Dipak Kumar Mishra

Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera

Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG). As genomic resources for this plant are limited, the genes involved in CG biosynthetic pathway remain largely unknown till date. Our study on stage and tissue specific metabolite accumulation showed that CG’s were maximally accumulated in stems of 3 month old seedlings. De novo transcriptome sequencing of same was done using high throughput Illumina HiSeq platform generating 44074 unigenes with average mean length of 1785 base pair. Around 66.6% of unigenes were annotated by using various public databases and 5324 unigenes showed significant match in the KEGG database involved in 133 different pathways of plant metabolism. Further KEGG analysis resulted in identification of 336 unigenes involved in cardenolide biosynthesis. Tissue specific expression analysis of 30 putative transcripts involved in terpenoid, steroid and cardenolide pathways showed a positive correlation between metabolite and transcript accumulation. Wound stress elevated CG levels as well the levels of the putative transcripts involved in its biosynthetic pathways. This result further validated the involvement of identified transcripts in CGs biosynthesis. The identified transcripts will lay a substantial foundation for further research on metabolic engineering and regulation of cardiac glycosides biosynthesis pathway genes.

Simultaneous quantitative determination of multiple bioactive markers in Ocimum sanctum obtained from different locations and its marketed herbal formulations using UPLC-ESI-MS/MS combined with principal component analysis.

INTRODUCTION Ocimum sanctum L., with phenolic acids, flavonoids, propenyl phenols and terpenoids as active pharmacological constituents, is a popular medicinal herb and is present as an ingredient in many herbal formulations. Therefore, development of a reliable analytical method for simultaneous determination of the pharmacologically active constituents of O. sanctum is of high importance. OBJECTIVE To develop and validate a new, rapid, sensitive and selective UPLC-ESI/MS/MS method for simultaneous determination of 23 bioactive markers including phenolic acids, flavonoids, propenyl phenol and terpenoid in the leaf extract and marketed herbal formulations of O. sanctum. METHODS An UPLC-ESI/MS/MS method using negative electrospray ionisation (ESI) in multiple-reaction-monitoring (MRM) mode was used for simultaneous determination. Chromatographic separation was achieved on an Acquity UPLC BEH C18 -column using a gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Principal component analysis (PCA) was applied to correlate and discriminate eight geographical collections of O. sanctum based on quantitative data of the analytes. RESULTS The developed method was validated as per International Conference on Harmonization guidelines and found to be accurate, with overall recovery in the range 95.09-104.84% (RSD ≤ 1.85%), precise (RSD ≤ 1.98%) and linear (r(2)  ≥ 0.9971) over the concentration range of 0.5-1000 ng/mL. Ursolic acid was found to be the most abundant marker in all the samples investigated, except for the marketed tablet. CONCLUSION The method established is simple, rapid and sensitive, hence it can be reliably utilised for the quality control of O. sanctum and derived herbal formulations.

Cucumis melo ssp. Agrestis var. Agrestis Ameliorates High Fat Diet Induced Dyslipidemia in Syrian Golden Hamsters and Inhibits Adipogenesis in 3T3-L1 Adipocytes

Background: Cucumis melo ssp. agrestis var. agrestis (CMA) is a wild variety of C. melo. This study aimed to explore anti-dyslipidemic and anti-adipogenic potential of CMA. Materials and Methods: For initial anti-dyslipidemic and antihyperglycemic potential of CMA fruit extract (CMFE), male Syrian golden hamsters were fed a chow or high-fat diet with or without CMFE (100 mg/kg). Further, we did fractionation of this CMFE into two fractions namely; CMA water fraction (CMWF) and CMA hexane fraction (CMHF). Phytochemical screening was done with liquid chromatography-mass spectrometry LC- (MS)/MS and direct analysis in real time-MS to detect active compounds in the fractions. Further, high-fat diet fed dyslipidemic hamsters were treated with CMWF and CMHF at 50 mg/kg for 7 days. Results: Oral administration of CMFE and both fractions (CMWF and CMHF) reduced the total cholesterol, triglycerides, low‐density lipoprotein cholesterol, and very low‐density lipoprotein-cholesterol levels in high fat diet-fed dyslipidemic hamsters. CMHF also modulated expression of genes involved in lipogenesis, lipid metabolism, and reverse cholesterol transport. Standard biochemical diagnostic tests suggested that neither of fractions causes any toxicity to hamster liver or kidneys. CMFE and CMHF also decreased oil-red-O accumulation in 3T3-L1 adipocytes. Conclusion: Based on these results, it is concluded that CMA possesses anti-dyslipidemic and anti-hyperglycemic activity along with the anti-adipogenic activity. SUMMARY The oral administration of Cucumis melo agrestis fruit extract (CMFE) and its fractions (CMWF and CMHF) improved serum lipid profile in HFD fed dyslipidemic hamsters. CMFE, CMWF and CMHF significantly attenuated body weight gain and eWAT hypertrophy. The CMHF decreased lipogenesis in both liver and adipose tissue. CMFE and CMHF also inhibited adipogenesis in 3T3-L1 adipocytes. Abbreviation used: CMA: Cucumis melo ssp. agrestis var. agrestis, CMFE: CMA fruit extract, CMWF: CMA water fraction, CMHF: CMA hexane fraction, FAS: Fatty acid synthase, SREBP1c: Sterol regulatory element binding protein 1c, ACC: Acetyl CoA carboxylase, LXR α: Liver X receptor α.

Callus culture and in vitro biosynthesis of cardiac glycosides from Calotropis gigantea (L.) Ait

Calotropis gigantea (L.) Ait., belonging to the family Asclepiadaceae, is a source of many cardiac glycosides (CGs) and their steroidal moieties (genins). These CGs have been reported to have anti-proliferative activity on tumor cell lines and are potential targets for cancer chemotherapy. However, the abundance of CGs in wild plants is particularly restricted and it is difficult to isolate the desired compound in required quantities. This study is the first attempt to standardize the induction and proliferation of callus from various explants of C. gigantea specifically for the production of CGs. Callus growth was accompanied by CG measurement using high-performance liquid chromatography-tandem mass spectrometry. Murashige and Skoog (MS) and modified Murashige and Skoog (MMS) media were optimized with various combinations and concentrations of auxin and cytokinin for induction and growth of calli from a range of explant sources. While leaves and stem explants resulted in greatest callus induction, MMS medium was found to be optimal. However, no CG was produced from callus grown on this medium. In contrast, the induction and proliferation of callus on MS medium were optimum at primary stages, but growth slowed during the third subculture. Therefore, calli were transferred to MMS medium to promote callus proliferation and production of CGs. As a result, three CGs and two genins were biosynthesized. Furthermore, the callus induction data in MS medium indicated that among different auxins, 2,4-dichlorophenoxyacetic acid was the best for callus induction compared to 1-naphthylacetic acid and indole-3-acetic acid. The data also revealed that the cytokinin/auxin ratio was critical rather than their independent presence for the induction of callus. Thus, the in vitro biosynthesis of targeted CGs may offer an alternative pathway for new source of anti-proliferative agents in required quantities.

Nymphaea rubra Ameliorates TNF-α-Induced Insulin Resistance via Suppression of c-Jun NH2-Terminal Kinase and Nuclear Factor-κB in the Rat Skeletal Muscle Cells

In this work, we demonstrated insulin signaling and the anti-inflammatory effects by the chloroform fraction of ethanolic extract of Nymphaea rubra flowers in TNF-α-induced insulin resistance in the rat skeletal muscle cell line (L6 myotubes) to dissect out its anti-hyperglycemic mechanism. N. rubra enhances the GLUT4-mediated glucose transport in a dose dependent manner and also increases the tyrosine phosphorylation of both IR-β and IRS-1, and the IRS-1 associated PI-3 kinase activity in TNF-α-treated L6 myotubes. Moreover, N. rubra decreases Ser307 phosphorylation of IRS-1 by the suppression of JNK and NF-κB activation. In conclusion, N. rubra reverses the insulin resistance by the inhibition of c-Jun NH2-Terminal Kinase and Nuclear-κB.

Bioactivity guided isolation of oxypregnane-oligoglycosides (calotroposides) from the root bark of Calotropis gigantea as potent anticancer agents

Bioactivity guided isolation of oxypregnane-oligoglycosides (calotroposides) from the ethanolic extract of root bark of Calotropis gigantea (L.) Dryand. with purple flowers has been performed. Dereplication by NMR and LC-MS analysis confirmed the presence of oxypregnane-oligoglycosides (calotroposides) in the ethyl acetate fraction. One new (7) and six known calotroposides (1–6) were isolated from the most active ethyl acetate fraction of ethanolic extract of Calotropis gigantea and structure elucidation was accomplished by spectroscopic methods (IR, UV, MS and NMR). Isolated calotroposides were investigated for cytotoxic activity against six cancer cell lines (MDA-MB-231, MCF-7, LNCaP, HeLa, K562, and Caco-2) and a non-cancer cell line (HEK-293), where calotroposides 2, 4 and 5 showed highest cytotoxicity (IC50 8.49 ± 0.41 μM, 6.49 ± 0.15 μM and 9.65 ± 0.44 μM respectively) against MDA-MB-231. Conclusively, calotroposides 2, 4 and 5 have promising anticancer effect against aggressive breast cancer cells and can serve as leads for further structural exploration as anticancer moiety.

Pyranocarbazoles from Murraya koenigii (L.) Spreng. as antimicrobial agents

Abstract The bioassay guided fractionation of methanolic extract of Murraya koenigii (L.) Spreng. leaves resulted in the isolation of seven pyranocarbazoles. These were evaluated against four bacterial strains and ten Candida sp. including two matched pair of fluconazole sensitive/resistant clinical isolates. Out of seven, three i.e. Koenine (mk279), Koenigine (mk309) and Mahanine (mk347) exhibited significant antibacterial activity MIC90 3.12–12.5 μg/mL against bacterial strains Streptococcus aureus and Klebsiella pneumonia compared with standard drug Kanamycin MIC90 12.5 μg/mL. However, only mk309 was found active against variety of Candida species MIC90 12.5–100 μg/mL. It was observed that hydroxylation at C-6 and C-7 positions in the studied pyranocarbazoles activate the bioactivity. Simultaneously, decrease in Log P value compares with −H and −O−CH3 substituted derivatives. The study is focused on selective antifungal and antibacterial activity of pyranocarbazoles on bacterial strains S. aureus, K. pneumonia and variety of Candida species with structure activity relationship observations.