Abstract
The study was designed to assess whether plant extracts / phytochemical (D-Pinitol) synergistically combine with antituberculosis drugs and act on Mycobacterium smegmatis (M. smegmatis) as well as assess their mode of action on Mycobacterium tuberculosis (M.tb) Filamenting temperature sensitive mutant Z (FtsZ) protein. Resazurin microtitre plate assay (Checker board) was performed to analyze the activity of plant extracts against M. smegmatis. Synergistic behaviour of plant extracts / D-Pinitol with Isoniazid (INH) and Rifampicin (RIF) were determined by time-kill and checker board assays. Elongation of M. smegmatis cells due to this treatment was determined by light microscopy. The effect of Hexane methanol extract (HXM) plant extracts on cell viability was determined using PI/SYTO9 dual dye reporter Live/Dead assay. Action of HXM plant extracts / D-Pinitol on inhibition of FtsZ protein was done using Guanosine triphosphatase (GTPase) light scattering assay and quantitative Polymerase Chain Reaction (qPCR). The Hexane-methanolic plant extract of Acacia nilotica, Aegle marmelos and Glycyrrhiza glabra showed antimycobacterial activity at 1.56 ± 0.03, 1.32 ± 0.02 and 1.25 ± 0.03 mg/mL respectively and that of INH and RIF were 4.00 ± 0.06 μg/mL and 2.00 ± 0.04 μg/mL respectively. These plant extracts and major phytochemical exudate D-Pinitol was found to act synergistically with antimycobacterial drugs INH and RIF with an FIC index ~ 0.20. Time-Kill kinetics studies indicate that, these plant extracts were bacteriostatic in nature. D-Pinitol in conjunction with INH and RIF exhibited a 2 Log reduction in the growth of viable cells compared to untreated. Attempt to elucidate their mode of action through phenotypic analysis indicated that these plant extracts and D-Pinitol was found to interfere in cell division there by leading to an abnormal elongated cellular morphology. HXM extracts and D-Pinitol synergistically combined with the first line tuberculosis drugs, INH and RIF, to act on M. smegmatis. The increase in the length of M. smegmatis cells on treatment with D-Pinitol and HXM extract of the plants indicated that they hinder the cell division mechanism thereby leading to a filamentous phenotype, and finally leading to cell death. In addition, the integrity of the bacterial cell membrane is also altered causing cell death. Further gene expression analysis showed that these plant extracts and D-Pinitol hampers with function of FtsZ protein which was confirmed through in vitro inhibition of FtsZ-GTPase enzymatic activity.
Highlights
Mycobacterium tuberculosis is a pathogenic organism which causes Tuberculosis
The presence of D-Pinitol in the three Hexane methanol extract (HXM) plant extracts were confirmed by both Gas Chromatography Mass Spectrophotometer (GCMS) and High Performance Liquid Chromatography (HPLC) analysis
The various active compounds as identified by the database with their peak number, concentration, and retention time (RT) of all three plant extracts and their biological activities of the phytochemicals identified by GCMS analysis are provided in the S1–S12 Figs and S1–S4 Tables
Summary
About a quarter of the global population is affected with this disease [1]. Due to emerging drug resistant strains, and reduced effectiveness of treatment due to failure in patient adherence to treatment regime leads to complication and failure of treatment [2]. There is a need to screen for novel antimycobacterial medicinal plant extracts to employ them as complementary and adjuvant medicine along with the conventional chemotherapy to increase the effectiveness and action of chemotherapeutic drugs. Plant extracts and their active components have been used to treat many diseases, and the structures of many phytochemicals have been the starting scaffold for the design of synthetic drugs, including aspirin and taxol [3]. Plant extracts possess phenolic compounds and their derivatives play an important role to protect the human body against the damage caused by free radicals [4]
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