22 Antibacterial and Antifungal Activities of the Essential Oil and Crude Extract of Piper betle L. Against Microorganism Contaminated in public toilets 1 Prapassorn Rugthaworn 1 Uraiwan Dilokkunanant 1 1 Udomlak Sukatta 1 Email-address : aapuls@ku.ac.th water steam distillation 10.53 0.18 10 Staphylococcus aureus, S. epidermidis, Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Shigella flexneri, Klebsiella pneumoniae, Proteus mirabilis, Vibrio cholerae Bacillus cereus 8 ; Aspergillus niger, A. flavus, Penicillium sp., Cladosporium cladosporioides, Chaetomium globosum, Rhizopus oligosporus Curvularia lunata Candida albicans disc diffusion method contact assay S.aureus Curvularia lunata 32-2,048 g/ml 512-4,096 g/ml 128-4,096 g/ml 128-1,024 g/ml
2 May - August 2010 ABSTRACT Ethanolic extraction of betel vine leaves by maceration technique gave 10.53 percentage yields, while betel oil getting from water and steam distillation technique gave 0.18 percentage yields. Extracts and essential oils betel leaf were tested for effectiveness against ten bacterial species; Staphylococcus. aureus, S. epidermidis, scherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Shigella flexneri, Klebsiella pneumoniae, Proteus mirabilis, Vibrio cholerae and Bacillus cereus and eight fungal species; Aspergillus niger, A. flavus, Penicillium sp., Cladosporium cladosporioides, Chaetomium globosum, Rhizopus oligosporus, Curvularia lunata and Candida albicans which are microorganism contaminated in public toilets. Both extracts, betel extract and betel oil, inhibited growth of all tested microorganism. Ethanolic betel extract showed higher tendency to inhibit bacterial growth than fungal growth, and S. aureus was the most sensitive to the extract. Betel oil showed higher inhibitory effect to fungal growth than bacterial growth and gave the highest inhibitor effect against Curvularia sp. Minimal Inhibitory Concentration (MIC) of betel extract against the bacteria and fungi ranged between 32-2,048 g/ml and 512-4,096 g/ml. MIC of betel oil ranged between 128-4,096 g/ml and 128-1,024 g/ml, respectively. KEYWORD : Piper betle, betel oil, betel extract, fungi, bacteria, toilet 23 Piper betle Linn. Piperaceae 1,2 3 7 Aspergillus flavus, Penicillium sp., Fusarium sp., Phoma sp., Cladosporium sp. Curvularia lunata 100,000 ppm. 31 Acinetobacter baumannii, Bacillus cereus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella spp., Shigella flexneri, Staphylococcus aureus, Streptococcus mutans Streptococcus pyogenes 4 8 Aspergillus japonicus, A. niger, Eurotium amstelodami, E. chevaleiri, Penicillium sp., P. camemberti Fusarium sp. 5
24 Staphylococcus aureus, S.epidermidis, Pseudomonas aeruginosa Escherichia coli 3,5,6,7,8,9,10,11 : 3 flask 500 3 3 3 rotary evaporator : 500 water steam distillation 8 sodium sulfate anhydrous sodium sulfate anhydrous 4 10 ; S. aureus (DMST 8840), S. epidermidis (DMST 15505),E. coli (TISTR 780), Salmonella typhimurium (TISTR 292), Pseudomonas aeruginosa (TISTR 781), Shigella flexneri (DMST 4423), Klebsiella pneumoniae (DMST 8216), Proteus mirabilis (DMST 8212), Vibrio cholerae (DMST 15778) Bacillus cereus (TISTR 687) 8 ; Aspergillus niger (TISTR 3245), Aspergillus flavus (TISTR 3366), Penicillium sp. (TISTR 3046), Cladosporium cladosporioides (TISTR 3286), Chaetomium globosum (TISTR 3093), Rhizopus oligosporus (TISTR 3301) Curvularia lunata (TISTR 3289) Candida albicans (TISTR 5779)
2 May - August 2010 disc diffusion method 12 swab (suspension) 1x10 8 cfu/ml Muller-Hinton Agar Potato Dextrose Agar (PDA) Dimethyl sulfoxide 100,000 ppm. 1 5 37 24 Db paper disc Da Antimicrobial index = (Db-Da)/Da Villaseñor 13 Contact assay 14 7 6 PDA ( 2 / ) cork borer 6 100,000 ppm. 10 L 37 3-5 ( PDA 5 ) Da Db (Antifungal Index) = (1-(Db/Da)) x 100 25 Resazurin Microtiter Assay Plate Method 15 Muller-Hinton Broth (MHB) Potato Dextrose Broth 100 65,536 g/ml two-fold dilution 1x10 5 cfu/ml 100 16,384, 8,192, 4,096, 2,048, 1,024, 512, 256, 128, 64, 32, 16, 8 g/ml 37 24 resazurin 0.02 30 resazurin ( ) water steam distillation 10.53
26 0.18 (Lovibond PFX 195 Tintometer) L* 2.26 a* 1.22 b* 2.11 100,000 ppm. 10 S. aureus, S. epidermidis, E. coli, Salmonella typhimurium,pseudomonas aeruginosa, Shigella flexneri, Klebsiella pneumoniae, Proteus mirabilis, Vibrio cholerae Bacillus cereus Candida albicans disc diffusion method 1 S.aureus Antimicrobial Index 4.95 3,5,7,8,9,11 Voravuthikhunchai 16 S.aureus ß-hemolytic streptococcus group A E. coli, S. aureus Salmonella derby Pseudomonas aeruginosa, Bacillus subtilis, Lactobacillus sp., Saccharomyces cerevisiae Aspergillus niger 17 Candida albicans Antimicrobial Index 0.22 Trakranrungsie 18, Trichophyton mentagrophyte, Microsporum canis, M. gypseum C.albicans 1 (.) Antimicrobial Index S. aureus (DMST 8840) 35.67 aa +0.58 13.67 ab +1.53 4.95 aa 1.28 ab S. epidermidis (DMST 15505) 10.33 da +0.58 7.00 efb +0.00 0.72 ca 0.17 efb E. coli (TISTR 780) 8.33 ga +0.29 7.00 efb +0.00 0.39 fa 0.17 efb Salmonella Typhimurium (TISTR 292) 7.83 ha +0.29 8.00 da +0.00 0.31 ga 0.33 da Pseudomonas aeruginosa (TISTR 781) 8.83 fa +0.76 7.00 efb +0.00 0.47 ea 0.14 fb Shigella flexneri (DMST 4423) 10.67 ca +0.58 9.00 cb +0.00 0.78 ca 0.50 cb
2 May - August 2010 27 (.) Antimicrobial Index Klebsiella pneumoniae (DMST 8216) 7.50i A +0.50 6.83 fa +0.29 0.25 ha 0.17 efb Proteus mirabilis (DMST 8212) 9.17 ea +0.29 7.33 eb +0.58 0.53 da 0.22 eb Vibrio cholerae (DMST 15778) 18.83 ba +0.29 10.67 bb +0.58 2.19 ba 0.78 bb Bacillus cereus (TISTR 687) 8.17 ga +0.76 6.83 fb +0.29 0.36 fa 0.17 efb Candida albicans (TISTR 5779) 7.33 ib +0.29 9.33 ca +0.58 0.22 B 0.56 ca a,b,c,...,i (p<0.05) A,B (p<0.05) S.aureus Antimicrobial Index 1.28 Pseudomonas aeroginosa Antimicrobial Index 0.14 Pseudomonas aeroginosa (200 ) S.aureus Salmonella enteridis 19 Candida sp. Antimicrobial Index 0.56 Li-Ching Jiau 20 S.aureus M.pachydermatis C.albicans hydroxychavicol, fatty acids (stearic and palmitic) hydroxy fatty acid esters (stearic, palmitic and myristic) plasma cell membrane 21 7 Aspergillus niger, A. flavus, Penicillium sp., Cladosporium cladosporioides, Chaetomium globosum, Rhizopus oligosporus Curvularia lunata Contact assay 2
28 2 (AFI) Aspergillus niger (TISTR 3245) 5.71 gb 17.60 da Aspergillus flavus (TISTR 3366) 6.53 fb 10.91 ea Penicillium sp. (TISTR 3046) 38.52 bb 54.88 ba Cladosporium cladosporioides (TISTR 3286) 35.37 cb 38.06 ca Chaetomium globosum (TISTR 3093) 20.28 db 37.66 ca Rhizopus oligosporus (TISTR 3301) 10.73 eb 17.15 da Curvularia lunata (TISTR 3289) 65.81 ab 68.45 aa a,b,c,...,i (p<0.05) A,B (p<0.05) Curvularia lunata 65.81 Aspergillus A.niger A.flavus 5.71 6.53 Alternaria alternata, Curvularia lunata, Fusarium equiseti, Macrophomina phaseolina, Botryodiplodia theobromae, Colletrotrichum corchori 50 22 13 A.niger, A.oryzae Penicillium sp. 3 100 2.2, 1.5 1.5 30-60 23 Curvularia sp. 68.45 2
2 May - August 2010 3 8 A.japonicus, A.niger, Eurotium amstelodami, E.chevaleiri, Penicillium sp. P.camemberti Fusarium sp. P.camemberti 5 A.niger, 29 A.flavus, Fusarium sp. Penicillium sp. 3 eugenol chavibetol acetate 24,25 69.7% eugenol (36.2%), chavibetol acetate (16.9%), 4-allylphenyl acetate (9.4%) 4-allylphenol (7.2%) 20 ( ) ( ) 1 ( ) S.aureus ( ) Curvularia lunata Resazurin Microtiter Assay Plate Method S.aureus 32 g/ml 128 g/ml
30 3 ( g/ml) Staphylococcus aureus (DMST 8840) 32 128 S. epidermidis (DMST 15505) 2,048 2,048 Escherichai coli (TISTR 780) 2,048 2,048 Salmonella Typhimurium (TISTR 292) 2,048 2,048 Pseudomonas aeruginosa (TISTR 781) 2,048 2,048 Shigella flexneri (DMST 4423) 512 1,024 Klebsiella pneumoniae (DMST 8216) 1,024 1,024 Proteus mirabilis (DMST 8212) 2,048 4,096 Vibrio cholerae (DMST 15778) 512 512 Bacillus cereus (TISTR 687) 1,024 1,024 Aspergillus niger (TISTR 3245) 4,096 1,024 Aspergillus flavus (TISTR 3366) 4,096 1,024 Penicillium sp. (TISTR 3046) 4,096 1,024 Cladosporium cladosporioides (TISTR 512 256 3286) Chaetomium globosum (TISTR 3093) 512 256 Rhizopus microsporus (TISTR 3301) 2,048 1,024 Curvularia lunata (TISTR 3289) 512 256 Candida albicans (TISTR 5779) 2,048 1,024 Chaetomium globosum, Cladosporium cladosporioides Curvularia lunata 512 g/ml 256 g/ml 3
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