Identification of isoflavone glycosides in Pueraria lobata cultures by tandem mass spectrometry

Isoflavones in the methanolic extracts of kudzu (Pueraria lobata) callus, suspension and root cultures were compared in order to develop an experimental system in which puerarin (daidzein 8-C-glucoside) and other isoflavones could be synthesised in vitro. Quantitative variation of puerarin and other known isoflavones was estimated in kudzu culture extracts using HPLC-UV. The highest and lowest amounts of puerarin (14.56 and 0.33 mg/g) were present in in vitro root cultures and leaf tissue-derived callus cultures, respectively. A total of 48 isoflavone metabolites were detected in extracts of kudzu root cultures by HPLC-MS/MS, and the structures of 33 of them were tentatively assigned. Amongst these, 12 isoflavone C-glycosides were identified. Hydroxyderivatives of puerarin in several isomeric forms were detected, some of which have not been previously reported in kudzu root. The molecular weights, interpretation of characteristic fragment ions obtained from HPLC-MS/MS and comparison with reported data allowed the putative identification of the isoflavone metabolites.     

Progress toward a Universal H5N1 Vaccine: A Recombinant Modified Vaccinia Virus Ankara-Expressing Trivalent Hemagglutinin Vaccine

Background

The rapid evolution of new sublineages of H5N1 influenza poses the greatest challenge in control of H5N1 infection by currently existing vaccines. To overcome this, an MVAtor vector expressing three H5HA antigens A/Vietnam/1203/04, A/Indonesia/669/06 and A/Anhui/01/05 (MVAtor-tri-HA vector) was developed to elicit broad cross-protection against diverse clades by covering amino acid variations in the major neutralizing epitopes of HA among H5N1 subtypes.

Methods

BALB/c mice and guinea pigs were immunized i.m. with 8×10⁷ TCID₅₀/animal of MVAtor-tri-HA vector. The immunogenicity and cross-protective immunity of the MVAtor-tri-HA vector was evaluated against diverse clades of H5N1 strains.

Results

The results showed that mice immunized with MVAtor-tri-HA vector induced robust cross-neutralizing immunity to diverse H5N1 clades. In addition, the MVAtor-tri-HA vector completely protected against 10 MLD₅₀ of a divergent clade of H5N1 infection (clade 7). Importantly, the serological surveillance of post-vaccinated guinea pig sera demonstrated that MVAtor-tri-HA vector was able to elicit strong cross-clade neutralizing immunity against twenty different H5N1 strains from six clades that emerged between 1997 and 2012.

Conclusions

The present findings revealed that incorporation of carefully selected HA genes from divergent H5N1 strains within a single vector could be an effective approach in developing a vaccine with broad coverage to prevent infection during a pandemic situation.     

Phytochemical and pharmacological studies in genus Berberis

The genus Berberis is well known for its diversity and pharmacological uses in traditional medicine system since ancient time. Exploring this medicinal plant with more prominence is the need of present day medicinal system. The present review highlighted the phytochemical and pharmacological studies reported from genus Berberis over the last two decades.     

Furanocoumarins: Novel topoisomerase I inhibitors from Ruta graveolens L.

Topoisomerase I inhibitors from Ruta graveolens are reported for the first time. Potent topoisomerase I inhibitory activity from in vitro culture extracts R. graveolens were observed. Stabilization of DNA–topoisomerase covalent complex was observed in all the tested extracts. The mechanism of topoisomerase inhibition was determined by preincubation studies. The irreversible topoisomerase I mediated relaxation of plasmid in enzyme–substrate preincubation study, indicated that the observed inhibitory activity of extract constituents was not mediated through conformational changes in the DNA. Furthermore, the affinity of inhibitors with the enzyme was tested by enzyme–extract preincubation study. Increase in inhibition of topoisomerase activity and promotion of DNA–enzyme complex was observed after enzyme–extract preincubation. The activity could be assigned to furanocoumarins—psoralen, bergapten and xanthotoxin, identifying them as novel, potent topoisomerase I inhibitors.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Identification and modeling of a drug target for Clostridium perfringens SM101

In the present study, comparative genome analysis between Clostridium perfringens and the human genome was carried out to identify genes that are essential for the pathogen's survival, and non-homologous to the genes of human host, that can be used as potential drug targets. The study resulted in the identification of 426 such genes. The number of these potential drug targets thus identified is significantly lower than the genome's protein coding capacity (2558 protein coding genes). The 426 genes of C. perfringens were further analyzed for overall similarities with the essential genes of 14 different bacterial species present in Database of Essential Genes (DEG). Our results show that there are only 5 essential genes of C. perfringens that exhibit similarity with 12 species of the 14 different bacterial species present in DEG database. Of these, 1 gene was similar in 12 species and 4 genes were similar in 11 species. Thus, the study opens a new avenue for the development of potential drugs against the highly pathogenic bacterium. Further, by selecting these essential genes of C. perfringens, which are common and essential for other pathogenic microbial species, a broad spectrum anti-microbial drug can be developed. As a case study, we have built a homology model of one of the potential drug targets, ABC transporter-ATP binding protein, which can be employed for in silico docking studies by suitable inhibitors.     

Novel technique for scaling up of micropropagated Ruta graveolens shoots using liquid culture systems: a step towards commercialization

Wide applications of Ruta graveolens L. in pharmaceutical industry has led to increased interest in large-scale plant production, with emphasis on use of in vitro cultures. Earlier reports describe use of in vitro germinated seedlings for raising shoot cultures and not regeneration. There is only a single regeneration protocol of R. graveolens; however, it employs conventional labour intensive techniques deterring automation. The aim of present investigation was to establish a cost effective protocol for large-scale plant production. We report for the first time a one-step protocol with improved regeneration efficiency for multiple shoots induction employing liquid culture systems. Effect of polyamines (putrescine and spermine) on growth and furanocoumarin was studied. Addition of spermine enhanced the number of multiple shoots formed (2.5-fold) and reduced the time taken by half. Spermine addition resulted in 1.47-fold in furanocoumarin production. The selected shoot line, RS2 was successfully scaled up to 5L in culture vessels, with 1.53-fold increase in biomass without affecting the productivity of these cultures. This proves to be a commercially feasible alternative to bioreactors for large-scale biomass and furanocoumarin production.     

<Emphasis Type="Italic">Withania somnifera</Emphasis> (Linn.) Dunal: a review of chemical and pharmacological diversity

Withania somnifera Dunal, is a commonly used herb in Indian Ayurvedic medicine system. Due to its pharmacological value and an inexhaustible source of novel biologically active compounds, it has been a great interest for researchers. The plant is known to possess anti-inflammatory, antitumor, antistress, antioxidant, immunomodulatory and hemopoetic properties. Various withanolides, steroidal lactones, have been isolated from W. somnifera and were known to have high therapeutic value. Based on the differences in the substitution patterns of withanolides the species has been classified into various chemotypes. So far, three different chemotypes have been identified, which have been further classified into ecotypes based on the contents of withanolides. Present review summarizes the phytochemical variability and pharmacological advances reported in literature.