Antioxidant and Free Radical Scavenging Properties of Traditionally Used Three Indian Medicinal Plants

Current Trends in Biotechnology and Pharmacy, Vol. 2 (4) 538-547 (2008) Antioxidant and Free Radical Scavenging Properties of Traditionally Used Three Indian Medicinal Plants Prateek Kumar Jain, V. Ravichandran and R. K. Agrawal* Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar (M.P) - 470 003, India * For Correspondence: dragrawal2001@yahoo.co.in Abstract The antioxidant properties of methanol extract of three medicinal plants viz. Withania somnifera, Asparagus racemosus and Centella asiatica that probably involve free radical mechanisms, were evaluated by the methods, namely the ABTS (2, 2 -azinobis-3-ethylbenzothiozoline-6-sulphonic acid) radical scavenging assay, Trolox equivalent antioxidant capacity assay (TEAC), Oxygen radical absorbance capacity (ORAC) assay, DPPH free radical scavenging activity assay, Super oxide scavenging activity assay and Nitric oxide radical scavenging activity assay. The methanol extract from Centella asiatica was most active in the ABTS assay with an IC 50 value of 185.18 μg/ml followed by Withania somnifera with IC 50 value of 214.78 μg/ml. The minimum activity showed by Asparagus racemosus with IC 50 value of 384.55 μg/ml. The Trolox equivalent antioxidant capacity, DPPH (1, 1-diphenyl-2-picryl hydrazyl) and Superoxide radical scavenging activity of the methanolic extracts was in order of Withania somnifera > Centella asiatica > Asparagus racemosus. In the Oxygen radical absorbance capacity assay the ORAC value of Withania somnifera was found maximum 443.5 μ moles TE # / g and followed by Centella asiatica and Asparagus racemosus showed 211.5 μ moles TE # / g and 31.3 μ moles TE # / g respectively. The Nitric oxide radical activity was found in order of Asparagus racemosus >Withania somnifera > Centella asiatica. The present studies suggest that all the tested plants have moderate to potent antioxidant activity. Keywords: Antioxidant activity, Withania somnifera, Asparagus racemosus, Centella asiatica, Gallic acid. Introduction The traditional medicine all over the world is nowadays revalued by an extensive activity of research on different plant species and their therapeutic principles. Experiment evidence suggests that free radicals (FR) and reactive oxygen species (ROS) can be involved in a high number of diseases (1, 2). As plants produce a lot of antioxidants to control the oxidative stress caused by sunbeams and oxygen, they can represent a source of new compounds with antioxidant activity. Numerous physiological and biochemical processes in the human body may produce oxygen-centered free radicals and other reactive oxygen species as byproducts. Overproduction of such free radicals can cause oxidative damage to biomolecules (e.g. lipids, proteins, DNA), eventually leading to many chronic diseases, such as atherosclerosis, cancer, diabetes, aging, and other degenerative diseases in humans (3, 4). Plants (fruits, vegetables, medicinal herbs, etc.) may contain a wide variety of free radical scavenging molecules, such as phenolic compounds (e.g. phenolic acids, flavonoids, quinones, coumarins, lignans, stilbenes, tannins), nitrogen compounds (alkaloids, amines, betalains), vitamins, terpenoids (including carotenoids), and some other endogenous

Current Trends in Biotechnology and Pharmacy, Vol. 2 (2) 538-547 (2008) 539 metabolites, which are rich in antioxidant activity (5,6). The intake of natural antioxidants has been associated with reduced risks of cancer, cardiovascular disease, diabetes, and other diseases associated with aging (7,8,9). Condensing the importance of this area, methanolic extract of Withania somnifera, Asparagus racemosus and Centella asiatica were tested for antioxidant activity. In vitro antioxidant activity was done by ABTS radical scavenging assay (10), Trolox equivalent antioxidant capacity, Oxygen radical absorbance capacity assay (11), DPPH free radical scavenging activity assay (12), Super oxide scavenging activity assay (13) and Nitric oxide radical scavenging activity assay (14). Experimental Materials: The root of the Withania somnifera and Asparagus racemosus were picked up in the month of February-March, 2005 from the University Campus of Dr. H.S. Gour Vishwavidyalaya, Sagar (M.P.) and the leaves of the Centella asiatica were purchased from the Local Market of Sagar (M.P.), India. The voucher herbarium specimen numbers of the selected plants are Withania somnifera Dunel. (1492), Asparagus racemosus Willd. (2222) and Centella asiatica Linn. (428). The identity of collected / procured drug was confirmed from Botany Department at Dr. H.S. Gour Vishwavidyalaya, Sagar (M.P.). Methanolic Extract: For the preparation of methanolic extract, 500 g of coarse powder of each drug was extracted in Soxhlet apparatus with two liters of methanol for Twenty four hours and filtered to yield the extract. The extract was then concentrated and finally dried to a constant weight. ABTS radical scavenging assay: ABTS (2, 2 - azinobis-3-ethyl-benzothiozoline-6-sulphonic acid) assay is based on the scavenging of light by ABTS radicals. An antioxidant with an ability to donate a hydrogen atom will quench the stable free radical, a process which is associated with a change in absorption which can be followed spectrophotometrically. The relatively stable ABTS radical has a green color and is quantified spectrophotometrically at 734 nm. Reagents / chemicals used: Ammonium persulphate (APS)[Rankem, India], ABTS (2,2'- azinobis-3-ethyl-benzothiozoline-6-sulphonic acid) [Sigma, USA], Positive control: Gallic acid (3,4,5-Trihydroxy benzoic acid)[sigma, USA] Procedure: Two and half milligram of extract per milliliter in methanol was prepared and used in the assay. ABTS radical cations were produced by reacting ABTS and APS and incubating the mixture at room temperature in dark for 16 h. Add 20 ìl of various concentrations of 10 mm PBS ph 7.4/ positive control / test solution and 230 ìl of ABTS radical solution (0.238 mm). The absorbance is measured immediately at 734 nm. A control reaction was carried out without the test sample. Linear graph of concentration Vs percentage inhibition was prepared and IC 50 values were calculated. The % inhibition was calculated according to the following equation % Inhibition = ((A 0 -A t ) / A 0 x 100) Where A 0 was the absorbance of the control (blank, without extract) and A t was the absorbance in the presence of the extract. Trolox Equivalent Antioxidant Capacity (TEAC): The assay relies on the ability of antioxidants in the sample to inhibit the oxidation of ABTS (2, 2-Azino-di-[3-ethylbenzthiazoline sulphonate] to ABTS radical cation by metmyoglobin. The amount of ABTS produced can be monitored by reading the absorbance at 600 nm to a degree which is proportional to their concentration. The capacity of the antioxidants

540 Indian Medicinal Plants in the sample to prevent ABTS oxidation is compared with that of Trolox, and is quantified as millimolar Trolox equivalents. Reagents / chemicals used: Total antioxidant status kit from RANDOX, UK (Cat. No. NX2332), lot no. 069156 Procedure: The extract (312.5 mg) was dissolved in 5 ml methanol and made up the volume to 100 ml with de-ionized water, filtered and the filtrate was used for the assay. The assay is carried out as per the instructions of Total antioxidant status manual from Randox (cat. No. NX2332); the final volume of the assay is 244 µl in all the wells. Four micro litters of solvent / positive control / test solution and 200 μl of chromogen are added, mixed and read at 600 nm. Following the initial reading, 40µl of substrate is added to all the wells, mixed and incubated at 37 o C for 3min. The absorbance is measured at 600 nm and TEAC value is calculated. Oxygen radical absorbance capacity (ORAC) assay. The ORAC assay depends on the free radical damage to a fluorescent probe through the change in its fluorescence intensity. In the present assay, AAPH is used as free radical generator to reduce the fluorescence characteristics of fluorescien, which is used as the fluorescence probe. The change of fluorescence intensity (reduction in fluorescence) is an index of the degree of free radical damage. In the presence of an antioxidant, there is decrease in the change of fluorescence induced by AAPH. In the ORAC assay, the antioxidant activity of a sample is expressed relative to TROLOX, a derivative of Vitamin - E. Reagents / chemicals used: Sodium di-hydrogen orthophosphate dihydrate (RM1255, Himedia, India, Store at R.T), Di-sodium hydrogen phosphate dihydrate (RM 257 Himedia, India, Store at R.T), Preparation of Phosphate buffer: 6.177 g of Sodium dihydrogen phosphate and 1.014 g of Di- Sodium hydrogen phosphate in 100 ml of distilled water, Fluorescein sodium salt (F6377, Sigma, USA, Store at R.T), 2,2 1 - azobis(2-methyl propionamidine) dihydrochloride [AAPH] (44091-4, Aldrich, USA, Store at R.T), (±) 6-hydroxy-2,5,7,8-tetra methylchromane-2-carboxylic acid [TROLOX] (56510, Fluka, Store at R.T) Procedure: A pre-incubation mixture of 140 µl contain 20 µl of test solution / TROLOX of various concentrations / 75 mm Sodium phosphate buffer, ph 7.4; 120 µl of Sodium fluorescein (117 nm), mixed and incubated at 37 C for 10 min. Following pre-incubation, 60 µl of AAPH (40 mm) is added and mixed for 15 sec. The reaction was carried out for 80 min at 37 C. The fluorescence measurements were taken at 485 nm excitation and 520 nm emission filters with the following settings: Mode: Fluorescence intensity, Optic: Top optic mode, Filters: Excitation 485 nm, emission 520 nm, Number of cycles: 80, Number of flashes: 10, Cycle time: 60 sec and Plate used: Costar 96, Code 3792 from Corning. TROLOX equivalents (TE) of antioxidant values were obtained by dividing the slopes of the regression curves. DPPH radical scavenging assay: DPPH (1, 1- diphenyl-2-picryl hydrazyl) is a stable free radical with purple color, the intensity of which is measured at 510 nm spectrophotometrically. Antioxidants reduces DPPH to 1,1-diphenyl-2- picryl hydrazine, a colorless compound. Reagents / chemicals used: DPPH [RM 2798, Himedia, India], Methanol (HPLC grade) [43602, Qualigens], Positive control (Gallic acid) [G7384, Sigma, USA]. Procedure: The extract (3.125 mg) was dissolved and made up to 10 ml with methanol. The sample was completely soluble. 200 l of methanol /

Current Trends in Biotechnology and Pharmacy, Vol. 2 (4) 538-547 (2008) 541 positive control /various concentration of test solution and 50 μl of DPPH (0.659 mm) solution are incubated at 25 C for 20 min. Following which the absorbance is read at 510 nm. A control reaction was carried out without the test sample. Linear graph of concentration Vs percentage inhibition was prepared and IC 50 values were calculated. Superoxide radical scavenging activity (PMS- NADH System): Superoxide anions were generated using PMS / NADH system. The superoxide anions are subsequently made to reduce nitroblue tetrazolium which yields a chromogenic product, which is measured at 560 nm. Reagents / chemicals used: Reduced nicotinamide adenine dinucleotide Sodium salt (NADH) [RM 393Himedia, India], Phenazine methosulphate (PMS) [5165, Loba Chemie, India], Nitroblue tetrazolium (NBT) [94060, S.d. fine Chemicals, India], Positive control: Gallic acid G7384, [Sigma, USA]. Procedure: Ten milligrams of the extract was dissolved in 0.1mL methanol and made up the volume to 10 ml with 0.1M phosphate buffer ph 7.4. 62.5 µl of test sample / positive control (various concentrations) in 0.1M phosphate buffer ph 7.4, 62.5 µl of 468 µm NADH solution, 62.5 µl of 150 µm NBT solution and 62.5 μl of 60µM PMS solution were added to a microwell plate and incubated at room temperature for 5 min. The absorbance was read at 560 nm. Linear graph of concentration vs percentage inhibition was prepared and IC 50 values were calculated. Nitric oxide radical scavenging assay: Sodium nitroprusside in aqueous solution at physiological ph spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions, which can be measured at 546 nm spectrophtometrically in the presence of Griess reagent. Reagents / chemicals used: Sodium nitroprusside [40190, S.d.fine Chemicals, India], Sulphanilamide [3164, NR Chem., India], Orthophosphoric acid [39416, S.d.fine Chemicals, India], N-(1- naphthyl) ethylenediamine [N5889, Sigma, USA], Positive control: Curcuminoids ( 33533, Synthite, Kochin, India). Procedure: Five milligrams of the extract was dissolved and made up to 10 ml with methanol. The sample was completely soluble. 50 l of 10 mm sodium nitroprusside and 50 μl of test solution/ positive control of various concentrations are illuminated (using fluorescence light/18w CDL 6500K) at room temperature (25-30 C) for 15 minutes. Following incubation, 125 μl of Griess reagent was added and incubated for 10 min at room temperature. Then the absorbance was measured at 546 nm. Linear graph of concentration vs percentage inhibition was prepared and IC 50 values were calculated. Results and Discussion In recent years there has been growing interest in understanding the role of free radicals in many diseases such as cancer, arteriosclerosis, ageing and their prevention using antioxidants (15). Our attention has been focused, in particular, on the parts of three commonly used Indian medicinal plants. DPPH scavenging activity has been used by various researchers as a quick and reliable parameter to assess the in-vitro antioxidant activity of crude plant extracts (16, 17, 18). Literature confirms the antioxidant activities of Withania somnifera, Asparagus racemosus and Centella asiatica in various in vitro models (19) but there are no or scanty reports on trolox equivalent antioxidant capacity and oxygen radical absorbance capacity assays for Withania somnifera, Asparagus racemosus and Centella asiatica. In the present context, we have evaluated these extracts in six in-vitro tests viz. ABTS radical scavenging assay, trolox equivalent antioxidant capacity and oxygen radical

542 Jain et al absorbance capacity, DPPH free radical scavenging assay, super oxide radical scavenging activity and Nitric oxide radical scavenging assay for antioxidant activity were used to assess the antioxidant properties of methanol extracts of each plant. Together all the six testes provide a better assessment of antioxidant properties. ABTS assay: In this assay, gallic acid was used as a standard and its IC 50 value determined to be 1.30 μg/ml. The methanol extract from Centella asiatica was most active in ABTS assay with an IC 50 value of 185.18 μg/ml followed by Withania somnifera with IC 50 value of 214.78 μg/ml. The minimum free radical scavenging activity was showed by Asparagus racemosus with IC 50 value of 384.55 μg/ml (Table 1). Trolox equivalent antioxidant capacity (TEAC)

Current Trends in Biotechnology and Pharmacy, Vol. 2 (4) 538-547 (2008) 543 assay: This assay measures the antioxidants in the sample and is compared with that of trolox, water soluble tocopherol analogue and is quantified as millimolar trolox equivalents. Superoxide is biologically important since it can be decomposed to form stronger oxidative species such as singlet oxygen and hydroxyl radicals, is very harmful to the cellular components in a biological system. The superoxide anion radical scavenging activity of the extracts was assayed by the PMS-NADH system. The trolox equivalent antioxidant capacity, DPPH and Superoxide radical scavenging activity of the methanolic extracts was in order of Withania somnifera > Centella asiatica > Asparagus racemosus. The gallic acid used as control. The results are shown in (Table 2, 4 and 5). Oxygen radical absorbance capacity (ORAC)

544 Indian Medicinal Plants assay: In Oxygen radical absorbance capacity (ORAC) assay the AAPH is used as free radical generator to reduce the fluorescence characteristics of fluorescien. ORAC value of Withania somnifera was found maximum 443.5μ moles TE # / g and followed by Centella asiatica and Asparagus racemosus showed 211.5 μ moles TE # / g and 31.3 μ moles TE # / g respectively, the results are shown in (Table 3). Nitric oxide radical scavenging assay: Nitric oxide (NO) is a potent pleiotropic mediator of physiological processes such as smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation and regulation of cell mediated toxicity. It is a diffusible free radical which plays many roles as an effectors molecule in diverse biological systems including neuronal messenger, vasodilation and antimicrobial and antitumor activities. The Nitric oxide radical activity was found in order of Asparagus racemosus >Withania somnifera > Centella asiatica. The curcumnoids was used as control. The results are shown in (Table 6).

Current Trends in Biotechnology and Pharmacy, Vol. 2 (4) 538-547 (2008) 545 Conclusion From all the above antixoidant models used, Withania somnifera was found to be better antioxidant in DPPH, superoxide, TEAC and ORAC models when compared to Asparagus racemosus and Centella asiatica. Whereas Centella asiatica showed potent activity in ABTS model when compared to Withania somnifera and Asparagus racemosus. Asparagus racemosus was shown to have superior antioxidant activity in nitric oxide model than Withania somnifera and Centella asiatica. Finally all the three extracts were found to have good antioxidant activity. Acknowledgements One of the authors Prateek Kumar Jain is thankful to M.P. Council of Science and Technology (MPCOST), Bhopal and Indian Council of Medical Research (ICMR), New Delhi, India for providing financial assistance.

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