ABSTRACT
Aim: Identification of anti-Mycobacterium tuberculosis agents of plant origin, against sensitive and multidrug resistant (MDR) strains. Study Design: Assessing anti-M. tuberculosis activity of five Indian medicinal plants, which have been reported in traditional literature for various uses including respiratory ailments. Place and Duration of Study: Mumbai, India; May 2009 – December 2011. Methodology of Study: The reference strain (H37Rv), three susceptible and three MDR clinical isolates of M. tuberculosis were used. Acetone, ethanol and aqueous extracts (prepared sequentially) of Acorus calamus L. (rhizome), Andrographis paniculata Nees. (leaf), Ocimum sanctum L. (leaf), Piper nigrum L. (seed) and Pueraria tuberosa DC. (tuber) were tested at 1, 10 and 100 μg/ml using the Microplate Alamar Blue Assay. The active extracts were assessed for cytotoxicity on the human lung epithelial cell line (A549) using the neutral red assay and a phytochemical analysis was made using High Performance Thin Layer Chromatography (HPTLC). Results: Among the plants tested, the acetone extract of P. nigrum appears promising. It was effective against H37Rv, all susceptible isolates and one MDR isolate at 100 μg/ml. The ethanol extract caused some inhibition of growth, though less than the cut-off of 99%. A combination of acetone and ethanol extracts at 50 μg/ml each was effective against all isolates tested. The known active phytoconstituent of P. nigrum, piperine (also an efflux pump inhibitor), was effective against H37Rv in the presence of suboptimal concentration of Rifampicin, but not against the clinical isolates tested. Presence of piperine in the acetone and ethanol extracts was confirmed by HPTLC. Extracts of P. nigrum and piperine were not cytotoxic to the A549 cell line. Conclusion: Amongst the five plants tested, P. nigrum was active. The acetone extract may have active components in addition to piperine. It is possible that the class and expression of efflux pumps in H37Rv is different from that in the clinical isolates, and hence piperine did not inhibit these isolates. Thus, it is necessary to screen clinical isolates in addition to reference strains. The observation of the increased efficacy of the combination of acetone and ethanol extracts is interesting.
ABSTRACT
The natural resistance-associated macrophage protein (NRAMP1), Vitamin-D receptor (VDR) and Tumor necrosis factor (TNF-¦Á) have been associated in susceptibility to tuberculosis, but the results have been inconsistent. This study aimed to determine the association of NRAMP1, VDR, and TNF-¨¢ variant with development of pulmonary tuberculosis (PTB) among Iranian patients. The single nucleotide polymorphisms (SNPs) at INT4, D543, 3'UTR of NRAMP1 gene, SNPs in restriction sites of BsmI, and FokI of the VDR gene and SNPs of TNF-¦Á at -238,-308, -244,857,-863 positions were analyzed by PCR-RFLP among two groups of individual; patients with PTB (n=117) and healthy controls (n=60). Thereafter, the frequencies of extended haplotypes and diplotypes were estimated. No statistically significant differences were observed in allele frequencies of INT4, D543, 3'UTR of NRAMPI, FokI of VDR and TNF-¦Á at -238, -244,-863 and -857 position. Although, the frequency of b allele of BsmI [ORs: 0.24 CI95 percent (0.07-0.67 (p=0.001)] and -308 A variant in TNF-¦Á promoter region [ORs:0.26 CI95 percent( 0.07-0.77) (p=0.006)] were significantly more in PTB patients than healthy controls. The frequency of extended diplotypes of NRAMP [GG TGTG++GA; 0.02(0.001-0.0035)], VDR [FFBB; 0.2(0.6-0.6] and TNF-¦Á [CCCCGGGGGG; 0.49(0.25-0.97)] were statistically different in patients and control subjects (p<0.05). This study confirmed the association of SNPs in BsmI (B/b + b/b) of VDR and SNPs in -308A (G/A +G/G) of TNF-¦Á genes with susceptibility to tuberculosis in Iranian PTB patients. Furthermore, the extended haplotypes and diplotypes analysis can be considered as an alternative way to determine the host susceptibility to TB.