Farzana Sabir

Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania somnifera L. (Dunal)

AbstractWithanolides are pharmaceutically important C28-phytochemicals produced in most prodigal amounts and diversified forms by Withania somnifera. Metabolic origin of withanolides from triterpenoid pathway intermediates implies that isoprenogenesis could significantly govern withanolide production. In plants, isoprenogenesis occurs via two routes: mevalonate (MVA) pathway in cytosol and non-mevalonate or DOXP/MEP pathway in plastids. We have investigated relative carbon contribution of MVA and DOXP pathways to withanolide biosynthesis in W. somnifera. The quantitative NMR-based biosynthetic study involved tracing of 13C label from 13C1-d-glucose to withaferin A in withanolide producing in vitro microshoot cultures of the plant. Enrichment of 13C abundance at each carbon of withaferin A from 13C1-glucose-fed cultures was monitored by normalization and integration of NMR signal intensities. The pattern of carbon position-specific 13C enrichment of withaferin A was analyzed by a retro-biosynthetic approach using a squalene-intermediated metabolic model of withanolide (withaferin A) biosynthesis. The pattern suggested that both DOXP and MVA pathways of isoprenogenesis were significantly involved in withanolide biosynthesis with their relative contribution on the ratio of 25:75, respectively. The results have been discussed in a new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids. Key message The study elucidates significant contribution of DOXP pathway to withanolide biosynthesis. A new connotation of biosynthetic load-based role of DOXP/MVA recruitment in isoprenoid biosynthesis has been proposed.

Effect of prolonged water stress on specialized secondary metabolites, peltate glandular trichomes, and pathway gene expression in Artemisia annua L.

Artemisia annua L. accumulates substantial quantities of unique sesquiternoid artemisinin and related phytomolecules and characteristic essential oil in glandular trichomes, present on its leaves and inflorescence. Water stress is a major concern in controlling plant growth and productivity. In this study, our aim was to find out the modulation of artemisinin and essential oil constituents in plants grown under prolonged water stress conditions. A. annua CIM-Arogya plants grown in pots were subjected to mild (60% ± 5) and moderate (40% ± 5) water stress treatment and continued during entire developmental period. Results revealed that artemisinin, arteannuin-B, artemisinic acid, dihydroartemisinic acid and essential oil content were positively controlled by the growth and development however negatively modulated by water deficit stress. Interestingly, some of minor monoterpenes, all sesquiterpenes and other low molecular weight volatiles of essential oil components were induced by water deficit treatment. Camphor which is the major essential oil constituents did not alter much while 1, 8 cineole was modulated during development of plant as well as under water stress conditions. Water deficit stress induces a decrease in glandular trichome density and size as well. The dynamics of various secondary metabolites is discussed in the light of growth responses, trichomes and pathway gene expression in plants grown under two levels of prolonged water stress conditions.

Biotransformation of withanolides by cell suspension cultures of Withania somnifera (Dunal)

The biotransformation potential of cell suspension cultures generated from Withania somnifera leaf was investigated, using withanolides, i.e. withanolide A, withaferin A, and withanone as precursor substrates. Interestingly, the cell suspension cultures showed inter-conversion of withanolides, as well converted to some unknown compounds, released to the culture media. The bio-catalyzed withanolide was detected and quantified by TLC and HPLC, respectively. There is noticeable conversion of withanolide A to withanone, and vice versa though at a lower level. The type of reaction of this biotransformation appears to be substitution of 20-OH group to 17-OH in withanolide A. In this paper, we present for the first time the possibility of biotransformation by inter-conversion of withanolides of pharmacological importance through cell suspension culture of W. somnifera. The possible role of putative cytochrome P450 hydroxylases is implicated in the conversion.

Withanolides from Withania somnifera Dunal: Development of Cellular Technology and their Production

Withania somnifera Dunal is one of the most commonly used plants in Ayurvedic and indigenous system of medicine in India for over thousands of years. In view of its varied therapeutic potential, the plant has also been the subject of considerable scientific attention. The major chemical constituents of the Withania genus, the withanolides, are a group of naturally occurring C28-steroidal lactones built on an intact or rearranged ergostane framework, in which C22 and C26 are oxidized to form a six-member lactone ring. In recent years, numerous pharmacological investigations have been carried out utilizing W. somnifera extracts and several patents have been filed on pharmacological and medicinal importance of withanolides and extracts of W. somnifera, individually or in combination. Considering the immense importance of withanolides for medicinal purposes, the establishment of strategies to improve withanolides yield are highly desirable. Under natural conditions, W. somnifera possesses restricted levels of withanolides then, alternatives for obtaining withanolides in better yields are imperative. In vitro approaches followed by metabolic engineering could be attractive tools to achieve this goal. Therefore, we present here an overview of the development of various protocols for in vitro tissue regeneration from W. somnifera and in vitro secondary metabolite production as well. The review also gives an account of selected patents on various important activities of phytochemicals and extracts of W. somnifera.

In vitro withanolide production by Withania somnifera L. cultures.

In vitro multiple shoots, root, callus and cell suspension cultures of Withania somnifera exhibited the potentiality to produce pharmacologically active withanolides. Multiple shoots cultures exhibited an increase in withanolide A accumulation compared to shoots of the mother plant. In vitro generated root cultures as well as callus and suspension cultures also produced withanolides albeit at lower levels.

Rapid Micropropagation of Withania somnifera L. Accessions from Axillary Meristems

ABSTRACT An efficient method of in vitro shoot propagation of six elite accessions of Withania somnifera collected throughout India was developed. Maximum numbers of shoots in all accessions were achieved from axillary explant on Murashige and Skoog (MS) medium supplemented with 1 mgL−1 BAP and 1 mgL−1 kinetin. Inclusion of kinetin increased shoot numbers in a shorter time-period and was effective on all the elite accessions. The highest number of shoots (60 ± 1.82 and 60.05 ± 2.03) was observed in two lines developed by CIMAP, Lucknow, while the lowest number of shoots (19.4 ± 1.63) occurred on a wild collected accession with bushy growth. The in vitro raised shoots of all the accessions could be easily rooted on MS medium supplemented with 2 mgL−1 IBA. Rooted shoots were successfully established in a garden soil-vermi-compost (3:1, w:w) medium in a glasshouse. Stable production of withanolides from in vitro regenerated shoots was comparable to the yields from field grown mother plants, indicating the in vitro methodology could be used successfully for the true-to-type plant regeneration of Withania somnifera accessions.

Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides

Gymnema sylvestre R.Br., a pharmacologically important herb vernacularly called Gur-Mar (sugar eliminator), is widely known for its antidiabetic action. This property of the herb has been attributed to the presence of bioactive triterpene glycosides. Although some information regarding pharmacology and phytochemical profiles of the plant are available, no attempts have been made so far to decipher the biosynthetic pathway and key enzymes involved in biosynthesis of steryl glucosides. The present report deals with the identification and catalytic characterization of a glucosyltransferase, catalyzing biosynthesis of steryl glycosides. The full length cDNA (2572 bp) contained an open reading frame of 2106 nucleotides that encoded a 701 amino acid protein, falling into GT-B subfamily of glycosyltransferases. The GsSGT was expressed in Escherichia coli and biochemical characterization of the recombinant enzyme suggested its key role in the biosynthesis of steryl glucosides with catalytic preference for C-3 hydroxyl group of sterols. To our knowledge, this pertains to be the first report on cloning and biochemical characterization of a sterol metabolism gene from G. sylvestre R.Br. catalyzing glucosylation of a variety of sterols of biological origin from diverse organisms such as bacteria, fungi, and plants.

Bioconversion of Artemisinin to its Nonperoxidic Derivative Deoxyartemisinin through Suspension Cultures of Withania somnifera Dunal

Biotransformation of artemisinin was investigated with two different cell lines of suspension cultures of Withania somnifera. Both cell lines exhibited potential to transform artemisinin into its nonperoxidic analogue, deoxyartemisinin, by eliminating the peroxo bridge of artemisinin. The enzyme involved in the reaction is assumed to be artemisinin peroxidase, and its activity in extracts of W. somnifera leaves was detected. Thus, the non-native cell-free extract of W. somnifera and suspension culture-mediated bioconversion can be a promising tool for further manipulation of pharmaceutical compounds.