Antioxidant activity of acetone extract/fractions of Terminalia bellerica Roxb. fruit

Transcription

1 Indian Journal of Biochemistry & Biophysics Vol. 47, April 2010, pp Antioxidant activity of acetone extract/fractions of Terminalia bellerica Roxb. fruit Sanjay Guleria 1*, A K Tiku 1 and Subhash Rana 2 1 Division of Biochemistry and Plant Physiology, Sher-e-Kashmir University of Agricultural Sciences and Technology, Chatha , Jammu, India 2 Herbal Garden and Herbarium Research Institute in Indian System of Medicine, Joginder Nagar, District Mandi , 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 Fe 2+ 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 EC 50 values) observed were µg/ml, 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 conditions 1. 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 antioxidants 2. 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 atherosclerosis 3. They also retard rancidity in foods caused by lipid oxidation 4. 1 Corresponding author guleria71@rediffmail.com Phone: Secondary metabolites from plants, mainly phenolics having antioxidant properties are currently estimated to be between 4000 and ,6. A direct relationship has been reported between the levels of phenolic compounds and antioxidant potential of plants 7. Phenolic compounds exhibit their protective action through various mechanisms like preventing the generation of carcinogens from precursors by acting as blocking agents Fruits, vegetables and medicinal plants are rich sources of phenolics, such as flavonoid compounds which are endowed with antioxidant properties 11,12. 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 acids 13. Leaves and fruits show antioxidant activity 14. The plant exhibits inhibitory effect on human immuno-deficiency virus-1 reverse trascriptase 15. A water soluble fraction from the defatted fruits has shown hepatoprotective activity against CCl 4 -induced hepatotoxicity 16. In this study, we

2 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 Na 2 CO 3, NaOH, sodium nitrite, AlCl 3 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 method 17. 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% Na 2 CO 3. 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 = gallic acid (µg) (R 2 = ) Flavonoid content in the acetone extract/fractions was determined by a colorimetric method 18. Plant extract (250 µl) was mixed with 1.25 ml of distilled water and 75 µl of a 5% NaNO 2 solution. After 5 min, 150 µl of 10% AlCl 3 H 2 O 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 = quercetin (µg) (R 2 = ) 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 elsewhere 19. 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 = [(A 0 A s )/A o ] 100 where A 0 and A s are the absorbance of the control (containing all reagents, except the test compound)

3 112 INDIAN J. BIOCHEM. BIOPHYS., VOL. 47, APRIL 2010 and test compound respectively. The extract/fraction concentration providing 50% of radical-scavenging activity (EC 50 ) 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 system 20. 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- (A s(0) A s(120) )/A c(0) A c(120) )] 100 where A s(0) the initial absorbance of the sample at 0 min, A s(120) the absorbance of the sample at 120 min, A c(0) the initial absorbance of the negative control at 0 min, and A c(120) the absorbance of the negative control at 120 min. The extract/fraction concentration providing 50% antioxidant activity (EC 50 ) 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 previously 21. 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 [K 3 Fe(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 (EC 50 ) 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 (Fe 2+ ) ions The chelating effect on Fe 2+ ions from acetone extract and fractions was estimated according to method described elsewhere 22. Briefly, 200 µl of different concentrations of extract/fractions and 740 µl of methanol were added to 20 µl of 2 mm FeCl 2. 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-fe 2+ 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 extracts 23. 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 (EC 50 ) 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

4 GULERIA et al: ANTIOXIDANT ACTIVITY OF TERMINALIA BELLERICA FRUIT 113 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] 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 electron 24. 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 system 3. 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 (EC 50 ) 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 system 25. 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 investigators 26,27. Antioxidant action of reductones has been shown to be based on breaking the radical chain by donation of hydrogen atom 28. 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 (R 2 = ) was observed between EC 50 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 (R 2 = ) well with the extent of antioxidant activity 29. Similarly, antioxidant activities of mung bean hull and burdock extracts were found to be concomitant with the development of reducing power 30. 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

5 114 INDIAN J. BIOCHEM. BIOPHYS., VOL. 47, APRIL 2010 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] 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 H 2 O 2 decomposition is an important mechanism of antioxidant activity 31. From the Fe 2+ data, it was evident that the acetone extract and fractions possessed Fe 2+ ions chelating activity and might play a protective role against oxidative damage by sequestering Fe 2+ ions which might otherwise participate in metal catalyzed H 2 O 2 decomposition reactions 32. 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 Fe 2+ ion to Fe 3+ and thereby retarding oxidative damage 33. In order to determine the antioxidant compounds in extract/fractions, the total phenolic and flavonoid Fig. 3 Phenolic and flavonoid content of acetone extract/fractions of T. bellerica fruit 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

6 GULERIA et al: ANTIOXIDANT ACTIVITY OF TERMINALIA BELLERICA FRUIT 115 of phenolic compounds, as these compounds exhibit important mechanism of antioxidant activity 34. It has been reported that fruits contain antioxidant nutrients, in addition to vitamins which contribute to their antioxidant potential 35. 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 donors 17,36. 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 (R 2 value of , and , respectively) with their phenolic content. Further, the extract/or fractions with higher polyphenol contents showed lower EC 50 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 studies 37. Flavonoid content was also correlated with EC 50 values obtained from DPPH free-radical scavenging, β-carotene bleaching antioxidant effect and reducing power assays, but with less coefficient of correlation (R 2 value of , and , respectively). This suggested that the principal antioxidant molecules in crude extract/fractions of T. bellerica fruit are nonflavonoid 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. References 1 Aruoma O I (1998) J Am Oil Chem Soc 75, Yeşilyurt V, Halfon B, Oztürk M & Topçu G (2008) Food Chem 108, Kinsella J E, Frankel E, German B & Kanner J (1993) Food Tech 47, Duthie G G (1993) Eur J Clin Nutr 47, Havsteen B H (2002) Pharmacol Therap 96, Wollgast J & Anklam E (2000) Food Res Int 33, Robards K, Prenzler P D, Tucker G, Swatsitang P & Glover W (1999) Food Chem 66, Wattenberg L W & Lam L K T (1983) In: Radioprotectors and Anticarcinogens (Nygaard O F & Simig M G, eds), pp , Academic Press, New York 9 Newmark H L (1996) Adv Exp Med Biol 401, Claudine M, Augustin S, Christine M, Christian R & Liliana J (2004) Am J Clin Nutr 79, Auddy B, Ferreira M, Blasina F, Lafon L, Arredondo F & Dajas F (2002) J Ethanopharmacol 84, Choi C W, Kim S C, Hwang S S, Choi B K, Ahn H J & Lee M Y (2002) Plant Sci 163, Rastogi P & Mehrotra B N (1999) Compendium of Indian Medicinal Plants, Drug Research Perspective, Vol. 2, CDRI, Lucknow & NISCOM, New Delhi 14 Bajpai S, Pande A, Tewari S K & Dhan P (2005) Int J Food Sci Nutr 56, El Mekkawy S, Meselhy M R, Kushmoto I T, Kodota S, Hattori, M & Namba T (1995) Chem Pharm Bull 43, Anand K K, Singh B, Saxena, A K, Chandan B K & Gupta V N (1994) Phytother Res 8, Chang S T, Wu J H, Wang S Y, Kang P L, Yang N S & Shyur L F (2001) J Agric Food Chem 49, Jia Z, Tang M & Wu J (1999) Food Chem 64, Abe N, Murata T & Hirota A (1998) Biosci Biotech Biochem 62, Kabouche A, Kabouche Z, Oztürk M, Kolak U & Topçu G (2007) Food Chem 102, Oyaizu M (1986) Jpn J Nutr 44, Dinis T C P, Madeira V M C & Almeida L M (1994) Arch Biochem Biophy 315, Porto C D, Calligaris S, Celloti E & Nicoli M C (2000) J Agric Food Chem 48, Shimada K, Fujikawa K, Yahara K & Nakamura T (1992) J Agric Food Chem 40, Jayaprakasha G K, Singh R P & Sakariah K K (2001) Food Chem 73, Duh P D, Tu Y Y & Yen G C (1999) Leb Wissen Tech 32, Yen G C & Chen H Y (1995) J Agric Food Chem 43, Gordon M H (1990) In: The Mechanism of Antioxidants Action In Vitro (Hudson B J F, ed), pp 1-18, Food Antioxidants, New York 29 Yen G C & Duh P D (1993) J Am Oil Chem Soc 70, Duh P D, Yen W J, Du P C & Yen G C (1997) J Am Oil Chem Soc 75, Manian R, Anusuya N, Siddhuraju P & Manian S (2008) Food Chem 107,

7 116 INDIAN J. BIOCHEM. BIOPHYS., VOL. 47, APRIL Dorman H J D, Kosar M, Kahlos K, Holm Y & Hilturien R (2003) J Agric Food Chem 51, Singh R, Singh S, Kumar S & Arora S (2007) Food Chem 103, Yildirim A, Mavi A, Oktay M, Kara A A, Algur O F & Bilaloglu V (2000) J Agric Food Chem 48, Cao G, Sofic E & Prior RL (1997) Free Radic Biol Med 22, Halliwell B, Aeschbach R, Loliger J & Aruoma O I (1995) Food Chem Toxicity 33, Barreira J C M, Ferreira I C F R, Oliveira M B P P & Peieira J A (2008) Food Chem 107,