1. INTRODUCTION:

Terminalia arjuna is an ancient promising medicinal plant of India. The bark, leaf and fruit of T. arjuna are indigenously used to treat many diseases.¹ The bark powder of this plant is extensively used as anti-ischemic.² Sushruta, father of Indian medicine in his compendium Sushruta Samhita referred to the various parts of the tree being used in the treatment of wound infections,³ which was later scientifically validated.⁴ Similarly the alcoholic extract of T. arjuna bark also acts as an antioxidant.^{5, 6} In humans, natural antioxidants play a vital role in lowering the risk of cardiovascular disease and cancer.^{7, 8} Natural antioxidants are mainly the phenolics and flavonoids,⁹ which are present in various parts of the tree. In case of T. arjuna, phenolics and flavonoids are present in stem bark.

In the present work, extracts from bark and core wood of T. arjuna were used for investigating their antioxidant property using reducing power, DPPH scavenging activity, beta-carotene bleaching and hydrogen peroxide methods. This is the first report to compare the antioxidant property of bark and core wood of T. arjuna.

2. MATERIALS AND METHODS:

2.1 Collection and Processing:

The bark and core wood (CW) of T. arjuna were collected from Tirunelveli Dt. Tamil Nadu, India in May 2009 and authenticated by Dr. V. Chelladurai, Research Officer (Retired) C.C.R.A.S., Government of India. The bark and core wood were shade dried and ground (bark – pulverized and core wood – coarse).

2.2 Extraction:

Based on the polarity, powdered material (100g) both bark and core wood were successively extracted with Hexane (250ml), Ethyl Acetate (250ml), Methanol (250ml) and Water (250ml) using Soxhlet apparatus. Each extraction was carried out for 12hrs. The extracts thus obtained were dried and stored.

2.3 Reagents and Chemicals:

Linoleic acid, Gallic Acid, DPPH, β carotene, organic solvents and all other chemicals were purchased from M/S Sigma Aldrich, USA.

All the extracts were subjected to antioxidant activity assays using Reducing Power, DPPH, β-carotene and hydroxyl radical scavenging methods. Ascorbic acid, Butylated Hydroxy Toluene (BHT) and Catechin were used as the reference standards. IC₅₀ values of the extracts for DPPH and Hydroxy radical scavenging method were determined using Mat-lab software.

2.4 Quantification of Total Phenolics:

Samples were dissolved in DMSO at concentration of 1mg/ml. To 500µl of extract, 500µl of 50% Folin ciocalteau reagent V/V and 1ml of 20% sodium carbonate w/v were added. The volume was made up to 3.5ml with distilled water. The mixture was incubated at room temperature for 20 minutes. Absorbance was measured at 720 nm using Gallic acid as a standard and quantity of phenolics was calculated and expressed in Gallic acid equivalence.¹⁰

2.5 Quantification of Total Flavonoids:

To 100 µl of sample in DMSO at 1mg/ml concentration, 3.7ml of 80% of ethanol, 0.1 ml of 1M potassium acetate and 0.1ml of 10% aluminum nitrate were added. The mixture was made up to 4ml using distilled water and kept for incubation at room temperature for 40min. Absorbance was measured at 415nm using Quercetin as standard and quantity of the flavonoids was calculated and expressed in Quercetin equivalence.¹¹

2.6 Reducing Power Assay:

All the crude extracts were screened for reducing power as described by Yen and Duh,¹² with some modifications. Various concentrations of crude extracts were made up to 500µl using DMSO. To each sample 1.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 1.5 ml potassium ferricyanide (1%) were added. The mixture was incubated at 50ºC for 20 min followed by addition of 5ml of 10% trichloroacetic acid. They were centrifuged at 6000rpm for 5 min at 4ºC. The upper layer of the solution (1.5ml) was diluted with 1.5 ml of distilled water. Finally 300µl of ferric chloride (0.1%) was added and absorbance read at 700nm. Ascorbic acid and BHT were used as standards and results expressed in terms of ascorbic acid and BHT equivalence.

2.7 DPPH Radical Scavenging Assay:

Free radical scavenging activity of the extracts was measured using the procedure described by Blois.¹³ The stock solutions of crude extracts at 1mg/mL were prepared in DMSO. To each of the reaction mixtures of different concentrations, 3.0 mL of freshly prepared solution of 2, 20-diphenyl-1-picrylhydrazyl (DPPH) at concentration 19g/500ml was added. Absorbance was measured at 515 nm after 30min. The remaining amounts of DPPH-radical were calculated from calibration curve. The scavenging activity of hydroxyl radical (%) was calculated according to the equation: [(A515blank - A515sample)/A515blank]*100. IC₅₀values were further calculated using Mat-lab software.

2.8 Beta-carotene Bleaching Assay:

Ten ml of Linoleic acid solution dissolved in ethanol (2 mg/ml) and 10 ml of β-carotene solution (2 mg/ml) in acetone were added to 10 ml of the molten agar (1.2% solution in boiling water). The mixture was shaken and poured into Petri dishes (25 ml per dish). The plates were kept away from light. Holes (8 mm diameter) were punched into the agar and 100µl of each extract (1 mg) in DMSO were transferred into each The Petri dishes were incubated at 45 ºC for 4 h. A zone of orange colour around the hole indicated positive for antioxidant activity. The diameter of the zone was measured in millimeters.¹⁴

2.9 Hydroxyl Radical Scavenging Assay:

Non site-specific hydroxyl radical scavenging activity of the extracts was measured using the deoxyribose assay.¹⁵ To 0.5 ml of each of the test samples (10, 50, 100, 250 and 500 µg) of the extract, 1.0 ml of 20 mM potassium phosphate buffer (pH 7.4) containing 28 mM 2-deoxy-ribose, 1.04 µM EDTA, 200 µM FeCl3, 100 µM ascorbate and 1 mM hydrogen peroxide were added. The mixtures were incubated at 37 ºC for 1 h followed by addition of 1ml of 2.8% trichloro acetic acid containing 1% thiobarbituric acid. The solutions were subsequently boiled at 100 ºC for 20 min. Deoxy-ribose mixed with buffer was used as blank. The absorbance was measured at 532 nm using catechin as standard. The scavenging activity of hydroxyl radical (%) was calculated according to the equation: [(A532blank - A532sample)/A532blank]*100 and IC₅₀ values were determined using Mat-lab software.

The procedure for measuring site-specific hydroxyl radical scavenging activity was identical to that detailed above but with EDTA replaced by an equal volume of buffer as reported previously.¹⁶

3. RESULTS AND DISCUSSION:

3.1 Total Phenolics and Flavonoids:

The amount of total phenolics and flavonoids present in the crude extracts are shown in Table 1. Methanol extract of core wood contained the highest amounts of phenolics and flavonoids. Hexane extract of the core wood also showed significant amount of phenolics and flavonoids whereas hexane extract of the bark did not show any. Siddhuraju and Becker in 2003 have reported that methanol and ethanol extracts generally yielded higher quantity of phenolics which are consistent with the results obtained in this study.¹⁷

3.2 Reducing Power Assay:

Reducing power assay, measures the reducing ability of extract antioxidant property against the oxidative effects of reactive oxygen species. The data for the reducing power of different bark and core wood extracts are presented in Table 2 and 3. The reducing power of the bark and core wood extracts showed increase in activity with increase in concentration up-to concentration from 10 µg up to 100 µg. Reducing power of the various solvent extracts ranged from 0 to 56 ascorbic acid equivalents and 0 to 57 BHT equivalents. Among the solvents, methanol extract of core wood showed the highest value followed by methanol extract of the bark. Hexane extract of the bark showed no activity, while hexane extract of core wood showed substantial antioxidant property. Ethyl acetate and water extracts showed a moderate response. Shridhar et al., in 2009 has reported that in reducing power assay, the methanolic extract of T. arjuna bark possessed slightly higher activity than that of the aqueous extract.¹⁸

Table 1: Phytochemical Quantification of T. arjuna bark and core wood extracts

*T. arjuna*	Solvents	Total Phenolics (mg/100mg) (Gallic Acid Equivalent)	Total Flavonoids (mg/100mg) (Quercetin Equivalent)
BARK	Hexane	0	0


	Ethyl Acetate	41	91
	Methanol	84	93
	Water	36	61
CORE WOOD	Hexane	30	83
	Ethyl Acetate	11	54
	Methanol	94	97
	Water	54	55

3.3 DPPH Free Radical Scavenging Activity:

DPPH, a relatively stable organic radical has been widely used in the determination of antioxidant activity of single compounds, as well as of different plant extracts. ¹⁹The IC₅₀ results of the DPPH scavenging activity of T. arjuna bark and core wood extracts are shown in Fig 1. The scavenging ability of all the extracts was compared with the standards ascorbic acid and BHT. Except hexane extract of bark all the other crude extracts exhibited significant DPPH scavenging efficacy. Ethyl acetate extract of core wood followed by water and methanol extract of bark exhibited significant DPPH radical scavenging activity with IC₅₀ values 11.86 μg/ml, 16.24 μg/ml and 29.89 μg/ml respectively compared to vitamin C (IC₅₀ 10.65 μg/ml). When the extracts were compared with the standards Ascorbic Acid and BHT, ethyl acetate extract of core wood and methanol extract of bark showed significant correlation. The results obtained were comparable with the data published by Bushra et al.²⁰

3.4 Beta-carotene Bleaching Assay:

In a β-carotene/linoleic acid model system, β-carotene undergoes rapid de-colorization in the absence of an antioxidant. All extracts except hexane extract of bark, were able to retain the color of beta-carotene. Methanol extract of core wood showed highest color retention of 17 mm mean zone. The values of all the extracts are shown in Fig 2. Interestingly in the bark sample ethyl acetate extract showed slightly higher color retention potential than that of the methanol where as in all the other antioxidant assays methanol extract showed higher activity irrespective of bark or core wood. This activity might be because of some other antioxidant compound present in ethyl acetate extract. This assay also confirms the presence of large amount of antioxidant compounds in the core wood than the bark.

Fig 1: Antioxidant activity of T. arjuna - DPPH Assay IC₅₀ value comparison

(H- Hexane, EA – Ethyl Acetate, M – Methanol, W – Water, B –Bark, CW – Core Wood, BHT – Butylated Hydroxy Tolune, As. A – Ascorbic Acid)

Fig 2: Antioxidant activity of T. arjuna - β-carotene Assay. [Conc – 1mg/ml]

(H- Hexane, EA – Ethyl Acetate, M – Methanol, W – Water, B –Bark, CW – Core Wood, BHT – Butylated Hydroxy Tolune)

Table 2: Reducing Power Assay - Antioxidant activity of T. arjuna [Ascorbic Acid equivalent]

*T. arjuna*	Concentration (µg/500 µl)	SOLVENTS
*T. arjuna*	Concentration (µg/500 µl)	Hexane	Ethyl Acetate	Methanol	Water
BARK	25	0	6.686	13.759	6.651
	50	0	15.202	28.609	13.478
	75	0	17.419	39.729	23.894
	100	0	23.823	43.776	30.298
CORE WOOD	25	0.739	1.795	12.774	6.334
	50	2.498	3.413	30.298	12.985
	75	4.856	5.138	39.236	20.903
	100	8.023	6.440	56.303	24.597

Table 3: Reducing Power Assay - Antioxidant activity of T. arjuna [Butylated Hydroxy Toluene equivalent]

*T. arjuna*	Concentration (µg)	SOLVENTS
*T. arjuna*	Concentration (µg)	Hexane	Ethyl Acetate	Methanol	Water
BARK	25	0	6.777572	13.94753	6.741901
	50	0	15.41006	29.00088	13.66216
	75	0	17.65736	40.27304	24.2209
	100	0	24.14956	44.37526	30.7131
CORE WOOD	25	0.7491001	1.819243	12.94873	6.420858
	50	2.532672	3.460129	30.7131	13.16276
	75	4.922658	5.208029	39.77365	21.18883
	100	8.133087	6.527872	57.0743	24.93433

3.5 Hydroxyl Radical Scavenging Assay:

Hydroxyl radical is a highly potent oxidant, which reacts with all bio-macromolecules in living cells. Determination of the scavenging property of this hydroxyl radical using a simple test tube assay would result in easy detection of anti-oxidant property.²¹ The site specific and non-site specific hydroxyl radical scavenging assays were performed for all the crude extracts and is shown in Fig 3. The values thus obtained were used to calculate the IC₅₀ and are shown in Fig 3. Both site specific and non-site specific hydroxyl radical scavenging activity were found in all the extracts except hexane extract of the bark. Methanol extract of the core wood showed the highest activity in both site specific and non-site specific assays. A comparison of the protocols showed higher values in the non-site specific protocol than site specific. The experimental data showed the extracts of T. arjuna possessed significant antioxidant property when compared with the standard catechin. Gupta, et al., in 2001 studied the antioxidant property of T. arjuna bark by measuring thiobarbituric acid reacting substances (TBARS) and the results were compared with Vitamin E. The results showed that T. arjuna significantly decreased the lipid peroxide levels.²²A similar study performed by Balamurugan et al.., on Caesalpinia sappan Linn, has employed similar antioxidant assays to determine antioxidant potential in the plant extract and its outcome is in accordance with present study results. ²³

Fig 3: Antioxidant activity of T. arjuna Hydroxyl scavenging Assay (Site Specific and Non Site Specific) IC₅₀ value comparison

(H- Hexane, EA – Ethyl Acetate, M – Methanol, W – Water, B –Bark, CW – Core Wood)

4. CONCLUSION:

This research on the in-vitro anti-oxidant property of Terminalia arjuna bark and core wood revealed that the core wood extract of the tree contained more antioxidant compounds than that of the bark. Similarly among the different solvent extracts of core wood, methanol extract of the core wood showed high antioxidant activity in major antioxidant assays proving methanol as best solvent to extract the antioxidant compounds. It is also evident from the study that the methanol extract of both bark and core wood possessing high total phenolic and flavonoids showed better antioxidant activity than the other extracts.

5. ACKNOWLEDGEMENT:

We would like to thank the VIT University for supporting this research.

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Received on 08.05.2013 Modified on 18.06.2013

Research J. Pharm. and Tech. 6(9): September 2013; Page 996-1000