The Application of Multiplate Resazurin Reduction Assay in The Screening for Anti-Mycobacterial Activity from Indonesian Medicinal Plants

Martha Sari,Wien Kusharyoto,Gita Syahputra

doi:10.14499/indonesianjpharm30iss3pp199

Martha Sari, Wien Kusharyoto + Show 1 more

Open Access

https://doi.org/10.14499/indonesianjpharm30iss3pp199

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Abstract

Tuberculosis (TB) is an airborne illness generated by Mycobacterium tuberculosis (Mtb), also one of the prominent infectious killers of adults worldwide. There is a pressing need to expand novel anti-mycobacterial drugs because of the increasing resistance of pathogenic mycobacteria to existing antibiotics. Native compounds acquired from microbial resources and medicinal cultivars have played an essential part as the origin of TB medications. The microplate resazurin reduction assay (MRRA) is generally utilized to assess natural and synthetic compounds for anti-mycobacterial activity. In our work, the MRRA method was employed to evaluate the anti-mycobacterial activity of extracts from curative plants using Mycobacterium smegmatis and Mycobacterium bovis BCG and to compare them to rifampicin as an anti-mycobacterial drug. The optimized MRRA utilized 2% aqueous DMSO and 62.5 μg/mL resazurin as an indicator compound in 5% aqueous Tween 80. The optimal incubation time for M. smegmatis was 24 hours, and for M. bovis BCG was 48 hours. The methanolic plant extracts were acquired from various Indonesian medicinal plants known to have anti-mycobacterial activity. The MRRA method using M. smegmatis or M. bovis BCG as anti-mycobacterial targets offers a distinct advantage such as low-cost, rapid, and safe screening for anti-mycobacterial activity in a middle to high-through-put-format.

Highlights

Tuberculosis (TB) is a worldwide infectious disease induced by Mycobacterium tuberculosis (Dheda et al, 2014)
In our effort to evaluate the antimycobacterial activity from Indonesian medicinal plants, we relied on the established microplate resazurin reduction assay (MRRA) protocol previously described using M. tuberculosis H37Rv as an antimycobacterial target (O’Neill et al, 2014)
Since M. smegmatis and M. bovis BCG were used as surrogate models in our experiments, the protocol had to be modified slightly to accommodate different growth characteristics of both strains compared to M. tuberculosis

Summary

INTRODUCTION

Tuberculosis (TB) is a worldwide infectious disease induced by Mycobacterium tuberculosis (Dheda et al, 2014). Indonesia is famously known for its rich and uncharacterized biodiversity, and Indonesian medicinal plants are frequently used to treat diseases as a means of reducing reliance on expensive imported and/or chemical drugs Such plants should be identified and screened on the basis of traditional knowledge for their effectiveness of the treatment of TB, and their molecular entities need to be isolated and further characterized since they might possess the high chance to become or generate new antimycobacterium substance (Garcia et al, 2012; Salomon et al, 2012). We reported our investigation on the anti-mycobacterium ability of thirty-three methanolic extracts of Indonesian medicinal plants based on resazurin reduction assay and searched out potential extract concentration for optimal growth inhibition towards M. smegmatis and M. bovis BCG, subsequently

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSIONS