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Antimicrobial activities of the ethanol extracs of capsium fruits with different pungencies

JMS Vol. 2 No. 2, hal. 57 - 63, Oktober 1997 Antimicrobial Activities of the Ethanol Extracts of Capsicum Fruits
with Different Levels of Pungency
S. Soetarno*, Sukrasno, E. Yulinah and Sylvia Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Jl. Ganesa 10, Bandung, 40132 - Indonesia Diterima tanggal 7 Mei 1997, disetujui untuk dipublikasikan 27 September 1997 abstract
Ethanol extracts of the fruits of three kinds of Capsicum showed similar potencies in their
antimicrobial activities against Gram (+) and Gram (-) bacterias, and fungi, although they
contained different level of capsaicin. Bioautographic tests demonstrated that capsaicin was
the main antimicrobial component. At least two other non-polar components of ethanol
extract also contributed in the antimicrobial activity and very likely that these compounds
were responsible for the activity toward Pseudomonas aeruginosa.

Antimicrobial activity, ethanol extracts, fruits, Capsicum frutescens L., Capsicum annuum L,

1. Introduction
Pepper fruits are added at a substantial quantity to produce a characteristic taste of cuisine of West Sumatran (Indonesia). It was observed that cuisine using a large amount of chilli pepper could stand at long period without significant deterioration. Only a special variety of chilli pepper was employed by this ethnic, i.e. Capsicum annuum L. var. longum (Solanaceae) which has a curly shape. Chilli tinctur was previously reported to be able to inhibit the growth of Staphyllococcus sp., Escherichia coli, Bacillus aureus and Bacillus subtilis1). In Madura island (Indonesia), chilli pepper (Capsicum frutescens) is traditionally used to treat ox cuts before race2). In Indonesia, chilli pepper is also traditionally used to treat oral thrush which is usually caused by Candida albicans3). This report presents the results of our study on the activity of the ethanol extracts of Capsicum frutescens and two cultivars of Capsicum annuum, var. longum. The first pepper fruit was very hot and one of the C. annuum was hot while the other one was moderately * To whom correspondence should be addressed 2. Materials and Methods
2.1. Plant Materials
Three different pepper fruits were purchased from local market. Fruit-1 was from Capsicum frutescens L. (chilli pepper), fruit-2 C. annuum L. var. longum (curly pepper), fruit- 3 C. annuum L. var. longum (sweet pepper). Taxonomic identification of fruits was conducted at The Herbarium Bandungense, Department of Biology, Institut Teknologi Bandung and Horticulture Research Center - Lembang, Indonesian Ministry of Agriculture. Voucher specimens were deposited in the Herbarium of the Department of Pharmacy. The fruits were dried at 60oC in an air oven and then ground to produce pepper powder. 2.2. Extraction
Chilli pepper fruit extracts were prepared by macerating pepper powder in ethanol for 24 h then filtered. The residue was washed twice with fresh ethanol and the filtrates combined with the first filtrate. Combined filtrate was then evaporated to dryness to produce sticky 2.3. Microorganisms
The microorganisms used in these tests include : Gram (+) bacteria : Staphylococcus aureus (ATCC-14154) Sarcina lutea (Collection of Microbiology Lab. of Chemical Engineering Department, Institut Teknologi Bandung) Pseudomonas aeruginosa (ATCC-23993) Microsporum gypseum (Biofarma-Bandung Collection) 2.4. Media
Bacteria were grown on nutrient agar (Oxoid). Inoculum for the assay was prepared by suspending bacterial cells 18-24 h old in nutrient broth (Oxoid) to yield 25% transmittance at 580 nm. Fungi were grown on Sabouraud Dextrose Agar (Oxoid) and inoculum for the assay prepared by suspending the 72 h old fungal cells in Sabouraud Dextrose Broth (SDB) to 2.5. Method of assay
The antimicrobial assay was performed by dispersing 100 µl of inoculum homogeneously in 15 ml liquified nutrient agar (NA) or Sabouraud Dextrose Agar (SDA) media and then left to solidify on Petri dishes. Six wells of 6.5 mm in diameter were prepared on plate of each dish. Respectively 10 µl solution of pepper extract in DMSO at the desired concentration was then added into each well. The assay dishes were then left for one hour and subsequently incubated for 24 h at 37oC for bacteria and 22oC for fungi. The diameter of inhibition was then observed and measured. As a comparison, tetracycline hydrochloride was used as standard for antibacteria, nystatine for the activity against C. albicans and M. gypseum, griseofulvin for A. niger. 2.6. Thin Layer Chromatography
Thin layer chromatography was performed on precoated silica gel GF-254 with 10 µm layer and benzene-acetic acid (9:2) as solvent system. Spot of capsaicin was visualized with diazotised sulfanilamide which was prepared by freshly mixing 5 ml of 1% sulfanilamide in 10% HCl, 5 ml of 5% sodium nitrite and 40 ml of metanol4)). Intensification of capsaicin spot was performed by spraying with 5% sodium carbonate which yielded red color following 2.7. Determination of capsaicin content in the extract
Sample was prepared by dissolving extract in ethanol at concentration of 5 mg/ml and 15 µl solution was applied on TLC plate. Capsaicin isolated from fruits was used as a standard for the measurement and applied in the range of 5 µg to 25 µg in each spot. After development and visualization, the intensity and the area of the spots was measured using Spectrophoto densitometer (Shimadzu CS-910) at 510 nm. 2.8. Bioautography
To improve the separation and increase the loading capacity of the plate, the extract was firstly fractionated into aqueous and dichlormethane fractions at pH 3 and the later fractions collected and dried with sodium sulfate. Dichlormethane fraction was evaporated to dryness at reduced pressure and redissolved in ethanol and applied on silica gel as a strip. TLC chromatogram was dried using spray drier to eliminate solvent remaining on the plate and then laid on the surface of NA/SDA media freshly inoculated with the assay microbes. The plate was left for15-30 min to facilitate diffusion of substances in the plate to the gel and then removed. Subsequently the assay gel was incubated at 37oC (bacteria) or 22oC (fungi) for 18- 3. Results and Discussion
In order to provide background information on the pepper fruits under our study, following is a brief description of the three fruits. The first pepper was identified as one cultivar of C. frutescens, since there are different type of fruits based on the color, size, form and pungency. The other two fruits were all from C. annuum var longum, but they are very different in shape and also pungency. Table 1 gives more detail description of the Capsicum All fruit extracts were active against most Gram (+) and Gram (-) bacteria tested. Differ from fruit-1 and fruit-3 extracts, fruit-2 extract was inactive against P. aeruginosa. Against fungi tested, i.e. C. albicans, M. gypseum and A. niger, the three fruit extracts were only active to the first microorganism. These results suggest that all kinds of Capsicum fruits tested are useful as antibacterial and anticandidal agents and not necessarily the most pungent pepper as in the tradisional use2,3). Consistent with their pungency, the sequence of capsaicin level in the three Capsicum fruit extracts was fruit-1 > fruit-2 > fruit-3 (see Table 1). Based on their minimum inhibition concentrations (MIC), the activity of fruit-1 and fruit-3 extracts to P. aeruginosa compared to the other bacteria tested and to C. albicans was the lowest, while fruit-2 extract was inactive. Although fruit-3 contained the lowest level of capsasicin, its extract exhibited the lowest MIC against S. lutea (see Table 1). Inactivity of fruit-2 against P. aeruginosa, and the similarity in the activity of the three fruit extracts to the other microrganisms suggest that capsaicin may not the only compound responsible for the Bioautography of chromatogram of the three extracts which was conducted by contacting the chromatogram with the assay gel for 15-30 min followed by incubation and observation the inhibion area showed that capsaicin was the main compound for antimicrobial activity. Bioautographic tests against S. aureus and B. subtilis gave the same pattern of results, while against C. albicans, good result was not obtained although several repetition had been made. Apart from capsaicin, in extract of fruit-1 another component having hRx 123.3 (relative to capsaicin) also showed antimicrobial activity. In fruit-2 capsaicin was the only antimicrobial compound, while in fruit-3, two other active components i.e. spots with hRx 153.8 and 165.4 were also active. The higher Rf value of the other active components than capsaicin in the TLC system used suggest that these compounds are less polar than capsaicin. The bioautographic data also suggest that these compounds may be responsible for the activity toward P. aeruginosa since fruit-2 extract which contained capsaicin as the main active component was inactive. Clearly further tests are needed to confirm this possibility. The similarity in the antimicrobial activity of the hottest and the least hot pepper fruit extracts tested suggests the presence of synergism between capsaicin and the other components in the Compared to the activity of the standard antibiotics, the activity of pepper fruit extracts was still much lower. Relative activity fruit-1 extract compared to tetracycline HCl for example only approximately 10-5 times and variation existed depended on the bacterial test used. The relative activity of Capsicum fruit extracts to nystatine was in the order of 10-3 time magnitude which was higher than to tetracycline. These results support the traditional use of Capsicum fruits to treat oral thrush caused by C. albicans3) . Table 1. Botanical description of the three Capsicum fruits, antimicrobial activity and bioautographic data of their ethanol extracts to B. subtilis and S. aureus Acknowledgement
The authors are gratefully thank The Herbarium Bandungense, Department of Biology, Institut Teknologi Bandung, and Horticulture Research Center - Lembang, Indonesian Ministry of Agriculture for the assistance in taxonomic identification. References
1. Siehta, A.S., Magdoub, M.W.I., Fayed, E.O., and Hoft, A.A. "Effect of added salt and Capsicum tincture on non lactic acid bacteria in pickled Domiati Cheese", Annual of Agricurtural Science, 29: 793-810 (1984).
2. Djarwaningsih and Uji. T. "The utilization of Indonesian traditional medicines for treatment of cattle diseases in three villages of East Java, in: National Seminar and Workshop Proceeding on Ethnobotany", Ministry of Education and Culture, Ministry of Agriculture, Indonesian Research Institute, Indonesian National Library, Cisarua-Bogor: 3. Syamsuhidayat, S.S., and Hutapea, J.R. "Inventarization of Indonesian Medicinal Plants", Vol. 1, Indonesian Ministry of Health, Jakarta: 112-115 (1991). 4. Medziak, I. and Waksmundski, A. "Selection of composition and concentration of the mobile phase for separation of phenol derivatives by TLC", Chem. Anal., 28: 251-257


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