Identification of Novel Inhibitors of Leishmania donovani γ-Glutamylcysteine Synthetase Using Structure-Based Virtual Screening, Docking, Molecular Dynamics Simulation, and in Vitro Studies.

Trypansomatids maintain their redox balance by the trypanothione-based redox system, enzymes of which exhibit differences from mammalian homologues. γ-Glutamylcysteine synthetase (Gcs) is an essential enzyme in this pathway that performs the first and rate-limiting step. l-Buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of Gcs, induces toxicity in hosts infected with Trypanosoma brucei, underlining the need for novel Gcs inhibitors. The present study reports identification of Leishmania donovani Gcs (LdGcs) inhibitors using computational approaches and their experimental validation. Analysis of inhibitor-LdGcs complexes shows modifications that could result in increased efficacy of these compounds.

Biotransformation of lignan glycoside to its aglycone by Woodfordia fruticosa flowers: quantification of compounds using a validated HPTLC method.

CONTEXT: Saraca asoca Linn. (Caesalpiniaceae) is an important traditional remedy for gynaecological disorders and it contains lyoniside, an aryl tetralin lignan glycoside. The aglycone of lyoniside, lyoniresinol possesses structural similarity to enterolignan precursors which are established phytoestrogens. OBJECTIVES: This work illustrates biotransformation of lyoniside to lyoniresinol using Woodfordia fruticosa Kurz. (Lythraceae) flowers and simultaneous quantification of lyoniside and lyoniresinol using a validated HPTLC method. MATERIALS AND METHODS: The aqueous extract prepared from S. asoca bark was fermented using W. fruticosa flowers. The substrate and fermented product both were simultaneously analyzed using solvent system:toluene:ethyl acetate:formic acid (4:3:0.4) at 254 nm. The method was validated for specificity, accuracy, precision, linearity, sensitivity and robustness as per ICH guidelines. RESULTS: The substrate showed the presence of lyoniside, however, it decreased as the fermentation proceeded. On 3rd day, lyoniresinol starts appearing in the medium. In 8 days duration most of the lyoniside converted to lyoniresinol. The developed method was specific for lyoniside and lyoniresinol. Lyoniside and lyoniresinol showed linearity in the range of 250-3000 and 500-2500 ng. The method was accurate as resulted in 99.84% and 99.83% recovery, respectively, for lyoniside and lyoniresinol. CONCLUSION: Aryl tetralin lignan glycoside, lyoniside was successfully transformed into lyoniresinol using W. fruticosa flowers and their contents were simultaneously analyzed using developed validated HPTLC method.

Development of Leishmania donovani stably expressing DsRed for flow cytometry-based drug screening using chalcone thiazolyl-hydrazone as a new antileishmanial target.

Green fluorescent protein produces significant fluorescence and is extremely stable, however its excitation maximum is close to the ultraviolet range and thus can damage living cells. Hence, Leishmania donovani stably expressing DsRed were developed and their suitability for flow cytometry-based antileishmanial screening was assessed by evaluating the efficacies of standard drugs as well as newly synthesised chalcone thiazolyl-hydrazone compounds. The DsRed gene was successfully integrated at the 18S rRNA locus of L. donovani and transfectants (LdDsRed) were selected using hygromycin B. Enhanced expression of DsRed and a high level of infectivity to J774A.1 macrophages were achieved, which was confirmed by fluorescence microscopy and flow cytometry. Furthermore, these LdDsRed transfectants were utilised for development of an in vitro screening assay using the standard antileishmanial drugs miltefosine, amphotericin B, pentamidine and paromomycin. The response of transfectants to standard drugs correlated well with previous reports. Subsequently, the suitability of this system was further assessed by screening a series of 18 newly synthesised chalcone thiazolyl-hydrazone compounds in vitro for their antileishmanial activity, wherein 8 compounds showed moderate antileishmanial activity. The most active compound 5g, with ca. 73% splenic parasite reduction, exerted its activity via generating nitric oxide and reactive oxygen species and inducing apoptosis in LdDsRed-infected macrophages. Thus, these observations established the applicability of LdDsRed transfectants for flow cytometry-based antileishmanial screening. Further efforts aimed at establishing a high-throughput screening assay and determining the in vivo screening of potential antileishmanial leads are required.

Optimization of microwave-assisted extraction conditions for preparing lignan-rich extract from Saraca asoca bark using Box-Behnken design.

CONTEXT: Lyoniside is the major constituent of Saraca asoca Linn. (Caesalpiniaceae) bark. There is an immediate need to develop an efficient method to isolate its chemical constituents, since it is a therapeutically important plant. OBJECTIVE: A rapid extraction method for lyoniside based on microwave-assisted extraction of S. asoca bark was developed and optimized using response surface methodology (RSM). MATERIALS AND METHODS: Lyoniside was analyzed and quantified by high-performance liquid chromatography coupled with ultraviolet detection (HPLC-UV). The extraction solvent ratio (%), material solvent ratio (g/ml) and extraction time (min) were optimized using Box-Behnken design (BBD) to obtain the highest extraction efficiency. The optimal conditions were the use of 1:30 material solvent ratio with 70:30 mixture of methanol:water for 10 min duration. RESULTS: The optimized microwave-assisted extraction yielded 9.4 mg/g of lyoniside content in comparison to reflux extraction under identical conditions which yielded 4.2 mg/g of lyoniside content. Under optimum conditions, the experimental values agreed closely with the predicted values. The analysis of variance (ANOVA) indicated a high goodness-of-fit model and the success of the RSM method for optimizing lyoniside extraction from the bark of S. asoca. DISCUSSION: All the three variables significantly affected the lyoniside content. Increased polarity of solvent medium enhances the lyoniside yield. CONCLUSION: The present study shows the applicability of microwave-assisted extraction in extraction of lyoniside from S. asoca bark.

Phytochemical, therapeutic, and ethnopharmacological overview for a traditionally important herb: Boerhavia diffusa Linn.

UNLABELLED: Boerhavia diffusa (BD) is a plant of rasayana category as per ayurvedic claims. It is reported to possess antiaging, disease prevention, and life strengthening activities which hold enormous influence in disease burden and affordability/availability of healthcare in the world. Objective. This paper has been compiled to comment on the studies reported for BD to highlight its chemical and therapeutic potential along with its ethnopharmacological considerations. METHODS: In the present paper, a detailed account of chemical constituents and pharmacological activities has been presented. All the findings were correlated with modern pharmacological activities to appraise the value of BD. RESULTS: Chemical analysis of BD gives a wide variety of chemical constituents, namely, rotenoids, flavonoids, xanthones, purine nucleoside, lignans, and steroids. Various ethnopharmacological reports emphasize its role in disorders of reproductive system, gastrointestinal system, respiratory system, urinary system, hepatic system/jaundice, cardiovascular system, and cancer. CONCLUSIONS: The studies on the therapeutic activities of BD range from studies on crude extracts to isolated compounds; however some of the studies require sophistication and validated results. BD is a plant of enormous importance in the purview of its chemical and therapeutic properties.

Formulation and evaluation of guggul lipid nanovesicles for transdermal delivery of aceclofenac.

CONTEXT: Most new drugs have low water solubility and liposome is an important formulation to administer such drugs; however, it is quite unstable and has negligible systemic absorption. OBJECTIVE: Aceclofenac nanovesicles were made using guggul lipid for formulating stable transdermal formulation. MATERIALS AND METHODS: Guggul lipid was formulated into vesicles along with cholesterol and dicetyl phosphate using film hydration method. The formulations were analyzed for physicochemical properties and stability. Then its skin permeation and anti-inflammatory activity were determined. RESULTS: Both categories of vesicles (PC and GL) showed optimum physicochemical properties; however, accelerated stability study showed considerable differences. GL-1 was appreciably stable for over 6 months at 4 °C. Corresponding gels (PCG-1 and GLG-1) showed C max values at 4.98 and 7.32 µg/mL along with the Tmax values at 4 and 8 hours, respectively. GLG-1 inhibited edema production by 90.81% in 6 hours. Discussion. PC liposomes are unstable at higher temperature and upon longer storage. The formulation with higher lipid content (GL-1) showed good drug retention after 24 hours and appreciable stability both at higher temperature and for longer duration. Guggul lipid being a planar molecule might be stacked in vesicle wall with cholesterol. CONCLUSION: The composition of the nanovesicle played an important role in stability and drug permeation. Guggul lipid is suitable for producing stable vesicles.