7th International Conference on Medical, Biological and Pharmaceutical Sciences (ICMBPS'2015) June 17-18, 2015 Pattaya (Thailand) Antibacterial Activity of Nymphaea Pubescens Willd. Leaves Kessaya Waidee, Surang Chankhamhaengdecha, and Praneet Damrongphol Pseudomonas aeruginosa, Bacillus subtilis, Bacillus cereus, Abstract—Antibacterial activity of Nymphaea pubescens Willd. Escherichia coli, Xanthomonas campestris, and Candida leaves against human pathogens was studied using agar well albicans [4]. The methanolic extract of N. alba showed diffusion method. The methanolic extract showed high antibacterial antibacterial activity against Streptococcus pyogenes, S. activity. The extract inhibited growth of six Gram-positive bacteria, aureus, S. epidermidis, and Enterobacter cloacae [5]. The i.e., Bacillus cereus, Bacillus cereus ATCC 14875, Staphylococcus ethanolic extract of N. lotus leaves inhibited growth of aureus 980, Staphylococcus aureus ATCC 25923, Methicillin- methicillin resistant S. aureus (MRSA) and vancomycin resistant Staphylococcus aureus (MRSA), and Enterococcus faecium, but did not inhibit growth of five Gram-negative bacteria tested, i.e., resistant S. aureus (VRSA), S. aureus, S. pyogenes, E. coli, P. Escherichia coli (ETEC), Escherichia coli 1175, Serratia aeruginosa, and Klebsiella pneumonia [6], [7]. Bioactive marcescens, Salmonella Typhi, and Shigella sonnei. B. cereus and B. compounds such as tannins, flavonoids, alkaloids, cereus 14875 were most sensitive to 100% methanolic extract. Thin anthraquinones, saponins, glycosides, ellagic acid, and phenols layer chromatography-bioautography of 100% methanolic leaf extract in the genus Nymphaea have been identified [7], [8]. The revealed three major antibacterial constituents at Rf 0.33, 0.38, and present study investigated antibacterial activity of N. 0.82. Purification of active constituents of N. pubescens leaf extract pubescens leaf extracts against human pathogens using agar in further study may lead to clinical application. well diffusion method. Furthermore, active constituents of the extract showing strong antibacterial activity were also Keywords—Antibacterial, Bioautography, Human pathogens, examined using thin layer chromatography (TLC) and Nymphaea pubescens, Thin layer chromatography bioautography method. I. INTRODUCTION II. METHODOLOGY YMPHAEA pubescens Willd (Water lily) is a floating N aquatic rhizomatous plant. The floating leaves are large A. Plant materials and round with a radial notch. The leaves are papery covered N. pubescens were collected from Bueng Boraphet wetland, with fine hairs. N. pubescens is widely distributed in tropical Nakhon Sawan province, central Thailand. The leaves were and temperate regions of Asia and is a dominant aquatic plant washed and dried at room temperature. The dried samples species in Bueng Boraphet wetland, central Thailand. were cut into small pieces and ground to powder. Leaves, flowers, roots and rhizomes of N. pubescens have a wide range of pharmaceutical activities; they have been used in B. Preparation of plant extracts treating diabetes, fever, liver disorders, jaundice, and tumors Maceration extraction method based on solvent polarity was in traditional medicine [1][3]. Plants in the Genus Nymphaea used. The powdered samples were extracted with 100% or show an ability to inhibit bacterial growth depending on plant 80% of acetone, diethyl ether, hexane, ethanol, or methanol. species, part of the plant and extraction method. The After shaking of the extraction solutions for 4 days, the methanolic extract of N. nouchali leaves showed antimicrobial solutions were filtered using Whatman No.1 filter paper. The activity against Staphylococcus aureus, Pseudomonas aureus, crude extracts were dried in a rotary evaporator (Multivapor P- 12, Buchi, Switzerland) and were re-dissolved in 10% dimethyl sulfoxide (DMSO) for determination of antibacterial activity. Kessaya Waidee is with the Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand (corresponding author to C. Antibacterial susceptibility test provide phone: +66-2201-5381; fax: +66-2354-7161; email: Crude extracts were screened for antibacterial activity using [email protected]) Surang Chankhamhaengdecha is with the Department of Biology, Faculty agar well diffusion method [9]. A total of 11 human pathogens of Science, Mahidol University, Bangkok 10400, Thailand (email: were tested: six Gram-positive bacterial species, i.e., B. cereus, [email protected]) B. cereus 14875, S. aureus 980, S. aureus 25923, MRSA, and Praneet Damrongphol is with the Department of Biology, Faculty of Enterococcus faecium, and five Gram-negative bacterial Science, Mahidol University, Bangkok 10400, Thailand (email: [email protected]) species, i.e., E. coli ETEC, E. coli 1175, S. marcescens, S. This work was funded by Mahidol University and partially by the Center Typhi and S. sonnei. Each bacterial type was streaked on a for Environmental Health, Toxicology and Management of Chemicals under nutrient agar (NA) and incubated at 37°C for 1624 h. A the Science and Technology Postgraduate Education and Research Office of single colony was selected and cultured in Muller-Hilton broth the Ministry of Education, Thailand. (MHB) for 36 h and then suspended in 0.85% saline. The 62 7th International Conference on Medical, Biological and Pharmaceutical Sciences (ICMBPS'2015) June 17-18, 2015 Pattaya (Thailand) density of bacterial suspension was adjusted to a 0.5 developed in a chamber saturated with eluting vapor. TLC McFarland turbidity standard that corresponds to 1×108 plates were visualized under ultraviolet light (254 and 360 CFU/ml. Seven wells in a Muller-Hilton agar (MHA) plate nm). were cut using a sterile cork borer. Bacteria from the inoculum F. Bioautography overlays were streaked in three directions on the surface of MHA plate using sterile cotton swab. A 50 µl of extract (5 mg/well), Antibacterial constituents of the active extract were positive control (ampicillin, 10 µg/well), or negative control analysed using bioautographic method [11]. Bacterial (10% DMSO) was loaded in the well. The plate was incubated suspension (B. cereus, B. cereus 14875, S. aureus 980, at 37°C for 1624 h. A clear zone indicated the inhibition of S.aureus 25923, MRSA, or E. faecium) in MHB was adjusted bacterial growth. The diameter of each zone of inhibition was to a 0.5 McFarland turbidity standard. The TLC developed measured using vernier ruler. plates were covered with bacterial suspension, and incubated at 37°C for 24 h. After incubation, the TLC plates were D. Determination of minimum inhibitory concentration and sprayed with aqueous solution of p-iodonitrotetrazolium violet minimum bactericidal concentration (2 mg/ml) and incubated at 37°C for 26 h. A clear zone Minimum inhibitory concentration (MIC) and minimum indicated the inhibition of bacterial growth. bactericidal concentration (MBC) of the active extract were determined using broth microdilution assay [10]. A 50 µl of tryptic soy broth (TSB) was pipetted into each well of a flat- III. RESULTS bottomed 96-well microplate. Then, 50 µl of various Antibacterial activity of various N. pubescens leaf extracts concentrations of extract, positive control, or negative control against Gram-positive pathogens is shown in Tables I. The was added. Ampicillin was used as positive control and 10% diameter of inhibition zone ranged between 10.18±0.25 mm DMSO was used as negative control. A 50 µl of bacterial and 20.50±0.35 mm. The methanolic extracts were found to be suspension (1×105 CFU/well) was added to each experimental most effective giving large zones of inhibition. The ethanolic well. The plate was covered and incubated at 37°C for 24 h. The MIC was determined from the concentration of the extract and acetone extracts were less effective. Whereas, the hexane in the well showing invisible growth. The MBC was and diethyl ether extracts did not inhibit growth of any determined by subculturing 50 µl of the solution from MIC bacterial species tested. All leaf extracts of different extraction well and spread on the MHA plates. The plates were incubated solvents did not inhibit growth of the five Gram-negative at 37 °C for 24 h. The MBC was determined as the lowest bacteria tested. concentration of extract that showed no visible growth of The 100% methanolic extract, the most effective extract bacteria in the plate. giving large zone of inhibition, was determined for the MIC and MBC values against various Gram-positive bacteria E. Thin layer chromatography (Table II and Table III respectively). The MIC values ranged The chemical profile of active extract was determined using between 0.45 mg/ml and 1.8 mg/ml. B. cereus, B. cereus TLC method. The extract was loaded on a 10 cm×10 cm 14875, and MRSA were most sensitive to 100% methanolic aluminum baked thin layer silica plates (Merck silica gel 60 extract showing MIC values of 0.45 mg/ml. B. cereus and B. F254, Germany) and developed in a solvent system of ethyl cereus 14875 were also killed at the same concentration of acetate: methanol: water (40:5.4:4,v/v/v).The plates were extract as shown by the MBC values of 0.45 mg/ml. But higher concentration of extract was required to kill MRSA as shown by higher MBC value of 0.9 mg/ml. TABLE I ANTIBACTERIAL ACTIVITY OF VARIOUS N. PUBESCENS LEAF EXTRACTS AGAINST GRAM-POSITIVE BACTERIA Extraction solvent Average diameter of inhibition zone±SD (mm) B. cereus B.cereus 14875 S. aureus 980 S. aureus25923 MRSA E. faecium Acetone 100% 13.24±0.12 12.62±0.16 – – – – 80% 13.10±0.24 13.43±0.80 17.18±0.06 15.45±0.11 18.26±0.13 – Diethyl ether 100% – – – – – – 80% – – – – – – Hexane 100% – – – – – – 80% – – – – – – Methanol 100% 15.34±0.65 15.88±0.23 20.13±0.33 19.43±0.29 20.50±0.35 12.56±0.24 80% 10.38±0.32 13.04±0.82 15.95±0.28 11.67±0.33 14.69±0.56 11.05±0.12 Ethanol 100% 14.24±0.26 12.55±0.64 12.27±0.44 14.45±0.33 11.53±0.74 – 80% 10.18±0.25 12.11±1.05 13.42±0.41 13.30±0.41 11.22±0.22 – Ampicillin 17.50±0.22 16.92±0.46 16.644±0.44 32.40±0.14 19.92±0.15 24.38±0.80 –, not inhibited.