Inshad Ali Khan

Piperine, a Phytochemical Potentiator of Ciprofloxacin against Staphylococcus aureus

ABSTRACT Piperine, a trans-trans isomer of 1-piperoyl-piperidine, in combination with ciprofloxacin markedly reduced the MICs and mutation prevention concentration of ciprofloxacin for Staphylococcus aureus, including methicillin-resistant S. aureus. The enhanced accumulation and decreased efflux of ethidium bromide in the wild-type and mutant (CIPr-1) strains in the presence of piperine suggest its involvement in the inhibition of bacterial efflux pumps.

Piperine as an inhibitor of Rv1258c, a putative multidrug efflux pump of Mycobacterium tuberculosis

OBJECTIVESnTo evaluate the role of piperine as an inhibitor of Rv1258c of Mycobacterium tuberculosis.nnnMETHODSnRifampicin, in combination with piperine, was tested against M. tuberculosis H37Rv and rifampicin-resistant (rif(r)) M. tuberculosis. A laboratory-generated rifampicin-resistant mutant (rif(r)) of M. tuberculosis was tested for drug susceptibility and the expression level of the putative efflux protein (Rv1258c) by real-time PCR. The three-dimensional (3D) structure of Rv1258c was also predicted using an in silico approach.nnnRESULTSnIn the present study, rifampicin in combination with piperine, a trans-trans isomer of 1-piperoyl-piperidine, reduced the MIC and mutation prevention concentration (MPC) of rifampicin for M. tuberculosis H37Rv, including multidrug-resistant (MDR) M. tuberculosis and clinical isolates. Moreover, piperine effectively enhanced the bactericidal activity of rifampicin in time-kill studies and also significantly extended its post-antibiotic effect (PAE). In the presence of rifampicin, M. tuberculosis rif(r) showed a 3.6-fold overexpression of Rv1258c. The 3D structure of Rv1258c, using in silico modelling, was analysed to elucidate the binding of piperine to the active site.nnnCONCLUSIONSnThe results of this study are suggestive of piperines involvement in the inhibition of clinically overexpressed mycobacterial putative efflux protein (Rv1258c). Piperine may be useful in augmenting the antimycobacterial activity of rifampicin in a clinical setting.

Novel structural analogues of piperine as inhibitors of the NorA efflux pump of Staphylococcus aureus

OBJECTIVESnEvaluation of novel synthetic analogues of piperine as inhibitors of multidrug efflux pump NorA of Staphylococcus aureus.nnnMETHODSnA library of piperine-derived compounds was evaluated for their potential to inhibit ethidium bromide efflux in NorA-overexpressing S. aureus SA 1199B. The active compounds were then individually combined with ciprofloxacin to study the potentiation of ciprofloxacins activity.nnnRESULTSnBased on the efflux inhibition assay, a library of 200 compounds was screened. Three piperine analogues, namely SK-20, SK-56 and SK-29, were found to be the most potent inhibitors of the NorA efflux pump. These inhibitors acted in a synergistic manner with ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. These analogues were 2- to 4-fold more potent than piperine at a significantly lower minimal effective concentration. Furthermore, these inhibitors also significantly suppressed the in vitro emergence of ciprofloxacin-resistant S. aureus.nnnCONCLUSIONSnA newly identified class of compounds derived from a natural amide, piperine, is more potent than the parent molecule in potentiating the activity of ciprofloxacin through the inhibition of the NorA efflux pump. These molecules may prove useful in augmenting the antibacterial activities of fluoroquinolones in a clinical setting.

In vitro antifungal activity of hydroxychavicol isolated from Piper betle L

BackgroundHydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies.MethodsThe minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species respectively. Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide.ResultsHydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 μg/ml for yeasts, 125 to 500 μg/ml for Aspergillus species, and 7.81 to 62.5 μg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 × MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 × MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 × to 8 × MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol.ConclusionsThe antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.

Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus

OBJECTIVESnTo delineate the role of capsaicin (8-methyl-N-vanillyl-6-nonenamide) as an inhibitor of the NorA efflux pump and its impact on invasion of macrophages by Staphylococcus aureus.nnnMETHODSnCapsaicin in combination with ciprofloxacin was tested for activity against S. aureus SA-1199B (NorA overproducing), SA-1199 (wild-type) and SA-K1758 (norA knockout). The role of NorA in the intracellular invasion of S. aureus and the ability of capsaicin to inhibit this invasion was established in J774 macrophage cell lines. The three-dimensional structure of NorA was predicted using an in silico approach and docking studies of capsaicin were performed.nnnRESULTSnCapsaicin significantly reduced the MIC of ciprofloxacin for S. aureus SA-1199 and SA-1199B. Furthermore, capsaicin also extended the post-antibiotic effect of ciprofloxacin by 1.1 h at MIC concentration. There was a decrease in mutation prevention concentration of ciprofloxacin when combined with capsaicin. Inhibition of ethidium bromide efflux by NorA-overproducing S. aureus SA-1199B confirmed the role of capsaicin as a NorA efflux pump inhibitor (EPI). The most significant finding of this study was the ability of capsaicin to reduce the intracellular invasion of S. aureus SA-1199B (NorA overproducing) in J774 macrophage cell lines by 2 log(10).nnnCONCLUSIONSnThis study, for the first time, has shown that capsaicin, a novel EPI, not only inhibits the NorA efflux pump of S. aureus but also reduces the invasiveness of S. aureus, thereby reducing its virulence.

Piperine analogs as potent Staphylococcus aureus NorA efflux pump inhibitors.

Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors.

Evaluation of the Antimicrobial, Antioxidant, and Anti-Inflammatory Activities of Hydroxychavicol for Its Potential Use as an Oral Care Agent

ABSTRACT Hydroxychavicol isolated from the chloroform extraction of aqueous extract of Piper betle leaves showed inhibitory activity against oral cavity pathogens. It exhibited an inhibitory effect on all of the oral cavity pathogens tested (MICs of 62.5 to 500 μg/ml) with a minimal bactericidal concentration that was twofold greater than the inhibitory concentration. Hydroxychavicol exhibited concentration-dependent killing of Streptococcus mutans ATCC 25175 up to 4× MIC and also prevented the formation of water-insoluble glucan. Interestingly, hydroxychavicol exhibited an extended postantibiotic effect of 6 to 7 h and prevented the emergence of mutants of S. mutans ATCC 25175 and Actinomyces viscosus ATCC 15987 at 2× MIC. Furthermore, it also inhibited the growth of biofilms generated by S. mutans and A. viscosus and reduced the preformed biofilms by these bacteria. Increased uptake of propidium iodide by hydroxychavicol-treated cells of S. mutans and A. viscosus indicated that hydroxychavicol probably works through the disruption of the permeability barrier of microbial membrane structures. Hydroxychavicol also exhibited potent antioxidant and anti-inflammatory activities. This was evident from its concentration-dependent inhibition of lipid peroxidation and significant suppression of tumor necrosis factor alpha expression in human neutrophils. Its efficacy against adherent cells of S. mutans in water-insoluble glucan in the presence of sucrose suggests that hydroxychavicol would be a useful compound for the development of antibacterial agents against oral pathogens and that it has great potential for use in mouthwash for preventing and treating oral infections.

Cytotoxicity and antimicrobial activity of the methanol extract and compounds from Polygonum limbatum.

The present study was designed to investigate the antimicrobial activity and the cytotoxicity of the methanol extract (PLA) as well as fractions (PLA1-4) and compounds [cardamomin (1), (±)-polygohomoisoflavanone (2), (S)-(-)-pinostrobin (3), 2,4-dihydroxy-3,6-dimethoxychalcone (4), (2S)-(-)-5-hydroxy-6,7-dimethoxyflavanone (5), and (2S)-(-)-5,7-dimethoxyflavanone (6)] obtained from leaves of Polygonum limbatum. The microbroth dilution was used to determine the minimal inhibitory concentration (MIC) of the samples against 11 microbial strains including Candida albicans, C. krusei, C. tropicalis, Aspergillus fumigatus, Pseudomonas aeruginosa, Escherichia coli, vancomycin-resistant Enterococcus faecalis (VRE), Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), S.epidermidis, and Mycobacterium tuberculosis H37Rv. The sulphorhodamine B cell growth inhibition assay was used to assess the cytotoxicity of the above samples on lung A549 adenocarcinoma, breast carcinoma MCF-7, prostate carcinoma PC-3, cervical carcinoma HeLa, and the acute monocytic leukemia cell line THP-1. The results of the MIC determination indicated that, apart from fraction PLA3, all other fractions as well as PLA and compound 3 were selectively active. MIC values were noted on 100 % of the 11 tested microorganisms for fraction PLA3, 72.7 % for PLA, fraction PLA2, and compound 4, 63.6 % for PLA1, and 54.5 % for fraction PLA4. The results of the cytotoxicity assay revealed that, except for A459 cells, more than 50 % inhibition of the proliferation was obtained with each of the tested samples on at least one of the four other cell lines. IC₅₀ values below 4 µg/mL were obtained with 1 and 4 on THP-1 cells. The overall results of the present study provided baseline information for the possible use of Polygonum limbatum as well as some of the isolated compounds for the control of cancer diseases and mostly leukemia.

Antistaphylococcal and biofilm inhibitory activities of acetyl-11-keto-β-boswellic acid from Boswellia serrata

BackgroundBoswellic acids are pentacyclic triterpenes, which are produced in plants belonging to the genus Boswellia. Boswellic acids appear in the resin exudates of the plant and it makes up 25-35% of the resin. β-boswellic acid, 11-keto-β-boswellic acid and acetyl-11-keto-β-boswellic acid have been implicated in apoptosis of cancer cells, particularly that of brain tumors and cells affected by leukemia or colon cancer. These molecules are also associated with potent antimicrobial activities. The present study describes the antimicrobial activities of boswellic acid molecules against 112 pathogenic bacterial isolates including ATCC strains. Acetyl-11-keto-β-boswellic acid (AKBA), which exhibited the most potent antibacterial activity, was further evaluated in time kill studies, postantibiotic effect (PAE) and biofilm susceptibility assay. The mechanism of action of AKBA was investigated by propidium iodide uptake, leakage of 260 and 280 nm absorbing material assays.ResultsAKBA was found to be the most active compound showing an MIC range of 2-8 μg/ml against the entire gram positive bacterial pathogens tested. It exhibited concentration dependent killing of Staphylococcus aureus ATCC 29213 up to 8 × MIC and also demonstrated postantibiotic effect (PAE) of 4.8 h at 2 × MIC. Furthermore, AKBA inhibited the formation of biofilms generated by S. aureus and Staphylococcus epidermidis and also reduced the preformed biofilms by these bacteria. Increased uptake of propidium iodide and leakage of 260 and 280 nm absorbing material by AKBA treated cells of S aureus indicating that the antibacterial mode of action of AKBA probably occurred via disruption of microbial membrane structure.ConclusionsThis study supported the potential use of AKBA in treating S. aureus infections. AKBA can be further exploited to evolve potential lead compounds in the discovery of new anti-Gram-positive and anti-biofilm agents.

Citral derived amides as potent bacterial NorA efflux pump inhibitors.

Monoterpene citral and citronellal have been used as starting material for the preparation of 5,9-dimethyl-deca-2,4,8-trienoic acid amides and 9-formyl-5-methyl-deca-2,4,8-trienoic acid amides. The amides on bioevaluation as efflux pump inhibitors (EPIs) against Staphylococcus aureus 1199 and NorA overexpressing S. aureus 1199B bacteria resulted in the identification of several of these as potent EPIs. Many of these amides have been shown to possess potency higher or equivalent to known EPIs such as reserpine, verapamil, carsonic acid, and piperine. In this communication, we report a convenient synthesis of alkenyl amides, their bioevaluation and identification as efflux pump inhibitors against S. aureus.