Santosh Kumar Srivastava

Pharmacophore, QSAR, and ADME based semisynthesis and in vitro evaluation of ursolic acid analogs for anticancer activity

In the present work, QSAR models for predicting the activities of ursolic acid analogs against human lung (A-549) and CNS (SF-295) cancer cell lines were developed by a forward stepwise multiple linear regression method using a leave-one-out approach. The regression coefficient (r2) and the cross-validation regression coefficient (rCV2) of the QSAR model for cytotoxic activity against the human lung cancer cell line (A-549) were 0.85 and 0.80, respectively. The QSAR study indicated that the LUMO energy, ring count, and solvent-accessible surface area were strongly correlated with anticancer activity. Similarly, the QSAR model for cytotoxic activity against the human CNS cancer cell line (SF-295) also showed a high correlation (r2 = 0.99 and rCV2 = 0.96), and indicated that dipole vector and solvent-accessible surface area were strongly correlated with activity. Ursolic acid analogs that were predicted to be active against these cancer cell lines by the QSAR models were semisynthesized and characterized on the basis of their 1H and 13C NMR spectroscopic data, and were then tested in vitro against the human lung (A-549) and CNS (SF-295) cancer cell lines. The experimental results obtained agreed well with the predicted values.

Synergistic action of cinnamaldehyde with silver nanoparticles against spore-forming bacteria: a case for judicious use of silver nanoparticles for antibacterial applications

Silver has long been advocated as an effective antimicrobial. However, toxicity issues with silver have led to limited use of silver in nanoform, especially for food preservation. With the aim of exploring combinatorial options that could increase the antibacterial potency of silver nanoparticles and reduce the effective dosage of silver, we evaluated the extent of synergy that a combination of silver nanoparticles and an essential oil representative (cinnamaldehyde) could offer. A battery of gram-positive and gram-negative bacterial strains was utilized for antibacterial assays, and extents of synergism were calculated from fractional inhibitory concentration indices. The activity of nanoparticles was greatly enhanced when utilized in the presence of cinnamaldehyde. We observed combinatorial effects that were strongly additive against all the bacterial strains tested, and genuine synergy was found against spore forming Bacillus cereus and Clostridium perfringens – bacterial strains associated with release of cytotoxins in contaminated food and known for their persistence. Bacterial kill curve analysis revealed a very fast bactericidal action when a combination of two agents was used. The electron and atomic force microscopy also revealed extensive damage to the bacterial cell envelop in the presence of both agents. We also performed hemolysis assays to investigate and approximate the extent of toxicity exhibited by the two agents, and observed no adverse effect at the concentrations required for synergy. This study shows that safe levels of silver in nanoform in combination with essential oil component cinnamaldehyde can be effectively used for controlling the spore-forming bacterial species.

Simultaneous determination of three steroidal glycoalkaloids in Solanum xanthocarpum by high performance thin layer chromatography

A new high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitation of three bioactive steroidal glycoalkaloid (SGA) markers, solasonine (SN), solamargine (SM) and khasianine (KN) in the plant Solanum xanthocarpum. Extraction efficiency of targeted SGAs from plant matrix using methanol and acidified methanol were studied using percolation, ultrasonication and microwave techniques. The separation was achieved on silica gel 60F(254) TLC plates using chloroform-methanol-water as mobile phase. The quantitation of SGAs was carried out using the densitometric reflection/absorption mode at 520 nm after post chromatographic derivatization using Dragendorffs reagent. The method was validated for peak purity, precision, accuracy, robustness, limit of detection (LOD) and quantitation (LOQ). Method specificity was confirmed using retention factor (R(f)), Vis spectral correlation and electrospray ionization mass spectra (ESI-MS) of marker compounds in the sample track.

Large-scale separation of clavine alkaloids from Ipomoea muricata by pH-zone-refining centrifugal partition chromatography

Centrifugal partition chromatography in the pH-zone-refining mode was successfully applied to the separation of alkaloids, directly from a crude extract of Ipomoea muricata. The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether (MtBE)-acetonitrile-water (4:1:5, v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and trifluoroacetic acid (10 mM) to the aqueous mobile phase as an eluter. From 4 g of crude extract, 210 mg lysergol and 182 mg chanoclavine were obtained in 97% and 79.6% purities. Total yield recovery was >95%. Isolated alkaloids were characterized on the basis of their (1)H, (13)C NMR and ESI-MS data.

In Silico and In Vivo Anti-Malarial Studies of 18β Glycyrrhetinic Acid from Glycyrrhiza glabra

Malaria is one of the most prevailing fatal diseases causing between 1.2 and 2.7 million deaths all over the world each year. Further, development of resistance against the frontline anti-malarial drugs has created an alarming situation, which requires intensive drug discovery to develop new, more effective, affordable and accessible anti-malarial agents possessing novel modes of action. Over the past few years triterpenoids from higher plants have shown a wide range of anti-malarial activities. As a part of our drug discovery program for anti-malarial agents from Indian medicinal plants, roots of Glycyrrhiza glabra were chemically investigated, which resulted in the isolation and characterization of 18β-glycyrrhetinic acid (GA) as a major constituent. The in vitro studies against P. falciparum showed significant (IC50 1.69µg/ml) anti-malarial potential for GA. Similarly, the molecular docking studies showed adequate docking (LibDock) score of 71.18 for GA and 131.15 for standard anti-malarial drug chloroquine. Further, in silico pharmacokinetic and drug-likeness studies showed that GA possesses drug-like properties. Finally, in vivo evaluation showed a dose dependent anti-malarial activity ranging from 68–100% at doses of 62.5–250mg/kg on day 8. To the best of our knowledge this is the first ever report on the anti-malarial potential of GA. Further work on optimization of the anti-malarial lead is under progress.

Design, synthesis and in vitro evaluation of 18β-glycyrrhetinic acid derivatives for anticancer activity against human breast cancer cell line MCF-7.

In the present work, QSAR model was derived by multiple linear regression method for the prediction of anticancer activity of 18β-glycyrrhetinic acid derivatives against the human breast cancer cell line MCF-7. The QSAR model for anti-proliferative activity against MCF-7 showed high correlation (r(2)=0.90 and rCV(2)=0.83) and indicated that chemical descriptors namely, dipole moment (debye), steric energy (kcal/mole), heat of formation (kcal/mole), ionization potential (eV), LogP, LUMO energy (eV) and shape index (basic kappa, order 3) correlate well with activity. The QSAR virtually predicted that active derivatives were first semi-synthesized and characterized on the basis of their (1)H and (13)C NMR spectroscopic data and then were in-vitro tested against MCF-7 cancer cell line. In particular, octylamide derivative of glycyrrhetinic acid GA-12 has marked cytotoxic activity against MCF-7 similar to that of standard anticancer drug paclitaxel. The biological assays of active derivative selected by virtual screening showed significant experimental activity.

QSAR and Docking Based Semi-synthesis and in vitro Evaluation of 18 β-glycyrrhetinic Acid Derivatives Against Human Lung Cancer Cell Line A-549

For the prediction of anticancer activity of glycyrrhetinic acid (GA-1) analogs against the human lung cancer cell line (A-549), a QSAR model was developed by forward stepwise multiple linear regression methodology. The regression coefficient (r(2)) and prediction accuracy (rCV(2)) of the QSAR model were taken 0.94 and 0.82, respectively in terms of correlation. The QSAR study indicates that the dipole moments, size of smallest ring, amine counts, hydroxyl and nitro functional groups are correlated well with cytotoxic activity. The docking studies showed high binding affinity of the predicted active compounds against the lung cancer target EGFR. These active glycyrrhetinic acid derivatives were then semi-synthesized, characterized and in-vitro tested for anticancer activity. The experimental results were in agreement with the predicted values and the ethyl oxalyl derivative of GA-1 (GA-3) showed equal cytotoxic activity to that of standard anticancer drug paclitaxel.

HPTLC method for the simultaneous determination of four indole alkaloids in Rauwolfia tetraphylla: a study of organic/green solvent and continuous/pulse sonication.

A new validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitation of four antipsychotic indole alkaloids (IAs), reserpiline (RP, 1), α-yohimbine (YH, 2), isoreserpiline (IRP, 3) and 10-methoxy tetrahydroalstonine (MTHA, 4) as markers in the leaves of Rauwolfia tetraphylla. Extraction efficiency of the targeted IAs from the leaf matrix with organic and ecofriendly (green) solvents using percolation, ultrasonication and microwave techniques were studied. Non-ionic surfactants, viz. Triton X-100, Triton X-114 and Genapol X-80 were used for extraction and no back-extraction or liquid chromatographic steps were used to remove the targeted IAs from the surfactant-rich extractant phase. The optimized cloud point extraction was found a potentially useful methodology for the preconcentration of the targeted IAs. The separation was achieved on silica gel 60F(254) HPTLC plates using hexane-ethylacetate-methanol (5:4:1, v/v/v) as mobile phase. The quantitation of IAs (1-4) was carried out using the densitometric reflection/absorption mode at 520 nm after post chromatographic derivatization using Dragendorffs reagent. The method was validated for peak purity, precision, accuracy, robustness, limit of detection (LOD) and quantitation (LOQ). Method specificity was confirmed using retention factor (R(f)) and visible spectral (post chromatographic scan) correlation of marker compounds in the samples and standard tracks.

QSAR, docking and in vivo studies for immunomodulatory activity of isolated triterpenoids from Eucalyptus tereticornis and Gentiana kurroo.

Two triterpenoids ursolic acid (1) and lupeol (2) isolated and characterized from Eucalyptus tereticornis and Gentiana kurroo were subjected to in silico QSAR modeling and docking studies and later the predicted results were confirmed through in vivo experiments. QSAR modeling results showed that both the triterpenoids possess immunomodulatory and anti-inflammatory activity comparable to boswellic and cichoric acids, but were less active than levamisol. Docking results suggested that both the triterpenoids (1 and 2) showed immune modulatory and anti-inflammatory activity due to high binding affinity to human receptors viz., NF-kappaB p52 (-50.549 kcal/mol), tumor necrosis factor (TNF-alpha) (-47.632 kcal/mol), nuclear factor NF-Kappa-B P50 (-16.798 kcal/mol) and cyclooxygenase-2 (-55.244 kcal/mol). Further both the triterpenoids (1 and 2) were subjected to in vivo immunomodulatory activity in female Swiss albino mice. The experimental mice were divided into nine groups, each comprised of six mice. These received oral treatment for a period of 28 days. The triterpenoids (1 and 2) showed significant increased in humoral immune function, but no significant changes were observed in cell mediated immune response and hematological parameters. The in silico and in vivo experimental data suggested that both the triterpenoids 1 and 2 may be considered as potential immunomodulatory drug-like molecules.

Drug Resistance Reversal Potential of Ursolic Acid Derivatives against Nalidixic Acid- and Multidrug-resistant Escherichia coli.

As a part of our drug discovery program, ursolic acid was chemically transformed into six semi‐synthetic derivatives, which were evaluated for their antibacterial and drug resistance reversal potential in combination with conventional antibiotic nalidixic acid against the nalidixic acid‐sensitive and nalidixic acid‐resistant strains of Escherichia coli. Although ursolic acid and its all semi‐synthetic derivatives did not show antibacterial activity of their own, but in combination, they significantly reduced the minimum inhibitory concentration of nalidixic acid up to eightfold. The 3‐O‐acetyl‐urs‐12‐en‐28‐isopropyl ester (UA‐4) and 3‐O‐acetyl‐urs‐12‐en‐28‐n‐butyl ester (UA‐5) derivatives of ursolic acid reduced the minimum inhibitory concentration of nalidixic acid by eightfold against nalidixic acid‐resistant and four and eightfold against nalidixic acid‐sensitive, respectively. The UA‐4 and UA‐5 were further evaluated for their synergy potential with another antibiotic tetracycline against the multidrug‐resistant clinical isolate of Escherichia coli‐KG4. The results showed that both these derivatives in combination with tetracycline reduced the cell viability in concentration‐dependent manner by significantly inhibiting efflux pump. This was further supported by the in silico binding affinity of UA‐4 and UA‐5 with efflux pump proteins. These ursolic acid derivatives may find their potential use as synergistic agents in the treatment of multidrug‐resistant Gram‐negative infections.