Bioautography Method Research Articles

Assessment of the Antibacterial Activity and the Antidiarrheal Function of Flavonoids from Bayberry Fruit

Chinese bayberry fruits are used as a folk medicine to cure diarrhea. However, the active compounds have not yet been reported. We found that bayberry fruit extract showed significant antibacterial activity against Salmonella, Listeria, and Shigella, and the minimal inhibitory concentration (MIC) ranged from 2.07 to 8.28 mg/mL. Positive relationships were found between the antibacterial activity and the total polyphenol (r = 0.88) and flavonoid contents (r = 0.92) of samples using different extraction times. The active compounds showed green or blue fluorescence under UV light using the bioautography method and were purified using a polyamide column. The fraction F1 with the most activity was comprised of flavonoids, which included cyanidin-3-O-glucoside, myricetin deoxyhexoside, quercetin-3-O-glucoside, and quercetin deoxyhexoside, and it also possessed an antidiarrheal activity (p < 0.10) at 80 mg/kg in mice. These findings provide scientific evidence for the antidiarrheal function of bayberry.

Screening of Medicinal Plants from Iranian Traditional Medicine for Acetylcholinesterase Inhibition

To find new herbal compounds with an acetylcholinesterase (AChE) inhibitory effect, this study focused on herbal drugs and resins which have been used in Iranian traditional medicine for the treatment of cognitive disorders. Forty drugs were selected from authoritative written documents of Iranian traditional medicine. Each drug was extracted by accelerated solvent extraction using dichloromethane followed by methanol. The 80 extracts were screened for AChE inhibitory activity by a TLC bioautography method. The inhibiting effect of the 32 most active extracts was measured by a microplate colorimetric assay. Due to the best activity, the seeds of Peganum harmala L. were investigated in detail. From the TLC bioautography assay the alkaloids harmaline and harmine were identified as active compounds. This result was confirmed by means of HPLC-DAD. The IC(50) values were 41.2 μg/mL for the methanol extract, 95.5 μg/mL for the dichloromethane extract, 8.4 μg/mL for harmaline and 10.9 μg/mL for harmine. The concentrations of active compounds in the extracts were determined by a fast and precise HPLC method. As the amounts of harmaline and harmine in the extracts were correlated with the IC(50) values of the extracts, it can be concluded that these two alkaloids are responsible for the AChE inhibitory activity of P. harmala.

Study on the antibacterial potential of Physalis Minima linn

Physalis minima is an important medicinal plant of Indian System of Medicine. This plant is reported for its diuretic, laxative and antiinflammatory activities. However, the plant is not well scrutinized for its antimicrobial potential. The major chemical constituents reported from the plant are phenolics and alkaloids, which suggest that the plant may turn out to be a potent antiinfective agent. The aim of the study was to find out the antibacterial potential of mature berries of P. minima using streak plate, well diffusion, determination of minimum inhibitory concentration and bioautographic methods against a battery of Gram positive and Gram negative bacterial strains. Results of the study showed that methanol and chloroform extracts of P. minima exhibited potent inhibitory activity against all the bacterial strains tested. Minimum inhibitory concentration found out was 100 μg in both the extracts. Bioautography assay showed polar compounds present in the crude extract are responsible for the antimicrobial action.

Antimicrobial and miticide activities of Eucalyptus globulus essential oils obtained from different Argentine regions

The biological activity of Eucalyptus globulus essential oils derived from plant material obtained from different geographic areas was analyzed in in vitro experiments on Paenibacillus larvae, Varroa destructor, and Apis mellifera. The physicochemical properties, composition, antimicrobial, and bioactivity of these essential oils were studied. The bioactivity against P. larvae was analyzed by means of two in vitro techniques (tube dilution and bioautography). Mite and bee lethality were estimated using a complete exposure method test with oils at different concentrations. Essential oils differed in their composition, albeit their similar physicochemical properties. The minimal inhibitory concentrations range for E. globulus essential oil lay between 600-700 µg mL–1 for Mar del Plata and 900-1,200 µg mL–1 for Valle de Conlara essential oils against all P. larvae strains. The bioautography method determined that limonene accounted for the greatest antimicrobial activity with respect to the other compounds. The complete exposure method at 24, 48, and 72 h yielded lower LC50 values for mites exposed to E. globulus essential oils from Conlara. LC50 values for acari after 72 h of treatment with E. globulus from Mar del Plata and Conlara were 47.1 and 11.7 µL capsule–1, respectively; whilst those for bees was &gt; 20 µL capsule–1 for both oils. The E. globulus essential oils tested in this study featured high efficiency against V. destructor, yet their antimicrobial activity against P. larvae proved to be lower, and innocuous to bees. The present experience promotes the use of active compounds for American fouldbrood and Varroosis management.

The possible role of medicinal plants in tackling resistant microbial pathogens in Limpopo Province, South Africa

The present study was conducted to investigate the antibacterial activities of nine selected medicinal plants against metheciline resistance coagulase negative Staphylococcus, metheciline resistance Staphylococcus aureus and extended spectrum beta-lactamase producing Escherichia coli, Enterobacter cloace and Pseudomonas aeroginosa. The grounded plant materials were extracted with different extractants and screened for anti-microbial activity using the disk diffusion, micro-dilution techniques and bioautographic methods. Preliminary screening revealed that methanol extracts were most active against all assayed bacteria. Metheciline resistance coagulase negative Staphylococcusand metheciline resistance S. aureus were found to be susceptible to all crude methanolic extracts of all tested plants (100%), followed by P. aeroginosa and extended spectrum beta-lactamase producing E. coli, E. cloace. Contact biautography indicated that Schotia brachypetala extracts possess one major anti-microbial component against extended spectrum beta-lactamase producingE. coli , E. cloace and four components against P. aeroginosa The lowest minimum inhibitory concentration recorded for the different crude methanol extracts against metheciline resistance coagulase negative Staphylococcus, metheciline resistance S. aureus and extended spectrum beta-lactamase producing E. coli, E. cloace and P. aeroginosa were 500, 125 and 250 ug/ml respectively. This study also confirm the antimicrobial potential of investigated plants and their usefulness in treatment of resistance microorganisms both gram-negative and gram-positive Key words: Metheciline resistance coagulase negative Staphylococcus,metheciline resistance Staphylococcus aureus, extended spectrum beta-lactamase producing Escherichia coli, Enterobacter cloace, Pseudomonas aeroginosa.

Antibacterial activity of extracts of three Croton species collected in Mpumalanga region in South Africa.

The antibacterial activities of three Croton species were compared using bioautography and the serial microdilution methods. The methanolic extracts of all the species had low activity against Escherichia coli. The highest activity was observed with Croton megalobotrys against Enterococcus faecalis with a minimal inhibitory concentration (MIC) value of 0.02 mg/ml. Croton steenkapianus extracts were the least active of the species investigated, only managing an MIC value of 0.625 mg/ml against Pseudomonas aeruginosa. Croton megalobotrys leaf powder was serially extracted using solvents of various polarities. The lowest MIC value (0.06 mg/ml) of the serially extracted fractions was observed with acetone against Pseudomonas aeruginosa. The liquid-liquid fractions of the methanol extract of Croton megalobotrys were also tested. The lowest MIC value of 0.02 mg/ml was observed with n-hexane fraction against Enterococcus faecalis. The carbon tetrachloride fraction was further fractionated using column chromatography with silica as the immobile phase. The resulting seven fractions were tested for activity following the bioassay-guided practice, and it emerged that the first three fractions had active compounds against Staphylococcus aureus when the bioautography method was used.

Antibacterial activity of extracts of Acacia aroma against methicillin-resistant and methicillin-sensitive Staphylococcus

Antibacterial activity of organic and aqueous extracts of Acacia aroma was evaluated against methicillin-resistant Staphylococcus aureus (MRSA), methicillin sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus epidermidis. Inhibition of bacterial growth was determined using agar diffusion and bioautographic methods. Among all assayed organic extracts only ethanolic and ethyl acetate extracts presented highest activities against all tested Staphylococcus strains with minimal inhibitory concentration (MIC) values ranging from 2.5 to 10 mg/ml and from 2.5 to 5 mg/ml respectively. The aqueous extracts show little antibacterial activity against Staphylococcus strains. The bioautography assay demonstrated well-defined growth inhibition zones against S. aureus in correspondence with flavonoids and saponins. A. aroma would be an interesting topic for further study and possibly for an alternative treatment for skin infections.

Chemical composition, acetylcholinesterase inhibitory and antifungal activities of Pera glabrata (Schott) Baill. (Euphorbiaceae)

Pera glabrata (Schott) Baill. was selected for this study after showing a preliminary positive result in a screening of Atlantic Forest plant species in the search for acetylcholinesterase inhibitors and antifungal compounds. The bioassays were conducted with crude ethanol extract of the leaves using direct bioautography method for acetylcholinesterase and antifungal activities. This extract was partitioned with hexane, chloroform and ethyl acetate solvents. The active chloroform fraction was submitted to silica gel chromatography column affording 12 groups. Caffeine, an alkaloid, which showed detection limits of 0.1 and 1.0 µg for anticholinesterasic and antifungal activities, respectively, was isolated from group nine. After microplate analyses, only groups four, nine, 10, 11 and 12 showed acetylcholinesterase inhibitory activity of 40% or higher. The group 12 was purified by preparative layer chromatography affording four sub-fractions. Two sub-fractions from this group were analyzed by gas chromatography-mass spectrometry and gas chromatography-flame ionization detector. The first sub-fraction showed anticholinesterasic activity and contained two major compounds: 9-hydroxy-4-megastigmen-3-one (84%) and caffeine (6%). The second sub-fraction presented five major compounds identified as 9-hydroxy-4-megastigmen-3-one, isololiolide, (-) loliolide, palmitic acid and lupeol and did not show activity.

Antibiotic pigment from desert soil actinomycetes; biological activity, purification and chemical screening

An actinomycete strain, Streptomyces hygroscopicus subsp. ossamyceticus (strain D10) was isolated from Thar Desert soil, Rajasthan during the year 2006 and found to produce a yellow color pigment with antibiotic activity. Crude pigment was produced from strain D10 by solid state fermentation using wheat bran medium followed by extraction with ethyl acetate. The antimicrobial activity of the crude pigment was evaluated against drug resistant pathogens such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, extended spectrum β-lactamase producing cultures of Escherichia coli, Pseudomonas aeruginosa and Klebsiella sp. About 420 mg of crude pigment was produced per 10 g of wheat bran medium. In the disc diffusion method the crude ethyl acetate extract showed a minimum of 10 mm inhibition against Klebsiella sp. and maximum of 19 mm of inhibition against Escherichia coli. The crude pigment was partially purified using thin layer chromatography with the solvent system chloroform:methanol (30:70) and the Rf value was calculated as 0.768. Antimicrobial activity of the partially purified compound from thin layer chromatography was determined using the bioautography method. The purified pigment showed minimum of 15 mm inhibition against Klebsiella sp. and a maximum of 23 mm of inhibition against vancomycin-resistant Staphylococcus aureus in the disc diffusion method. Based on the results of chemical screening, the pigment was tentatively identified as group of sugar containing molecules.

Atividade antibacteriana de floroglucinóis e do extrato hexânico de Hypericum brasiliense Choysi

Three phloroglucinols were obtained from Hypericum brasiliense: japonicine A (1), isouliginosin B (2) and uliginosin B (3). Bioautography and disk diffusion methods were used to determine antibacterial activity of the hexanic extract. Strains of the Coagulase Negative Staphylococcus and American Methicillin Resistant Staphylococcus aureus clones showed a growth inhibition zone ranging from 10 to 12 mm and 7 to 15 mm, respectively. Minimal inhibitory concentration (MIC) values were used to measure antistaphylococcal activity for all phloroglucinols. Isouliginosin B and uliginosin B presented MIC values of 1.5 and 3.0 µg/mL, respectively, while japonicine A displayed MIC value of 50.0 µg/mL.

Ankara Piyasasında Satılan Sütlerde Bazı Antibiyotik Kalıntılarının İnce Tabaka Kromatografisi ve Biyootografik Yöntemle Saptanması

Summary In this study, ampicillin, amoxycillin, cloxacillin, danofloxacin, enrofloxacin, erythromycin, florfenicol residue analysis were carried out in a total of 240 milk samples consisting of 120 raw and 120 pasteurized milk obtained from 7 commercial firms separate dairying units. Residue analysis were performed by bioautography method based on the combination of thin layer chromatography and microbiologic disc diffusion test. According to the results, one pasteurized milk samples was found pozitive for ampicillin. In 239 samples weren’t detected any antibiotic residues. It was calculated that the mean ampicillin residue incidence of contamination was %0.4 in all of the samples.

Searching for Natural Bioactive Compounds in Four Baccharis species from Bolivia

The volatile oil composition of four Baccharis species ( Baccharis papilosa, B. polycephala, B. buxifolia, and B. convawyi) growing wild in Bolivia were studied by GC and GC/MS. The antimicrobial activity of the oils was determined by the agar diffusion and bioautography methods. Solvent extracts, polar and non-polar, of the same species were also tested for antimicrobial and antioxidant activities. Extracts from B. polycephala, B. papilosa and B. convawyi presented significant antioxidant activity, determined using the DPPH radical scavenging method, showing EC50 values of 4.74, 7.17 and 7.97 μg/mL, respectively. Screening for antimicrobial activity was conducted using the agar diffusion test. The most active extracts (% inhibition ≥ 50) were further examined by the dilution method. All the plant extracts studied showed higher antimicrobial activity against S. aureus (Gram-positive) than against E. coli and P. fluorescens (Gram-negative).

Activities of 2,4-Dihydroxy-6-n-pentylbenzoic Acid Derivatives

Esters of 2-hydroxy-4-methoxy-6-n-pentylbenzoic acid (2-8) (methyl, ethyl, butyl, pentyl, isopropyl, sec-butyl and benzyl), olivetol (9), methyl, ethyl, butyl perlatolates (10-12), 2,4-dihydroxy-6-n-pentylbenzoic acid (15), and methyl and ethyl esters of (15) were prepared through structural modifications of perlatolic acid (1) with the aim to detect new antifungal and antibacterial substances and also to evaluate the toxicity by the brine shrimp lethality assay against Artemia salina. The antifungal assays were carried out against the fungus Cladosporium sphaerospermum through the bioautography method, and methyl 2,4-dihydroxy-6-n-pentylbenzoate (13) showed the highest antifungal activity (2.5 yg). Olivetol (9) and 2,4-dihydroxy-6-n-pentylbenzoic acid (15) are also potent inhibitors of the growth of the fungus (5.0 microg). Except for methyl (10), the ethyl (11) and butyl (12) perlatolates were less active than perlatolic acid (1). The activities presented by methyl (2) and ethyl (3) 2-hydroxy-4-methoxy-6-n-pentylbenzoates and methyl (13) and ethyl (14) 2,4-dihydroxy-6-n-pentylbenzo-ates suggest that compounds with a free hydroxy group in the aromatic ring (C-4) have a more pronounced effect against C. sphaerospermum. Antibacterial activities were tested by the disc diffusion method using pathogenic strains of S. aureus and E. coli. The compounds were weakly active with inhibition zones between 9-15 mm. The 2-hydroxy-4-methoxy-6-n-pentylbenzoic esters 2-8 and alkyl perlatolates 10-12 were selective against E. coli. Perlatolic acid (1) and methyl 2-hydroxy-4-methoxy-6-n-pentylbenzoate (2) were the most active with LD50 values of 24.1 microM and 27.2 microM, respectively. The other compounds were not toxic to Artemia salina larvae.

AVALIAÇÃO DA ATIVIDADE ANTIBACTERIANA DE Aster lanceolatus Willd. (ASTERACEAE) CONTRA Staphylococcus aureus

Utilizando o método de bioautografia, avaliou-se a atividade antibacteriana de frações de Aster lanceolatus Willd contra cepas de Staphylococcus aureus. Os resultados demonstraram que a fração hexano e fração diclorometano apresentam atividade antibacteriana contra o microrganismo testado.

In vitro antimicrobial activity of the leaf extract of Harungana madagascariensis Lam. ex Poir. (Hypericaceae) against strains causing otitis externa in dogs and cats

Otitis externa in dogs and cats is always caused by a combination of yeasts and bacteria, among which the most important are Malassezia pachydermatis, Staphylococcus intermedius and Pseudomonas species. These organisms often develop resistance to classical antimicrobial agents. Therefore, the aim of this study was to investigate the antimicrobial activities of an ethyl acetate leaf extract of Harungana madagascariensis against the organisms cited, to carry out the phytochemical investigation of this extract and to determine its bioactive chemical class using dilution techniques, the bioautography method and the standard phytochemical method described by Harborne (1973). Phytochemical analysis revealed the presence of saponins, tannins, flavonoids, alkaloids and anthracenic derivatives. The bioassay showed that the antimicrobial properties may be attributed to astilbin, a flavanone derivative identified on the basis of its spectroscopic data. The results suggest that the extract could be used in an antimicrobial preparation effective against the whole range of organisms incriminated in otitis externa in dogs and cats, with a minimal inhibitory concentration (MIC) of 250 microg/ml.

Antibacterial activity of Zuccagnia punctata Cav. ethanolic extracts

The present study was conducted to investigate antibacterial activity of Zuccagnia punctata ethanolic extract against 47 strains of antibiotic-resistant Gram-negative bacteria and to identify bioactive compounds. Inhibition of bacterial growth was investigated using agar diffusion, agar macrodilution, broth microdilution and bioautographic methods. Zuccagnia punctata extract was active against all assayed bacteria ( Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Morganella morganii, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia) with minimal inhibitory concentration (MIC) values ranging from 25 to 200 μg/mL. Minimal bactericidal concentration (MBC) values were identical or two-fold higher than the corresponding MIC values. Contact bioautography, indicated that Zuccagnia punctata extracts possess one major antibacterial component against Pseudomonas aeruginosa and at least three components against. Klebsiella pneumoniae and Escherichia coli. Activity-guided fractionation of 1he ethanol extract on a silica gel column yielded a compound (2′,4′-dihydroxychalcone), which exhibited strong antibacterial activity with MIC values between 0.10 and 1.00 μg/mL for Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Morganella morganii, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia. These values are lower than imipenem (0.25–16 μg/mL). Zuccagnia punctata might provide promising therapeutic agents against infections with multi-resistant Gram-negative bacteria.

Strategy in the Search for New Lead Compounds and Drugs from Plants

Plants represent an extraordinary reservoir of novel molecules and there is currently a resurgence of interest in the vegetable kingdom as a provider of new lead compounds for introduction into therapeutic screening programs. Plant constituents of interest are usually isolated following a bioactivity-guided fractionation procedure. In order to render this approach more rapid and efficient, the dereplication of crude plant extracts with LC-hyphenated techniques (LC/UV-DAD, LC/MS and LC/NMR) represents a strategic element to avoid finding known constituents and to target the isolation of new bioactive compounds. The development of simple and rapid bioassays is also crucial for an efficient localisation of active principles. In this respect, the use of bioautographic methods, which enable a direct screening of bioactive constituents on TLC is of great interest. The combination of chemical and biological screening has proved to be very efficient for the survey of an important number of extracts and for the discovery of new lead compounds and several examples of this approach will be discussed in this paper.

The diversity of antifungal compounds of six South African Terminalia species (Combretaceae) determined by bioautography

A bioautography method was developed to determine the number of antifungal compounds in Terminalia species extracts. Acetone, hexane, dichloromethane and methanol leaf extracts of six Terminalia species (T. prunioides, T. brachystemma, T. sericea, T. gazensis, T. mollis and T. sambesiaca) were tested against five fungal animal pathogens (Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Microsporum canis and Sporothrix schenkii). The Rf values and relative activities of separated compounds were determined. Hexane and dichloromethane extracts had at least three times more antifungal compounds than the other extracts indicating the nonpolar character of the antifungal compounds. From the Rf values, the non-polar character of the antifungal compounds was confirmed indicating that the antifungal activity is not due to tannins. M. canis had the highest number, up to ten, of antifungal compounds. All Terminalia species contained a compound (Rf =0.46 in benzene/ethanol/ammonium hydroxide (90/10/1) active against all tested pathogens. T. sericea and T. brachystemma were the most promising candidates for isolating antifungal compounds. The results demonstrate the value of bioautography in examining plant extracts with antifungal activity, selecting species for further study and dereplicating the isolation of compounds.

Stilbene derivatives with antifungal and radical scavenging properties from the Stem Bark of artocarpus nobilis

Antifungal activity-guided fractionation of the n-butanol extract from the methanol extract of the stem bark of Artocarpus nobilis furnished two stilbene derivatives (E)-4-isopentenyl-3,5,2′,4′-tetrahydroxystilbene and (E)-4-(3-methyl-E-but-1-enyl)-3,5,2′,4′-tetrahydroxystilbene. Both compounds showed strong antifungal activity against Cladosporium cladosporioides and high radical scavenging activity towards the DPPH radical in TLC bio-autography method.

Geranyl chalcone derivatives with antifungal and radical scavenging properties from the leaves of Artocarpus nobilis

Antifungal activity guided fractionation of the n-butanol extract from the methanol extract of the leaves of Artocarpus nobilis furnished 2 ′,4 ′,4-trihydroxy-3 ′-geranylchalcone ( 1), 2 ′,4 ′,4-trihydroxy-3 ′-[6-hydroxy-3,7-dimethyl-2( E),7-octadienyl]chalcone ( 2), 2 ′,4 ′,4-trihydroxy-3 ′-[2-hydroxy-7-methyl-3-methylene-6-octaenyl]chalcone ( 3), 2 ′,3,4,4 ′-tetrahydroxy-3 ′-geranylchalcone ( 4), 2 ′,3,4,4 ′-tetrahydroxy-3 ′-[6-hydroxy-3,7-dimethyl-2( E),7-octadienyl]chalcone ( 5). The chalcones 3 and 5 are new natural products whereas 1 and 2 are reported first time from the family Moraceae. All these compounds showed good fungicidal activity against Cladosporium cladosporioides and high radical scavenging activity towards the 2,2 ′-diphenyl-1-picrylhydrazyl (DPPH) radical in TLC bio-autography method.