http://bma.org.in/ijfas.aspx Int. J. Fund. Appl. Sci. Vol. 4, No. 3 (2015) 81-86 ORIGINAL ARTICLE Open Access ISSN 2278-1404 International Journal of Fundamental & Applied Sciences In vitro antioxidant profiling of Bambusa tulda Roxb. aqueous methanolic leaf extract growing in the forests of Kokrajhar district, BTAD, Assam, India Swarna Kamal Dey 1, Sushil Kumar Middha 2, Talambedu Usha 3, Birendra Kumar Brahma 1 and Arvind Kumar Goyal 1* 1Bamboo Technology, Department of Biotechnology, Bodoland University, Kokrajhar-783370, B.T.A.D, Assam, India; 2Department of Biotechnology, MLA College for Women, Bangalore-560012, Karnataka, India; 3 Department of Biochemistry, MLA College for Women, Bangalore-560012, Karnataka, India Manuscript received 11th May, 2015, revised 12th June, 2015, accepted 14th June, 2015 Abstract Background: From an age-old period bamboo has been an important ingredient of traditional Asian medicines in general and Chinese medicine in particular. Today people are searching over the use of herbal medicine instead of the synthetic drugs. Objective: The a im of this study was to evaluate phytochemical, biochemical constituent and in vitro antioxidant activity of aqueous methanolic leaf extract of Bambusa tulda. Bambusa tulda (Poaceae) is widely distributed in North Eastern parts of the Country. Methodology: Phytochemical constituents of leaves were extracted by hot extraction method. Aqueous methanolic extract showed that the presence of phenolic compound, alkaloids, saponnins, flavonoids, steroids, anthraquinones, glycosides. The total phenolic, flavonoid and flavonol content in the aqueous methanolic extract were determined by calorimetric assay as well as their antioxidant activity through various chemical assays like DPPH radicals, hydrogen peroxide scavenging and reducing power assay. Results: The total phenolic, flavonoids and flavonol content in aqueous methanolic extract were 17.494±0.01 mg GAE/g, 176.35±0.03 mg QE /g and 96.2±0.01 mg QE /g respectively. Conclusion: This may be first report to provide evidence that the crude aqueous metanolic extract of Bambusa tuld a leaf is a potential source of natural antioxidant. Keywords : Bambusa tulda ; antioxidants; DPPH, Kokrajhar; Jati banh ; correlation; FRP, phenolics, Owa gubwai @2015 BioMedAsia All right reserved 1. Introduction used world-wide as rich antioxidant resources and specific functional factor 3. Previous authors insisted In spite of diverse Innovations and improvements in different species of bamboo such as Phyllostachys remedial sciences, modern world is yet to overcome edulis 4, Dendrocalamus hamiltonii 1, Sasa borealis 5, various diseases like HIV, neurodegenerative, diabetes Dendrocalamus strictus 3, Bambusa vulgaris ‘Vittata’ 6,7 and many more. Many of these ailments are because of are rich source of antioxidants 8. overproduction of ROS and RNS due to various metabolic reactions which in turn damages our tissues as Bambusa tulda Roxb., (Family: Poaceae; Bengal a result of oxidative stress. Therefore, the secondary bamboo) 9 locally addressed as Owa gubwai (Bodo) Jati metabolites of plants as phytomedicine that could banh (Assamese) 10 , is considered to be one of the most produce a definite physiological action on human body useful bamboo species and is extensively in paper pulp have been an imperative element of the diet since the industry in India. It can grow up to 15m height, 8cm days of yore 1. Thus researchers are attempting to find thickness 11 and commonly found in southeastern Asia. In cure using these natural antioxidants occurring in food India, it is endemic North Eastern parts of the country and medicinal resources to substitute synthetic ones. and West Bengal 12 . In North-eastern part of India, this species of bamboo is also valued as medicine. The fresh Thus in recent years interest on natural antioxidant juice of shoots is applied to the injury of nails while especially of plant origin has increased many folds2. decoction of fermented shoots is prescribed for tumors 12 . Bamboo species is a source of traditional medicines, However, as per our knowledge there are no studies on leaf extract of Bambusa tulda from North Eastern *Corresponding author province, India on its antioxidant potential. So, the aim Full Address : Bamboo Technology, Department of Biotechnology, Bodoland of this study was to evaluate in vitro antioxidant activity University, Kokrajhar-783370, B.T.A.D, Assam, India of aqueous methanolic extract of Bambusa tulda leaves Phone no. +91 8256002212 along with preliminary phytochemical analysis. Attempts E-mail: [email protected] 81 Dey et al. Int. J. Fund. Appl. Sci. Vol. 4, No. 3 (2015) 81-86 have also been made to quantitatively estimate the concentrations of BME (0.2–2.0 mg/mL) in 1 mL of phenolics and flavonoids content. DDW was mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 2. Materials and Methods mL, 1%). The mixture was incubated at 50°C for 20 min. 2.1 Plant material collection and extraction A portion (2.5 mL) of TCA (10%) was added to the Bambusa tulda leaves were collected from the forests of mixture, which was then centrifuged at 3,000 rpm for 10 Kokrajhar District, BTAD, Assam, India during winter min. The upper layer of the solution (2.5 mL) was mixed session, 2013. Identified and authenticated by a with DDW (2.5 mL) and FeCl3 (0.5 mL, 0.1%) and the taxonomist in university and a voucher specimen absorbance was measured at 700 nm. Ascorbic acid was (Voucher No. DBT/BU/ Bamboo/007) was deposited to used as a reference standard. Phosphate buffer (pH 6.6) Bamboo technology, Bodoland University, Kokrajhar, was used as blank solution. BTAD, Assam, India. 10 grams of mechanically grinded 2.4.3 Hydrogen peroxide scavenging activity dry leaf samples were extracted in a Soxhlet apparatus The ability of the extracts to scavenge hydrogen peroxide using 150ml of 70% aqueous methanol ( w/v ) separately was determined according to the method of Ruch et al. 21 . at boiling temperature for 6 h. The extract was then An aliquot of H O (2mM) and sample at various filtered using whatman filter paper (no 42). The obtained 2 2 concentrations (0.2–2.0 mg/mL) were mixed (1:0.6 v/v) extract was reduced at rotary evaporator at 60-80°C and incubated for 10 min at room temperature. After under reduced pressure, followed by lyophilization using incubation, absorbance was read at 230 nm was a freeze drier until constant weight was obtained then determined against a blank solution containing phosphate stored at 4°C until required 13 . Before use, the methanolic buffer without hydrogen peroxide. The percentage extract (BME) was dissolved in double-distilled water scavenging activity of hydrogen peroxide was calculated (DDW) in desired concentrations. using the following equation. 2.2 Preliminary Phytochemical Screening H2O2 activity (%)= Abs (control)- Abs (sample)/ Abs The presence or absence of the preliminary (control) *100 phytochemical constituents such as reducing sugars, Where, Abs (control): Absorbance of the control and carbohydrates, saponnins, tannins, alkaloids, anthraquinones, steroids, flavonoids, glycosides of the Abs (sample) : Absorbance of the extracts/standard. sample were analyzed using previously explained 2.5 Statistical analysis standard methods 14,15 . Results were calculated as the mean_s.d. for each 2.3 Determination of Biochemical Constituents sample. Statistical analysis was done with one-way The total soluble phenolics (TPC) were determined by ANOVA with Graph Pad Prism, ver. 4.0 (Graph Pad Singleton and Rossi 16 method with slight modifications Software, San Diego, CA, USA). A significant difference using gallic acid equivalent (mg GAE/g) as a standard 6. was judged to exist at a level of p < 0.05. The total flavonoid content (TFC) was determined 3. Results according to Zhishen et al. 17 with minor modifications 3.1 Preliminary phytochemical screening using quercetin equivalent (mg QE/g) as a standard 18 . The modified method of Goyal et al. 19 was used to The preliminary phytochemical screening of aqueous estimate the total flavonols using quercetin equivalent methanolic exact of B. tulda leaf was depicted in table I . (mg QE/g) as a standard. Table I: Preliminary phytochemical screening of 2.4 In vitro Antioxidant properties of the extracts Bambusa tulda leaf extract Three different in vitro test systems viz. DPPH Chemical compounds Results scavenging activity, ferric reducing power assay and Saponins + hydrogen peroxide scavenging activity were used to Steroids + access the antioxidant potential of the BME. Alkaloids + 2.4.1 Free Radical Scavenging Activity (DPPH Method) Tannins + The antioxidant activity of B. tulda leaf extracts and standard were assessed on the basis of the radical Carbohydrates + scavenging effect of the stable DPPH free radical as per Flavonoid + the modified protocol by Goyal et al. 6. Anthraquinone + 2.4.2 Ferric reducing power assay (FRP) Glycosides + The reducing power of the extracts was determined Reducing sugars + according to the method of Oyaizu 20 . Different - = Compound not detected; + = Compound detected 82 In vitro antioxidant profiling of Bambusa tulda Roxb. Int. J. Fund. Appl. Sci. Vol. 4, No. 3 (2015) 81-86 Figure I: DPPH scavenging activity of aqueous methanolic extract of Bambusa tulda leaf Figure II: Reducing power assay of aqueous methanolic extract of Bambusa tulda leaf 3.2 Determination of Biochemical constituents compared to the standard ascorbic acid is shown in The biochemical constituent such as total phenolic, figure I . flavonoid, flavonol content in aqueous methanolic 3.4 Reducing power assay extract of Bambusa tulda were found to be 17.494±0.01 The ferric reducing power capability of plant extracts mg GAE /g, 176.35±0.03 mg QE /g and 96.2±0.01 mg compare to BHT as depicted in figure II . QE /g respectively. 3.5 H 2O2 Scavenging activity 3.3 DPPH Scavenging activity The DPPH scavenging The H O scavenging activity of different concentration activity of different concentration of leaf extracts 2 2 of leaf extracts compound to standard ascorbic acid is 83 Dey et al.