International Journal of Science and Research (IJSR) ISSN (Online): 2319-7064 Impact Factor (2012): 3.358 Volume 3 Issue 11, November 2014 www.ijsr.net Licensed Under Creative Commons Attribution CC BY HPLC, Antioxidant and Antibacterial Activities of Methanolic Extract of Berberis aristata Stem Ovas Dar 1 , Reena Lawrence 2 , Sikander Dar 3 1, 3 Research scholar, Chemistry Department, Sam Higginbottom Institute of Agriculture, Technology and Sciences, Allahabad, U.P, India 2 Assistant Professor, Chemistry Department, Sam Higginbottom Institute of Agriculture, Technology and Sciences, Allahabad, U.P, India Abstract: The components in methanolic extract of Berberis aristata stem were identified by HPLC. The three components which were identified are Berberine (95.52%), Palmitine (2.64%) and Jatrorrhizine (1.83%). The antioxidant activity of the methanolic extract were determined by DPPH (1, 1-Diphenyl-2-picryl hydrazyl) assay and Nitric oxide scavenging method. The methanolic extract was able to reduce stable purple colored DPPH reaching 50% of reduction with IC 50 values as – IC 50 for methanolic extract=0.90 mg/ml, IC 50 for Gallic acid = 0.61 mg/ ml IC 50 for BHT = 0.41 mg/ml. The methanolic extract reduced the concentration of DPPH free radical with an efficacy near to that standard Gallic acid. In case of Nitric oxide scavenging assay, IC 50 values have been found to be 0.80 mg/ml, 0.65 mg/ml and 0.60mg/ml for the methanolic extract, gallic acid and BHT respectively. The antibacterial activity of methanolic extract was evaluated on bacteria strains like Escherichia coli, Staphylococcus aureus and Bacillus subtilis by using agar well diffusion method. The results were compared with ampicillin, a commercial antibacterial agent. Methanolic extract showed maximum (19mm) zone of inhibition against Bacillus subtilis. The MIC values of methanolic extract for different pathogenic bacteria ranged from 37 to 111μg/ml. Keywords: Methanolic extract, HPLC, IC 50 , Zone of inhibition, MIC 1. Introduction Berberis aristata commonly known as “Daru haldhi and chitra” is spinous shrub native to northern Himalayan region. The plant is widely distributed from Himalayas to Srilanka, Bhutan, and hilly areas of Nepal in Himalaya region (1). Berberis aristata is used in ayurvedic medicines from very long time. The plant is used traditionally in inflammation, wound healing, skin disease, menohrrhagia, diarrhoea, jaundice and diabetes (2). Berberis aristata contains protoberberine and bis isoquinoline type of alkaloid. Root of plant B. aristata contains alkaloids which are berbamine, berberine, oxycanthine, epiberberine, palmatine, dehydrocaroline, jatrorhizine and columbamine (3), karachine (4), dihyrokarachine, alkaloids, pakistanine, 1- Omethylpakistanine, pseudopalmatine chloride and pseudoberberine chloride were also isolated from Berberis aristata (5). Berberis aristata roots have been used in treatment of jaundice in Ayurveda. Hypoglycemic effect of Berberis aristata root was evaluated. Dried and powdered root extracted with water and methanol was administrated to normal and alloxan induced diabetic albino rabbit. The results show that B. aristata roots contain potent and orally effective antidiabetic component which either triggers the formation of insulin or shows insulin like effect (6). Ethanolic root extract of B.aristata shows antifungal activity was found against Candida species and Aspergillus species (7). Thus, the aim of this study is to investigate the chemical composition, antioxidant and antibacterial activities of methanolic extract of Berberis aristrata stem. 2. Material and Methods 2.1 Sample Preparation The dried stem of Berberis aristata was available in pieces of variable length and thickness. The dried stem was pulverized using sterile laboratory mortar and pestle to obtain the powdered form. These were stored in airtight glass containers protected from sunlight until required for analysis. 2.2 Preparation of Extract The dried and powdered plant material was extracted with methanol using soxhlet extractor for 5 h at a temperature not exceeding the boiling point of the Solvent (8). The extract was filtered and then concentrated to dryness. 2.3 HPLC HPLC fingerprinting and content determination methods were applied to evaluate methanol extract of Berberis aristata. The sample was analyzed in model Shimadzu CLASS –VP V6.14 SPI, AYOTO Japan/LC/20 ATPC. Two pumps were used STP-20 and SPD. Detector used was AVP- UV. Reverse phase HPLC column (C-18) was used with length 260X4.6mm, thickness being 5µ. Column Temperature was 36ºC. Injected volume was 10 µl. Mobile phase was methanol-water (water containing 4% acetic acid). The ratio of methanol: water being 66:34 v/v. Flow rate 1ml/min. Two detectors were of 290 and 250nm. 2.4 Antioxidant activities assay 2.4.1 DPPH Free Radical Scavenging Assay The DPPH radical scavenging activity assay elucidated by [9] was followed. The hydrogen atom or electron donating ability of the corresponding methanolic extract was Paper ID: OCT141038 1137