Nitric Oxide Radical Scavenging Activity of Terminalia Belericabark

The total phenolic contents and total flavonoid contents have also been determined. The results of the present study show that the aqueous extract of TBB possesses antioxidant activity through the DPPH free radical scavenging activity. The preliminary phytochemical investigation indicates the presence of flavonoids and polyphenols. The results are found to be significant when compared with the standard ascorbic acid. Further studies are required to determine the mechanism and isolation of active constituents involved in the antioxidant activity. Keywords: Terminalia belerica, DPPH, Flavonoid, Phenolic contents.

INTRODUCTION

Free radicals had been implicated in several human diseases e. g. atherosclerosis, arthritis, ischemia and reperfusion injury of many tissues, central nervous system injury, gastritis, ageing, inflammatory response syndrome, respiratory diseases, liver diseases, cancer, and AIDS1-4. Many herbal plants contain antioxidant compounds and these compounds protect cells against the damaging effects of reactive oxygen species (ROS), such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals, and peroxynitrite5, 6. Moreover, these synthetic antioxidants also show low solubility and moderate antioxidant activity7. Therefore, many researchers are in search of antioxidants of natural origin. Terminalia belerica is a large deciduous tree with characteristic bark traditionally. Nitric Oxide Radical Scavenging…. called as Baheda or Bibitaki. It is grown throughout India. Its principal constituents are gallic acid, ellagic acid,  -sitosterol and chebulagic acid8. It is astringent, tonic, expectorant, and laxative. Its pulp is used in dropsy, piles and diaorrhea.

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It is also useful in leprosy, fever, and hair care. It is also used in oxalic acid and preparation of ink. It is used in the case of rheumatism. Seed oil is applied in skin diseases and premature graying of hair9, 10. In the present investigation, we have attempted to investigate the antioxidant potential of the barks of Terminalia belerica.

EXPERIMENTAL

Material and methods Plant material and extraction The barks of Terminalia belerica were collected from Bhopal, cut into small pieces, and dried in shade for 5 days. The dried barks were then grinded.

This material was macerated in water for 72 hours with occasional shaking in dark. Macerate was decanted and filtered. The marc was pressed and filtration was done 2-3 times. The macerates were concentrated to give aqueous extract (22. 13% w/w). Chemicals Various chemicals used were DPPH (1, 1-diphenyl-2-picryl-hydrazyl) (Sigma Chemicals, USA) and aluminum chloride. Ascorbic acid obtained from Sisco Research Laboratories Pvt. Ltd. , India. Folin-Ciocalteus's phenol reagent and sodium carbonate were from Merck Chemical Supplies (Damstadt, Germany). All chemicals and solvents were of analytical grade.

Preliminary phytochemical investigation

The preliminary phytochemical screening of the extract was carried out to know the different constituents present in aqueous fractions of Terminalia belerica as per the standard procedures. The extract was tested for alkaloids11, 12, sterols13, triterpenes14, saponins15, flavonoids16, tannins12,17, carbohydrates12, glycosides, and amino acids12. Shinoda test and thin layer chromatography was also carried out to confirm the presence of flavonoids16.

Nitric oxide scavenging method Sodium nitroprusside (final concentration 5 mM) in phosphate buffer saline was incubated with 0. mL of different concentrations of drug at 25oC for 5 hours. The control experiment was done without test compounds, but with equivalent amounts of buffer in an identical manner. After 5 hours of incubation, Griess reagent (1% sulphanilamide, 2% H3PO4 and 0. 1% napthyl ethylene diamine dihydrochloride) was added to 0. 5 mL of the incubated solution and absorbance was immediately measured at 546 nm using the JASCO V530 UV-VIS spectrophotometer. Percentage inhibition of nitric oxide free radical was calculated by using the following equation: % Inhibition= {1- (Abs. of sample/ Abs. f blank)} x 100 ... (1) The experimental results were expressed as mean ± standard error of mean (SEM) of three replicates18 (Table 1). Table 1: Nitric oxide (NO) scavenging activity of aqueous extract of terminalia belerica bark (tbb) S. No.

1. 2. 3. 4. 5. Conc. (µg/mL) 100 200 300 400 500 % Scavenging Ascorbic acid 48.

82 53. 64 58. 98 82. 28 92. 23 Aq. TBB 29. 52 ± 0.

079 31. 17 ± 0. 112 38. 86 ± 0. 115 58. 65 ± 0. 021 65.

10 ± 0. 047 (n = 3, X ± SEM) Determination of total phenolics Total phenolic contents (C) in the extracts were determined by the modified FolinCiocalteu method18, 19.An aliquot of the extract was mixed with 5 mL Folin-Ciocalteu reagent (previously diluted with water 1 : 10 v/v) and 4 mL (75 g/L) of sodium carbonate. The tubes were vortexed for 15 sec and allowed to stand for 30 min at 40°C for color development. Absorbance was then measured at 765 nm using the JASCO V530 UV-VIS spectrophotometer. Samples of extract were evaluated at different concentrations. Total phenolic contents was expressed as mg/g gallic acid equivalent.

The experimental results were expressed as mean ± standard error of mean (SEM) of three replicates (Table 2). C = c. V /m ... (2) Akhlesh Singhai et al. Nitric Oxide Radical Scavenging….

Where C = Total phenolic content (mg/g) plant extract; c = Conc. of gallic acid established via calibration curve; V = Volume of extract; m = Wt. of plant extract (g) Table 2: Total phenolic and total flavanoid contents in terminalia belerica bark (tbb) S. No. 1. 2. a Sample Total phenolic content a Total flavanoid contentb Aqueous TBB (Aq.

TBB) 0. 1226 ± 0. 77 2. 244 ± 0. 02 (n = 3, X ± SEM) Expressed as mg gallic acid/g of dry plant material b Expressed as mg quercetin/g of dry plant material Determination of total flavonoids Total flavonoid contents (TFC) were determined using the method of Ordon et al. 20), of sample solution. A volume of 1.

5 mL of 2% AlCl3 ethanol solution was added to 1. 5 mL of sample solution. After one hour at room temperature, the absorbance was measured at 420 nm. A yellow color indicated the presence of flavonoids. Total flavonoid content was expressed as mg/g quercetin equivalent. The experimental results were expressed as mean ± standard error of mean (SEM) of three replicates (Table 2). TFC = (Abs.x Dilution factor x 100)/ E1% x Wt. of extract (g) ... (3)

RESULTS AND DISCUSSION

Preliminary photochemical investigations of Terminalia belerica bark showed the presence of carbohydrates, tannins and flavonoids.Shinoda test and thin layer chromatography for flavonoids using mobile phase n-butanol : water : glacial acetic acid (40 : 50 : 10) and use of spraying reagent ferric chloride solution confirmed its presence. Nitric oxide exhibits numerous physiological properties and it is also implicated in several pathological states.

The interaction of nitric oxide with other radicals leads to the formation of more hazardous radical such as peroxy nitrile anion and hydroxyl radical. The absorption maximum of a stable NO radical in methanol was at 546 nm. The IC50 values were found to be 363 g/mL and 144 g/mL for aqueous Terminalia belerica (Aq.TBB) and ascorbic acid, respectively. The extract had shown the activity in dose dependent manner (Fig. 2). Nitric oxide (NO) scavenging activity of aqueous extract of terminalia belerica bark (TBB) % Radical Scavenging Capacity 100 80 60 40 20 0 0 100 200 300 400 500 600 Ascorbic acid Aq. TBB Conc. ( g/mL) Fig. 1: A significant decrease in the concentration of NO radical due to the scavenging ability of both Terminalia belerica and ascorbic acid Total phenolic and total flavanoid content Aq. TBB 2. 5 Conc. (mg/g) 2 1. 5 1 0.

5 0 Total phenolic content Total flavanoid content Fig. : Total phenolic and flavanoid contents of Terminalia belerica bark Polyphenols are the major plant compounds with antioxidant activity. This activity is believed to be mainly due to their redox properties21, which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides. The results from this study strongly suggest that phenolics are Singhai et al. : Nitric Oxide Radical Scavenging…. important components of these plants, and some of their pharmacological effects could be attributed to the presence of these valuable constituents.The total phenolic content in mg/g gallic acid equivalent (GAE) was found to be 0.

1226 in aqueous extract of TBB. The total flavonoid content in mg/g quercetin equivalent was found to be 2. 244 in aqueous extract of TBB (Fig. 2). CONCLUSION The results of the present study show that the aqueous extract of the barks of Terminalia belerica possess antioxidant activity (based on the DPPH free radical scavenging activity). The preliminary phytochemical investigation indicates the presence of flavonoids in Terminalia belerica bark. Polyphenols like flavonoids and tannins are the well known natural antioxidants.

So, the antioxidant potential of Terminalia belerica may be due to the presence of flavonoids and phenolic contents. Although in most cases, the biological activities of the extracts from the barks of Terminalia belerica are not as high as those of the standard compounds used in this study; the present results indicate clearly that the aqueous extract of Terminalia belerica bark possess antioxidant properties and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants.

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