HPLC Coupled with Chemometric Analysis and LC-MS Studies of Three Flavonoids in Tephrosia purpurea (L.) Pers Revealed Impact of Chemodiversity on the Quest for the Chemical Markers.

Globally, Tephrosia purpurea (L.) Pers is used as an important component in herbal drug formulations for liver health. The present study is aimed to develop a suitable analytical approach for simultaneous analysis of three flavonoids (rutin, deguelin and rotenone) to establish quality control methods for plant. A novel High-performance liquid chromatography photodiode array detector (HPLC-PDA) method has been developed to quantify these flavonoids in T. purpurea. The method was validated, and data were subjected to chemometric analysis to select most optimal marker compound. The method that was found linear with R2 values ranges from 0.996 to 0.998 with good recoveries. Intra- and inter-day precision values were <2. HPLC analysis revealed high level of chemodiversity. Quantity of all the three chemical markers was found significantly disparate in samples from different locations. Deguelin was detectable only in three out of total eight samples. However, liquid chromatography-mass spectrometry analysis was found sufficiently sensitive to detect all the compounds in all samples. Thus, results suggest to apply combination of approaches to enhance confidence in chromatographic methods for quality control of herbal drugs. Principal component analysis ranked the markers as Rutin>Rotenone>Deguelin. This comprehensive approach employing multichromatography platforms can be successfully utilized in analysis of these bioactive markers and routine standardization of herbal material and formulations containing T. purpurea.

Investigation on apposite chemical marker for quality control of Tephrosia purpurea (L.) Pers. by means of HPTLC-chemometric analysis.

Application of phytochemicals as the markers for quality assurance of the herbal medicinal material is one of the prominently used approach. In the present study, six major chemical compounds i.e. rutin, quercetin, lupeol, ß-sitosterol, rotenone, deguelin of therapeutically important plant Tephrosia purpurea were tested for their significance to be used as chemical markers in analytical methods. Plants were collected from different locations. Extraction procedures as well as HPTLC analytical methods have been optimized for each compound. All these methods have been validated in terms of linearity, intraday-interday precision, LOD, LOQ, accuracy and repeatability. Metabolites have been quantified and quantitative data has been subjected to chemometric analysis. Results revealed that Rf values of all the compounds are between 0.3 and 0.4. Rutin, lupeol and ß-sitosterol gave desirable response and other three compounds were found undetectable in HPTLC. Content of rutin, beta-sitosterol and lupeol ranged from 0.095 to 0.84, 0.043 to 0.125, 0.023 to 0.045 w/w % respectively. Among them, rutin content was highest in all samples. Quantitative measurements combined with chemometric analysis displayed chemodiversity in the samples. In addition, principal component analysis ranked the marker as in order of their significance rutin > ß-sitosterol > lupeol. Results indicate rutin to be most preferable chemical marker for the Tephrosia. Furthermore, application of all the three compounds combined as the multimarker system should be preferred for standardization of T. purpurea.

Enhancing epi-cedrol production in Escherichia coli by fusion expression of farnesyl pyrophosphate synthase and epi-cedrol synthase.

Terpene synthase catalyses acyclic diphosphate farnesyl diphosphate into desired sesquiterpenes. In this study, a fusion enzyme was constructed by linking Santalum album farnesyl pyrophosphate synthase (SaFPPS) individually with terpene synthase and Artemisia annua Epi-cedrol synthase (AaECS). The stop codon at the N-terminus of SaFPPS was removed and replaced by a short peptide (GSGGS) to introduce a linker between the two open reading frames. This fusion clone was expressed in Escherichia coli Rosseta DE3 cells. The fusion enzyme FPPS-ECS produced sesquiterpene 8-epi-cedrol from substrates isopentenyl pyrophosphate and dimethylallyl pyrophosphate through sequential reactions. The K m values for FPPS-ECS for isopentyl diphosphate was 4.71 µM. The fusion enzyme carried out the efficient conversion of IPP to epi-cedrol, in comparison to single enzymes SaFPPS and AaECS when combined together in enzyme assay over time. Further, the recombinant E. coli BL21 strain harbouring fusion plasmid successfully produced epi-cedrol in fermentation medium. The strain having fusion plasmid (pET32a-FPPS-ECS) produced 1.084 ± 0.09 mg/L epi-cedrol, while the strain harbouring mixed plasmid (pRSETB-FPPS and pET28a-ECS) showed 1.002 ± 0.07 mg/L titre in fermentation medium by overexpression and MEP pathway utilization. Structural analysis was done by I-TASSER server and docking was done by AutoDock Vina software, which suggested that secondary structure of the N- C terminal domain and their relative positions to functional domains of the fusion enzyme was greatly significant to the catalytic properties of the fusion enzymatic complex than individual enzymes.

Methyl jasmonate mediates upregulation of bacoside A production in shoot cultures of Bacopa monnieri.

Methyl jasmonate (MJ) enhances the production of a range of secondary metabolites including triterpenoid saponins in a variety of plant species. Here, it enhanced production of bacoside A, a valuable triterpenoid saponin having nootropic therapeutic activity in in vitro shoot cultures of Bacopa monnieri, the only known source of bacoside A. The highest yield was with 50 µM MJ giving 4.4 mg bacoside A/g dry wt; an 1.8-fold increase (compared to control) after 1 week.