Surjeet Verma

A clerodane diterpene from Polyalthia longifolia as a modifying agent of the resistance of methicillin resistant Staphylococcus aureus

BACKGROUND Staphylococcus aureus infections are raising serious concern across the world. The effectiveness of conventional drugs is continuously decreasing due to global emergence of multidrug resistance (MDR) and therefore, new resistance-modifying agents (RMAs) are highly needed. HYPOTHESIS Clerodane diterpene 16α-hydroxycleroda-3,13(14)-Z-dien-15,16-olide (CD) from leaves of Polyalthia longifolia (Sonn.) Thwaites (Annonaceae) as RAM will be useful in improving the current treatment strategies for staphylococcal infections. STUDY DESIGN In the present study, we determine the resistance-modifying activity of CD using clinical isolates of MRSA. Further, the influence of CD on innate immune response was also evaluated in vitro and in vivo. The nature of potential interactions was determined by fractional inhibitory concentration indices (FICIs) calculated from microdilution assays and time-kill curves. RESULTS The result of in vitro combination study showed that CD significantly reduced MIC of fluoroquinolones up to 16-folds (FICI 0.315-0.500), while in S. aureus infected Swiss albino mice model, combination of CD with norfloxacin, significantly (p<0.01, p<0.001) lowered the systemic microbial burden in blood, liver, kidney, lung and spleen tissues in comparison to CD, norfloxacin alone as well as untreated control. Flow cytometry analysis clearly showed that CD significantly inhibited EtBr efflux and extended post-antibiotic effect. In qRT-PCR analysis, CD alone as well as in combination, significantly modulated the expression of various efflux pump genes including norA up to 2-fold in clinical isolate MRSA-ST2071. Further, the in vitro combination study of the CD (10, 5, 2.5µg/ml) along with the norfloxacin (10µg/ml) depicted a significant decline in the pro-inflammatory cytokines, IL6 and TNF-α. In septic shock mice model, CD did not exhibit any significant changes in the level of pro-inflammatory cytokines. CONCLUSION This is the first report on drug resistance-modifying potential of CD through inhibition of MDR efflux pump.

Bioactivity-guided isolation of antiplasmodial constituents from Conyza sumatrensis (Retz.) E.H. Walker.

Conyza sumatrensis (Retz.) E.H. Walker (Cs) leaves are used for traditional treatment of malaria in Cameroon. However, the antimalarial activity of the leaf constituents of this plant is still unexplored. The aim of our investigation was to evaluate the antiplasmodial activity of some bioactive constituents from Cs leaves. Compounds were isolated from Cs leaves and structurally elucidated using extensive spectroscopic analysis. The in vitro antiplasmodial activity of the extracts and pure compounds were evaluated on chloroquine-sensitive strain (NF54) of Plasmodium falciparum. The in vivo assay was done by administering seven doses of extracts in mice infected with Plasmodium berghei K173 through oral route. Cytotoxicity of pure compounds on murine macrophage cells was performed through [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] (MTT) test. Hemolysis and lactate dehydrogenase assays were also carried out using standard procedures. The in silico prediction of bioactive constituents was performed through Autodock Vina. Polarity-based extracts from Cs were found to be active against P. falciparum (NF54) and P. berghei (K173) in vitro and in vivo respectively. Further, bioactivity-guided isolation of n-hexane fraction yielded three compounds, (1), (2) and (3) with IC50 of 34, 17.9 and 18μg/ml, respectively, while the ethyl acetate fraction afforded the fourth compound with an IC50 of 25μg/ml, indicating anti-malarial potential of Cs through PfLDH interaction without compromising normal cell growth. This study reports for the first time, the antiplasmodial activity of bioactive constituents from Cs and confirms its traditional use.

Antifertility chromene from Blepharispermum subsessile

The anti-implantation activity of the methanol extract of Blepharispermum subsessile rhizomes and therefrom isolated active constituent desmethylisoencecalin (1) is reported.

Activity guided isolation and characterization of antioxidant and antibacterial agents from some local Nigerian plants

This study aimed to present the activity guided fractionation, isolation and characterization of antioxidants and antibacterial agents from combined mixture of plants (Vitex doniana, Diospyros mesipiliformis, Acacia polycantha, Pirinari macrophylla, Ficus sycomorus and Parkia biglobosa) and that of Pergularia tomentosa. Combined Mixture of Plants (CMP) is used locally in ratio of 1:1 for the treatment of bacterial infections. The CMP and P. tomentosa were extracted with methanol separately; the residues obtained were also separately suspended in water and successively fractionated with hexane, ethylacetate and n-butanol. All the fractions obtained were screened for antimicrobial and antioxidant activities. For CMP, only the ethyl acetate fraction (EF) indicated marginal antibacterial activity with 8.0, 7.0 and 7.0 mm zone of inhibition against Micrococcus luteus (MTCC 2470), Bacillus subtilis (MTCC 121) and Salmonella typhimurium, respectively. Minimum inhibitory concentration (MIC) for the CMP was greater than 1000 for M. luteus and S. typhimurium and 87.5 μg/ml for B. subtilis. The CMP fraction was subjected to chromatographic separations which resulted in the isolation and characterization of five bioactive constituents, gallic acid, 3β-OH-α-amyrin, 5,7,3’.4’,5’pentahydroxy-3-O-glucophyranoside flavones (myricetin 3-O-β-rhamnopyranoside), 5,7,3’,4’ tetrahydroxy-3O-glucopyranoside flavone (quercetin 3-O-β-rhamnopyranoside) and 3,5,7,3’,4’-pentahydroxy flavones (quercetin). They were characterized with the help of ESI-MS, IR, 1 H C 13 , HMBC/HSQC and COSY-NMR data. These compounds did not show antibacterial activity when tested separately but exhibited appreciable antioxidant activities in different manner. Chromatographic fractionation of hexane extract of P. tomentosa resulted in the isolation of lupeol acetate (LA) with marginal but selective activity against M. luteus and the activity is due to LA rather than the combined constituents. These findings suggest that the fractions of the extracts and pure compounds possess antibacterial and antioxidant properties.

RP-HPLC-DAD method for the identification of two potential antioxidant agents namely verminoside and 1-O-(E)-caffeoyl-β-gentiobiose from Spathodea campanulata leaves

A simple and reliable high-performance liquid chromatographic method was successfully developed for the study of fingerprint chromatograms of extract and fractions from the leaves of Spathodea campanulata (SC) using verminoside (1) and 1-O-(E)-caffeoyl-β-gentiobiose (2) as marker compounds. Antioxidant activity of SC was determined by using free radical of 2,2-diphenyl-1-picryl-hydrazyl-hydrate as an experimental model. The docking study of selected target, tyrosinase and ligands (ascorbic acid, compounds 1 and 2) was performed through Autodock Vina v0.8. Fingerprints of methanol, chloroform, ethylacetate, n-butanol and water extracts could resolve 13, 11, 22, 16 and 5 peaks, respectively. Extract, fractions and compounds 1 and 2 previously isolated from SC displayed remarkable antioxidant activity with radical-scavenging activity ranging from 2.5 to 6.7 μg/mL. In silico study identified compounds 1 and 2 as potential inhibitors of tyrosinase correlating with the observed antioxidant activity in vitro.

Membrane stabilisation: a possible anti-inflammatory mechanism for the extracts and compounds from Spathodea campanulata

This study was undertaken to evaluate the efficiency of extract, fractions and pure molecules from Spathodea campanulata (SC) towards inflammation. Polarity-based extracts of SC were found active in stabilising red blood cell (RBC) membrane indicating anti-inflammatory potential. Bioactivity-guided isolation of SC produced 1-O-(E)-caffeoyl-β-gentiobiose and (2S)-1,2-di-O-[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl]-3-O-[α-d-galctopyranosyl-(1″ → 6′)-O-β-d-galactopyranosyl] glycerol as the active constituents with 65.91% and 67.41% of membrane stability, respectively. Activity of the third compound (verminoside) could not be ascertained owing to extremely low recoverability. Furthermore, the isolated compounds were subjected to in silico studies. The compounds showed good binding affinity towards cyclooxygenase-2. Absorption, distribution, metabolism & excretion (ADME)-toxicity studies illustrated that the isolated compounds are free of toxicity. These observations help us to conclude that SC might exert its anti-inflammatory activity by soothing the RBC membrane as it is the case for non-steroidal anti-inflammatory drugs towards lysozomal membranes. Therefore, SC might be considered as a potential candidate for development of anti-inflammatory drugs.