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Radical scavenging potential of Terminalia arjuna bark.


Free radicals have been implicated in many diseases such as cancer, atherosclerosis, diabetes, neurodegenerative disorders and aging [1]. The value of nutraceuticals in food has long been recognized for their health benefits [2]. Green tea [3], red wine [4] and ginseng [5] are known to have beneficial effects on the prevention or progression of diseases related to oxidative stress on account of their high antioxidant activity. It is believed that higher intake of antioxidant rich food is associated with decreased risk of degenerative diseases particularly cardiovascular diseases and cancer [6]. Vegetables contain several antioxidant nutrients in addition to vitamin C, E and carotenoids which contribute to their total antioxidant capacity [7]. Several studies have shown that plant derived antioxidant nutraceuticals scavenge free radicals and modulate oxidative stress-related degenerative effects [8]. Nutraceuticals are becoming widely incorporated in functional food owing to their therapeutic effects in enhancing the well-being. There is a great deal of interest in newer natural bioactive molecules with health promoting potential.

The arjun tree Terminalia arjuna (Roxb.) (family: Combretaceae) is a well-known medicinal plant, wherein the bark is extensively used in ayurvedic medicine, particularly as cardiac tonic [9]. The bark is also prescribed in biliousness, sores, as an antidote to poison, it is believed to have an ability to cure hepatic, congenital, venereal viral diseases and is protective against gastric ulcers as a juice for its alleged health promoting properties [10]. However, the bark of T. arjuna have not been investigated for their health promoting potential. Earlier studies have shown that the bark contain aldelydes, saponins, arjunic acid, arjungenin and arjunetin [11]. This paper reports the antioxidant potential of the aqueous and methanolic extracts of the T. arjuna bark employing various in vitro assay systems, such as DPPH/hydroxyl radical scavenging, inhibition of lipid peroxidation (LPO), reducing power and metal chelating activity.

Materials and Methods


Butylated hydroxyl anisole (BHA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), thiobarbituric acid (TBA), bovine serum albumin (BSA) and ethylenediamine tetraacetic acid (EDTA) were purchased from M/s Sigma Chemicals Co. (St. Louis, MO). Trichloroacetic acid (TCA), deoxyribose, ascorbic acid and other chemicals were purchased from M/s Sisco Research Laboratories, Mumbai, India. All the reagents were analytical grade.

Preparation of bark powder and extraction

Bark (5 kg) of T. arjuna were purchased from the local suppliers. They were washed with water and then crushed with a roller to separate the inner woody core and the outer fleshy layer. The fleshy portion was collected, dried at 40[degrees]C in a hot air oven and then finely powdered. The powder (1.9 kg) was used for extraction. Sequential extraction of the bark powder was done with different solvents with increasing polarity i.e., hexane, chloroform, ethyl acetate, acetone, methanol and water. A total of 50 g of bark powder was extracted in 0.5 L of the solvent in glass conical flask on a shaker for 24h at room temperature. The extract was filtered with Whatman paper no. I and dried by flash evaporation/lyophilization.

The methanolic extract was prepared by soxhlet extraction in methanol. A 75 g of bark powder was extracted (1 L) 50[degrees]C for 24h. Methanol was flash evaporated and the extract weighed (9.95 g). The aqueous extract was prepared by homogenizing the bark powder (75 g) in 1 L of warm water (50[degrees]C) and allowed to stand for 24h, filtered with Whatman paper no. I and the filtrate was lyophilized and weighed (7.36 g).

DPPH radical scavenging assay

DPPH radical scavenging activity was done according to [12]. Briefly, 1 ml of DPPH solution (0.1 mmol/1, in 95% ethanol (v/v) was incubated with different concentrations of the extract. The reaction mixture was shaken and incubated for 20 min at room temperature and the absorbance was read at 517 nm against a blank. The radical scavenging activity was measured as a decrease in the absorbance of DPPH and calculated using the following equation:

Scavenging effect (%) = [ 1 - [A.sub.sample(517nm)]/[A.sub.control(517nm)]] X 100.

Hydroxyl radical scavenging assay

The reaction containing different concentrations of the extract was incubated with deoxyribose (10mmol/1), [H.sub.2][O.sub.2] (10mmol/1), Fe[Cl.sub.3] (5 mmol/1), EDTA (1 mmol/1) and ascorbic acid (5 mmol/1) in potassium phosphate buffer (50 mmol/1, pH 7.4) for 60 min at 37[degrees]C [13]. The reaction was terminated by adding TCA (5g/100m1 water) followed by the addition of TBA (0.2 g/100 ml water) and boiled in water bath for 15 min. The absorbance of the color was measured at 535 nm against the reagent blank and the inhibition of the oxidation of deoxyribose was calculated with respect to the control.

Lipid peroxidation assay

Lipid peroxidation inhibitory activity of T. arjuna was measured according to [14]. Egg lecithin (3 mg [ml.sup.-1] in phosphate buffer, pH 7.4) was sonicated (Hielscher GmbH UP 50H ultra-challprozessor sonicator) for 30 min to obtain small membrane liposome vesicles. Different concentrations of T. arjuna were added to 0.5 ml of liposome mixture. Lipid peroxidation was induced by adding 10 x.11 of 400 MM Fe[Cl.sub.3] and 10 [micro]l of 200mM L-ascorbic acid. After 60 min reaction at 37[degrees]C, the reaction was stopped by the addition of 1 ml 0.25N HCl containing 15% TCA and 0.375% TBA and incubation in a boiling water bath for 15 min. The absorbance of the supernatant was measured at 532 nm after centrifugation at 10,000 rpm. BHA was used as a positive control.

Measurement of reducing power

The reducing power of the extracts was measured by incubating the reaction mixture (1 ml) containing the extract in phosphate buffer (0.2 mol/1, pH 6.6) with potassium ferricyanide (1 g/100 ml water) at 50[degrees]C for 20 min. The reaction was terminated by adding TCA solution (10 g/100 ml water), centrifuged at 3000 rpm for 10 min and the supernatant was mixed with ferric chloride (0.1 g/100 ml water), the absorbance measured at 700 nm [15]. Increased absorbance of the reaction mixture indicated increased reducing power.

Metal ion chelating assay

The [Fe.sup.2+]-chelating ability of the extract was measured by the ferrous iron-ferrozine complex at 562 nm [16]. The reaction mixture containing Fe[Cl.sub.2] (2 mmol/1) and ferrozine (5 mmol/1) along with extracts was adjusted to a total volume of 0.8 ml with methanol, mixed and incubated for 10 min at room temperature. The absorbance of the mixture was read at 562 nm against a blank. EDTA was used as positive control. The ability of the extract to chelate ferrous ion was calculated using the equation described for DPPH.

Total phenolic content

Total phenolic content was estimated by Folin-Ciocalteau method [17]. To 6 ml double distilled water, a 0.1 ml sample and 0.5 ml Folin-Ciocalteau reagent was mixed followed by the addition of 1.5 ml [Na.sub.2]C[O.sub.3] (20 g/100 ml water) and the volume was made up to 10 ml with distilled water. After incubation for 30 min at 25[degrees]C, the absorbance was measured at 760 nm and the phenolic content was calculated with a guaicol standard as expressed as guaicol equivalents.

Statistical analysis

All experiments were carried out in triplicates and repeated in three independent sets of experiments. Data were shown as mean [+ or -] standard error (SE). SPSS 10.0.5 version for windows (SPSS software Inc., USA) computer programme was used for statistical analysis. The significance of the study was assessed by one way ANOVA, followed by Post hoc comparison test. Correlations between quantitative properties were evaluated by calculating the Duncan and Dunnett's coefficient. Statistical significance value set at <0.05.

Results and Discussion

Antioxidant activity of the extracts

Antioxidant activity of the sequential extracts is presented in Table I. Among the extracts, maximum antioxidant activity was shown by the methanolic and aqueous extracts and therefore, they were chosen for the further study. The results indicate the choice of the solvent for obtaining the extract with high antioxidant activity.

DPPH radical scavenging activity

A high radical scavenging activity was observed in both the aqueous and methanolic extracts in a concentration dependent manner. The methanolic extract was slightly more active than the aqueous extract ([IC.sub.50] = 0.1 and 0.12 mg/ml respectively) (Table II and Fig. I). Proton-radical scavenging action is an important attribute of antioxidants, which is measured by DPPH radical scavenging assay. DPPH, a protonated radical has characteristic absorbance maxima at 517 nm which decreases with the scavenging of the proton radical [12]. Hydrogen-donating ability of the antioxidant molecule contributes to its free radical scavenging nature [18].


Hydroxyl radical scavenging activity

Both aqueous and methanolic extracts of T arjuna displayed hydroxyl radical scavenging activity. [IC.sub.50] values for the methanolic and aqueous extract were 1.22 and 1.85 mg/ml, respectively (Table II and Fig. II). The hydroxyl radical is an extremely reactive free radical formed in biological systems and has been implicated as a highly damaging species in free radical pathology, capable of damaging biomolecules of the living cells [19, 20]. Hydroxyl radical has the capacity to cause DNA strand breakage, which contributes to carcinogenesis, mutagenesis and cytotoxicity [21]. In addition, this radical species is considered as one of the quick initiators of the LPO process, abstracting hydrogen atoms from unsaturated fatty acids [22].


Lipid peroxidation assay

As shown in Fig. III, both aqueous and methanolic extracts were potent in inhibiting LPO of small membrane liposome vesicles. [IC.sub.50] values for the aqueous and methanolic extract were 0.49 and 0.29 mg/ml, respectively (Table II and Fig. III). LPO has been broadly defined as the oxidative deterioration of polyunsaturated lipids [22]. Initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid is due to abstraction of a hydrogen atom from the double bond in the fatty acid. The free radical tends to stabilize by a molecular rearrangement to produce a conjugated diene, which then readily reacts with oxygen molecule to give a peroxy radical [23]. Peroxy radicals can abstract a hydrogen atom from another molecule to give lipid hydroperoxide, R-OOH. A probable alternative fate of peroxy radicals is to form cyclic peroxides; these cyclic peroxides, lipid peroxides and cyclic endoperoxides fragment to aldehydes such as malondialdehyde (MDA) and polymerization products. MDA and 4- hydroxy nonenal are the major break down products of LPO. MDA is usually taken as a marker of LPO and oxidative stress [24].


Reducing power

The reducing power of T.arjuna extracts was concentration dependent (Table II and Fig. IV). Methanolic extract was being slightly more active than the aqueous extract. It is believed that antioxidant activity and reducing power are related [25, 26]. Reductones inhibit LPO by donating a hydrogen atom and thereby terminating the free radical chain reaction [15].


Metal ion chelating activity

The ferrous ion-chelating effect was shown by both of methanolic and aqueous extracts of T arjuna with [IC.sub.50] values of 11 and 13.25 mg/ml, respectively (Table II and Fig. V). Iron is known to generate free radicals through the Fenton and Haber-Weiss reaction [27]. Metal ion chelating activity of an antioxidant molecule prevents oxyradical generation and the consequent oxidative damage. Metal ion chelating capacity plays a significant role in antioxidant mechanism since it reduces the concentration of the catalysing transition metal in LPO [26]. It is reported that chelating agents, which form 6-bonds with a metal, are effective as secondary antioxidants since they reduce the redox potential thereby stabilizing the oxidized form of the metal ion [19].


Total phenolic content

Phenolics content in the methanolic extract of T. arjuna was higher than that of the aqueous extract (5.98 [+ or -] 3.4 and 5.04 [+ or -] 2.3 mg guaicol equivalents/g respectively). Antioxidant activity of the plant extract is often associated with the phenolic compounds present in them. Hydrogen donating property of the polyphenolic compounds is responsible for the inhibition of free radical induced LPO [28]. In our study, there seemed to be correlation between the phenolic content and antioxidant activity of the extracts since methanolic extract with higher phenolic content showed higher antioxidant activity. However, it is known that phenolic antioxidants could also contribute to the antioxidant activity of an extract [29].


In order to characterize antioxidant activity of a plant extract, it is desirable to subject it to a battery of tests that evaluates the range of activities such as scavenging of the reactive oxygen species and metal ion chelation. Antioxidant-rich plant extracts serve as sources of nutraceuticals that alleviate the oxidative stress and therefore prevent or slow down the degenerative diseases [3, 5, 6, 30]. This study is the first to report the antioxidant activity of the bark of T arjuna which may be associated with their alleged health benefits. The results showed that metal ions and inhibiting oxidation of lipids by breaking the chain reaction due to [Fe.sup.+3]. Atherosclerosis is characterized by the accumulation of cholesterol, lipid peroxides and oxysterols in the arterial wall and it is main cause of heart attack and stroke [31]. The broad range of antioxidant activity of the extracts indicates the potential of the bark as a source of natural antioxidants or nutraceuticals with potential application to reduce oxidative stress with consequent health benefits. Our efforts are underway to isolate and identify the antioxidant molecules in the bark of T arjuna and study their health promoting potential and mammalian safety. Since, Dual activity of methanolic extract to inhibit lipid peroxidation and scavenge free radical showed from T arjuna for application to prevent atherosclerosis.


The authors wish to thank Dr. Mahesh. B, DOS in Biotechnology and Mr. Farhan Zameer, ICMR-Fellow, DOS in Microbiology for their kind support in this study.


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Meghashri Shridhar and Shubha Gopal * University of Mysore, Dept. of Studies in Microbiology, Mysore, India * E-mail:
Table I: Antioxidant activity (a) of the sequential extracts of T.

                Extract     DPPH radical         Hydroxyl radical
Solvent         yield (g)   Scavenging  (%)      scavenging (%)

Hexane          2.78          3.4 [+ or -] 0.6   0
Chloroform      3.76        35.08 [+ or -] 2.8   0
Ethyl acetate   1           46.41 [+ or -] 3.4   13.7 [+ or -]2.4
Acetone         1.19        56.47 [+ or -] 4.6   26.2 [+ or -]3.4
Methanol        9.95        74.54 [+ or -] 4.9   86.2 [+ or -]4.8
Water           7.36        67.77 [+ or -] 4.6   49.9 [+ or -]4.6

                LPO (%
Solvent         inhibition)

Hexane          43.3 [+ or -] 2.8
Chloroform      64.7 [+ or -] 3.6
Ethyl acetate   71.4 [+ or -] 4.2
Acetone         76.5 [+ or -] 5.1
Methanol        68.8 [+ or -] 3.9
Water             60 [+ or -] 3.3

(a) Antioxidant activity was assayed at a concentration of 1 mg/ml
for all the extracts.

Table II: Antioxidant activity of the extracts of T. arjuna bark

Extracts             I[C.sub.50] (mg/ml)

                     Hydroxyl          Metal
             DPPH    radical    LPO    chelation

Methanolic   0.1     1.22       0.29   11
Aqueous      0.12    1.85       0.49   13.25
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Author:Shridhar, Meghashri; Gopal, Shubha
Publication:International Journal of Biotechnology & Biochemistry
Article Type:Report
Geographic Code:9INDI
Date:Sep 1, 2009
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