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Epaltes Divaricata L Glorybai et al. Annals of Clinical Microbiology and Antimicrobials (2015) 14:18 DOI 10.1186/s12941-015-0074-4 RESEARCH Open Access Some biological activities of Epaltes divaricata L. - an in vitro study Leela Glorybai1, Barathi Kannan K2, Mariadhas Valan Arasu3*, Naif Abdullah Al-Dhabi3 and Paul Agastian2* Abstract Background: Novel chemical molecules recovered from endangered medicinal plants have wide applications and have the potential to cure different diseases caused by microorganisms. The aim of this study was to investigate In vitro antimicrobial, α-glucosidase inhibition and antioxidant activity of different solvent extracts of Epaltes divaricata L. Methods: Antimicrobial activity of hexane, ethyl acetate and methanol extract of Epaltes divaricata was determined against bacteria and fungi using disc diffusion and microdilution method respectively. α-glucosidase inhibition, Total phenolic content (TPC), Reducing power activity, DPPH radical scavenging assay, hydroxyl radical scavenging activity, nitric oxide scavenging activity, superoxide scavenging activity and lipid peroxidation assay of plant extracts were performed according to standard protocol. Compound detection from the potential solvent extract was done through GC-MS analysis. Results: Epaltes divaricata ethyl acetate extracts (EDEa) (1.25 mg/disc) showed significant inhibition for E. lentum (23 mm), E. aerogenes (18 mm), P. fluorescence (15 mm) and A. baumanii (15 mm). Minimum inhibitory concentration (MIC) of EDEa was found to be 31.25 μg/ml, 62.5 μg/ml and 62.5 μg/ml against A. flavus, A. niger and T. rubrum respectively. EDEa showed more α-glucosidase inhibition and antioxidant activity compared to hexane and methanol. EDEa showed 50% α-glucosidase inhibition at the concentration of 525.20 ± 2.37 μg/ml. The TPC of EDEa was 412.0 ± 2.21 mg of catechol equivalents/g extract. EDEa showed great scavenging activity on 2,2-diphenyl-picrylhydrazyl (DPPH) (IC50 560 ± 2.02 μg/ml), hydroxyl (IC50 314.75 ± 2.56 μg/ml), nitric oxide (IC50 648.20 ± 2.09 μg/ml) and superoxide (IC50 361.14 ± 1.45 μg/ml) radicals, as well as high reducing power. EDEa also showed a more suppressive effect on lipid peroxidation. Using Antioxidant β-carotene linoleate method, the scavenging values of EDEa was significantly lower than BHT. GC-MS analysis of EDEa showed maximum amount of 2-butenamide, N-(4-fluorophenyl)-3-methyl trans-cinnamyl tiglate silane and trichlorocyclohexyl silane (36.86%). Conclusion: The results obtained in this study clearly indicate that EDEa can be used as a natural antimicrobial, α-glucosidase inhibition and antioxidant agent. Keywords: Antimicrobial activity, α-glucosidase inhibition, Antioxidants activity, Epaltes divaricata,GC-MSanalysis Background marked rise in scientific interest in the pharmacological Epaltes divaricata (L.) (Family: Compositae), a plant activities of medicinal plants, particularly in relation to used in traditional Ayurvedic medicine, is found in Sri folklore medicine [1]. In recent years, methicillin and Lanka, India, Myanmar, Java and China. It is used to multiple antibiotic drug resistant strains of Staphylococ- alleviate jaundice, urethral discharges and acute dyspep- cus aureus (MRSA) have caused outbreaks in hospital sia, and is also regarded as a diaphoretic, diuretic and a related infections throughout the world, and the WHO stimulating expectorant. Currently, there has been a puts the international prevalence of nosocomial infec- tions at 9%. Across the world, infectious disease is the * Correspondence: [email protected]; [email protected] primary cause of death, accounting for approximately 3Department of Botany and Microbiology, Addiriyah Chair for Environmental one-half of all fatalities in tropical countries; infectious Studies, College of Science, King Saud University, Riyadh 11451, Saudi Arabia disease mortality rates are all increasing in developing 2Department of Plant Biology and Biotechnology, Loyola College, Chennai 600034, India countries and between 1981 and 1992, deaths from Full list of author information is available at the end of the article © 2015 Glorybai et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Glorybai et al. Annals of Clinical Microbiology and Antimicrobials (2015) 14:18 Page 2 of 11 infectious disease increased to some 58% [2]. These soaked in hexane, ethyl acetate and methanol for 72 h negative health trends obviously point out the need for respectively with intermittent shaking [7]. After 72 h, renewed interest and novel strategies for the prevention the solution was filtered and the filtrate was concentrated and treatment of infectious diseases focused on the de- under reduced pressure using a rotary vacuum evaporator; velopment of new antimicrobials [3]. the crude extracts were then collected and stored at 4°C in Antioxidants are substances that delay or inhibit oxida- an air tight container until further use. tive damage to a target molecule. Antioxidant molecules react with single free radicals and can neutralize free radi- Assessment of antimicrobial activity cals by donating one of their own electrons, thereby end- Test organisms ing the carbon-stealing reaction. Antioxidants also act as The following bacterial cultures were used to test anti- scavenger and so prevent cell and tissue damage and cells bacterial test- Staphylococcus aureus (MRSA), Staphylo- provide defense against excessive free radicals by initiating coccus aureus (Methicillin sensitive), Eubacterium preventative and repair mechanisms, repair mechanisms lentum, Bacillus subtillis, Enterobacter aerogenes, Vibrio and physical and antioxidant defenses. A wide variety of fishcherii, Acinetoactor baumanii (ICMR), E.coli (ATCC plant constituents have been shown to be free-radical 25922), Erwinia amylovora (MTCC 2760), Pseudomonas scavengers [4] a fact that has encouraged the work de- fluorescence, Klebsiella pneumonia. The following fungal scribed here, in which solvent extracts of the medicinal cultures were used for antifungal test- Aspergillus flavus, herbs have been evaluated for both antimicrobial and anti- Botrytis cinerea, Curvularia lunata 46/01, Aspergillus oxidant activity [5,6]. Epaltes divaricata L. is a seasonal niger MTCC 1344, Trichophyton rubrum 57/01 and T. medicinal plant occurs only during the month December, mentagrophytes 66/01. All cultures were obtained from not much explored for its bioactivities; very scanty work IMTECH, Chandigarh, India and the clinical isolates has been carried out in the test plant Epaltes divaricata were obtained from the Department of Microbiology, (L.). Hence the present study, is taken up to investigate the Christian Medical College, Vellore, Tamil Nadu, India. solvent extracts for antimicrobial, antioxidant, anti- diabetic followed by identification of active compounds Disc diffusion method using GC-MS. Antibacterial assay was carried out using disc diffusion method [8]. Petri plates were prepared using 20 ml of Materials and methods sterile MHA (Hi-media, Mumbai). The test culture Chemicals and reagents (100 μl of suspension containing 108 CFU/ml bacteria) DPPH (1,1-diphenyl,2-picrylhydrazyl), NBT (nitro blue was swabbed on the surface of the solidified media and tetrazolium), NADH (nicotinamide adenine dinucleotide allowed to dry for 10 minutes. Sterile discs (6 mm diam.) phosphate reduced), PMS (phenazine methosulphate), were impregnated with 25 μl (2.5 mg) of hexane, ethyl TCA (trichloro acetic acid), ferric chloride and BHT acetate and methanol extracts of the test plants. The (butylated hydroxyl toluene) were obtained from Sigma discs were then dried at 37°C in laminar air flow cham- chemical co., USA. Ascorbic acid was obtained from SD ber before use. Streptomycin (10 μg/disc) was used as a fine chem. Ltd., Biosar, India. b-Carotene, ferrozine, positive control. Plates were incubated overnight at 37°C folin– phenol reagent and Tween 40 were purchased for the measurement of zone of inhibition (in mm) from Hi-Media Pvt. Ltd. Mumbai, India. All the other around the disc. All experiments were repeated in chemicals were of analytical grade. triplicate. Collection of Epaltes divaricata L Antifungal assays using broth micro dilution method Epaltes divaricata L. plants were collected on the banks of The filamentous fungi were grown on SDA slants at 28°C Oragadam fresh water lake near Chennai, Tamil Nadu, for 7 days. The spores were collected using sterile double India. Epaltes divaricata L. was identified and authenti- distilled water and stored in refrigerator until used. Anti- cated by Dr. J. Jeyajothi (Taxonomist), Department of fungal activity was performed by following the method of Plant Biology and Biotechnology, Loyola College, Chennai, Rejiniemon et al. 2014 [9]. The extracts were dissolved in India. A voucher specimen (LCH-71) was deposited at the water with 2% DMSO (concentration of the extract was Loyola College Herbarium (LCH) in Loyola College, 2 mg/ml). The initial test concentration was serially di- Chennai. luted two-fold in 96 well plate. Each well was inoculated with 5 μl of suspension containing approximately
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