Investigation of Cytotoxic Constituents from Seed Pulp of Entada Phaseoloides and Metabolite Profiling Using UPLC-QTOF-MSE.

BACKGROUND: Entada phaseoloides (Linn.) Merr. (Family: Fabaceae) is a well-known, traditional, medicinal plant that has been extensively used in the Ayurvedic system of medicine for centuries to combat a wide range of ailments. OBJECTIVE: The goal of this work was to investigate the bioactive constituents from n-butanol extracts of Entada. phaseoloides and develop a method for the comprehensive characterization of saponins using liquid chromatography with an electrospray ionization quadrupole time-of-flight mass spectrometer (LC-ESI-QTOF-MS). METHODS: A hyphenated technique, ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS), has been proposed to integrate LC and MS together with NMR for structural elucidation. This method allowed comprehensive characterization of saponin glycosides from E. phaseoloides based on their MS/MS fragmentation study. RESULTS: The phytochemical study of E. phaseoloides resulted in the isolation and identification of three bio-active constituents. Further, the UPLC-QTOF-MS/MS method led the structure elucidation of saponin constituents directly from crude extracts via comparison of the exact molecular masses from their MS/MS spectra. Identified common fragments m/z 648, 630, 498, 366, and 204 were used for the screening of saponin components. CONCLUSIONS: The present study summarizes the isolation and identification of bio-compounds from n-butanol extract and the demonstration of UPLC-QTOF-MS/MS analysis for the characterization of compounds in complex crude extracts. To the best of our knowledge, this is the first systematic study in structural characterization on complex saponins and other metabolites from crude extract of E. phaseoloides using UPLC-ESI-QTOF-MSE. HIGHLIGHTS: Rapid analysis and characterizations of three new saponins from E. phaseoloides using UPLC-ESI-QTOF-MSE were tentatively identified based on the mass fragmentation study.

Comparative functional characterization of eugenol synthase from four different Ocimum species: Implications on eugenol accumulation.

Isoprenoids and phenylpropanoids are the major secondary metabolite constituents in Ocimum genus. Though enzymes from phenylpropanoid pathway have been characterized from few plants, limited information exists on how they modulate levels of secondary metabolites. Here, we performed phenylpropanoid profiling in different tissues from five Ocimum species, which revealed significant variations in secondary metabolites including eugenol, eugenol methyl ether, estragole and methyl cinnamate levels. Expression analysis of eugenol synthase (EGS) gene showed higher transcript levels especially in young leaves and inflorescence; and were positively correlated with eugenol contents. Additionally, transcript levels of coniferyl alcohol acyl transferase, a key enzyme diverting pool of substrate to phenylpropanoids, were in accordance with their abundance in respective species. In particular, eugenol methyl transferase expression positively correlated with higher levels of eugenol methyl ether in Ocimum tenuiflorum. Further, EGSs were functionally characterized from four Ocimum species varying in their eugenol contents. Kinetic and expression analyses indicated, higher enzyme turnover and transcripts levels, in species accumulating more eugenol. Moreover, biochemical and bioinformatics studies demonstrated that coniferyl acetate was the preferred substrate over coumaryl acetate when used, individually or together, in the enzyme assay. Overall, this study revealed the preliminary evidence for varied accumulation of eugenol and its abundance over chavicol in these Ocimum species. Current findings could potentially provide novel insights for metabolic modulations in medicinal and aromatic plants.

Comparative structural modeling and docking studies of uricase: possible implication in enzyme supplementation therapy for hyperuricemic disorders.

Uricase (EC 1.7.3.3, UC) catalyzes the oxidation of uric acid (UA) to more soluble allantoin thereby lowering plasma UA levels. In humans, when concentration of UA exceeds >7mg/dl, it leads to hyperuricemia, gout, nephrolithiasis and urolithiasis. A new remedy to cure such metabolic diseases is the enzyme supplementation therapy by UC but with high degree of antigenic independence. Therefore screening of new uricase sources to expand its usefulness and reduced antigenecity is needed. Present study employed cheminformatics approach to construct models of reported UC from different sources viz. Bacillus megaterium, Streptomyces bingchenggensis BCW-1, Paenibacillus sp, Solibacter usitatus Ellin6076, Truepera radiovictrix DSM 17093 and Ktedonobacter racemifer DSM 4496 in order to study their structure-function relationship for enzyme mass production and modification for improved characteristics. BioMed CAChe version 6.1 was further used to study enzyme-substrate interactions of models with uric acid using docking approach. Results indicated that models for UC of Streptomyces bingchenggensis BCW-1 accounted for better regio-specificity towards UA, supporting the interested metabolism and thus may further be implicated in enzyme supplementation therapy for hyperuricemic associated disorders.