Sanmoy Karmakar

Improvement of cellular uptake, in vitro antitumor activity and sustained release profile with increased bioavailability from a nanoemulsion platform

Paclitaxel, a potential anticancer agent against solid tumors has been restricted from its oral use due to poor water solubility as well as Pgp efflux property. The present study was aimed to improve the oral bioavailability of paclitaxel through development of (o/w) nanoemulsion consisting of Capryol 90 as internal phase with Tween 20 as emulsifier with water as an external phase. Formulations were selected from the nanoemulsion region of pseudo-ternary phase diagrams, formulated by aqueous titration method. The developed nanoemulsion has been characterized by its thermodynamic stability, morphology, droplet size, zeta potential, viscosity where in vitro release was evaluated through dialysis. Paclitaxel nanoemulsion exhibited thermodynamical stability with low viscosity, nano-sized oil droplets in water with low poly-dispersity index. The shelf life of the paclitaxel nanoemulsion was found to be approximately 2.38 years. Increased permeability through the Caco-2 cell monolayer and decreased efflux is great advantageous for nanoemulsion formulation. The effects of paclitaxel nanoemulsion on breast cancer cell proliferation, morphology and DNA fragmentation were analyzed in vitro which showed significant anti-proliferation and decreased IC50 values in nanoemulsion group which may be due to enhanced uptake of paclitaxel through the oil core. Moreover, the absolute oral bioavailability and sustained release profile of the paclitaxel nanoemulsion evaluated in mouse model was found to improve up to 55.9%. The concentration of paclitaxel in mice plasma was determined by our validated LC-MS/MS method. By reviewing the significant outcome of the present investigation based on stability study, Caco-2 permeability, cell proliferative assay and pharmacokinetic profile it may be concluded that the oral nanoemulsion has got encouraging advantages over the presently available formulations of this injectable chemotherapeutic drug.

Alteration of Zeta potential and membrane permeability in bacteria: a study with cationic agents

In the present study, we have tried to establish the correlation between changes in Zeta potential with that of cell surface permeability using bacteria (Escherichia coli and Staphylococcus aureus). An effort has been made to establish Zeta potential as a possible marker for the assessment of membrane damage, with a scope for predicting alteration of cell viability. Cationic agents like, cetyl trimethyl ammonium bromide and polymyxin B were used for inducing alteration of Zeta potential, and the changes occurring in the membrane permeability were studied. In addition, assessment of poly-dispersity index (PDI), cell viability along with confocal microscopic analysis were performed. Based on our results, it can be suggested that alteration of Zeta potential may be correlated to the enhancement of membrane permeability and PDI, and it was observed that beyond a critical point, it leads to cell death (both Gram-positive and Gram-negative bacteria). The present findings can not only be used for studying membrane active molecules but also for understanding the surface potential versus permeability relationship.

Nanoemulsion strategy for olmesartan medoxomil improves oral absorption and extended antihypertensive activity in hypertensive rats.

Olmesartan medoxomil (OM) is hydrolyzed to its active metabolite olmesartan by the action of aryl esterase to exert its antihypertensive actions by selectively blocking angiotensin II-AT1 receptor. Poor aqueous solubility and uncontrolled enzymatic conversion of OM to its poorly permeable olmesartan limits its oral bioavailability. The aim of the current study was to formulate a novel nanoemulsion of OM to improve its pharmacokinetics and therapeutic efficacy. The oil-in-water (o/w) nanoemulsion of OM was developed using lipoid purified soybean oil 700, sefsol 218 and solutol HS 15. We have characterized the nanoemulsions by considering their thermodynamic stability, morphology, droplet size, zeta potential and viscosity and in vitro drug release characteristics in fasting state simulated gastric fluid (pH 1.2) and intestinal fluid (pH 6.5). The thermodynamically stable nanoemulsions comprises of spherical nanometer sized droplets (<50 nm) with low polydispersity index showed enhanced permeability through the Caco-2 cell monolayer. The concentration of active olmesartan in rat plasma following oral absorption study was determined by our validated LC-MS/MS method. The result of the pharmacokinetic study showed 2.8-fold increased in area under the curve (AUC0-27) of olmesartan upon oral administration of OM nanoemulsion and sustained release profile. Subsequent, in vivo studies with nanoemulsion demonstrated better and prolonged control of experimentally induced hypertension with 3-fold reduction in conventional dose. By analysing the findings of the present investigations based on stability study, Caco-2 permeability, pharmacokinetic profile and pharmacodynamic evaluation indicated that the nanoemulsion of OM (OMF6) could significantly enhance the oral bioavailability of relatively insoluble OM contributing to improved clinical application.

Anti-inflammatory activity of Acanthus ilicifolius

Acanthus ilicifolius Linn, is a perennial herb (Acanthaceae) widely found in the Sundarban mangroves and is popularly used for its wound healing effects. In the present study an attempt was made to evaluate the anti-inflammatory activity of the Acanthus ilicifolius leaves. The methanolic fraction of Acanthus ilicifolius leaf extract produced significant inhibition of rat paw oedema, when administered both prior to and after carrageenan administration, in a manner similar to BW755C a synthetic cyclooxygenase (COX) and lipoxygenase (LOX) inhibitor. The extract decreased protein exudation and leukocyte migration in the peritoneal fluid, thereby indicating its effectiveness towards inhibiting peritoneal inflammation. It also produced significant inhibition of COX (1 and 2) and 5-LOX activity. Preincubation of the extract inhibited the production of proinflammatory cytokines (TNFalpha and IL-6) in lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs). The methanolic fraction of the extract was also found to possess significant free radical (DPPH, ABTS, superoxide and hydroxyl radical) scavenging activity. The extract on intraperitoneal administration augmented the endogenous antioxidant status, as evident from the significant increase of ferric reducing ability of plasma (FRAP) and total peroxyl radical trapping activity of plasma (TRAP).

Anti-biofilm activity of Marula - a study with the standardized bark extract.

ETHNOPHARMACOLOGICAL RELEVANCE Marula (Sclerocarya birrea; family - Anacardiaceae) is an African plant, which enjoys wide socio-economic importance particularly in southern part of Africa. The fruits are consumed as food and also as alcoholic beverage (cream liquor). In different parts of Africa, the decoction of the bark is traditionally used for the treatment of dysentery, diarrhoea, and various other infectious conditions. The aim of the study was to investigate the anti-biofilm properties of the methanol extract of Marula bark (stem bark of Sclerocarya birrea), with a view towards combating the emergence of antimicrobial resistance often associated with bacterial biofilms. MATERIALS AND METHODS The standardized methanol extract was initially tested for its antimicrobial property. The crystal violet assay was used for evaluating anti-biofilm (biofilm formation by Pseudomonas aeuginosa) activity. Further in order to study the mechanism of anti-biofilm activity, the same was evaluated for understanding its role on various quorums sensing mediated phenomenon (swarming motility assay, protease and pyoverdin assay) that are known to be associated with the formation of biofilms and pathogenicity. RESULTS The methanol extract showed no inhibition of bacterial growth up to a concentration of 200 µg/ml. Interestingly, the sample produced anti-biofilm activity (around 75% decrease; 100 µg/ml) at sub-lethal concentration. Further it also significantly reduced the QS mediated swarming motility. The release of various virulent factors (protease and pyoverdin) was found to be lowered when pre-treated with the extract. CONCLUSION The present study illustrates the anti-biofilm property Sclerocarya birrea. The standardized extract significantly disrupted the quorum sensing mediated production of biofilm formation and also inhibited swarming ability of the cells. The extract displayed a regulatory role on the secretion of protease and pyoverdin, two QS dependent pathogenic factors found in Pseudomonas aeruginosa. This study also validates the ethnobotanical use of Marula.

The Gastroprotective Role of Acanthus ilicifolius - A Study to Unravel the Underlying Mechanism of Anti-Ulcer Activity.

Acanthus ilicifolius (Acanthaceae), a mangrove medicinal plant, is widely used by the local inhabitants of the Sundarbans (India) to treat a variety of diseases. As a part of our continued search for novel bioactive products from mangrove medicinal plants, we were able to document the anti-inflammatory effects of this plant. In the present study, we have performed a detailed evaluation of the gastroprotective activity of the methanolic extract of Acanthus ilicifolius using different models of gastric ulceration. Unlike the conventional non-steroidal anti-inflammatory drugs, a methanolic extract of Acanthus ilicifolius leaves (MEAL) possessing significant anti-inflammatory properties, as revealed from our previous studies displayed in rats in dosages of 200 mg and 400 mg/kg BW after intraperitoneal administration, showed significant protective activity (anti-ulcer activity) against the gastric lesions induced by aspirin, indomethacin, stress, ethanol, and pylorus ligation. In pylorus-ligated rats, administration of Methanolic extract of Acanthus ilicifolius leaves (MEAL) significantly decreased gastric volume, acidity, and peptic activity. Moreover, pre-treatment with MEAL significantly restored the levels of reduced glutathione (GSH) and the antioxidant enzyme superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), along with significant inhibition of both lipid peroxidation and myeloperoxidase (MPO) activity in pylorus-ligated animals. Ulceration induced with ethanol was significantly inhibited with MEAL, and the extract also resulted in the reduction of both lipid peroxidation and myeloperoxidase activity. Furthermore, in this experimental model, administration of MEAL improved the activities of SOD, CAT, GSH, and GPX. A similar pattern of action was also noticed in cold-restraint stress-induced (CRS) ulceration, where MEAL pre-treatment inhibited CRS-induced ulceration, improved the status of antioxidant enzymes, and also reduced the level of lipid peroxides. These results suggest that extracts of the leaves of Acanthus ilicifolius may exhibit anti-ulcer activities additional to the anti-inflammatory properties.

Development and Validation of RP-HPLC Method: Scope of Application in the Determination of Oil Solubility of Paclitaxel

A simple, reproducible, feasible and innovative reversed-phase high-performance liquid chromatographic method was developed and validated for the quantitative determination of paclitaxel dissolved in various oils. The method was validated after extraction of the analyte from capryol 90, triacetin and olive oil. The method was conducted on a Hypersil BDS C18 column, 250 × 4.6 mm, 5 µm particle size, with a mobile phase composed of acetonitrile: 10 mM KH2PO4 buffer (pH 3.5) (55:45, v/v) and detection at 227 nm. The linearity, in the range of 5 to 50 µg/mL, presented determination coefficients of 0.9983, 0.9997 and 0.9990 in capryol 90, triacetin and olive oil, respectively, calculated by the least-squares regression method. Intra-day precision values for percentages recovered were 0.68 to 0.80, 0.83 to 1.13 and 0.97 to 1.88, and inter day precision values were 1.52 to 1.92, 1.43 to 1.83 and 1.26 to 2.06 for capryol 90, triacetin and olive oil, respectively. The recovery of paclitaxel from the capryol 90, triacetin and olive oil ranged from 97.94 to 103.55, 96.85 to 103.27 and 97.14 to 103.64%, respectively. This developed and validated method was successfully applied to quantitatively assess paclitaxel dissolved in various oils. The solubility of paclitaxel was found to be higher in triacetin than in other tested oils.

Effect of amitriptyline on gastric ulceration

Amitriptyline significantly inhibited alcohol, aspirin, indomethacin and cold‐restraint stress‐induced ulceration. Secretory studies conducted in pyloric‐ligated rats revealed that the drug, at the doses employed, significantly reduced total acidity and protein content. However, significant reductions of the gastric volume were only observed at the highest dose of the drug. In another set of experiments, when 50% alcohol (v/v) was administered to the pyloric‐ligated rats pretreated with amitriptyline, it was observed that the drug significantly reduced the pH, total acidity and protein content.

Synthesis, Characterization, and Biological Evaluation of 99mTc(CO)3-Labeled Peptides for Potential Use as Tumor Targeted Radiopharmaceuticals

During the past decade, several peptides containing Arg‐Gly‐Asp sequence have been conjugated with different chelating agents for labeling with various radionuclides for the diagnosis of tumor development. In this study, we report the synthesis of two tetrapeptides (Asp‐Gly‐Arg‐His and Asp‐Gly‐Arg‐Cys) and one hexapeptide [Asp‐Gly‐Arg‐D‐Tyr‐Lys‐His] by changing the amino acid sequence of the Arg‐Gly‐Asp motif. Peptide synthesis was initiated from aspartic acid. Aspartic acid placed at C‐terminal end of the peptide chain can be conjugated with different drug molecules facilitating their transport to the site of action. The peptides were synthesized in excellent yield and labeled using freshly prepared [99mTc(CO)3(H2O)3]+ intermediate. A complexation yield of over 97% was achieved under mild conditions even at low ligand concentrations of 10−2 m. Radiolabeled peptides were characterized by HPLC and were found to be substantially stable in saline, in His solution as well as in rat serum and tissue (kidney, liver) homogenates. Internalization studies using Ehrlich ascites carcinoma cell line showed rapid and significant internalization (30–35% at 30 min of incubation attaining maximum value of about 40–60% after 2–4 h incubation). A good percentage of quick internalization was also observed in αvβ3‐receptor‐positive B16F10 mouse melanoma cell line (14–16% after 30 min of incubation and 25–30% after 2–4 h incubation). Imaging and biodistribution studies were performed in Swiss albino mice bearing Ehrlich ascites tumor in right thigh. Radiolabeled peptides exhibited fast blood clearance and rapid elimination through the urinary systems. 99mTc(CO)3‐tetra‐Pep2 exhibited remarkable localization at tumor site (1.15%, 1.17%, and 1.37% ID/g at 2, 4, and 6 h p.i., respectively) which could be due to slow clearance of the radiolabeled peptide from blood in comparison with the other two radiolabeled peptides. However, 99mTc(CO)3‐hexa‐Pep exhibited the highest tumor to muscle and tumor to blood ratios among the three. The preliminary results with these amino acid–based peptides are encouraging enough to carry out further experiments for targeting tumor.

Pharmacological studies on Buchanania lanzan Spreng.-A focus on wound healing with particular reference to anti-biofilm properties

OBJECTIVE To evaluate the wound healing activity of the methanolic root extract of Buchanania lanzan Spreng. (B. lanzan), with a focus on antimicrobial and anti-biofilm properties. METHODS The extract was evaluated for its wound healing properties (excision and incision models) as evident from the analysis of tensile strength and wound contraction. The extract was also screened for antibacterial properties against different Gram positive and Gram negative bacterial strains. B. lanzan was also studied for its effect on biofilm formation and disruption of preformed biofilms. The synergistic effect of B. lanzan was determined in combination with gentamicin. RESULTS Topical application of B. lanzan (10% w/w ointment) significantly increased (40.84%) the tensile strength in the incision wound model. B. lanzan also showed significant wound healing activity in excision model and such significant activity was observed from the 9th day. Whereas Soframycin displayed significant wound healing activity from the 6th day. It was found that root extracts of B. lanzan revealed significant inhibition against all tested pathogens. B. lanzan displayed antimicrobial activity against Gram positive (MIC 0.625 mg/mL) and Gram negative (MIC 0.625-1.25 mg/mL). B. lanzan was able to reduce biofilm formation and also caused disruption of preformed biofilms in a manner similar to ciprofloxacin. However, gentamicin was found to be ineffective against biofilms formed by Gram negative organism. According to the fractional inhibitory concentration index, B. lanzan displayed synergistic activity when it was combined with gentamicin. CONCLUSIONS From this study it may be concluded that the root extract of B. lanzan revealed significant wound healing potential, which was supported and well correlated with pronounced antibacterial activity of the tested plant parts.