Ashwagandha: Advances in plant biotechnological approaches for propagation and production of bioactive compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera is one of the most extensively delved Ayurvedic medicine. Apart from rejuvenation and increasing longevity, it has several other properties such as immunomodulation, anti-cancer, anti-stress and neuroprotection. Because of its prevailing use and increasing demand, it becomes prudent to scientifically evaluate and document both its propagation and production of desired phytoconstituents. AIM OF THE STUDY: This review aims to highlight the research progress achieved on various biotechnological and tissue culture aspects of Withania somnifera and to cover up-to-date information regarding in-vitro propagation and production of withanolides. MATERIALS AND METHODS: Significant published studies were identified for the years 2000-2018 using Elsevier-Science Direct, Pubmed and Google scholar and several research studies in our laboratory. Following keywords such as "plant extracts", "in vitro cultures", "callus and suspension culture", "micropropagation", "hairy root cultures" were used. Further, "Withania somnifera", "secondary metabolites specially withanolides", "molecular techniques" and "in vitro conservation" were used to cross-reference the keywords. RESULTS: Ashwagandha comprises a broad spectrum of phytochemicals with a wide range of pharmacological properties. W. somnifera seeds have reduced viability and germination rates; thus, its regular cultivation method fails to achieve commercial demands mainly for the production of desired phytoconstituents. Cultivation of plant cells/tissues under in vitro conditions and development of various biotechnological strategies will help to build an attractive alternative to provide adequate quality and quantity raw materials. Recently, a large number of in vitro protocols has developed for W. somnifera not only for its propagation but for the production of secondary metabolites as well. Present work highlights a variety of biotechnological strategies both for prompt propagation and production of different bioactive secondary metabolites. CONCLUSION: The present review focuses on the development and opportunities in various biotechnological approaches to accomplish the global demand of W. somnifera and its secondary metabolites. This review underlines the advances in plant biotechnological approaches for the propagation of W. somnifera and production of its bioactive compounds.

Organic cultivation of Ashwagandha with improved biomass and high content of active Withanolides: Use of Vermicompost.

Withania somnifera (Ashwagandha) has recently been studied extensively for its health-supplementing and therapeutic activities against a variety of ailments. Several independent studies have experimentally demonstrated pharmaceutical potential of its active Withanolides, Withaferin A (Wi-A), Withanone (Wi-N) and Withanolide A (Wil-A). However, to promote its use in herbal industry, an environmentally sustainable cultivation and high yield are warranted. In modern agriculture strategies, there has been indiscriminate use of chemical fertilizers to boost the crop-yield, however the practice largely ignored its adverse effect on the quality of soil and the environment. In view of these, we attempted to recruit Vermicompost (Vcom, 20-100%) as an organic fertilizer of choice during the sowing and growing phases of Ashwagandha plants. We report that (i) pre-soaking of seeds for 12 h in Vermicompost leachate (Vcom-L) and Vermicompost tea (Vcom-T) led to higher germination, (ii) binary combination of pre-soaking of seeds and cultivation in Vcom (up to 80%) resulted in further improvement both in germination and seedling growth, (iii) cultivated plants in the presence of Vcom+Vcom-L showed higher leaf and root mass, earlier onset of flowering and fruiting and (iv) leaves from the Vcom+Vcom-L cultivated plants showed higher level of active Withanolides, Withanone (Wi-N), Withanolide A (Wil-A) and Withaferin A (Wi-A) and showed anticancer activities in cell culture assays. Taken together, we report a simple and inexpensive method for improving the yield and pharmaceutical components of Ashwagandha leaves.

Virus-Induced Silencing of Key Genes Leads to Differential Impact on Withanolide Biosynthesis in the Medicinal Plant, Withania somnifera.

Withanolides are a collection of naturally occurring, pharmacologically active, secondary metabolites synthesized in the medicinally important plant, Withania somnifera. These bioactive molecules are C28-steroidal lactone triterpenoids and their synthesis is proposed to take place via the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways through the sterol pathway using 24-methylene cholesterol as substrate flux. Although the phytochemical profiles as well as pharmaceutical activities of Withania extracts have been well studied, limited genomic information and difficult genetic transformation have been a major bottleneck towards understanding the participation of specific genes in withanolide biosynthesis. In this study, we used the Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) approach to study the participation of key genes from MVA, MEP and triterpenoid biosynthesis for their involvement in withanolide biosynthesis. TRV-infected W. somnifera plants displayed unique phenotypic characteristics and differential accumulation of total Chl as well as carotenoid content for each silenced gene suggesting a reduction in overall isoprenoid synthesis. Comprehensive expression analysis of putative genes of withanolide biosynthesis revealed transcriptional modulations conferring the presence of complex regulatory mechanisms leading to withanolide biosynthesis. In addition, silencing of genes exhibited modulated total and specific withanolide accumulation at different levels as compared with control plants. Comparative analysis also suggests a major role for the MVA pathway as compared with the MEP pathway in providing substrate flux for withanolide biosynthesis. These results demonstrate that transcriptional regulation of selected Withania genes of the triterpenoid biosynthetic pathway critically affects withanolide biosynthesis, providing new horizons to explore this process further, in planta.

Novel Methods to Generate Active Ingredients-Enriched Ashwagandha Leaves and Extracts.

Ashwagandha (Withania somnifera) is an Ayurvedic herb commonly used in world-renowned traditional Indian home medicine system. Roots of Ashwagandha have been traditionally known to possess a variety of therapeutic and health promoting potentials that have not been sufficiently supported by laboratory studies. Nevertheless, most, if not all, of the preventive and therapeutic potentials have been assigned to its bioactive components, steroidal alkaloids and lactones. In contrast to the traditional use of roots, we have been exploring bioactivities in leaves of Ashwagandha. Here, we report that the leaves possess higher content of active Withanolides, Withaferin-A (Wi-A) and Withanone (Wi-N), as compared to the roots. We also established, for the first time, hydroponic cultivation of Ashwagandha and investigated the effect of various cultivation conditions on the content of Wi-A and Wi-N by chemical analysis and bioassays. We report that the Withanone/Withaferin A-rich leaves could be obtained by manipulating light condition during hydroponic cultivation. Furthermore, we recruited cyclodextrins to prepare extracts with desired ratio of Wi-N and Wi-A. Hydroponically grown Ashwagandha and its extracts with high ratio of withanolides are valuable for cancer treatment.

In Vitro Propagation of Withania somnifera (L.) Dunal.

Withania somnifera (L.) Dunal known as Ashwagandha is commonly used in traditional Indian medicine system. It possesses immense therapeutic value against a large number of ailments such as mental diseases, asthma, inflammation, arthritis, rheumatism, tuberculosis, and a variety of other diseases including cancer. The therapeutic potential of W. somnifera is due to the presence of secondary metabolites mainly, tropane alkaloids and withanolides (steroidal lactones). The growing realization of commercial value of the plant has initiated a new demand for in vitro propagation of elite chemotypes of Withania. Micropropagation which is an important tool for rapid multiplication requires optimization of number of factors such as nutrient medium, status of medium (solid and liquid), type of explant, and plant growth regulators. Similarly, an efficient and reproducible in vitro regeneration system which is a prerequisite for the development of genetic transformation protocol requires precise manipulation of various intrinsic and extrinsic factors.

Anticancer activities of Withania somnifera: Current research, formulations, and future perspectives.

CONTEXT: Cancer, being a cause of death for major fraction of population worldwide, is one of the most studied diseases and is being investigated for the development of new technologies and more accurate therapies. Still the currently available therapies for cancer have many lacunae which affect the patient's health severely in the form of side effects. The natural drugs obtained from the medicinal plants provide a better alternative to fight against this devastating disease. Withania somnifera L. Dunal (Solanaceae), a well-known Ayurvedic medicinal plant, has been traditionally used to cure various ailments for centuries. OBJECTIVES: Considering the immense potential of W. somnifera, this review provides a detail account of its vital phytoconstituents and summarizes the present status of the research carried out on its anticancerous activities, giving future directions. METHODS: The sources of scientific literature were accessed from various electronic databases such as PubMed, Google Scholar, Science Direct, and library search. RESULTS: Various parts of W. somnifera especially the roots with its unique contents have been proved effective against different kinds of cancers. The most active components withanolides and withaferins along with a few other metabolites including withanone (WN) and withanosides have been reported effective against different types of cancer cell lines. CONCLUSION: This herb holds an important place among various anticancer medicinal plants. It is very essential to further screen and to investigate different formulations for anticancer therapy in vitro as well as in vivo in combination with established chemotherapy.

Reclamation of petrol oil contaminated soil by rhamnolipids producing PGPR strains for growing Withania somnifera a medicinal shrub.

Soil contaminated by hydrocarbons, cannot be used for agricultural intents due to their toxic effect to the plants. Surfactants producing by plant growth promotory rhizobacteria (PGPR) can effectively rig the problem of petroleum hydrocarbon contamination and growth promotion on such contaminated soils. In the present study three Pseudomonas strains isolated from contaminated soil identified by 16S rRNA analysis were ascertained for PGPR as well as biosurfactants property. Biosurfactants produced by the strains were further characterized and essayed for rhamnolipids. Inoculation of the strains in petrol hydrocarbon contaminated soil and its interaction with Withania somnifera in presence of petrol oil hydrocarbons depict that the strains helped in growth promotion of Withania somnifera in petrol oil contaminated soil while rhamnolipids helped in lowering the toxicity of petrol oil. The study was found to be beneficial as the growth and antioxidant activity of Withania sominfera was enhanced. Hence the present study signifies that rhamnolipids producing PGPR strains could be a better measure for reclamation of petrol contaminated sites for growing medicinal plants.

2,3-Dihydrowithaferin A-3beta-O-sulfate, a new potential prodrug of withaferin A from aeroponically grown Withania somnifera.

Preparations of the roots of the medicinal plant Withania somnifera (L.) Dunal commonly called ashwagandha have been used for millennia in the Ayurvedic medical tradition of India as a general tonic to relieve stress and enhance health, especially in the elderly. In modern times, ashwagandha has been shown to possess intriguing antiangiogenic and anticancer activity, largely attributable to the presence of the steroidal lactone withaferin A as the major constituent. When cultured using the aeroponic technique, however, this plant was found to produce a new natural product, 2,3-dihydrowithaferin A-3beta-O-sulfate (1), as the predominant constituent of methanolic extracts prepared from aerial tissues. The characteristic bioactivities exhibited by 1 including inhibition of cancer cell proliferation/survival, disruption of cytoskeletal organization and induction of the cellular heat-shock response paralleled those displayed by withaferin A (2). The delayed onset of action and reduced potency of 1 in cell culture along with previous observations demonstrating the requirement of the 2(3)-double bond in withanolides for bioactivity suggested that 1 might be converted to 2 in cell culture media and this was confirmed by HPLC analysis. The abundant yield of 1 from aeroponically cultivated plants, its good aqueous solubility and spontaneous conversion to 2 under cell culture conditions, suggest that 1 could prove useful as a readily formulated prodrug of withaferin A that merits further evaluation in animal models.