Anupam Maurya

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.

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.

pH‐Zone‐refining centrifugal partition chromatography for preparative isolation and purification of steroidal glycoalkaloids from Solanum xanthocarpum

pH-Zone-refining centrifugal-partition chromatography (CPC) was successfully applied in the separation of complex polar steroidal glycoalkaloids of close Rf values, directly from a crude extract of Solanum xanthocarpum. The experiment was performed with a two phase solvent system composed of ethyl acetate/butanol/water (1:4:5 by volume) where triethylamine (5 mM) was added to the upper organic mobile phase as an eluter and TFA (10 mM) to the aqueous stationary phase as a retainer. Separation of 1 g of crude extract over CPC resulted in two distinct pH-zones. The fractions collected in pH-zone i afforded 72 mg of solasonine while the fractions collected in pH-zone ii were slightly impure, hence were purified over medium pressure LC, which afforded 30 mg of solasonine and further 15 mg of solamargine (SM). The steroidal glycoalkaloids, SM and solasonine were isolated in 93.3 and 91.6% purity, respectively. The isolated alkaloids were characterized on the basis of their (1)H, (13)C-NMR, and ESI-MS data.

Antibacterial and Synergy of Clavine Alkaloid Lysergol and its Derivatives Against Nalidixic Acid‐Resistant Escherichia coli

Antibacterial activity of lysergol (1) and its semi‐synthetic derivatives (2–14) and their synergy with the conventional antibiotic nalidixic acid (NA) against nalidixic acid‐sensitive (NASEC) and nalidixic acid‐resistant (NAREC) strains of Escherichia coli were evaluated. Lysergol (1) and derivatives (2–14) did not possess antibacterial activity of their own, but in combination, they significantly reduced the minimum inhibitory concentration (MIC) of NA. All the derivatives showed two‐ to eightfold reduction in the MIC of NA against NAREC and NASEC. Further, lysergol (1) and its derivatives 10 and 11 brought down eightfold reductions in the MIC of tetracycline (TET) against multidrug‐resistant clinical isolate of E. coli (MDREC). Treatment of these strains with the combinations of antibiotics and lysergol and its derivatives 10 and 11 (at reduced concentrations) significantly decreased the viability of cells. In an another observation, lysergol and its derivatives 10 and 11 inhibited ATP‐dependent efflux pumps, which was evident by ATPase inhibition and down‐regulation of multidrug ABC transporter ATP‐binding protein (yojI) gene. These results may be of great help in antibacterial drug development from a very common, inexpensive, and non‐toxic natural product.

Bioactivity guided isolation of antipsychotic constituents from the leaves of Rauwolfia tetraphylla L.

This study was undertaken to ascertain the antipsychotic properties of Rauwolfia tetraphylla L. leaves and to isolate and characterize the antipsychotic constituents. Among the MeOH extract and some alkaloidal fractions at different pHs, the alkaloidal CHCl(3) fraction at pH-9 (2C) showed the highest antipsychotic activity against dopaminergic (DA-D(2)) and serotonergic (5-HT(2A)) receptors in-vitro and amphetamine induced hyperactive mouse model in-vivo. The activity guided isolation of CHCl(3) fraction (2C) afforded six indole alkaloids: 10-methoxytetrahydroalstonine (1), isoreserpiline (2), an isomeric mixture of 11-demethoxyreserpiline (3) and 10-demethoxyreserpiline (4), α-yohimbine (5) and reserpiline (6). Given orally, alkaloids 3-6 showed significant antipsychotic activity in a dose dependent manner. None of the extract, alkaloidal fractions or alkaloids showed any extra pyramidal symptoms at the tested doses. It was also observed that MeOH extract was behaving similar to other clinically used novel atypical antipsychotics in having 5-HT(2A) occupancy greater than the DA-D(2) receptor at the tested doses. Further toxicity and safety evaluation studies of MeOH extracts of R. tetraphylla leaves at different doses (10, 100, 300 and 2000 mg/kg) on female Swiss albino mice showed that MeOH extract is non toxic. The isolated alkaloids, 3-6 could serve as a promising lead structure for drug development of treating psychotic conditions in human.

Preparative-Scale Separation of Anticancer Triterpenes from Eucalyptus hybrid by Centrifugal Partition Chromatography

Centrifugal partition chromatography was successfully applied in the separation of close R f complex anticancer triterpenes directly from a fraction of Eucalyptus hybrid (Myrtaceae). The experiment was performed with a two-phase solvent system composed of hexane/ethyl acetate/methanol/water (1:2:1.5:1 v/v/v) where 2% ammonia solution was added in lower aqueous mobile phase to achieve pH 9.5. From 1.5 g of a fraction, 145 mg of ursolic acid and 72 mg of ursolic acid lactone were obtained in 95.4% and 94.8% purities. The total yield recovery was >94% and the isolated triterpenes were characterized on the basis of their 1H, 13C-NMR, and ESI-MS data.

Quantitative determination of bioactive alkaloids lysergol and chanoclavine in Ipomoea muricata by reversed-phase high-performance liquid chromatography

A rapid, simple, sensitive, gradient and reproducible, reverse-phase high-performance liquid chromatographic method was developed for the quantitative estimation of bioactive alkaloids, lysergol and chanoclavine in the seeds of Ipomoea muricata. The clavine alkaloid, lysergol, is a bioenhancer for the drugs and nutrients. The samples were analyzed by reverse-phase chromatography on a Waters spherisorb ODS2 column (250 × 4.6 mm, i.d., 10 µm) using binary gradient elution with acetonitrile and 0.01 m phosphate buffer (NaH₂PO₄) containing 0.1% glacial acetic acid at a flow rate of 0.8 mL/min, a column temperature of 25 °C and UV detection at λ 254 nm. The limits of detection (LOD) and quantitation (LOQ) were 0.035 and 0.106 µg/mL for lysergol and 0.039 and 0.118 µg/mL for chanoclavine, respectively. Standard curves were linear in the range of 2-10 µg/mL (r > 99) for both analytes. Good results were achieved with respect to repeatability (RSD < 2%) and recovery (99.20-102.0). The method was validated for linearity, accuracy repeatability, LOQ and LOD. The method is simple, accurate and precise, and may be recommended for routine quality control analysis of I. muricata seed extracts containing these two clavine alkaloids (1, 2) as bioactive principles of the herb.

Synergy Potential of Indole Alkaloids and Its Derivative against Drug-resistant Escherichia coli

Antibacterial and synergy potential of naturally occurring indole alkaloids (IA): 10‐methoxy tetrahydroalstonine (1), isoreserpiline (2), 10 and 11 demethoxyreserpiline (3), reserpiline (4), serpentine (5), ajmaline (6), ajmalicine (7), yohimbine (8), and α‐yohimbine (9) was evaluated using microbroth dilution assay. Further, α‐yohimbine (9) was chemically transformed into six semisynthetic derivatives (9A‐9F), and their antibacterial and synergy potential in combination with nalidixic acid (NAL) against E. coli strains CA8000 and DH5α were also evaluated. The IA 1, 2, 4, 5, 9 and the derivative 9F showed eightfold reduction in the MIC of NAL against the DH5α and four‐ to eightfold reduction against CA8000. These alkaloids also reduced MIC of another antibiotic, tetracycline up to 8folds, against the MDREC‐KG4, a multidrug‐resistant clinical isolate of E. coli. Mode of action study of these alkaloids showed efflux pumps inhibitory potential, which was supported by their in silico binding affinity and downregulation of efflux pump genes. These results may be of great help in the development of cost‐effective antibacterial combinations for treating patients infected with multidrug‐resistant Gram‐negative infections.

Simple and reliable methods for the determination of three steroidal glycosides in the eight species of Solanum by reversed-phase HPLC coupled with diode array detection.

INTRODUCTION Solanum species are important ingredients of many traditional Indian medicines and thus the quality control of their herbal formulations is of paramount concern. OBJECTIVE To establish a simple and effective high-performance liquid chromatographic (HPLC) method to evaluate the quality of Solanum species and their herbal formulations. METHODOLOGY A rapid, simple, sensitive, robust and reproducible HPLC method was developed for the determination of three steroidal glycosides (SG); indioside D, solamargine and α-solanine in eight species of the genus Solanum. The analytes were separated on a monolithic performance RP-18e column (100 mm × 4.6 mm i.d.) using a gradient elution of acetonitile-water containing 0.1% trifluoroacetic acid (TFA) as the mobile phase with a flow rate 0.4 mL/min and UV detection at λ 210 nm. RESULTS The method was linear over the range 3-15 µg/mL (r > 9994). Accuracy, precision and repeatability were all within the required limits. The mean recoveries measured at the three concentrations were higher than 98.8% with RSD < 2% for the targets. CONCLUSION The established method is simple and can be used as a tool for quality control of plant material or herbal formulation containing SG.