Indian Journal of Biochemistry & Biophysics

Vol. 47, April 2010, pp 110-116

Antioxidant activity of acetone extract/fractions of Terminalia

bellerica Roxb. fruit

Sanjay Guleria1*

, A K Tiku1 and Subhash Rana

2

1Division of Biochemistry and Plant Physiology,

Sher-e-Kashmir University of Agricultural Sciences and Technology, Chatha 180 009, Jammu, India 2Herbal Garden and Herbarium Research Institute in Indian System of Medicine,

Joginder Nagar, District Mandi 176120, Himachal Pradesh, India

Received 20 October 2009; revised 04 March 2010

Terminalia bellerica Roxb. (Family: Combretaceae) has been valued in Indian system of medicine for treatment of

wide range of diseases and reported to have antioxidant properties. In the present study, the free radical scavenging activity

and antioxidant potential of acetone extract/fractions of its fruit was investigated using in vitro assays, including scavenging

ability against 2,2′-diphenyl-2-picrylhydrazyl (DPPH), β-carotene bleaching inhibition, reducing power and chelating ability

on Fe2+ ions. The fruit powder was extracted at room temperature with different solvents in the order of increasing and

decreasing polarity to obtain crude acetone extract which was further partitioned with ethyl acetate and water (1:1). It was

found that ethyl acetate fraction was more effective than crude acetone extract in all antioxidant assays, except chelating

power which was highest in water fraction. Maximum antioxidant activities (expressed as EC50 values) observed were

14.56 µg/ml, 27.81 µg/ml and 67.8 µg/ml in DPPH, β-carotene bleaching and reducing power assays, respectively. The

antioxidant potential was compared with known antioxidant (butylated hydroxyl toluene) and correlated with total phenolic

and flavonoid content in crude extract and fractions. Fractions rich in polyphenolic content were more effective than the

crude extract.

Keywords: Antioxidant activity, Flavonoids, Free radical, Phenols, Terminalia bellerica

Reactive oxygen species (ROS) are responsible for

variety of pathological conditions1. Innate defence

system of the human body may not be sufficient for

curing the damage caused by continued oxidative

stress. Thus, there is a need to supply the antioxidants

exogenously to balance their levels in the human

body. Many synthetic antioxidants, such as butylated

hydroxyl toluene (BHT), butylated hydroxyanisole

(BHA) and tertiary butylhydroquinone (TBHQ) are

commonly used for preservation of fats and oily

foods. However, growing scientific evidences have

shown adverse side effects of synthetic antioxidants2.

Therefore, recently there has been an upsurge of

interest in natural products as antioxidants, as

they can inhibit the free radical reactions and protect

the human body from various diseases, such as

cancer, inflammation, rheumatoid arthritis and

atherosclerosis3. They also retard rancidity in foods

caused by lipid oxidation4.

Secondary metabolites from plants, mainly

phenolics having antioxidant properties are currently

estimated to be between 4000 and 60005,6

. A direct

relationship has been reported between the levels of

phenolic compounds and antioxidant potential of

plants7. Phenolic compounds exhibit their protective

action through various mechanisms like preventing

the generation of carcinogens from precursors by

acting as blocking agents8-10

. Fruits, vegetables and

medicinal plants are rich sources of phenolics, such as

flavonoid compounds which are endowed with

antioxidant properties11,12

.

Terminalia bellerica Roxb. (Family: Combretaceae)

is used in the Indian system of medicine for the

treatment of several ailments, such as fever, cough,

diarrhoea, skin diseases and oral thrush. The fruits

contain β-sitosterol, ethylene gallate, galloyl glucose

and gallic, belleric and chebulinic acids13

. Leaves and

fruits show antioxidant activity14

. The plant exhibits

inhibitory effect on human immuno-deficiency virus-1

reverse trascriptase15

. A water soluble fraction from the

defatted fruits has shown hepatoprotective activity

against CCl4-induced hepatotoxicity16

. In this study, we

_________________ 1Corresponding author

E-mail: guleria71@rediffmail.com

Phone: 0191-2450221

GULERIA et al: ANTIOXIDANT ACTIVITY OF TERMINALIA BELLERICA FRUIT

111

have investigated the antioxidant activity of acetone

extract and its derived fractions from the fruits and its

relationship to the presence of phenolic and flavonoid

compounds.

Materials and Methods

Chemicals

2,2′-Diphenyl-2-picrylhydrazyl (DPPH), β-carotene,

linoleic acid, BHT, ferrozine, ferrous chloride and

Folin-Ciocalteu reagent were purchased from HiMedia

Lab. Pvt. Ltd., Mumbai, India. Ferric chloride,

potassium ferricyanide, trichloroacetic acid, sodium

dihydrogen orthophosphate, di-sodium hydrogen

orthophosphate dehydrate and sodium acetate were

purchased from Merck India Ltd. Other chemicals,

namely Na2CO3, NaOH, sodium nitrite, AlCl3 and

solvents were procured from SD Fine-Chem Ltd.,

Mumbai, India and were of analytical grade.

Plant material and preparation of extract and fractions

Terminalia bellerica fruits were collected from

Herbal Garden and Herbarium Research Institute

(HGHRI) in Indian System of Medicine (ISM),

Joginder Nagar, District Mandi, H.P, India during

February, 2008 and identified by Mr S K Sharma,

Botanist. Voucher specimen (no. 406) was deposited

in the herbarium of the institute. The fruits were dried

in shade and crushed to fine powder before processing

further for assaying antioxidant activity. 300 g of

dried and fine powdered fruit material was subjected

to solvent extraction (1500 ml solvent) in increasing

order of polarity, namely hexane, chloroform, ethyl

acetate and acetone and decreasing order of polarity

namely water, methanol and acetone. The process was

repeated twice with each solvent.

The crude acetone extract obtained from decreasing

and increasing order of solvent polarity extractions was

filtered through Whatman No. 1 filter paper and dried

under vacuum using rotary vacuum evaporator. 1 g of

crude extract obtained from decreasing and increasing

order of solvent polarity extractions was then

partitioned with 200 ml of double-distilled water and

ethyl acetate (1:1) which yielded 302 mg and 257 mg

freeze dried (Freeze dryer model: FD5508, Ilshin Lab

Ltd., S. Korea) water fractions (WF) and 418 mg and

355 mg rotary vacuum-dried ethyl acetate fractions

(EAF), respectively. For assaying antioxidant activity,

crude acetone extract/fractions were re-dissolved in

methanol.

Determination of total phenols and flavonoids

Total phenolic content was determined according

to Folin-Ciocalteu method17

. Briefly, 0.5 ml of

extract/fractions solution was mixed with 0.5 ml of 1 N

Folin-Ciocalteu reagent. The mixture was kept for

5 min at room temperature, followed by the addition of

1 ml of 20% Na2CO3. After 10 min of incubation at

room temperature, the absorbance was measured at 730

nm using double beam UV-VIS spectrophotometer.

Gallic acid was used as a standard. The concentration

of phenolic compounds was calculated according to the

following equation obtained from the standard gallic

acid (5 to 50 µg) graph:

Absorbance = 0.0291 gallic acid (µg) - 0.2561

(R2 = 0.9972)

Flavonoid content in the acetone extract/fractions

was determined by a colorimetric method18

. Plant

extract (250 µl) was mixed with 1.25 ml of distilled

water and 75 µl of a 5% NaNO2 solution. After 5 min,

150 µl of 10% AlCl3·H2O solution was added. After

6 min, 500 µl of 1 M NaOH and 275 µl of distilled

water were added to prepare the mixture. The solution

was mixed well and the absorbance was read at 510 nm

using double beam UV-VIS spectrophotometer.

Quercetin was used as a standard. The concentration of

flavonoid compounds was calculated according to the

following equation obtained from the standard

quercetin (20 to 100 µg) graph:

Absorbance = 0.001 quercetin (µg) + 0.0238 (R2 = 0.9965)

DPPH radical scavenging assay

In this assay, free radical scavenging activity of

crude extract/fractions was determined by measuring

the bleaching of purple-coloured methanol solution of

DPPH. The radical scavenging activity was determined

as described elsewhere19

. One millilitre from a 0.5 mM

methanol solution of the DPPH radical was mixed to

2.0 ml of different concentrations of acetone

extract/fractions and was added 2.0 ml of 0.1 M

sodium acetate buffer (pH 5.5). The mixtures were well

shaken and kept at room temperature in the dark for 30

min. The absorbance was measured at 517 nm using a

double beam UV-VIS spectrophotometer. BHT was

used as positive control, whereas methanol was used as

negative one. The radical scavenging activity (RSA)

was calculated as a percentage of DPPH discolouration

using the equation:

% RSA = [(A0 – As)/Ao] × 100

where A0 and As are the absorbance of the control

(containing all reagents, except the test compound)

INDIAN J. BIOCHEM. BIOPHYS., VOL. 47, APRIL 2010

112

and test compound respectively. The extract/fraction

concentration providing 50% of radical-scavenging

activity (EC50) was calculated from the graph of RSA

percentage against extract/fraction concentration.

β-Carotene bleaching inhibition assay

The antioxidant activity of crude extract/fractions

was evaluated using β-carotene-linoleic acid model

system20

. β-Carotene (0.5 mg) in 1 ml of chloroform

was added to 25 µl of linoleic acid, and 200 mg of

Tween-40 emulsifier mixture. Chloroform was

evaporated at 40°C by a rotary vacuum evaporator.

Then, 100 ml of distilled water saturated with oxygen

were slowly added to the residue and the solution was

vigorously agitated to form a stable emulsion. The

4000 µl of this mixture were transferred into test tubes

containing 0.2 ml portion of the extract/fractions

prepared in methanol at different concentrations. As

soon as the emulsion was added to each tube, zero

time absorbance was measured at 470 nm using a

spectrophotometer. The emulsion system was

incubated for 120 min at 50°C. A blank devoid of

β-carotene was used for background subtraction.

Antioxidant activity was calculated as percent of

inhibition (I%) relative to the control using the

following equation:

I% = [1- (As(0) – As(120) )/Ac(0) – Ac(120) )] × 100

where As(0) the initial absorbance of the sample at

0 min, As(120) the absorbance of the sample at 120 min,

Ac(0) the initial absorbance of the negative control at

0 min, and Ac(120) the absorbance of the negative

control at 120 min. The extract/fraction concentration

providing 50% antioxidant activity (EC50) was

calculated from the graph of antioxidant activity

percentage against extract/fraction concentration.

BHT was used as standard.

Reducing power assay

The reducing power of crude extract/fractions was

determined using the method as described previously21

.

Different concentrations of extracts were mixed with

2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml

of potassium ferricyanide [K3Fe(CN)6] (1%). The

mixture was incubated at 50°C for 20 min. Aliquots

(2.5 ml) of 10% trichloroacetic acid were added to the

mixture. The above mixture was then centrifuged at

1036 x g for 10 min. The upper layer of the solution

(2.5 ml) was mixed with 2.5 ml of distilled water and

2.5 ml of 1% ferric chloride solution. The absorbance

was measured at 700 nm in a double beam UV-VIS

spectrophotometer. Increased absorbance of the

reaction mixture indicated increased reducing power.

The extract concentration providing 0.5 of absorbance

(EC50) was calculated from the graph of absorbance at

700 nm against extract/fraction concentration and

compared with those of standard antioxidant (BHT).

Chelating power on ferrous (Fe2+) ions

The chelating effect on Fe2+

ions from acetone

extract and fractions was estimated according to

method described elsewhere22

. Briefly, 200 µl of

different concentrations of extract/fractions and 740

µl of methanol were added to 20 µl of 2 mM FeCl2.

The reaction was initiated by the addition of 40 µl of

5 mM ferrozine into the mixture, which was then left

at room temperature for 10 min before determining

the absorbance of mixture at 562 nm. The ratio of

inhibition of ferrozine-Fe2+

complex formation was

calculated using the equation:

% Inhibition = [(Absorbance of control – Absorbance

of test sample)/Absorbance of control)] × 100.

Quercetin was used as positive control.

Statistical analysis

For all the experiments, three samples were

analyzed and all the assays were carried out in

triplicate. The results were expressed as mean ±

standard deviation (SD).

Results and Discussion The DPPH radical has been widely used to test the

potential of compounds as free radical scavengers of

hydrogen donors and to investigate the antioxidant

activity of plant extracts23

. In the present study, the

acetone extract/fractions of T. bellerica fruit showed

DPPH radical scavenging activity. As the

concentration of extract/fractions increased, the

DPPH radical scavenging activity also increased in

both increasing and decreasing orders of solvent

polarity (Fig. 1a, b). The order of effectiveness (EC50)

of crude extract and fractions was: Ethyl acetate

fraction (16.92 µg/ml) > crude extract (18.97 µg/ml) >

water fraction (25.39 µg/ml) for increasing order of

solvent polarity and ethyl acetate fraction (14.56

µg/ml) > crude extract (17.37 µg/ml) > water fraction

(19.27 µg/ml) for decreasing order of solvent polarity.

The above results showed that extract/fractions

prepared in decreasing order of solvent polarity were

better scavengers of DPPH free radical than those

GULERIA et al: ANTIOXIDANT ACTIVITY OF TERMINALIA BELLERICA FRUIT

113

prepared in reverse order. A possible explanation of the

free radical scavenging activity is the neutralization of

DPPH free radical by the antioxidant components of

crude extract/fractions, either by transfer of hydrogen

or of an electron24

.

The bleaching inhibition was measured by the

peroxidation of β-carotene (Fig. 1c, d). Antioxidants

can reduce the extent of β-carotene destruction by

neutralizing the linoleate-free radical and other free

radicals formed in the system3. Accordingly, the

absorbance decreased rapidly in reaction mixtures

without extract/fractions, whereas in the presence of

extract/fractions the reaction mixtures retained their

colour and thus absorbance for a longer time. The

efficacy (EC50) of crude extract/fractions in inhibiting

the bleaching of β-carotene was in the order: ethyl

acetate fraction (37.13 µg/ml) > crude extract (55.27

µg/ml) > water fraction (70.82 µg/ml) and ethyl

acetate fraction (27.81 µg/ml) > crude extract (39.80

µg/ml) > water fraction (56.38 µg/ml) for increasing

and decreasing order of solvent polarity respectively.

Bleaching inhibition in the presence of crude

extract/fractions increased with increase in

concentration. It is probable that the presence of

different antioxidant molecules in the crude

extract/fractions might be responsible for inhibition of

β-carotene destruction by neutralizing the effect of

linoleate-free radical and other free radicals formed in

the system25

.

Results obtained in reducing power assay are

shown in Figs 2a, b. Ethyl acetate fraction had higher

reducing power potential (67.8 µg/ml, 70.4 µg/ml)

than the crude extract (73.1 µg/ml, 73.4 µg/ml) or

water fraction (83.7 µg/ml, 85.4 µg/ml) in increasing

and decreasing order of solvent polarities,

respectively. Antioxidant activity has been reported to

be related to reducing power by some

investigators26,27

. Antioxidant action of reductones has

been shown to be based on breaking the radical chain

by donation of hydrogen atom28

. Therefore,

polyphenolic constituents of the crude extract /

fractions appear to function as good electron and

hydrogen atom donors and should be able to convert

free radicals to stable products by terminating radical

chain reaction. Significant correlation (R2

= 0.9044)

was observed between EC50 values obtained from free

radical scavenging and reducing power assays. This

result was in agreement with previous report that

reducing power of peanut hull extract, increased with

increase in concentration and correlated (R2 = 0.9793)

well with the extent of antioxidant activity29

.

Similarly, antioxidant activities of mung bean hull

and burdock extracts were found to be concomitant

with the development of reducing power30

.

Figure 2c, d depicts the effect of extract/fractions

in the chelating power assay. The water fraction

(74.88%) showed higher chelating potential than the

Fig. 1—DPPH radical scavenging and β-carotene bleaching inhibition potential of acetone extract/fractions of T. bellerica fruit by DPPH

(a, b) and β-carotene bleaching assay (c, d), respectively [Values are mean of three replicates ± SD]

INDIAN J. BIOCHEM. BIOPHYS., VOL. 47, APRIL 2010

114

crude extract (50.08%) or the ethyl acetate fraction

(46.88%) obtained by increasing order of solvent

polarity. Similarly, with the extract/fractions of

decreasing order of solvent polarity, maximum

chelating power was exhibited by water fraction

(80.90%) as compared to crude extract (59.04%) and

ethyl acetate fraction (50.88%) at 500 µg/ml

concentration.

Chelation/deactivation of transition metals which

possess the ability to catalyze H2O2 decomposition is

an important mechanism of antioxidant activity31

.

From the Fe2+

data, it was evident that the acetone

extract and fractions possessed Fe2+

ions chelating

activity and might play a protective role against

oxidative damage by sequestering Fe2+

ions which

might otherwise participate in metal catalyzed H2O2

decomposition reactions32

. The higher chelating

power of water fraction as compared to acetone

extract and ethyl acetate fraction might be due to the

presence of higher concentration of certain phenolic

compounds having properly oriented functional

groups that can chelate metal ions. This study was in

conformity with the observation that binding of iron

to phenolic antioxidants can reduce the interaction of

iron with oxygen molecules by changing the redox

potential, thus converting Fe2+

ion to Fe3+

and thereby

retarding oxidative damage33

.

In order to determine the antioxidant compounds in

extract/fractions, the total phenolic and flavonoid

content in crude extract and derived fractions were

determined. As shown in Fig. 3, total phenolic content

in ethyl acetate fraction (655 mg of GAE/g and 634

mg of GAE/g) was highest, followed by acetone

extract (600 mg of GAE/g and 570 mg of GAE/g) and

water fraction (565 mg of GAE/g and 470 mg of

GAE/g) in decreasing and increasing order of solvent

polarities, respectively. Flavonoid content (Fig. 3)

was in the order: ethyl acetate fraction (278 mg of

QE/g) > crude extract (210 mg of QE/g) > water

fraction (160 mg of QE/g) for increasing order of

solvent polarity and acetone extract (204 mg of QE/g)

> ethyl acetate fraction (188 mg of QE/g) > water

fraction (174 mg of QE/g) for decreasing order of

solvent polarity.

The free radical scavenging activity of crude

extract and fractions may be atributed to the presence

Fig. 2—Reducing power and chelating power potential of acetone extract/fractions of T. bellerica fruit by reducing power (a, b) and

chelating power assay (c, d), respectively [Values are mean of three replicates ± SD]

Fig. 3—Phenolic and flavonoid content of acetone

extract/fractions of T. bellerica fruit

GULERIA et al: ANTIOXIDANT ACTIVITY OF TERMINALIA BELLERICA FRUIT

115

of phenolic compounds, as these compounds exhibit

important mechanism of antioxidant activity34

. It has

been reported that fruits contain antioxidant nutrients,

in addition to vitamins which contribute to their

antioxidant potential35

. Antioxidant activity of the

phenolic compounds is probably due to their redox

properties, which allow them to act as reducing

agents, singlet oxygen quenchers, metal ion chelators

and hydrogen donors17,36

.

The higher phenolic content and antioxidant

activity exhibited by the ethyl acetate fraction

suggested that this fraction might serve as a source of

dietary phenolic compounds which might help in

disease prevention and health promotion through

improved nutrition. The results demonstrated that

DPPH free-radical scavenging, β-carotene bleaching

antioxidant effect and reducing power of acetone

extract and derived fractions correlated closely

(R2 value of 0.9727, 0.9369 and 0.9653, respectively)

with their phenolic content. Further, the extract/or

fractions with higher polyphenol contents showed

lower EC50 values in antioxidant assays, confirming

that polyphenolics are likely to contribute to the

antioxidant activity of these extract/fractions, as has

been reported in other studies37

. Flavonoid content

was also correlated with EC50 values obtained from

DPPH free-radical scavenging, β-carotene bleaching

antioxidant effect and reducing power assays, but

with less coefficient of correlation (R2 value of

0.2434, 0.2704 and 0.3118, respectively). This

suggested that the principal antioxidant molecules in

crude extract/fractions of T. bellerica fruit are non-

flavonoid polyphenolic compounds.

In conclusion, the results of the present study

indicated the presence of compounds possessing

significant antioxidant activity in acetone

extract/fractions of T. bellerica fruits. The differences

in antioxidant activity of water, ethyl acetate fractions

and crude extract could be attributed to the difference

in their phenolic content. However, further

investigation of these extract/fractions is required to

isolate and elucidate the structures of active principles

responsible for the antioxidant activity.

Acknowledgement

The authors are grateful to the Department of

Science and Technology (DST), Ministry of Science

and Technology, Government of India, New Delhi for

financial support (Grant no. SR/SO/PS-34/07). The

authors also thank Mr. S K Sharma, Botanist, Herbal

Garden and Herbarium Research Institute in ISM,

Joginder Nagar, District Mandi, H.P., India for the

collection and identification of plant material.

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