cells, macrophages, neurons and it is involved in the regulation of various

physiological processes. Excess concentration of nitric oxide is associated with several

diseases[20]. The ability of extracts to scavenge the nitric oxide in invitro model using sodium

nitroprusside was studied. Both the extracts were capable of scavenging nitric oxide in a dose

dependant manner. Superoxide anion scavenging radical assay is based on the generation of

superoxide anion radical in PMS, NADH system by the oxidation of NADH. The capacity of the

extract to reduce NBT to blue pharmazone was studied with BHT as the standard. The

superoxide anion indirectly initiates lipid oxidation serving as precursor of singlet oxygen and

hydroxyl radicals. Hydroxyl radicals are one among the reactive oxygen species that are

generated in living cells. It is highly reactive form of free radical which is generated from

superoxide anion and hydrogen peroxide in presence of metal ions such as iron or copper which

in turn reacts with various biomolecules such as lipid, protein and DNA leading to cellular

damage[21]. The reducing ability is generally associated with the presence of reductones, which

breaks the free radical chain by donating a hydrogen atom. In our study both the extracts were

capable of scavenging free radicals in a dose dependant manner and it is found that GC was

more potential than BV.

CONCLUSION:

It is concluded from this study that the fruit rinds of Garcinia cambogia and Bauhinia variegata

have antioxidant property. This could justify its claims as antimicrobial, anti-inflammatory,

antiobesity, anticancer, antiulcer, hepatoprotective and hypoglycemic. The activity was found

due to the presence of hydroxycitric acid, polar phenolic compounds flavonoids and tannins.

Garcinia cambogia and Bauhinia variegata can be considered as a model herbal drug for

experimental studies related to free radical induced disorders like cancer, diabetes,

atherosclerosis and many other diseases.

Conflict of interest:

Conflict of interest declared none.



Figure 1: DPPH Free Radical Scavenging Activity

Figure 2: Super oxide anion radical scavenging activity

Figure 3: Nitric oxide radical scavenging activity



Figure 4: Hydroxyl radical scavenging activity

Figure 5: Hydrogen peroxide scavenging activity

Figure 6: Reducing activity



Table 1: IC50 values of GC and BV

Free Radicals Extracts IC50 (µg/ml) Ethyl Acetate Extract

Garcinia cambogia Bauhinia variegata

DPPH 42 ± 1.2 43 ± 2.3

Nitric Oxide 43.3 ± 1.7 47 ± 2.1

Super Oxide 40 ± 2.1 45.1 ± 2.8

Hydrogen Peroxide 40 ± 1.5 41.3 ± 1.9

Hydroxyl radical 39.8 ± 1.3 41.8 ± 2.1

Reducing Activity 28.6 ± 1.6 26.2 ± 1.3

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