A COMPARATIVE EVALUATION OF IN VITRO ANTIOXIDANT PROPERTIES OF BAMBOO BAMBUSA ARUNDINACEA LEAVES EXTRACTS

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1 Journal of Cell and Tissue Research Vol. 1(3) (21) ISSN: (Available online at Original Article A COMPARATIVE EVALUATION OF IN VITRO ANTIOXIDANT PROPERTIES OF BAMBOO BAMBUSA ARUNDINACEA LEAVES EXTRACTS MACWAN, C., PATEL, H. V. AND KALIA, K. Laboratory of Biochemistry, BRD School of Biosciences, Vallabh Vidhya Nagar , Gujarat. E. mail: kirankalia_in@yahoo.com Received: November 3, 21; Accepted: December 15, 21 Abstract: Medicinal plants, as source of remedies, are widely used as alternative therapeutic utensil for the anticipation or treatment of many diseases. In present study, we aspire to assess total phenolic and flavonoid content and in vitro antioxidant and metal chelating property of aqueous extract (AE), methanolic extract (ME) and butanolic extract (BE) of mature bamboo leaves by various assays. Study shows that methanol was most effective solvent to extract phenolic (14.6 mg GAE/g powder) and flavonoid (6.71 mm quercetin equivalent/g powder) compounds as compared to water and butanol. The phenolic content of water and butanolic extract was 2.79 and 4.26 mg GAE/g powder respectively, whereas, the flavonoid content of aqueous extract was higher ( 6.17 mm quercetin equivalent/g powder ) than butanolic extract ( 2.54 mm quercetin equivalent/g powder). The methanolic extract have shown the highest antiradical scavenging, metal chelating and nitric oxide scavenging capacity as specified by lower IC 5 value indicative of higher antioxidative capacity. Ferric reducing antioxidant power was comparatively higher for methanolic extract than the other two solvents used in the study. Over all study indicates that in vitro antioxidative properties of methanolic extract of bamboo leaves was comparatively higher. Key words: Bamboo leaves, Antioxidant properties, Metal chelation? INTRODUCTION In past decades, free radicals have aroused considerable inquisitiveness among scientists. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) like superoxide anion (O 2- ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH-), nitric Oxide (NO-) are natural by products produced by endogenous metabolic route and exogenous chemicals in the human body. Under the normal circumstances, the natural antioxidant defense system (enzymatic and non-enzymatic) counteract these free radicals by scavenging or binding with radicals and chelating metals [1]. In unsympathetic condition, there is an imbalance between free radical formation and antioxidant shield system leads to potential oxidative damage to lipid, protein and DNA which subsequently causes tissue erosions [2]. The overproduction of ROS and RNS leads to condition called oxidative stress which is an important etiological factor implicated in several chronic diseases like diabetes mellitus, neurodegerative disease, cancer, atherosclerosis and ageing process [3]. Food and antioxidants supplementation have their positive contribution as health promoters in several pathophysiological conditions and help to restrict the oxidative damage in the body. Plants contain assortment of phytochemical such as phenols, flavonoids, terpenoids, vitamins which possess antioxidant activity [4]. These phytochemicals and natural antioxidants exhibit a wide range of beneficial biological effect and could neutralize oxidation of biological molecules by scavenging free 2413

2 J. Cell Tissue Research radicals and chelating free catalytic metals [1]. Numerous studies have carried out to assess antioxidant properties of various plants result in the development of herbal medicine and nutritional supplementation in nutraceuticals [5]. Much attention has therefore been focused in finding naturally occurring antioxidants form medicinal plant and food because they are biodegradable non-toxic products that replace synthetic antioxidants which are having limited use because of their adverse side effects [6]. Bambusa arundinacea, locally known as Bans or bamboo, a perennial fastest-growing plant on earth is presumed to have origin in Asia. Bamboo, an ancient Chinese medicine and an Indian folk medicine, has more than 7 genera divided into about 1, species in the world, among which 13 growing in India [7]. Bamboo is considered as a rich source of flavones glycosides having ability to interact with lipid bilayers by influencing their incorporation rate into cells. Different parts of bamboo plant possess various biological functions due to the presence of different compounds. The stilbene gulcosides from the root of bamboo had been shown to possess various medicinal properties [8]. Estuko et al. [9] reported the presence of poly phenols viz., p-(hydroxyphenyl) propionic acid, ferulic acid, caffeic acid, and chlorogenic acid in bamboo shoot. The phenolic compounds derived from whole-plant extract showed inhibition of P- glycoprotein in adriamycin resistant human breast cancer cells [1]. Bamboo leaves have been used clinically in the treatment of hypertension, arteriosclerosis, cardiovascular disease, and cancer [11]. The purpose of present study was to study efficiency of various extracts of bamboo leaves to hinder free radical and transition metal ion induced deterioration of macromolecules in vitro. MATERIALS AND METHODS Chemicals: Ascorbic acid (Vitamin-C), 2,2- diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tri[2- pyridyl]-s-triazine (TPTZ), ferrous sulfate, ferrous chloride, gallic acid, 3-(2-pyridyl)-5,6-bis (4-phenylsulfonic acid) -1,2,4- triazine (Ferrozine), sodium nitroprusside (SNP), sulfanilamide, N-(1-naphthyl) ethylenediamine dihydrochloride (NED), Quercetin were used Plant material collection and extraction: The mature bamboo leaves were collected from botanical garden of Department of Biosciences, Vallabh Vidhyanagar, Anand, Gujarat in the autumn season. The specimens were botanically identified as Bambusa arundinacea. The collected leaves were washed 4-5 times by distilled water, shade-dried completely and then powdered with a mixture grinder and stored in an air-tight container in the refrigerator before use. The dried bamboo leaf powder was subjected to extraction, three times with 1 volumes of each solvent [aqueous (AE), methanol (ME) and butanol (BE)] in electrical shaker for 24 hours at room temperature. All extracts were separately filtered, through whatmann filter paper 1. The residue was re-extracted with 1 volumes of each solvent using same procedure. After procedure was repeated thrice, all the extracts were pooled and evaporated to dryness at 5 C. The resulting gummy mass was suspended in respective solvents and brought to the volume. Total phenolic content: Total phenolic content of all the three extracts was determined using a series of gallic acid as standard solutions (.5-.35mg/ml) as described by Slinkard and Singleton [12] with some modifications..1 ml of sample or standard was mixed with 2.ml of 2% (w/v) Na 2 CO 3 vortexed vigorously and after 3 minutes mixed with.1ml of (1N) Folinciocalteu s phenol reagent. Reaction mixture was incubated for 3 min at room temperature and the absorbance was measured at 75 nm. Results were expressed as mg of total phenolic content per g of dry leaf powder as gallic acid equivalent (GAE). Flavonoid content: Flavonoid content of was determined by the method of Chang et al. [13]. Briefly, 1.5 ml methanol was added to.5 ml extract and mixed with.1 ml of 1% aluminum chloride and.1 ml of 1. M potassium acetate. Final volume was made up to 5. ml with distilled water and incubated at room temperature for 3 min. The absorbance was measured at 415 nm with spectrophotometer. Results were expressed as mm of flavonoid content per g of dry leaf powder as quercetin equivalent. Ferric reducing antioxidant power (FRAP): Ferric reducing antioxidant power assay was performed by the method of Benzie and strain [14]. Briefly, 1µl standard (Ferrous sulfate or vitamin C) or sample was added to 1.5ml freshly prepared FRAP reagent (3mM acetate buffer, ph 3.6; 1 mm TPTZ in 4 mm HCl and 2mM FeCl 3 6H 2 O in the ratio of 1:1:1). The absorbance was read against 2414

3 reagent blank (1.5ml FRAP reagent+1µl D/W) at 593 nm after 1 min incubation at 37 C. Results were expressed as mm ferrous sulfate or vitamin C equivalent/g of dry leaf powder. Radical scavenging activity: The radical scavenging activity was assayed spectrophotometrically using DPPH as free radical [15]. Various concentration of sample extracts (.1-1mg/ml) were added to 1µM 2, 2-diphenyl-1-picrylhydrazyl in methanol. The reaction mixture was shaken vigorously and incubated at 37 C for 3 min in dark. The decrease in absorbance was measured at 517 nm. Vitamin C was used as a positive control. The scavenging activity of DPPH radical (%) was calculated following the equation: (A -A 1 ) / A 1%, where A =Concentration of DPPH without extract and A 1= Absorbance of DPPH in the presence of sample. Metal (Fe +2 ) chelating ability: Ferrous ion (Fe +2 ) chelating ability of each extract was measured using the method described previously [16]. The extract (1. ml) was mixed with.1 ml of 2mM FeCl 2 and after 3 minutes reaction was initiated by the addition of.2 ml of 5mM ferrozine. The mixture was vortexes and incubated for 1 min. The absorbance was measured at 562 nm using EDTA as standard. The % inhibition of ferrozine-fe +2 complex formation were given by the following formula: Ferrous ion chelating activity (%) = [1-(A 1 -A 2 )/A ] x 1%, Where A was the absorbance of the control (the mixture without extract), A1 was the absorbance of the mixture in the presence of the extract and A 2 was the absorbance without ferrozine. Nitric oxide scavenging assay: Nitric oxide scavenging activity of bamboo leaves different solvent extracts was examined using Griess reagent by the method of Marcocci et al. [17]. 1 ml sample extract at different concentration was added with Table 1: Comparison of antiradical, metal chelating and nitric oxide scavenging activities of various extracts of bamboo leaves. IC 5 denoted for concentration at which standard and/or extract showed 5% inhibition/ scavenging activity. Parameters Standard ME AE BE Antiradical activity (IC 5) Metal chelating activity (IC 5) NO scavenging activity (IC 5).44 µg Vit C /ml.111 µg EDTA/ml µg Vit C/ml 273 µg/ml 964µg/ml 113 µg/ml 148 µg/ml 713µg/ml 526 µg/ml 433 µg/ml 644 µg/ml 664 µg/ml.5ml 5mM sodium nitropruside and kept at 37 C for 6 min. An aliquot (.5ml) of the incubation solution was pipetted out and mixed with.5ml Griess reagent [1% sulfanilamide in 5% H 3 PO 4 and.1% N-(1-naphthyl) ethylenediamine dihydrochloride (NED)]. The absorbance was recorded immediately at 54 nm. Vitamin C was used as positive control. The efficiency to scavenge NO radicals was calculated using the following equation: Scavenging activity (%) = [1-(A 1 -A 2 )/A ] 1%, Where A was the absorbance of the control (the mixture without extract), A 1 was the absorbance of the mixture in the presence of the extract and A 2 was the absorbance without Griess reagent. RESULTS AND DISCUSSION Interaction between various phytochemicals i.e. phenols, flavonoids, vitamins contribute to the overall antioxidant activity of edible plants; therefore it is difficult to evaluate total antioxidant activity on the basis of individual active components [18]. By taking this fact in concern, in the present study we aimed to investigate the antioxidants in the form of total activity of different solvent extracts. Different solvents give different yields due to their differential polarity. The yield depends upon the nature of solvent being used and the kind of compound present in the plant material. Phenols and glycosides of many flavonoids like polar antioxidants are extracted using water, alcohol or a mixture of water and alcohols. In the present study, percentage yield of the bamboo extracts using different solvents were found to be 1.8%, 13.7% and 3.9% for water, methanol and butanol respectively, suggested methanol as most efficient solvent in extraction due to highest yield followed by water and butanol respectively. Bamboo is a rich source of phenolic compounds which process diverse bioactivities [19]. Phenol, a very important constituent present in medicinal plants, can scavenge a wide range of reactive oxygen and nitrogen species because of their scavenging ability owing to their hydroxyl groups [2]. These phytochemicals act as free radical terminators as they are a class of antioxidant agents [21]. Phenols also play a critical role in chelating iron like transition metal ions. As shown in figure 1 ME was found to have highest phenolic content (14.6 mg GAE/g of powder) followed by BE (4.26 mg GAE/g of powder), and AE (2.79 mg GAE/ g of powder). Earlier reports have demonstrated the effective role of various fruits 2415

4 J. Cell Tissue Research mg GAE/g powder Fig. 1: Total phenolic content % Inhibition Activity Fig. 5: Metal (Fe +2 ) chelating activity Conc in mg/ml mm QAE/g powder Fig. 2: Flavonoid content %Scavenging activity Fig. 6: Nitric oxide radical scavenging activity Con in mg/ml FRAP activity % Scavenging activity Fig. 3: Ferric reducing antioxidant power (FRAP) assay mm FeSO4/ g powder mm Vit C/ g powder Fig. 4: Free radical (DPPH) scavenging activity Conc in mg/ ml and vegetables due to the presence of antioxidant flavonoids in them [22]. Hydroxyl radical have high affinity with aromatic compounds like flavonoids. The antioxidant power of flavonoids depends upon the number and configuration of phenolic hydroxyl groups in the molecules and also upon glycosylation and configuration of other substituent. Flavonoids are potent antioxidants having characteristics of scavenging free radical, chelating metal and inhibiting lipid peroxidation. Our study proved ME as a rich source of flavonoids compare to AE and BE. ME, AE and BE were found to have 6.71, 6.17 and 2.54 mg quercetin equivalent flavonoid/ g powder respectively as shown in Fig. 2. The FRAP assay is simple, fast and reproducible which helps estimate the antioxidant potentials of the bamboo leaves extracts from their power to diminish the TPTZ-Fe(III) complex to TPTZ-Fe(II) complex [23]. Because FRAP is versatile, it can be readily practiced to both aqueous and alcohol extracts of different plants. As indicated in figure 3, the values of FRAP of BE, AE and ME were 73.2, 39.3 and 231 mm Vitamin C/g powder and 164.7, 88.8 and 519 mm Fe(II)/g powder respectively, indicating the highest ferric reducing power of ME as compared to BE and AE. These results have shown that ME was 2416

5 more effective for the total antioxidant and metal chelating capacity with respect to its total phenolic content compared with other two extracts. Bamboo extract may act as electron or hydrogen donors to scavenge free radicals and convert them to more stable products and terminate radical chain reaction. Compared with other methods, DPPH radical scavenging assay is widely used method to assess antioxidant activities in a relatively short time. DPPH is a stable free radical and accepts an electron or hydrogen radical to turn into a stable diamagnetic molecule [24]. Conversion of colour from purple to yellow showed the decline in absorbance of DPPH radical at 517nm caused by reaction between antioxidants present in bamboo leaves and free radical. The IC 5 value of antiradical, metal chelating and nitric oxide scavenging activities of each bamboo extracts are presented in Table 1. It is proved from figure 4 that the ME had higher inhibitory activity (IC 5 =273 µg/ml) against DPPH radical formation as compared to BE having IC 5 value 113 µg/ml and AE having IC µg/ml. Vitamin C used as a positive control and found to have an IC 5 value.44 μg/ml. So, descending order of antioxidant power of different extract: ME > AE > BE. Fe +2 can form complexes with ferrozine quantitatively. The transition metal ion Fe +2 can move single electrons to allow the formation and propagation of many radical reactions [25]. Antioxidant form insoluble complexes with ferrous ion to inhibit interaction between metal and lipid [26] thus inhibit disruption of cell membrane. The observation of the present study has shown the chelating activity and ferrous ion capturing before ferrozine. The formation of ferrous and ferrozine complex has helped in inhibition of lipid peroxidation. In the presence of chelating agents, red colour of the complex was decreased due to the disruption in the complex formation. In the present study it was seen that all the bamboo leaves extracts interfered with the ferrous- ferrozine complex formation, suggesting that it has chelating activity and captured ferrous ion before ferrozine. Figure 5 shows that IC 5 of the AE, ME and BE for metal chelating activity as 713, 148 and 526 ìg/ml respectively which is higher than the positive standard EDTA with IC 5 value.111 ìg/ml. All the solvent extracts of bamboo leaves showed a weaker potency than EDTA, where ME was most effective than AE and BE. Nitric oxide is an important chemical mediator generated by vascular endothelial cells, macrophages and neurons to yield nitrite and peroxynitrite anions but in reaction independent of transition metal ions it decompose to form OH radical [27]. Surplus concentration of nitric oxide was coupled with several oxidative stress related diseases [28]. Amount of nitric oxide radical scavenged was determined by the decrease in intensity of pink colored complex chromophore in the form of IC 5 values at 54 nm. Lower IC 5 value indicates higher antioxidant activity. From figure 6 it is evident that the IC 5 value of vitamin C, AE, ME, BE were found to be , 644, 433, 664 µg/ml respectively. The ME has lower IC 5 value among the extracts and thus maximum antioxidant activity. The nitric oxide radical scavenging activity of all the extracts was in the order: vitamin C > ME > AE > BE. CONCLUSION The different extracts of bamboo leaves proved to possess radical scavenging, metal chelating, ferric reducing and nitric oxide scavenging capability. The present study has verified the usefulness of bamboo leaves as effective antioxidant and a rich source of phenol and flavonoids. However, further investigations are necessary to identify the responsible components of bamboo leaves extract needed for its effective role as therapeutic plant and additional work should be done to fractionate the bamboo extract to elicit a better understanding of the unique mixture of plant antioxidants. The work in this respect is in progress in our laboratory. REFERENCES [1] Sanchez-Moreno, C.: Food Sci. Tec. Intl., 8: 122 (22). [2] Halliwell, B. and Gutteridge, J.M.C.: Free Radicals in Biology and Medicine. 4th Edn., Clarendon Press, Oxford (26). [3] Winyard, P.G., Moody, C.J. and Jacob, C.: Trends Biochem. Sci., 3: (25). [4] Cai, Y.Z., Sun, M. and Corke, H.: J. Agric. Food Chem., 51: (23). [5] Miliauskas, G., Venskulonis, P.R. and Beak, T.A.: Food Chem., 85: (24). [6] Imaida, K., Fukushima, S., Shivai, T., Ohtani, M., Nakanishi, K. and Ito, N.: Carcinogen, 4: (1983). [7] Vastano Bret, C., Chen, Y., Zhu, N.Q.H.O., Zhou, Z. and Rosen,R.T.: J. Agric. Food Chem., 48 (2): (2). 2417

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