Resazurin Microplate Assay Research Articles

Chemical Composition and Biological Activities of the Essential Oil from Leaves of Cleidion javanicum Bl.

The essential oil from the leaves of Cleidion javanicum Bl. was isolated by hydrodistillation with the percentage yield of 0.003 % as a pale yellow liquid. The composition of the essential oil was analysed by means of GC-(FID) and GC-MS. Ten constituents accounting for 92.60% total oil were identified. The major components were ethyl linoleolate (32.12 %), hexadecanoic acid (26.77 %), trans-phytol (24.64 %) and iso-phytol (4.80 %). The antimicrobial, anticancer, antioxidant activities and cytotoxicity test of this essential oil were investigated. The oil showed non-cytotoxic effects against Vero cells (African green monky kidney) because it inhibited more than 50 % cell growth. The anticancer activity of the essential oil was performed using the Resazurin Microplate Assay (REMA). The oil showed anticancer activity against three cancerous human cell lines; KB-Oral Cavity Cancer, MCF7-Breast Cancer and NCI-H187-Small Cell Lung Cancer with the IC50 of 47.16 μgmL-1, 40.23 μgmL-1 and 49.95 μgmL-1 respectively. The oil also showed antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa with the Minimum Inhibition Concentration (MIC) values of 25.00 mg/mL and 12.50 mg/mL respectively, using agar diffusion method. In addition, the essential oil also showed significant antioxidant activity with the IC50 of 27.05 mg mL-1 by means of DPPH radical scavenging assay.

English

A series of 2,5-dimercapto-1,3,4-thiadiazole derivatives (compounds 1 to 10) were prepared by nucleophilic substitution reaction between 2,5-dimercapto-1,3,4-thiadiazole and chloroheterocyclic compounds in methanol and in the presence of potassium carbonate (compounds 1 to 5 and 8) or metallic sodium (compounds 6, 7, 9 and 10) at room temperature. The cytotoxic activity was determined by green fluorescent protein (GFP)-based assay and anti-candida activity was determined by resazurin microplate assay (REMA). Compounds 1 to 4, 8 and 9 showed in vitro cytotoxic activities against Vero cells (African green monkey kidney). Compounds 4 and 10 exhibited anti-candida activities against Candida albicans (ATCC 90028) with IC50 values of 1.94 and 19.10 µg/ml, respectively. Docking studies on the catalytic site of cytochrome P450 14a-demethylase were used to identify the chemical structures in the molecule responsible for cytotoxic and anti-candida activities of the synthesized compounds.   Key words: 2,5-dimercapto-1,3,4-thiadiazol, docking, cytotoxicity, antifungal.

Characterization of n-Hexane sub-fraction of Bridelia micrantha (Berth) and its antimycobacterium activity

BackgroundTuberculosis, caused by Mycobacterium tuberculosis (MTB), is the most notified disease in the world. Development of resistance to first line drugs by MTB is a public health concern. As a result, there is the search for new and novel sources of antimycobacterial drugs for example from medicinal plants. In this study we determined the in vitro antimycobacterial activity of n-Hexane sub-fraction from Bridelia micrantha (Berth) against MTB H37Ra and a clinical isolate resistant to all five first-line antituberculosis drugs.MethodsThe antimycobacterial activity of the n-Hexane sub-fraction of ethyl acetate fractions from acetone extracts of B. micrantha barks was evaluated using the resazurin microplate assay against two MTB isolates. Bioassay-guided fractionation of the ethyl acetate fraction was performed using 100% n-Hexane and Chloroform/Methanol (99:1) as solvents in order of increasing polarity by column chromatography and Resazurin microtiter plate assay for susceptibility tests.ResultsThe n-Hexane fraction showed 20% inhibition of MTB H37Ra and almost 35% inhibition of an MTB isolate resistant to all first-line drugs at 10 μg/mL. GC/MS analysis of the fraction resulted in the identification of twenty-four constituents representing 60.5% of the fraction. Some of the 24 compounds detected included Benzene, 1.3-bis (3-phenoxyphenoxy (13.51%), 2-pinen-4-one (10.03%), N(b)-benzyl-14-(carboxymethyl) (6.35%) and the least detected compound was linalool (0.2%).ConclusionsThe results show that the n-Hexane fraction of B. micrantha has antimycobacterial activity.

A novel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents

A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200 μM of XTT and 60 μM of menadione in 250 μl of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nm whereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG, M. tuberculosis and M. smegmatis respectively. The calculated Z′ factors were > 0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether, XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli.

Antimycobacterial screening of traditional medicinal plants using the microplate resazurin assay

Multidrug-resistant Mycobacterium tuberculosis strains have rapidly become a global health concern. North American First Nations communities have used traditional medicines for generations to treat many pulmonary infections. In this study, we evaluated the antimycobacterial activity of 5 medicinal plants traditionally used as general therapeutics for pulmonary illnesses and specifically as treatments for tuberculosis. Aqueous extracts of Aralia nudicaulis, Symplocarpus foetidus, Heracleum maximum, Juniperus communis, and Acorus calamus were screened for antimycobacterial activity against Bacillus Calmette-Guérin, Mycobacterium avium, and M. tuberculosis H37Ra using the colorimetric microplate resazurin assay. Extracts of Acorus calamus and H. maximum root demonstrated significant antimycobacterial activity comparable to that of the rifampin control (2 microg/mL). Evaluation of the cytotoxicity of these 2 extracts using the MTT assay also showed that the extracts were less toxic to 3 human cell lines than was the DMSO positive control. This study demonstrates that aqueous extracts of the roots of H. maximum and Acorus calamus possess strong in vitro antimycobacterial activity, validates traditional knowledge, and provides potential for the development of urgently needed novel antituberculous therapeutics.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis in Cotonou (Benin) using two low-cost colorimetric methods: resazurin and nitrate reductase assays

We have evaluated two simple, rapid and low-cost colorimetric methods for the detection of multidrug-resistant Mycobacterium tuberculosis. A total of 151 M. tuberculosis strains were tested for resistance to rifampicin (RMP) and isoniazid by resazurin microplate assay (REMA) and nitrate reductase assay (NRA) in comparison with the conventional proportion method (PM) on Löwenstein-Jensen medium. A complete agreement was found between NRA and PM, while one false RMP-susceptible result was found by REMA. REMA and NRA tests are rapid and inexpensive, and could be good alternatives to the conventional PM in low-resource countries.

Resazurin reduction assays for screening of anti-tubercular compounds against dormant and actively growing Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis

To develop simple, rapid, low-cost and robust assays for screening drugs against dormant and actively growing mycobacteria. Actively growing aerobic and hypoxia-adapted dormant cultures of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis were tested for susceptibility to standard antimicrobial drugs by resazurin reduction assay. The visual and fluorimetric MICs were compared with those obtained by the standard cfu assay. Drug MICs for M. tuberculosis and M. bovis BCG were determined by the aerobic resazurin microplate assay (REMA) and correlated well with those obtained by the cfu assay. Metronidazole and nitrofurans showed comparable bactericidal activity in the hypoxic resazurin reduction assay (HyRRA). The HyRRA assay was noted to be superior to the cfu assay in that it distinguished between metabolically active dormant bacteria and non-viable organisms, unlike the cfu assay that could not differentiate between these two populations. The HyRRA assay performed with good concordance in both fluorimetric and visual formats to distinguish between bactericidal and bacteriostatic effects of a drug. The REMA and HyRRA assays will be useful for anti-tubercular anti-dormancy compound screening and drug susceptibility testing in a safe, reliable, easy and cost-effective manner particularly in low resource countries. The application of the assays in M. smegmatis or M. bovis BCG offers the distinct advantage of rapidly and safely screening anti-tubercular compounds in a high-throughput format.

A fluorometric study of relative ocular lens cytosensitivity to multipurpose contact lens solutions using the resazurin assay method

This study investigated the relative ocular lens cytosensitivity to three multipurpose contact lens solutions using lens culture and resazurin microplate assay method. Comparison in the cell viability levels of the cultured bovine lenses as influenced by 3 h experimental exposure to three contact lens multipurpose solutions (OPTI-FREE Express, Alcon; ReNu MultiPlus, Bausch & Lomb; and COMPLETE Comfort Plus, Allergan) was performed. The pre- and post-exposure fluorescence emission levels of the lenses were obtained, and findings were compared to baseline and control measurements. The solutions demonstrated varying levels of sensitivity ( P < 0.1), exhibiting non-significant to significant reversible disruption of the lenses’cell viability levels as revealed by the fluorescence intensity levels in the following ranked order (COMPLETE < ReNu < OPTI-FREE solutions). The findings show that OPTI-FREE Express and ReNu MultiPlus solutions exhibited more cytosensitive effect compared to COMPLETE Comfort Plus. These results suggest that the resazurin assay together with lens culture would be a reliable system for in vitro investigation of the effect of contact lens solutions and other ophthalmic formulations on ocular tissues.