Antimicrobial activity of Calophyllum inophyllum crude extracts ...

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Antimicrobial activity of Calophyllum inophyllum crude extracts obtained by pressurized liquid extraction / AsianJournal of Traditional Medicines, 2009, 4 (4)abundance in Vietnam.There is an explosion of interest in naturalantimicrobials from plant sources due to the rise ofmultidrug resistant strains of bacteria such as MRSA,VRE and multidrug resistant tuberculosis [2] . Thus,rapid screening techniques are essential to quicklyidentify and isolate these compounds. Conventionalsolvent extraction techniques have severaldisadvantages in terms of high volumes of organicsolvents consumed and long extraction times.Modern extraction techniques, such as pressurizedliquid extraction (PLE), seek to circumvent theselimitations. At the high pressures employed,extraction can be carried out above the boiling pointof the solvent, which remains in the liquid phase,allowing better diffusion and improved mass transferkinetics [3] . This drastically reduces the amount oforganic solvent used as well as the extraction time,from a period of hours to only minutes. PLE alsooffers the possibility of performing extractions inan inert atmosphere protected from light, whichrepresents an advantage since phenolic compounds arevery sensitive to these two factors [4] .Calophyllum inophyllum fruit is used to extractoil. The oil has been proven to have vulnerary andcicatrising effects [5] . The fruit peel is often discardedas waste. To our knowledge, there is no studycurrently available on the antimicrobial propertiesof the Calophyllum inophyllum fruit peel. Thus,this paper describes a study aimed to establish (1)PLE as a rapid extraction method for obtainingbioactive principles from botanical material, and (2)to investigate the potential of Calophyllum inophyllumfruit peel as a source of antimicrobial compounds.Materials and methodsPlant materialsThe dried fruit peels of Calophyllum inophyllumwere collected in December 2008 from Ben Treprovince which is situated about 80 km from Ho ChiMinh City, Vietnam.Pressurized liquid extractionThe plant material was pulverized using a Fitzmill (M5A, Fitzpatrick, USA) and sieved to obtaina powder with a median particle size of 1.84 mm [6] .Then, 5 g of the milled material was extracted byPLE (ASE-100, Dionex, USA) and the dead space inthe extraction vessel was minimized using 1-2 mmglass beads. Glass wool and 10 µm frits were placedat each end of the extraction vessel to prevent finescontaminating the extract. Extraction was carriedout at a fixed pressure of 1500 psi, a temperature of120 °C, and for a period of 15 min. A single cycleof extraction was used. Flush volume of 60 % anda nitrogen purge time of 90 s were used to rinse theequipment of any entrapped extract to enable a moreaccurate quantitative recovery.Soxhlet extractionSoxhlet extraction was conducted usingan automated Soxhlet extractor (B-811, Buchi,Switzerland). For this, 1 g milled material wasaccurately weighed and placed in a cellulose thimble(25 mm×27 mm×100 mm, Whatman, UK) andextraction was performed using methanol and n-hexanefor 8 h.Preparation of dried extractsThe extracts obtained from both PLE and theSoxhlet extraction were dried under reduced pressureunder ambient conditions using a rotary evaporator(Eyela, Japan). The yield of extract, expressed as theweight ratio over the weight of raw material used, wascalculated. The extracts were stored in airtight vialsand kept refrigerated (2-8 °C) until further analysis.Screening of phenolic compoundsThin-layer chromatography was carried outusing thin-layer chromatography (TLC) plates of precoatedsilica-gel 60 F 254on aluminum sheets (Merck,Germany). Development of the TLC plates was142
Antimicrobial activity of Calophyllum inophyllum crude extracts obtained by pressurized liquid extraction / AsianJournal of Traditional Medicines, 2009, 4 (4)carried out vertically in a 20 cm×20 cm twin troughchromatographic tank, pre-saturated for 10 min(with filter paper) with an appropriate developingsolvent. The separation was allowed to run adistance of 100 mm from the lower edge of the plate.After developing, the plates were dried at ambienttemperature. Mobile phase systems used were: (A)n-hexane: ethyl acetate (7:3), (B) toluene: ethylacetate (8:2), and (C) chloroform: methanol (9:1).Detection of the compounds on the TLC plates wasmade under UV light at 254 and 366 nm and theywere subsequently sprayed with 5 % ferric chloridesolution.Antimicrobial assayStandard cultureStandard strains used were Staphylococcus aureus(ATCC 6538 P), Pseudomonas aeruginosa (ATCC9027), and Mycobacterium smegmatis (ATCC 14468)purchased in the form of inoculation loops from Oxoid(England). These micro-organisms were cultivated onnutrient agar until the 6 th generation and then used forthe study.Preparation of impregnated discsSterile paper discs (Whatman No.54) wereindividually impregnated with diluted plant extractsusing the respective solvent (methanol or n-hexane) toobtain 100 μg of dried extract per disc. These discswere then dried at 40 ºC overnight before use.Then, 20 ml sterilized Mueller Hinton agar (3.8% w/v) was transferred to 90 mm Petri dishes andallowed solidify. Sterile water was used to wash thesurface of the colonies from fresh subcultures of thetest microorganisms. The turbidity of the microbialsuspension was standardized against McFarland tube0.5 (equivalent to 1.5×10 8 CFU/ml). The standardizedsuspension was diluted 10 times and 200 μl (3×10 6 CFU) was inoculated at the centre of the solidmedium. The suspension was evenly spread using aglass spreader on the surface of the agar and allowedto dry for 15 min. The extract-impregnated discsand a standard antibiotic disc (Table 1) were placedequidistant from each other on the surface of theinoculated agar and a period of 30 min was allowedfor diffusion of the compounds. The plates were thenincubated at 37 °C for 18 h (S. aureus, Ps. aeruginosa)and 48 h (M. smegmatis). The diameters of the zonesof inhibition around the discs were measured to thenearest mm and recorded at the end of the incubationperiod. Two diameter readings, perpendicular to eachother, were recorded for each zone (d1 and d2), andthe average readings were calculated. The ratio of theaverage (dextract)/average (dstandard) was calculated foreach extract, and for each particular microorganism.Table 1. Microorganisms tested and the corresponding positivecontrolsMicroorganismStandard antibiotic discStaphylococcus aureus Methicillin 5 µgPseudomonas aeruginosa Carbenicillin 100 µgMycobacterium smegmatis Streptomycin 10 µgResultsExtraction method and yieldThe yields of respective extracts using methanoland n-hexane are shown in Table 2.Screening of phenolic compoundsExtracted compounds were separated by TLC andthe results are shown in Table 3. These spots produceda violet coloration with 5 % ferric chloride solutionindicating the presence of phenolic compounds.Antimicrobial activityThe methanolic and n-hexane extracts weretested for their activity against Staphylococcusaureus, Mycobacterium smegmatis, and Pseudomonasaeruginosa and the results of their anti-microbialproperties are shown in Table 4.143
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