TLC Bioautography Research Articles

In vitroBioactivity Screening ofDesmostachya bipinnata

Plant metabolites are proven source to perform biological activities such as antimicrobial, anticancer, antioxidant etc. In this study, Desmotachya sp was collected from Chennai, Tamil Nadu, India. The crude extracts were obtained using various solvents like acetone, ethanol, chloroform and petroleum ether. The extracts obtained were subjected for qualitative analysis for phytochemicals as well as TLC bio autography analysis for antioxidants and antibacterial activity. Further the crude extracts were fractionized using column chromatography and all the fractions were subjected for spot assay for antioxidant activity.

THE STUDY OF ANTIFUNGAL ACTIVITY FROM INDIGENOUS PLANTS FROM INDONESIA: AN IN VITRO STUDY

Objective: This study aimed to look for new agents from extracted Indonesia herbs which have antifungal activity with better safety profile against Candida albicans, Microsporum gypseum, and Trichophyton mentagrophytes. Methods: Screening of eleven herbs was perform to determine the highest antifungal activity. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of selected extracts, rosemary (Rosmarinus officinalis) leaves, Java turmeric (Curcuma xanthorriza) rhizome, and ginger (Zingiber officinalis) rhizome; were determined by using microdilution and agar diffusion methods. Extracts were then combined to evaluate further activities. Selected extracts were fractionated by using liquid-liquid extraction, analyzed by TLC bioautography.Results: ethanolic extract of rosemary (Rosmarinus officinalis) leaves, Java turmeric (Curcuma xanthorriza) rhizome, and ginger (Zingiber officinalis) rhizome in combination showed the highest activity and synergistic interaction against Trichophyton mentagrophytes. There were several components from fractions were actively inhibiting corresponding fungi according to TLC bioautography method. Conclusion: ethanolic extracts of rosemary, Java turmeric, and ginger had the highest antifungal potency, both as extract and in combination comparable to ketoconazole as reference drug. These selected extracts are potential to be used as new antifungal agents.

Scapaundulin C, a novel labdane diterpenoid isolated from Chinese liverwort Scapania undulate, inhibits acetylcholinesterase activity

In the present study, scapaundulin C (1), a new labdane diterpenoid, and four related known compounds scapaundulin A (2), 5α, 8α, 9α-trihydroxy-13E-labden-12-one (3), 5α, 8α-dihydroxy-13E-labden-12-one (4), and (13S)-15-hydroxylabd-8 (17)-en-19-oic acid (5), were isolated from the Chinese liverwort Scapania undulate (L.) Dum., using column chromatography. The structures of these compounds were determined on the basis of 1D- and 2D-NMR analyses. The acetylcholinesterase (AchE) inhibitory activity was evaluated using a bioautographic TLC assay and the cytotoxic activity was evaluated by the MTT method. All the compounds were reported for the first time to exhibit moderate AchE inhibitory activity with minimal inhibitory quantities ranging from 250 to 500 ng. All the compounds were tested for their cytotoxicity against five human tumor cell lines, A549, K562, A2780, Hela, and HT29, and compounds 3 and 4 exhibited moderate inhibitory effects on the growth of A2780 cells.

A new TLC bioautographic assay for qualitative and quantitative estimation of lipase inhibitors.

Lipase inhibitory assays based on TLC bioautography have made recent progress; however, an assay with greater substrate specificity and quantitative capabilities would advance the efficacy of this particular bioassay. To address these limitations, a new TLC bioautographic assay for detecting lipase inhibitors was developed and validated in this study. The new TLC bioautographic assay was based on reaction of lipase with β-naphthyl myristate and the subsequent formation of the purple dye between β-naphthol and Fast Blue B salt (FBB). The relative lipase inhibitory capacity (RLIC) was determined by a TLC densitometry with fluorescence detection, expressed as orlistat equivalents in millimoles on a per sample weight basis. Six pure compounds and three natural extracts were evaluated for their potential lipase inhibitory activities by this TLC bioautographic assay. The β-naphthyl myristate as the substrate improved the detection sensitivity and specificity significantly. The limit of detection (LOD) of this assay was 0.01 ng for orlistat, the current treatment for obesity. This assay has acceptable accuracy (92.07-105.39%), intra-day and inter-day precisions [relative standard deviation (RSD), 2.64-4.40%], as well as intra-plate and inter-plate precisions (RSD, 1.8-4.9%). The developed method is rapid, simple, stable, and specific for screening and estimation of the potential lipase inhibitors.

Acetylcholinesterase inhibitors and compounds promoting SIRT1 expression from Curcuma xanthorrhiza

Curcuma xanthorrhiza is a well-known traditional medicine with anti-inflammatory and anticancer activities, as well as protective effects against neurodegenerative disorders. A previous study revealed the acetylcholinesterase (AChE) inhibitory activity of some sesquiterpenoids from C. xanthorrhiza. In this study, further bioassay-guided isolation led to the identification of nine compounds for the first time from C. xanthorrhiza, including a new Guaiane-type sesquiterpene, zedoaraldehyde (1). Their structures were elucidated using NMR and MS techniques. The AChE inhibitory activities of compounds 1, 3, 4 and 7 were detected as minimum inhibitory quantities of 3, 4, 6 and 1μg, respectively, using a TLC bioautography assay. Meanwhile, compounds 1, 3, 4 and 8 could promote SIRT1 expression by 1.37-, 1.71-, 1.73- and 1.27-fold, respectively, in HEK293 cell lines exposed to compounds at a concentration of 20μM for 24h. SIRT1 is becoming an important drug target for new therapies in the treatment of neurodegenerative diseases. This study indicates the potential of sesquiterpenoids from C. xanthorrhiza for use against Alzheimer's disease.

Isolation and Identification of antibacterial compound producing Streptomyces parvulus ICN698 from a Wetland Ecosystem of Kanyakumari

Objective: The alarming threats of drug resistant pathogens have created the search of new antibiotics from different environmental niches. The goal of this study was to identify the antibacterial compound producing actinomycete from Therur pond wetland ecosystem and to isolate the active principle by bioassay-directed isolation. Methods: Actinomycete strain ICN698 was isolated and screened through double layer agar against drug resistant bacterial pathogens. Identification of the antagonistic species was carried out through 16S rDNA sequencing analysis. Production, extraction and TLC bioautography based partial purification was carried out and the Minimal inhibitory concentration value was determined using broth dilution method. Results: The antibacterial active strain was identified as Streptomyces parvulus ICN698. TLC bioautography revealed the Rf value of the active principle were detected at 0.11 and 0.29. Minimal inhibitory concentration of the partially purified compound showed 16 ?g/ml against MRSA, K. pneumonia, A. baumannii and Enterobacter sp. and 64 ?g/ml of MIC against P. aeruginosa. Conclusion: The antibacterial active strain ICN698 from Kanyakumari wetland ecosystem proves to be a good source of antimicrobial agent for further studies. Key Words: Wetland ecosystem, TLC bioautography, 16S rDNA sequencing, Enterobacter sp., Streptomyces parvulus

TLC Bioautography Guided Detection and Biological Activity of Antifungal Compounds from Medicinal Plant Acorus calamus Linn

TLC Bioautography Guided Detection and Biological Activity of Antifungal Compounds from Medicinal Plant Acorus calamus Linn

Isolation and identification of anti-methicillin resistant Staphylococcus aureus compounds from Phyllanthus columnaris stem bark

Aim: Phyllanthus columnaris Mull.Arg. was found to possess anti-methicillin resistant Staphylococcus aureus (antiMRSA) activities. This study aimed at isolating, identifying and evaluating the active compounds from the stem bark of Phyllanthus columnaris Mull.Arg. against MRSA. Methodology and results: Stem bark extracts (methanol, acetone and aqueous) of Phyllanthus columnaris were subjected to anti-MRSA screening by disc diffusion method. MIC and MBC tests were carried out to compare the lowest concentration to inhibit and kill the sixteen MRSA tested among the three extracts. TLC bioautography were performed to detect the bioactive compounds. Isolation of the two active compounds was performed by means of preparative TLC. Morphological and ultra-structure alterations of the MRSA treated with bioactive compounds after 24 h were revealed by scanning and transmission electron microscopy. Both methanol and acetone extracts exhibited good anti-MRSA activity with the lowest minimum inhibitory concentration (MIC) value for both extracts were 0.78 mg/mL and the lowest minimum bactericidal concentration (MBC) were 1.56 mg/mL. Bioassay-guided chromatography by bioautography revealed two active anti-MRSA compounds from both tannin-free methanol and acetone extracts and characterized as stigmasterol and lupeol by nuclear magnetic resonance (NMR) spectral data. Scanning and transmission electron microscopy of MRSA treated with stigmasterol and lupeol showed cell wall disruption, release of cytoplasmic compounds and decreased in cellular volume. Conclusion, significance and impact of study: Results obtained herein, may suggest that the stem bark of Phyllanthus columnaris possess anti-MRSA and the two of the active compounds isolated were stigmasterol and lupeol. Their anti-MRSA effects up to the morphological and ultra-structure studies were not reported earlier.

Acetylcholinesterase inhibitors from galbanum, the oleo gum-resin of Ferula gummosa Boiss.

Due to the correlation of Alzheimer's disease with a cholinergic deficit, acetylcholinesterase (AChE) inhibitors from herbal drugs or herbal medicinal preparations have gained a lot of scientific interest.In a previous study, several substances with AChE inhibitory activity were detected in galbanum by TLC bioautography and two of them were identified as auraptene and farnesiferol A. The aim of present study was the isolation and characterization of the other active compounds in galbanum.In an activity-guided approach, six components were isolated via column chromatography and solid phase extraction. The compounds were identified as deacetylkellerin, kellerin, umbelliferone, 5′-acetoxyauraptene, 5′-hydroxyauraptene and farnesiferol B and their concentrations in galbanum were determined by HPLC analysis with external standardization.Kellerin showed the best AChE inhibition with an IC50 value of 10.6μM which was about one third of the positive control physostigmine (IC50 2.91μM).

Antibiotic susceptibility of Genistein and Alkaloids from Rhizophora apiculata

Abstract Antimicrobial prospective of dichloromethane and methanolic extracts of Rhizophora apiculata was to evaluate against urinary tract infective strains such as Escherichia coli , Staphylococcus aureus , Pseudomonas aeurginosa , Proteus mirabilis and Klebsiella pneumoniae , methanolic extract showed better quality results at 250–350 µg/ml against all strains. And minimum inhibitory concentration and minimum bactericidal concentration were recorded at 160 µg/ml concentration level against for four strains excluding S. aureus at 240 µg/ml. Six phytochemicals (cardiac glycosides, saponins, tannins, coumarins, quinines and steroids) present in methanolic extract. Two spots were separated in thin layer chromatography and identified as genistein and unidentified alkaloids in addition to showed antibacterial activity in the TLC bioautography overlay assay screening. Together with characterized the genistein and alkaloids for FT-IR spectrum and displayed the occurrence of active functional groups such as hydrogen, carbonyl, carboxylic and ketones. These findings advise that the mangrove metabolites might be a potential candidate for the individualization of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

Chemical Composition of the Bark of Tetrapterys mucronata and Identification of Acetylcholinesterase Inhibitory Constituents

The secondary metabolite content of Tetrapterys mucronata, a poorly studied plant that is used occasionally in Brazil for the preparation of a psychotropic plant decoction called "Ayahuasca", was determined to establish its chemical composition and to search for acetylcholinesterase (AChE) inhibitors. The ethanolic extract of the bark of T. mucronata exhibited in vitro AChE inhibition in a TLC bioautography assay. To localize the active compounds, biological profiling for AChE inhibition was performed using at-line HPLC-microfractionation in 96-well plates and subsequent AChE inhibition bioautography. The analytical HPLC-PDA conditions were transferred geometrically to a preparative medium-pressure liquid chromatography column using chromatographic calculations for the efficient isolation of the active compounds at the milligram scale. Twenty-two compounds were isolated, of which six are new natural products. The structures of the new compounds (9, 10, 16-18, and 20) were elucidated by spectroscopic data interpretation. Compounds 1, 5, 6, 9, and 10 inhibited AChE with IC50 values below 15 μM.

An approach based on antioxidant fingerprint–efficacy relationship and TLC bioautography assay to quality evaluation of Rubia cordifolia from various sources

In this work, an approach based on antioxidant fingerprint-efficacy relationships and TLC bioautography assay was developed for quality evaluation of traditional Chinese medicine (TCM). First, chemical fingerprints of 20 batches of different sources of Rubia cordifolia were established by HPLC. The antioxidant activities of these samples were evaluated by 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. Then, five active components from fingerprint peaks were determined by both multiple correlation analysis and TLC bioautography assay. Similarity analysis and hierarchical clustering analysis (HCA) based on these active peaks were chosen to evaluate the quality of R. cordifolia from different sources. The samples with similarities below 0.9 had poorer antioxidant activities and those having stronger activities fell into the same cluster in HCA. Finally, five batches of commercial samples were processed in the same way to verify the feasibility of this method. The results suggested that chemical fingerprinting combined with similarity analysis based on fingerprint-efficacy relationship and TLC bioautography could evaluate the antioxidant activity of R. cordifolia from various sources. The established technique would also provide an easy method of evaluating the quality of other TCMs.

Characterization of Corrinoid Compounds in the Edible Cyanobacterium <i>Nostoc flagelliforme</i> the Hair Vegetable

Vitamin B12 contents in the edible cyanobacterium Nostoc flagelliforme, also known as hair vegetable, were assayed using a microbiological method. We detected high vitamin B12 contents in samples of naturally grown cells (109.2 ± 18.5 μg/100g dry weight) and cultured cells (120.2 ± 53.6 μg/100g dry weight). However, commercially available hair vegetable samples, which comprised fake substitutes and Nostoc, had variable contents (4.8 - 101.6 μg/100g dry weight) because concomitant fake items contain very low vitamin B12 contents. To evaluate whether natural and cultured N. flagelliforme samples contained vitamin B12 or pseudovitamin B12, corrinoid compounds were purified and identified as pseudovitamin B12 (approximately 72%) and vitamin B12 (approximately 28%) using silica gel 60 TLC bioautography and LC/MS. The results suggested that N. flagelliforme contains substantial amounts of pseudovitamin B12, which is inactive in humans.

A marine sponge associated strain of Bacillus subtilis and other marine bacteria can produce anticholinesterase compounds

BackgroundAcetylcholinesterase (AChE) inhibitors or anticholinesterases reduce the activity of enzyme acetylcholinesterase that degrades the neurotransmitter acetylcholine in the brain. The inhibitors have a significant pharmacological role in neurodegenerative diseases like Alzheimer’s and Parkinson’s etc. Although plants have been a significant source of these compounds, there are very few sporadic reports of microorganisms producing such inhibitors. Anticholinesterase activity in bacterial associates of marine soft corals and sponges were not previously reported.ResultsWe screened 887 marine bacteria for the presence of acetylcholinesterase inhibitors, in a microplate based assay, and found that 140 (15.8%) of them inhibit the electric eel enzyme, acetylcholinesterase. Majority of the active isolates were bacterial associates of soft corals followed by sediment isolates while most of the potent inhibitors belonged to the bacterial associates of marine sponges. Maximum inhibition (54%) was exhibited by a bacterial strain M18SP4P (ii), isolated from the marine sponge Fasciospongia cavernosa. Based on phenotypic characterization and 16S rDNA sequencing, the strain was identified as Bacillus subtilis - revealing yet another activity in a strain of the model organism that is considered to be a cell factory. TLC bioautography of the methanol extract of this culture, showed the presence of two major components having this activity, when compared to Galanthamine, the positive control.ConclusionFrom the results of our study, we conclude that acetylcholinesterase inhibitors are quite prevalent in marine bacteria, particularly the bacterial associates of marine invertebrates. Several potential AChE inhibitors in marine bacteria are waiting to be discovered to provide easily manipulable natural sources for the mass production of these therapeutic compounds.

Cholinesterase inhibitory potential of different Alternaria spp. and their phylogenetic relationships

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors are used for the treatment of various disorders related to decline in acetylcholine levels in the brain by inhibiting the activity of the neurotransmitter AChE. The present study reports the potential of endophytic Alternaria spp. for their potential to produce cholinesterase inhibitors active against both acetylcholine and butyrylcholine. Twenty-nine isolates belonging to Alternaria spp. were isolated from different plants and screened. Variation with respect to inhibitor production was observed in different isolates. Out of 29 cultures screened, good cholinesterase (both AChE and BChE) inhibitory activity in range of 70-85% was observed in three isolates, whereas three showed only AChE inhibition. No correlation was observed in AChE and BChE inhibitor production. TLC bioautography for the inhibitor in the selected cultures evinced different Rf values of inhibitors indicating different nature of the compounds produced. In order to analyze evolutionary relationships between producer and non-producer strains, phylogenetic analysis of six producer and five non-producer strains was carried out using amplified ITS-I-5.8SrDNA- ITS-II region. Phylogenetic analysis revealed majority of the non-producer strains to be present on different clades indicating different evolutionary origins. The dual cholinesterase inhibitory activity and the diversity in the inhibitors produced by different isolates could prove to be novel sources of pharmaceutical as well as agriculturally important biomolecules after purification and characterization.

Simultaneous isolation of seven compounds fromGlehnia littoralisroots by off-line overpressured layer chromatography guided by a TLC antioxidant autographic assay

A combinative method using overpressured layer chromatography (OPLC) and TLC bioautography against O2(•-) was developed to separate antioxidants from Glehnia littoralis roots. Through target-directed isolation by the TLC bioautographic method, seven compounds including five antioxidants were rapidly isolated by OPLC and identified as 1-linoloyl-3-palmitoylglycerol, facarindiol, panaxynol, isoimperatorin, β-sitosterol, scopoletin, and umbelliferone from G. littoralis roots. This OPLC method is a very suitable separation technique for light-sensitive polyacetylenes (panaxynol and facarindiol) with higher isolated yields compared to conventional open column chromatography. This is the first report on the separation of polyacetylenes by OPLC.

Extensive phytochemical investigation of the polar constituents of Diospyros bipindensis Gürke traditionally used by Baka pygmies

The water maceration and methanolic extract of the stem barks of Diospyros bipindensis, which is a medicinal plant used in Cameroon by Baka pygmies, revealed a complex high-performance liquid chromatography (HPLC) profile primarily composed of coumarin and naphthoquinone glycosides. The methanolic and apolar extracts also exhibited significant antifungal activity on a TLC bioautography assay against Candida albicans. HPLC-microfractionation in 96-well plates combined with bioautography enabled the rapid localization of the antifungal compound that was identified by HPLC-PDA and HPLC-MS analysis as plumbagin. These on-line structural information were also used to dereplicate four known compounds. The isolation of the polar constituents from the methanolic extract enabled the identification of eleven other compounds also present in the traditional preparation, nine of which are reported for the first time. The structures of those compounds were elucidated by UV, NMR and HR-MS analysis.

Isolation and characterization of antimicrobial metabolite producing endophytic Phomopsis sp. from Ficus pumila Linn. (Moraceae)

Abstract Background/Aim An endophytic fungus belongs to the genus Phomopsis was isolated from stem of Ficus pumila (Moraceae) and identified based on morphological and molecular characteristics. Methods Antimicrobial potential was investigated by agar plug, disc diffusion and TLC bioautography agar overlay methods. Detection of polyketide synthase (PKS) genes using DOP-PCR techniques. Results Endophytic Phomopsis sp. identified by morphological as well as molecular techniques, which exhibited broad a spectrum antimicrobial activity against Gram positive and Gram negative human and phytopathogenic bacteria and fungi. TLC bioautography detects the production of antimicrobial metabolites. Conclusion This preliminary study revealed that endophytic Phomopsis species are prolific producer of antimicrobial metabolites with wide range of antimicrobial activity.

TLC Bioautography: High Throughput Technique for Screening of Bioactive Natural Products

TLC bioautography is a method that combines chromatographic separation and in situ biological activity determination. The antimicrobial, antioxidant and enzyme inhibitory activities can be performed on TLC bioautography. This method is mainly used for preliminary screening natural products possessing these biological activities and for the bioactivity-directed fractionation and isolation of active components from complex extracts. This review covers the mechanisms and methodology of the antimicrobial, antioxidant activities, and enzyme inhibition adopted on TLC. It will be in particular discuss the recent advances in these technologies and assess their applications in targeting natural products.

Compounds from Gum Ammoniacum with Acetylcholinesterase Inhibitory Activity

The use of herbal medicinal preparations in dementia therapy has been studied based on experience from traditional medicine. A dichloromethane extract of gum ammoniacum, the gum-resin from Dorema ammoniacum D. Don had shown acetylcholinesterase (AChE) inhibitory activity in a previous study. The aim of this study was the isolation and characterization of the active compounds from this resin. The extract was investigated by a respective colorimetric microplate assay and the active zones were identified via TLC bioautography and isolated using several chromatographic techniques. The structures of the active components were characterized by one- and two-dimensional 1H and 13C NMR spectroscopy and mass spectrometry as (2′S,5′S)-2′-ethenyl-5′-(3-hy-droxy-6-methyl-4-oxohept-5-en-2-yl)-7-methoxy-2′-methyl-4H-spiro[chromene-3,1′-cyclopentane]-2,4-dione (1), which is an analogue of doremone A and a new natural compound, and as (2′S,5′R)-2′-ethenyl-5′-[(2R,4R)-4-hydroxy-6-methyl-3-oxohept-5-en-2-yl]-7-methoxy-2′-methyl-4H-spiro[chromene-3,1′-cyclo-pentane]-2,4-dione (2 = doremone A), (4E,8E)-1-(2,4-dihydroxyphenyl)-5,9,13-trimethyltetradeca-4,8,12-trien-1-one (3 = dshamirone), and 4,7-dihydroxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-2H-chromen-2-one (4 = am-moresinol). Dshamirone turned out to be the most active compound with an IC50 value for AChE inhibitory activity of 23.5 μM, whereas the other substances showed weak activity. The concentrations of the analytes in the resin were determined by HPLC as 3.1%, 4.6%, 1.9%, and 9.9%, respectively.