Bioautography Method Research Articles

α-Glucosidase Enzyme Inhibitory Activity of Extracts from the Fermentation of Endophytic Bacteria from Periwinkle (Catharanthus roseus (L.) G. Don) Leaves

Catharanthus roseus (L.) G. Don contain bioactive compounds that act as inhibitors against the α-glucosidase enzyme. This study aims to test the α-glucosidase enzyme inhibitor activity of fermented extract of endophytic bacteria from tapak dara leaves and identify the bacterial species. Endophytic bacteria were isolated using a direct planting method, resulting in eight isolates. The bacteria were fermented for 24-72 hours in nutrient broth media. Ethyl acetate was used to extract the fermented bacteria. Screening for α-glucosidase enzyme inhibitory activity was conducted using the TLC bioautography method. Four bacterial isolates (TD 1, TD 3, TD 4, TD 7) produced clear zones, qualitatively indicating that the isolates have inhibitory activity against the α-glucosidase enzyme. The IC50 results from the eight isolates showed that three isolates provided strong IC50 values: isolate TD 1 at 19.93 µg/ml, isolate TD 2 at 92.34 µg/ml, and isolate TD 4 at 13.94 µg/ml, compared to acarbose at 48.85 µg/ml). Molecular identification showed that isolate TD 1 was Pseudomonas oryzihabitans and isolate TD 4 was Staphylococcus warneri. This study concluded that endophytic bacteria from tapak dara leaves have the potential as a source of α-glucosidase enzyme inhibitor compounds for the development of antidiabetic drugs.

ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF ETHYL ACETATE EXTRACT OF ACTINOMYCETES ISOLATED FROM TERMITE NESTS

Actinomycetes are an important source for the discovery of bioactive secondary metabolite compounds. Over 10,000 bioactive metabolite compounds have been isolated from terrestrial actinomycetes with various biological activities, such as antibiotic, antiviral, anti-inflammatory, antitumor, anticancer, and antioxidant. One source of origin for actinomycetes is soil, including termite nests. Utilization of natural sources such as actinomycetes from termite nests as antioxidants and antibacterials can be an alternative source of production of new secondary metabolite compounds. This research aims to evaluate the antioxidant and antibacterial activities of actinomycete extracts from termite nests. Antioxidant activity using the DPPH free radical scavenging method, and antibacterial activity against S. aureus. Antioxidant and antibacterial activity were performed by using the TLC dot-blot, TLC bioautography, and microdilution methods to determine the inhibitory concentration of 50% (IC50), antioxidant activity index (AAI), and minimum inhibitory concentration (MIC). From the 33 extracts tested, 16 extracts showed antioxidant activity with IC50 values range of 76.64-126.22 µg/mL or AAI value > 0.05 (moderate), and 8 extracts had moderate antibacterial activity against S. aureus (MIC values range of 256-512 µg/mL). Future research for scaling-up of actinomycete culture, isolating of active compounds, determining of the chemical structure of active compounds, and further testing as antioxidants and antibacterials still need to be carried out.

Fluorescent probe for the detection of acetylcholinesterase inhibitors using high performance thin layer chromatography effect-directed assay in complex matrices

In recent years Bioautography has become an efficient bioassay for finding active compounds in complex matrices including extracts of; bacteria, plants or fungi. High Performance Thin Layer Chromatography (HPTLC) is a technique which allows effect-directive analysis (EDA) through the separation and identification of biologically active substances on a Thin Layer Chromatography (TLC) plate and can be run as a high throughput screening assay for enzymes. This paper presents a new bioautography method using a novel fluorescent probe derived from coumarin and its validation with Acetylcholinesterase (AChE) inhibition. This method combines the advantages of bioautography and the high sensitivity of fluorescence for detection. Combining these advantages, the limit of detection (LOD) and limit of quantification (LOQ) limits usually obtained with traditional chromophores has been reduced by three times. Therefore our new method has been applied on 14 mushroom extracts and we highlight a compound (variegatic acid) as a potentially new AChE inhibitor in X. Chrysenteron.

Antibacterial effect of essential oils and their components against Xanthomonas arboricola pv. pruni revealed by microdilution and direct bioautographic assays.

Bacterial spot of stone fruits caused by Xanthomonas arboricola pv. pruni (Xap) is one of the most significant diseases of several Prunus species. Disease outbreaks can result in severe economic losses while the control options are limited. Antibacterial efficacy of essential oils (EOs) of thyme, cinnamon, clove, rosemary, tea tree, eucalyptus, lemon grass, citronella grass, and lemon balm was assessed against two Hungarian Xap isolates. The minimal inhibitory concentration (MIC) was determined by broth microdilution assay and for the identification of active EOs' components a newly introduced high-performance thin-layer chromatography (HPTLC)-Xap (direct bioautography) method combined with solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) was applied. All EOs inhibited both bacterium isolates, but cinnamon proved to be the most effective EO with MIC values of 31.25 µg/mL and 62.5 µg/mL, respectively. Compounds in the antibacterial HPTLC zones were identified as thymol in thyme, trans-cinnamaldehyde in cinnamon, eugenol in clove, borneol in rosemary, terpinen-4-ol in tea tree, citral (neral and geranial) in lemon grass and lemon balm, and citronellal and nerol in citronella grass. Regarding active compounds, thymol had the highest efficiency with a MIC value of 50 µg/mL. Antibacterial effects of EOs have already been proven for several Xanthomonas species, but to our knowledge, the studied EOs, except for lemon grass and eucalyptus, were tested for the first time against Xap. Furthermore, in case of Xap, this is the first report demonstrating that direct bioautography is a fast and suitable method for screening anti-Xap components of complex matrices, like EOs.

Optimization of aqueous extraction of henna leaves (Lawsonia inermis L.) and evaluation of biological activity by HPLC-based profiling and molecular docking techniques

Due to the antimicrobial capabilities of henna leaf extract (Lawsonia inermis L.), this study intends to identify environmentally friendly ideal conditions for extracting bioactive compounds from henna leaf powder. For this purpose, the maceration method with a 100% aqueous solvent was selected for further analysis. Optimization of the extraction method was carried out by central composite design (CCD) protocol as a subset of Response Surface Methodology (RSM). The temperature of 40–90 °C, extraction time of 2–24 h, and the solvent-plant weight ratio ranging from 10 to 100 mL/mg were all considered independent variables. Also, the amount of lawsone, as an active component, and the total phenolic content were tracked as responses. Finally, a yield of 30.59% was achieved under the best extraction condition at 40 °C, 2 h, and a solvent-plant weight ratio of 1:100. The biological activity of an aqueous extract was evaluated using an HPLC-based activity profiling and TLC bioautography method. Peaks collected as fractions between 3 and 30 min were tested for antibacterial activity against Staphylococcus aureus ATCC 25923, and micro fractions from 5, 6, 7.5, 9, 10.5, 12, 13.5, 15, 19, and 30 min exhibited significant inhibitory effects. All spotted fractions on the TLC plate inhibited the growth of Escherichia coli ATCC 25922, except for micro fractions from 3, 18, and 21 min. Moreover, molecular docking analysis of quinones in the extract identified 3a, 4a-Dihydroxy-a-tetralone (C-2) as the most potent compound in interacting with bacteria sterol 14-demethylase protein, with docking scores of − 6.715 and − 7.196 kcal/mol, respectively.

Significance of Astragaloside IV from the Roots of Astragalus mongholicus as an Acetylcholinesterase Inhibitor-From the Computational and Biomimetic Analyses to the In Vitro and In Vivo Studies of Safety.

The main aim of the study was to assess the acetylcholinesterase-inhibitory potential of triterpenoid saponins (astragalosides) found in the roots of Astragalus mongholicus. For this purpose, the TLC bioautography method was applied and then the IC50 values were calculated for astragalosides II, III and IV (5.9 μM; 4.2 μM, and 4.0 μM, respectively). Moreover, molecular dynamics simulations were carried outto assess the affinity of the tested compounds for POPC and POPG-containing lipid bilayers, which in this case are the models of the blood-brain barrier (BBB). All determined free energy profiles confirmed that astragalosides exhibit great affinity for the lipid bilayer. A good correlation was obtained when comparing the logarithm of n-octanol/water partition coefficient (logPow) lipophilicity descriptor values with the smallest values of free energy of the determined 1D profiles. The affinity for the lipid bilayers changes in the same order as the corresponding logPow values, i.e.,: I > II > III~IV. All compounds exhibit a high and also relatively similar magnitude of binding energies, varying from ca. -55 to -51 kJ/mol. Apositive correlation between the experimentally-determined IC50 values and the theoretically-predicted binding energies expressed by the correlation coefficient value equal 0.956 was observed.

ISOLATION OF ENDOPHYTIC FUNGI FROM PATCHOULI LEAVES (POGOSTEMON CABLIN BENTH) AS ANTIBACTERIAL AGAINST PATHOGENIC BACTERIA BY BIOAUTOGRAPHY AND AGAR DIFFUSION

Patchouli (Pogostemon cablin Benth) is a plant used as a substance in the pharmaceutical industry such as the manufacture of anti-inflammatory, antifungal, anti-insect and anti-inflammatory drugs. This study aimed to obtain endophytic fungi isolates having the potential as antibacterial by using TLC- Bioautography and agar diffusion methods. This research was conducted by isolating the endophytic fungi from Patchouli leaves and after that, purification and macroscopic tests were carried out and ten isolates were obtained pure. The results of the isolate screening test with codes IFDN 1, IFDN 3, IFDN 4, IFDN 6, IFDN 7, IFDN 8 and IFDN 9, showed the highest inhibition. Furthermore, the antibacterial activity was assayed by TLC-Bioautography. The results showed active spots (Rf 3 = 0.54; Rf 4 = 0.61; Rf 6 = 0.58; Rf 7 = 0.45; Rf 8 = 0.54; Rf 9 =0.47) against Eschericia coli, Staphylococcus aureus and Staphylococcus epidemics. The results of the antibacterial activity test by diffusion method obtained an inhibition zone for each bacterium, namely Staphylococcus aureus of 8.66 mm, Staphylococcus epidermidis of 8.38 mm, and Eschericia coli of12.23 mm.Key Words: Agar diffusion; Antibacterial; Endophytic fungi; skin infections; TLC Bioautograph.

Evaluation of antimicrobial activity of Streptomyces pactum isolated from paddy soils and identification of bioactive volatile compounds by GC-MS analysis.

The objective of this study was to isolate Streptomyces sp. from north paddy soils of Iran and investigation and identification of the bioactive compounds by carrying out GC-MS analysis. Antimicrobial activity was investigated by well diffusion agar technique against the pathogenic microorganisms including Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 4698, Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 9144, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis ATCC 43071. Based on the results of gene sequencing of gene 16S rRNA and phylogenetic analysis, the isolated sample belongs to the genus Streptomyces with the highest degree of resemblance (99/87%) to the Streptomyces pactum strain ACT12. The isolate showed a broad spectrum of antibacterial activity against test microorganisms. This isolate showed maximum antibacterial activity against M. luteus (25 ± 0.5 mm) and the most resistant microorganism against antibacterial activity of this isolate was P. aeruginosa (9 ± 0.5 mm). The contact bioautography method was used to detect compounds that were responsible for antimicrobial activity and showed the active compounds with Rf values of 0.8-0.9. The identification of bioactive metabolites were performed using gas chromatography-mass spectrometry (GC-MS). GC-MS analysis of the extract showed the presence of 15 volatile compounds. The main compounds were Methyl-3-(3,5-ditertbutyl-4-hydroxyphenyl) (10.88%) and Dibutyl phthalate (8.34%) in comparison with other bioactive compounds. The results showed that north paddy soils of Iran are a rich source of microbial flora for the production of antimicrobial compounds and useful for antimicrobial compounds discovery from Streptomyces sp.

Agar-free high-performance thin-layer chromatography–bioautography method for the qualitative estimation of α-amylase inhibitor in Syzygium cumini seed extract and formulation

Agar-free high-performance thin-layer chromatography–bioautography method for the qualitative estimation of α-amylase inhibitor in Syzygium cumini seed extract and formulation

High-performance thin-layer chromatography – antibacterial assay first reveals bioactive clerodane diterpenes in giant goldenrod (Solidago gigantea Ait.)

The present work introduces a high-performance thin-layer chromatography (HPTLC)–direct bioautography method using the Gram-positive plant pathogenic bacterium, Rhodococcus fascians. The screening and isolation procedure comprised of a non-targeted high-performance thin-layer chromatography-effect-directed analysis (HPTLC–EDA) against Bacillus subtilis, B. subtilis subsp. spizizenii, R. fascians, and Aliivibrio fischeri, a targeted HPTLC–mass spectrometry (MS), and bioassay-guided column chromatographic (preparative flash and semi-preparative HPLC) fractionation and purification. The developed new separation methods enabled the discovery of four bioactive cis-clerodane diterpenes, solidagoic acid H (1), solidagoic acid E (2), solidagoic acid I (3), and solidagoic acid F (4), in the n-hexane extract of giant goldenrod (Solidago gigantea Ait.) leaf for the first time. These compounds were identified by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. The initially used HPTLC method (chloroform – ethyl acetate – methanol 15:3:2, V/V/V) was changed (to n-hexane – isopropyl acetate – methanol – acetic acid 29:20:1:1, V/V/V/V) to achieve the separation of the closely related isomer pairs (1–2 and 3–4). Compounds 1 and 3 exhibited moderate antibacterial activity against the Gram-positive B. subtilis subsp. spizizenii and R. fascians bacterial strains in microdilution assays with half-maximal inhibitory concentration (IC50) values in the range of 32.3–64.4 µg/mL. The mass spectrometric fragmentation of the isolated compounds was interpreted and their previously published NMR assignments lacking certain resonances were completed.

ANTIBACTERIAL ACTIVITY OF PATCHOULI OIL REFINERY WASTE AGAINTS STAPHYLOCOCCUS AUREUS BACTERIA

Patchouli oil refining waste is biomass or organic waste which has chemical contect so that it has antibacterial potential. This study aims to determine the antibacterial activity of patchouli oil refining waste against Staphylococcus aureus and knowing the groups of compounds identified as having antibacterial activity after the TLC Bioautography test was carried out. The sample was extracted using the maceration method with 96% ethanol solvent. Antibacterial activity testing was carried out with a concentration variant of 50%, 25% and 10% with the well method. Thin layer Chromatography (TLC) using n-hexane : ethyl acetate (3:1) elucants. TLC Bioautography using contact bioautography method. From the research that has been done, it is found that the ethanol extract of patchouli oil refining can inhibit Staphylococcus aureus which has the largest diameter of resistance with a concentration of 50% which is categorized as very strong. The results of the TLC Bioautography study showed the saponin compound class after being sprayed with H2SO4 10% reagent, it was found that the ethanol extract of patchouli oil refined pulp has antibacterial activity against Staphylococcus aureus and compounds to play a role in antibacterial activity are saponins.

Testing The Antibacterial Activity of Crude Extracts of Tithonia diversifolia Leaves, Flowers, and Roots in Various Solvent Fraction

Medicinal plants are natural sources of effective antimicrobials from natural products. Tithonia diversifolia ( Asteraceae ) is a conventional herb that can be used for the treatment of malaria, wounds, and other diseases with biological properties such as antioxidant, antimalarial, antidiabetic, antibacterial, and anti-hyperglycemic. Research on Tithonia diversifolia leaf extract as an antibacterial has been widely carried out. However, its use as a natural antibacterial is rarely studied, especially on flowers and roots. This study aimed to test the antibacterial activity of leaf, flower, and root extracts of the Tithonia diversifolia plant in various solvent fractions. The methods used in this research were Antibacterial Testing with Agar Diffusion and Direct Bioautography Methods, while the solvent fractions used were petroleum ether, chloroform, and ethyl acetate. The results showed that the crude extract of Tithonia diversifolia leaves, flowers, and roots for petroleum ether, chloroform, and ethyl acetate fractions contained antibacterial compounds. They could inhibit the growth of E. coli, P. aeruginosa, S. aureus , and B. subtilis bacteria, with inhibitory activity in chloroform fraction, which was better than in petroleum ether and ethyl acetate fractions. The results of the bioautography test showed that the inhibition was good on gram-positive bacteria with the highest number of spots in the chloroform fraction for the crude leaf and flower extract samples.

New robustaflavone from Garcinia latissima Miq. leave and Its antibacterial activity.

Isolation and determination of antibacterial compounds from plants are essential to obtain a new antibacterial as a substitute for conventional resistant antibiotics. This study aims to isolate and identify a new robustaflavone as antibacterial activity from Garcinia latissima Miq. leave. In this study, the isolation process was carried out using column chromatography followed by preparative thin layer chromatography (TLC) based on the TLC profile. The fraction D was tested for anti-bacterial Bacillus subtilis using the TLC bioautography method. The isolates obtained were then identified using 1H-NMR, 13C-NMR, distortionless enhancement by polarization transfer, heteronuclear single quantum coherence, and heteronuclear multiple bond coherence. The Activity assay of the isolate was performed using the microdilution method. A pure compound obtained the result of the separation process with eluent n-hexane: Ethyl acetate (3:2) with Rf 0.6. This spot follows the spot in the contact bioautographic result of fraction D, the spot with Rf 0.6 gives an inhibition zone. After identifying and purifying the isolate were known as Robustaflavone, this compound has activity against B. subtilis with a (minimum inhibitory concentration) value of 2500 ppm. Robustaflavone successfully isolated and identified from G. latissima leave and its antibacterial activity.

TLC-contact bioautography and disc diffusion method for investigation of the antibacterial activity of Melastoma malabathricum L. leaves

The emergence of multi-resistant strains of bacteria reinforces the need to discover new antibacterial agents that are able to combat resistant microorganisms. Medicinal plants are a valuable natural source of bioactive substances against various infectious diseases. Melastoma malabathricum L. is an important herb that is traditionally used to treat several ailments associated with microbial infection diseases such as wounds, diarrhea, dysentery, and toothache. This study investigated extracts of M. malabathricum L. for antibacterial properties against Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MRSA). Disc diffusion and TLC-contact bioautography techniques were employed to examine antibacterial properties of n-hexane, ethyl acetate, and ethanol extracts with observations of diameter inhibition zones and Rf values. Investigation of active compounds in TLC-bioautography used several reagents including citroboric, cerium (IV) sulfate and 2,2-diphenyl-1-picrylhydrazyl (DPPH), continued by identification of chromatogram profiles through densitometry analysis. The three extracts showed good inhibition against bacterial strains with diameter inhibition zones in the range of 8.0 – 14.0 mm with a number of active spots on TLC-contact bioautography for each extract. This plant may serve as useful source of antibacterial agents for resistant microorganisms and further investigation is needed of its bioactive pure compounds as well as their particular therapeutic potentials and applications.

Anti-microbial Activities of <em>Pseudomonas</em> Metabolites Methanolic Extracts against Different MDR Bacterial Pathogens

Background: A Multiple drug resistance (MDR) in pathogenic bacteria has become a significant public health issue for treatment. Among bacteria, Pseudomonas is another important genus except Streptomyces and Bacillus in production of antimicrobial compounds. The current study aim to to determine the antibacterial activity and preliminary characterization of antibacterial compounds produced by Pseudomonas species such as BB1D11, BN2D41, TG1D11, TR1D41, LH1D11 and TN1D41. Method: The antibacterial activity was checked by using bio autography method as well as agar well diffusion method, against four multiple drug resistant bacteria including three Gram negative bacteria (E.coli, Acinetobacter and Pseudomonas) and one Gram positive bacterium (Methicillin Resistant Staphylococcus aureus). Isolation test showed good activity against all the four MDR bacteria, by producing clear zone of diameter from 2mm up to 20mm. Optimum temperature for growth and antibiotic production of Pseudomonas BB1D11, BN2D41, TG1D11, TR1D41, LH1D11 and TN1D41 was 37C0. It produces more metabolites when subjected to shaking incubation. In thin layer chromatography, the extracts were repeatedly inserted on a silica gel coated plate, which was run in mobile phase. Normal HPLC was perform to reveal the presence of antibacterial compounds. Results: By well diffusion assay a zone of inhibition range from 2-18 mm of diameter against different test bacteria. The components were separated, resulting in the formation of bands with different colors, each showing a different compounds. Biological screening was performed by bio autography, metabolites showed a significant activity at retention factor of 0.89. While HPLC at retention time 2.50-2.90 showed presence of significant antibacterial compounds. Conclusion: Pseudomonas BB1D11, BN2D41, TG1D11, TR1D41, LH1D11 and TN1D41 showed promising anti-microbial activity against different MDR bacteria. It is concluded that HPLC revealed the presence of DAPG at retention time 2.90 which inhibit the growth of MDR bacterial strains.

Bioprospection of Enggano Macroscopic Fungi as Antibacterial and Antioxidant Agents

Macrofungi in Indonesia have not been widely studied for their pharmacological activity, especially as a source of antibacterial and antioxidant properties, even though Indonesia as a tropical country has quite a high diversity of macrofungi. This study aims to reveal the potential of macrofungi from the Enggano forest as a source of antibacterial and antioxidant compounds. Four types of macrofungi were collected and their metabolites were extracted using four types of organic solvents. Antibacterial and antioxidant activity assay of the extract was carried out using the TLC Bioautography method. From the sixteen macrofungal extracts, there is one extract that has the strongest antibacterial activity compared to the others, namely n-hexane Coriolopsis polyzona. It showed moderate antibacterial activity against Staphylococcus aureus and Escherichia coli with MIC values of 256–128 μg/mL, respectively. Meanwhile, the antioxidant activity of the macrofungal extracts showed weak activity with IC50 values of 3080–7370 μg/mL (AAI values of 0.033–0.079).

Biological activity survey of Pereskia aculeata Mill. and Pereskia grandifolia Haw. (Cactaceae)

Background: Pereskia aculeata and P. grandifolia are non-traditional Brazilian vegetables with high nutritional value used in traditional medicine. The antioxidant, anticholinesterase, molluscicidal, cytotoxic, and antiproliferative properties of hydroethanolic extracts of P. aculeata and P. grandifolia leaves (PAL, PGL) and fruits (PAF, PGF) are investigated in this study. Methods: All extracts were prepared by maceration with ethanol 70%. Their antioxidant properties were assessed through DPPH, ABTS, FRAP, and β-carotene bleaching inhibition assays. A TLC bioautography method was employed to evaluate the inhibiting capacity of the acetylcholinesterase enzyme. The molluscicidal activity was tested against the snail Biomphalaria glabrata, which serves as an intermediate host for Schistosoma mansoni. The cytotoxic activity was assessed by a Artemia salina lethality test and the antiproliferative properties against seven human carcinoma cell lines. Results: Compounds with anticholinesterase activity were found in all extracts. Polar compounds present in PAF and PGL extracts were the most active (IC50 < 25 μg of dry mass) and had an adequate inhibition capacity of the AChE. PGF and PGL were classified as moderate (LC50 = 19.2 μg/ml) and modest molluscicidal agents (LC50 = 66.6 μg/ml), respectively. All extracts exhibited selective antiproliferative activity against human chronic myeloid leukemia cell lines (K562). PAL, PGL, and PGF presented potent antiproliferative activity (TGI ≤ 5 μg/ml). Conclusion: Both species exhibited anticholinesterase, cytotoxic and antiproliferative properties. This research supports the potential of these species as sustainable sources of nutraceutical compounds.

Evaluation of the Antibacterial Activities of Isolated Bioactive Components from the plant Adenodolichos paniculatus

The three bioactive components isolated included: component A (major phytochemicals were Bis (2-ethylhexyl) phthalate (16.36 %), 9,12-Octadecadienoic acid, ethyl ether (14.77 %) and 9.cis., 11.trans.-octadecadie noate (14.77 %), component B (major phytochemicals were 9,12-Octadecadienal (Linoleic acid) (40.98 %), Octadecanoic acid (Stearic acid) (9.26 %), Undecanoic acid, 10-bromo- (10-bromoudecanoic acid) (9.26 %) and n-Hexadecanoic acid (Palmitic acid) and component C (cis-9-octadecenoic acid (Oleic Acid) (30.45 %), Octadecanoic acid (Stearic acid) (17.33 %)). These components isolated from the chloroform fraction of Adenodolichos paniculatus are used by traditional medicinal practitioners for the management of mouth and throat infections. The antibacterial activities against Streptococcus pyogenes, Staphylococcus aureus and Pseudomonas aeruginosa were evaluated using bioautography and agar-well diffusion methods. The bioautogram result showed that component A had inhibited spots against S. pyogenes (17.50 mm) and P. aeruginosa (16.00 mm), corresponding to the TLC spots with Rf values of 0.594, 0.55 and 0.26, respectively. Component B showed inhibition spots against Streptococcus pyogenes (36.50 mm), Staphylococcus aureus (16.00 mm) and Pseudomonas aeruginosa (11.00 mm), corresponding to the TLC spots with Rf values 0.891, 0.87, 0.85 and 0.25, respectively. Component C showed inhibition spots against Streptococcus pyogenes (16.50 mm), Staphylococcus aureus (15.00 mm) and Pseudomonas aeruginosa (10.50 mm), corresponding to the TLC spots Rf values of 0.938, 0.44, 0.21 and 0.90, respectively. For the agar-well diffusion method, component A at 1 mg/ml inhibited Streptococcus pyogenes, Staphylococcus aureus and Pseudomonas aeruginosa growths with zones of inhibition 23.0, 19.5 and 17.50 mm, respectively. MIC and MBC of component A were 125, 250 and 250 and 250, 500 and 500 μg/ml, respectively. Component B at 1 mg/ml inhibited Streptococcus pyogenes, Staphylococcus aureus and Pseudomonas aeruginosa growth with zones of inhibition 30.0, 28.0 and 18.5 mm, respectively. MIC and MBC of the compound B were 31, 62 and 125 and 62, 125 and 250 μg/ml, respectively. Component C at 1 mg/ml inhibited Streptococcus pyogenes, Staphylococcus aureus and Pseudomonas aeruginosa growth with zones of inhibition 24.5, 20.5 and 17.0 mm, respectively. MIC and MBC of the component C were 62, 125 and 250 and 125, 250 and 500 μg/ml, respectively. This study confirmed that bioactive compounds of A. paniculatus root have antibacterial properties and support the use of this part of the plant as a traditional remedy for mouth and throat infections possibly caused by the test bacteria.

ANTIFUNGAL ACTIVITY OF ACTINOBACTERIA WITH A POTENTIAL TO INHIBIT RICE BLAST FUNGUS MAGNAPORTHE ORYZAE (ANAMORPH PYRICULARIA ORYZAE)

Objective: The aims of the present study were to screen the actinobacteria with high potential ability to produce secondary metabolites that have inhibitory activity against plant pathogenic fungi, Magnaporthe oryzae. Production of secondary metabolites was analysis by thin-layer chromatography and bioautography assay.
 Methods: Screening and selection of potential Streptomyces sp. morphological, cultural, physiological, and biochemical characterization of the screened isolate was carried out. Antifungal compound was confirmed by bioautography assay.
 Results: Bioautography method use in this study was found to be antifungal fraction from the crude extract. Antifungal secondary metabolites can be readily located on the plates by observing clear zones where active compounds inhibit fungal growth.
 Conclusion: The bioautography assay shows that this isolates can produce antifungal compound. Therefore, this isolate proves to be a promising microbe which can be further studied for its applications a biocontrol agent against rice blast fungi.

Validation of TLC-contact bioautography and TLC-densitometry methods for simultaneous limit test of streptomycin sulfate and kanamycin sulfate residues in fresh fish meat

A simple, sensitive, and accurate TLC method was combined and validated for a simultaneous limit test of streptomycin sulfate and kanamycin sulfate residues in fresh fish meat. The optimum mobile phase was 10% potassium dihydrogen phosphate. TLC-contact bioautography used Staphylococcus aureus ATCC 29737 as a test organism. For TLC-densitometry, analyte spots were detected using 1% w/v ninhydrin in ethanol and maximum absorbance at 400 nm. TLC-contact bioautography and TLC-densitometry methods were validated according to USP guidelines for the limit test with respect to selectivity and limit of detection (LOD) added with the accuracy of a recovery test of streptomycin sulfate and kanamycin sulfate in fresh fish samples was developed and validated. The result of LOD from the TLC-contact bioautography method for streptomycin sulfate and kanamycin sulfate were obtained 0.7028 μg and 0.8032 μg, respectively. Meanwhile, LOD of the TLC-densitometry method for streptomycin sulfate and kanamycin sulfate were obtained 0.0631 μg and 0.0685 μg, respectively. In this study, TLC-contact bioautography method showed better results than TLC-densitometry. TLC-contact bioautography appears a good choice for the simultaneous limit test of streptomycin sulfate and kanamycin sulfate residues in fresh fish samples.