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Evaluation of Antioxidant and Antimicrobial Activities of Bergenia Ciliata Sternb (Rhizome) Crude Extract and Fractions

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Evaluation of Antioxidant and Antimicrobial Activities of Bergenia Ciliata Sternb (Rhizome) Crude Extract and Fractions Evaluation of antioxidant and antimicrobial activities of Bergenia ciliata Sternb (Rhizome) crude extract and fractions Aman Khan,1,2 Gul Jan,3 Afsar Khan,4 Farzana Gul Jan3 and Muhammad Danish1 1Department of Botany, Hazara University, Mansehra, Pakistan 2School of Life Science, Lanzhou University, Lanzhou, China 3Department of Botany, Abdul Wali Khan University, Mardan, Pakistan 4Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad, Pakistan Abstract: Biologically, screening of medicinal plants extracts have been since pre-historic to determine the antioxidants and antimicrobial profile. The present study was aimed to investigate and evaluate crude extract and different fractions of Bergenia ciliata Sternb (rhizome) for bioactivity which is most considerable medicinal plants. The chloroform fraction was found to be highly anti-oxidative with the IC50 value (4.15±0.82) as compare to ethyl acetate and n-hexane fraction. In addition, neither crude extract nor any fraction showed inhibition against fungal strains, i.e. Aspergillus flavus and Aspergillus niger. Furthermore, the crude extract and fractions of B. ciliata (rhizome) exhibiting promising activities against Bacillus atrophoeus, Bacillus subtilis, Kleibsiella pneumonia and Pseudomonas aeruginosa strains. In summary, B. ciliata is recommended as a source of bioactive compounds which might be used against oxidative stress and drug resistance bacteria. Keywords: Bergenia ciliata, antioxidant, antifungal, antibacterial. INTRODUCTION protection to human health against infectious and degenerative disease (Rajkumar et al. 2010). Furthermore, The northern areas of Pakistan are rich in biodiversity and the crude extract of genus Bergenia was more active contain more than 400 species of medicinal plant. The against microorganisms such as Bacillus cereus, evergreen perennial herb Bergenia ciliata Sternb is tall Escherichia coli, Staphylococcus aureus, Pseudomonas from 30 cm to 150 cm (Rajkumar et al. 2010), belong to aeruginosa, and Candida albicans (Kokoska et al. 2002). family saxifragaceae (Jan et al. 2014), the genus consist Therefore, the Bergenin compound which isolated from of six species found in the Himalayas and Central and Bergenia was found highly anti-oxidative, antimicrobial East Asia (Siddiqa et al. 2012), while with two species and as well as for xanthine oxidase inhibitory activities namely Bergenia ciliata and Bergenia stracheyi were (Nazir et al. 2011). The evaluations of plant extracts were found in Pakistan temperate Himalayan region (Awan et usually done through different techniques to ascertain al. 2011). In the traditional medicine, the genus Bergenia antioxidants and inhibition effects on pathogenic and non- is in the curing of several disease Nepal, India, Pakistan, pathogenic microorganism (Sati & Joshi 2010). Thus the Bhutan and some others developing countries (Roselli et aim of the present study was to evaluate antioxidant and al. 2012). Mostly the rhizome of Bergenia is used as an antimicrobials properties of crude extract and fractions of ideal tool for kidney and gall bladder stone, cough, cold, B. ciliata (rhizome). wound, cuts, burn and inflammation and wounds (Hamayun 2005; Chowdhary et al., 2009). Moreover, the MATERIALS AND METHODS Bergenia ciliata used as folk medicine to cure diabetic mellitus (Mazhar-Ul-Islam et al., 2002), anti-diabetic, Plant material expectorant, body tonic, sunburn, pimple, stomachache, The Bergenia ciliata rhizome was collected from the muscular pain and running eyes (Bano et al., 2014) and Kaghan valley Khyber Pakhtunkhwa, Pakistan (fig. 1). for stomachic, diuretic, cardio active and skin diseases The plant species was deposited in Herbarium of Hazara (Ahmad et al., 2009). University Mansehra, Pakistan with the voucher number 3556. Plants have been source of medicinal compound since several decades and it is well established that all parts of Extraction and fractionation plants were used in traditional medicine system for The collected plant material was dried out at room centuries. However, the discovery of new herbal products temperature and grinded into fine powder. The powder led popularity as compare to synthetic drugs in the disease plant material soaked in methanol (commercial grade) for therapy (Sajad et al. 2010). Similarly, the antioxidant and 12 days three times. The extract was filtered using antimicrobial agents from natural resources provides Whitman filter paper (No.1). The residue plants material was soaked again in methanol and the solvent were *Corresponding author: e-mail: [email protected] Pak. J. Pharm. Sci., Vol.31, No.1, January 2018, pp.031-035 31 Evaluation of antioxidant and antimicrobial (fungal and bacteria) activities of Bergenia ciliata Sternb (Rhizome) crude vaporized at 40°C through rotary evaporator (Sengul et al. C. Petri plates and tips were also autoclaved. Two strains 2009). The process repeated three times and the methanol of fungus were used i.e. Aspergillus niger and Aspergillus crude extract was obtained. The methanolic crude extract flavus. The plant extract was prepared in the dimethyl was suspended in distilled water with constant stirring and sulfoxide (DMSO) by dissolving 1mg/ml and 25ml of fractionated with n-hexane, chloroform, ethyl acetate as media was poured in each Petri plate. Media was allowed well as n-Butanol respectively (Rahman et al. 2008; Nisar to solidify. Spores of fungus were applied in the center of et al. 2010). The solvents were evaporated at 40°C the plate. Sterile paper discs were placed in front of the through Rotary Evaporator and different fractionations growing end of the mycelium. Sample was applied on the were obtained. sterile paper discs. Each sample was applied in triplicate. The culture was allowed to grow for four to seven days at Chemicals 26°C. Fluconazole was used as standard (positive Propyl gallate (PG), 1,1-diphenyl-2-picrylhydrazyl control). Dimethyl sulfoxide (DMSO) was used as (DPPH), 3-Tert-Butyl-4- Hydroxyanisole (TBH), negative control. Crescent shape of fungi was appeared in Dimethyl sulfoxide (DMSO), Sabourud dextrose agar front of paper discs, which shows an inhibition. Zone of (SDA), Nutrient agar and Nutrient broth. All chemical inhibition was measured according to (Nisar et al. 2011). Linear growth in test (mm) and reagents were purchased from Merck (Germany) and Inhibition of fungal growth = 100 - ×100 Sigma Aldrich (USA). Linear growth in control (mm) Antibacterial assay Preparation of stock solution Antibacterial activities of the plant crude extract and Methanolic crude extract and different fractions (n- different fractions were determined by using paper disc Hexane, Chloroform, Ethyl acetate and n-Butanol) of the diffusion method (Jagessar et al. 2008). Media was plant was dissolved in Dimethyl sulfoxide (DMSO) with prepared by dissolving 28g of nutrient agar and 13g of the concentration of 1 mg/ml evaluated for biological nutrient broth in 1000ml of distilled water in flask. The potential (Jacinto et al., 2011). nutrient broth was taken approximately 7-8ml per test tube. All the apparatus and media viz Petri plates, tips, Antioxidant assay Whitman filter paper disc and normal saline etc used in The plant extract was examined for their antioxidant the activity were autoclaved for 20min at 121°C. After activity on the basis of scavenging effects using stable 1, sterilization nutrient agar were poured into the Petri plates 1-diphenyl-2-picrylhydrazyl (DPPH) method (Miliauskas in the laminar flow hood allowed to solidify and placed in et al. 2004). Freshly DPPH solution were prepared and the incubator at 37°C to avoid any contamination. The reserved in dark at 4°C 1, 1-diphenyl-2-picrylhydrazyl bacterial stock culture were freshened on the nutrient agar (DPPH) (3.2µl) dissolved in 25ml methanol steering plates by streaking with sterile inoculation loop in laminar regularly up to 30 minutes. Methanolic solution of DPPH flow hood culture were incubated at 37°C for 24 hours. (90 µl) was added to 10µl of plant extract solution with The bacterial culture were inoculated into the sterilized different concentration. The mixtures were incubated for nutrient broth in the flask containing approximately 20 - 30 minutes at 37°C. After incubation the absorbance 25 ml broth media were then incubated in the shaking were measured at 490 nm using Multiplate reader (Bio- water bath (Model GLSC-SBR-04-28) for 16 hours at 200 Tek Elx800 TM, Instruments, Inc. USA). Propyl gallate rpm at 37°C. The bacterial cultures from the flask were (PG) and 3-tert-butyl-4-hydroxyanisol (TBH) were used diluted in test tube containing sterilized nutrient broth for as standard while dimethyl sulfoxide (DMSO) as negative standardization by comparing with 0.5 McFarland control. All determination was performing in triplicate. (turbidity) standards. 50µl of standardized bacterial Percentage inhibition of the radical scavenging activity of culture were spread on each nutrient ager plates with the test sample as compare with the standard was measure help of glass spreader. These impregnated plates were according to the equation. refrigerated for 10 mints for absorption after refrigeration (Absorbance of control Absorbance of sample) impregnated plates brought to laminar flow to place the Inhibition (%) = ×100 Absorbance of control filter paper disc (6mm in diameter) with the help of sterilized forceps. The samples (6µml) were applied on The extracts concentration giving 50% inhibitions (IC50) each paper disc. Azithromycin and
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