Akhilesh K. Yadav

Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal

The medicinal plant Withania somnifera is researched extensively to increase the quantity of withanolides and specifically withaferin A, which finds implications in many pharmacological activities. Due to insufficient knowledge on biosynthesis and unacceptability of transgenic approach, it is preferred to follow alternative physiological methods to increase the yield of withanolides. Prior use of elicitors like salicylic acid, methyl jasmonate, fungal extracts, and even mechanical wounding have shown to increase the withanolide biosynthesis with limited success; however, the commercial viability and logistics of application are debatable. In this investigation, we tested the simple nitrogeneous fertilizers pertaining to the enhancement of withaferin A biosynthesis. Application of ammonium sulfate improved the sterol contents required for the withanolide biosynthesis and correlated to higher expression of pathway genes like FPPS, SMT1, SMT2, SMO1, SMO2, and ODM. Increased expression of a gene homologous to allene oxide cyclase, crucial in jasmonic acid biosynthetic pathway, suggested the involvement of jasmonate signaling. High levels of WRKY gene transcripts indicated transcriptional regulation of the pathway genes. Increase in transcript level could be correlated with a corresponding increase in the protein levels for WsSMT1 and WsWRKY1. The withaferin A increase was also demonstrated in the potted plants growing in the glasshouse and in the open field. These results implicated simple physiological management of nitrogen fertilizer signal to improve the yield of secondary metabolite through probable involvement of jasmonate signal and WRKY transcription factor for the first time, in W. somnifera besides improving the foliage.

Acacetin 7-O-α-l-rhamnopyranosyl (1–2) β-D-xylopyranoside Elicits Life-span Extension and Stress Resistance in Caenorhabditis elegans

The advancements in the field of gerontology have unraveled the signaling pathways that regulate life span, suggesting that it might be feasible to modulate aging. To this end, we isolated a novel phytomolecule Acacetin 7-O-α-l-rhamnopyranosyl (1-2) β-D-xylopyranoside (ARX) from Premna integrifolia and evaluated its antiaging effects in Caenorhabditis elegans The spectral data analysis revealed the occurrence of a new compound ARX. Out of the three tested pharmacological doses of ARX, viz. 5, 25, and 50 µM, the 25-µM dose was able to extend life span in C. elegans by more than 39%. The present study suggests that ARX affects bacterial metabolism, which in turn leads to dietary restriction (DR)-like effects in the worms. The effect of ARX on worms with mutations (mev-1, eat-2, sir-2.1, skn-1, daf-16, and hsf-1) indicates that ARX-mediated life-span extension involves mechanisms associated with DR and maintenance of cellular redox homeostasis. This study is the first time report on longevity-promoting activity of ARX in C. elegans mediated by stress and DR-regulating genes. This novel phytomolecule can contribute in designing therapeutics for managing aging and age-related diseases.

Simultaneous quantification of the anti-inflammatory phytoconstituents betulinic acid, 24β-ethylcholesta-5,22E,25-triene-3β-ol, and lupeol in Clerodendrum phlomidis

A new validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous determination of anti-inflammatory compounds betulinic acid (BA, 1), 24β-ethylcholesta-5,22E,25-triene-3β-ol (ECTO, 2), and lupeol (LU, 3) in the roots of Clerodendrum phlomidis. Extraction efficiency of marker compounds was studied using normal (cold and hot), ultrasonic, as well as microwave-assisted extraction techniques with various solvents. Well-resolved separation of marker compounds was achieved on silica gel 60F254 plates using the mobile phase consisting of chloroform—methanol (98:2, v/v). Marker compounds were scanned using the densitometric reflection—absorption mode after post-chromatographic derivatization with vanillin—sulfuric acid reagent. Validation of method was performed as per the International Conference on Harmonization (ICH) guidelines. Report on the occurrence of betulinic acid for the first time in C. phlomidis is of chemotaxonomic importance. In addition, anti-inflammatory potential of the rare sterol ECTO (2) on lipopolysaccharide (LPS)-stimulated production of pro-inflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukin-6 [IL-6]) was also evaluated as it was not reported earlier.

Novel anti-inflammatory phytoconstituents from Desmodium gangeticum.

A new aliphatic enone, (17Z,20Z)-hexacosa-17,20-dien-9-one (3), and one new bisindole alkaloid, gangenoid (6), together with seven known compounds were isolated from the roots and aerial parts of Desmodium gangeticum (family: Leguminosae). All the compounds except 2 and 7 were isolated from this plant for the first time, which may be of chemotaxonomic importance. Structures of compounds 3 and 6 were determined on the basis of their detailed spectroscopic analyses (NMR, IR and mass). In addition, compounds 3 and 6 were investigated for their effects on lipopolysaccharide-stimulated macrophages for the production of pro-inflammatory cytokines such as tumour necrosis factor-α and interleukin-6.

A VALIDATED STABILITY-INDICATING HPLC-PDA METHOD FOR ANALYSIS OF DESMODIUM GANGETICUM: AN IMPORTANT INGREDIENT OF AYURVEDIC DRUG “DASHMOOL”

A stability-indicating reversed-phase liquid chromatographic (RPLC) method has been established for analysis of rutin (1), kaempferol-3-O-robinobioside (2), and nicotiflorin (3) in Desmodium gangeticum. The study was performed in the presence of the degradation products generated in the study of forced degradation. Marker compounds were subjected to stress by hydrolysis (acidic and basic), oxidation, photolysis, and thermal treatment. Under the optimized conditions, well-resolved separation of pure compounds from the degradation products with significantly different Rt values was achieved on a Spherisorb ODS2 column (250 mm × 4.6 mm, 10 µm) using isocratic elution of methanol and water (0.5% acetic acid); with acceptable validation results such as linearity, sensitivity, and recovery in terms of RSD (%). The calibration curves were linear in the concentration range of 20–100 µg/mL. Method was validated as per ICH guidelines. The reproducible and robust method may be applied for assays and stability tests of D. gangeticum and phytopreparations containing D. gangeticum.

Quantitation of Antitubercular Compounds in Oroxylum indicum, a Thai Vegetable Used in the Indian System of Medicine

Oroxylum indicum (Bignoniaceae) is an edible plant, and its young pods are eaten in Asian countries, especially in Thailand and Laos. In a recent study, we have reported its flavonoids as potent antitubercular chemicals of the plant. Here, we are reporting a high-performance thin-layer chromatographic method for simultaneous detection and quantification of these important flavonoids of O. indicum. Well resolved separation of marker compounds was achieved on silica gel 60 F254 plates using the mobile phase consisting of chloroform-methanol-water-formic acid (97:3:0.25:0.25, v/v) with acceptable limit of validation parameters such as linearity, sensitivity, recovery, etc. The developed method is reproducible, robust, and inexpensive without any derivatization process.

Syntheses of conformationally restricted benzopyran based triarylethylenes as growth inhibitors of carcinoma cells

A series of conformationally restricted benzopyran based triarylethylenes has been synthesized and characterized as potential growth inhibitors of breast carcinoma cells. The synthesized compounds (14a-b, 15a and 16a-e) presented significant growth inhibition of ER+ and ER- breast cancer cells within the range of IC50 0.55-9.96µM. Amongst other, 16c showed potent anticancer activity at IC50 0.95µM in MCF-7 cells with good selectivity (Selectivity Index 4.47) towards healthy cells. The mechanistic studies for 16c were performed to elucidate possible mode of action which showed 16c elicited anticancer activity through necroptosis process.

Validated RP-HPLC and HPTLC methods for determination of anti-inflammatory bis-indole alkaloid in Desmodium gangeticum

Here, two simple and accurate methods, namely high-performance liquid chromatography and high-performance thin-layer chromatography for the detection of gangenoid, an anti-inflammatory alkaloid, in a well-known Indian medicinal plant Desmodium gangeticum, are described. The proposed methods were successfully used for the estimation of gangenoid in D. gangeticum root.

Validated HPTLC method for the simultaneous quantification of diterpenoids in Vitex trifolia L.

Vitex trifolia L. is an important Indian medicinal plant with diverse pharmacological properties. In a recent study, we reported the isolation and antitubercular activity evaluation of three new diterpenoids from its leaves; here we have developed a validated rapid, simple, precise, and accurate high-performance TLC method for the simultaneous quantification of isolated diterpenoids in V. trifolia. Diterpenoids, 6α,7α-diacetoxy-13-hydroxy-8(9),14-labdadien (A), 13-hydroxy-5(10),14-halimadien-6-one (B), and 9-hydroxy-13(14)-labden-16,15-olide (C) were separated on silica gel 60F254 high-performance TLC plates using chloroform/acetone (98:2, v/v) as mobile phase. The quantitation of diterpenoids was carried out using densitometric reflection/absorption mode at 610 nm after postchromatographic derivatization using a vanillin/sulfuric acid reagent. A precise and accurate quantification can be performed for compounds A and B in the linear working concentration range of 333-1000 ng/band and for C in the range of 670-2000 ng/band with good correlations (r = 0.9984, 0.9991, and 0.9994, respectively). The method was validated for peak purity, precision, accuracy, robustness, LOD, and LOQ, as per the ICH guidelines. The method reported here is simple, reproducible and may be applied for the quantitative analysis of the above diterpenoids in the leaves of V. trifolia.