The class of steroid-like compounds designated cardiac glycosides includes well-known drugs such as digoxin, digitoxin, and ouabain. Their continued efficacy in treatment of congestive heart failure and as anti-arrhythmic agents is well appreciated. Less well known, however, is the emerging role of this category of compounds in the prevention and/or treatment of proliferative diseases such as cancer. New findings within the past five years have revealed these compounds to be involved in complex cell-signal transduction mechanisms, resulting in selective control of human tumor but not normal cellular proliferation. As such, they represent a promising form of targeted cancer chemotherapy. New clinical studies of their anticancer potential as single or adjuvant treatments may provide insight into these potentially valuable therapeutic options. This review focuses on recent findings on cellular pharmacology of cardiac glycosides as they relate to treatment of human cancer and attempts to explain why these agents have been overlooked in the past.

Cardiac glycosides as novel cancer therapeutic agents

Plectranthus : A review of ethnobotanical uses

Withania somnifera, commonly known as Ashwagandha, is an important medicinal plant that has been used in Ayurvedic and indigenous medicine for over 3,000 years. In view of its varied therapeutic potential, it has also been the subject of considerable modern scientific attention. The major chemical constituents of the Withania genus, the withanolides, are a group of naturally occurring C28-steroidal lactone triterpenoids built on an intact or rearranged ergostane framework, in which C-22 and C-26 are appropriately oxidized to form a six-membered lactone ring. In recent years, numerous pharmacological investigations have been carried out into the components of W. somnifera extracts. We present here an overview of the chemical structures of triterpenoid components and their biological activity, focusing on two novel activities, tumor inhibition and antiangiogenic properties of withaferin A and the effects of withanolide A on Alzheimer's disease. The most recent attempts in biotechnological production of withanolides are also discussed.

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Steroidal lactones from Withania somnifera, an ancient plant for novel medicine.

The role of activated charcoal in plant tissue culture.

Physician's Desk Reference

Elicitation is a possible aid to overcome various difficulties associated with the large-scale production of most commercially important bioactive secondary metabolites from wild and cultivated plants, undifferentiated or differentiated cultures. Secondary metabolite accumulation in vitro or their efflux in culture medium has been elicited in the undifferentiated or differentiated tissue cultures of several plant species by the application of a low concentration of biotic and abiotic elicitors in the last three decades. Hairy root cultures are preferred for the application of elicitation due to their genetic and biosynthetic stability, high growth rate in growth regulator-free media, and production consistence in response to elicitor treatment. Elicitors act as signal, recognized by elicitor-specific receptors on the plant cell membrane and stimulate defense responses during elicitation resulting in increased synthesis and accumulation of secondary metabolites. Optimization of various parameters, such as elicitor type, concentration, duration of exposure, and treatment schedule is essential for the effectiveness of the elicitation strategies. Combined application of different elicitors, integration of precursor feeding, or replenishment of medium or in situ product recovery from the roots/liquid medium with the elicitor treatment have showed improved accumulation of secondary metabolites due to their synergistic effect. This is a comprehensive review about the progress in the elicitation approach to hairy root cultures from 2010 to 2019 and the information provided is valuable and will be of interest for scientists working in this area of plant biotechnology.

Elicitation: A biotechnological tool for enhanced production of secondary metabolites in hairy root cultures

Developmental variability was introduced into Withania somnifera using genetic transformation by Agrobacterium rhizogenes, with the aim of changing withasteroid production. Inoculation of W. somnifera with A. rhizogenes strains LBA 9402 and A4 produced typical transformed root lines, transformed callus lines, and rooty callus lines with simultaneous root dedifferentiation and redifferentiation. These morphologically distinct transformed lines varied in T-DNA content, growth rates, and withasteroid accumulation. All of the lines with the typical transformed root morphology contained the TL T-DNA, and 90% of them carried the TR T-DNA, irrespective of the strain used for infection. Accumulation of withaferin A was maximum (0.44% dry weight) in the transformed root line WSKHRL-1. This is the first detection of withaferin A in the roots of W. somnifera. All of the rooty callus lines induced by strain A4 contained both the TL and the TR-DNAs. In contrast, 50% of the rooty-callus lines obtained with strain LBA 9402 contained only the TR T-DNA. All the rooty callus lines accumulated both withaferin A and withanolide D. The callusing lines induced by LBA 9402 lacked the TL T-DNA genes, while all the callusing lines induced by strain A4 contained the TL DNA. Four of these callus lines produced both withaferin A (0.15–0.21% dry weight) and withanolide D (0.08–0.11% dry weight), and they grew faster than the transformed root lines. This is the first report of the presence of withasteroids in undifferentiated callus cultures of W. somnifera.

Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera.

We have developed an efficient transformation system for Tylophora indica, an important medicinal plant in India, using Agrobacterium rhizogenes strains LBA9402 and A4 to infect excised leaf and stem explants and intact shoots at different sites. The induction of callus and transformed roots was dependent on the bacterial strain, explant type and inoculation site used. Transformed roots were induced only in explants infected with A. rhizogenes strain A4, while an optimal transformation frequency of up to 60% was obtained with intact shoots inoculated at the nodes. The presence of the left-hand transferred DNA (TL-DNA) in the genome of T. indica roots induced by A. rhizogenes was confirmed by PCR amplification of the rooting locus genes of A. rhizogenes. Root growth and the production of tylophorine, the major alkaloid of the plant, varied substantially among the nine root clones studied. Both parameters increased over time in liquid cultures, with maximum biomass and tylophorine accumulation occurring within 4–6 weeks of growth in fresh medium. Interestingly, in liquid culture, the culture medium also accumulated tylophorine up to concentrations of 9.78±0.21 mg l−1.

Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones

Multiple shoot cultures of Withania somnifera were established from single shoot tip explants and their potential for the production of two principle withanolides, withaferin A and withanolide D was investigated. Shoot tips grown on MS medium supplemented with BA (1 mg l-1) induced 10.0 ± 1.15 microshoots per explants and shoot cultures accumulated both withanolides (withaferin A = 0.04 %, withanolide D = 0.06 %). Supplementation of MSSM (solid) agar medium with 4 % sucrose enhanced accumulation of both withaferin A (0.16 %) and withanolide D (0.08 %). Reduction of the agar concentration to 0.16 % increased the number of microshoots induced per explant to 25.5. MSSM liquid medium containing 10 % coconut milk favoured a maximum increase in biomass (27 fold); number of microshoots induced (37.6 ± 1.45) as well as accumulation of withaferin A (0.14 %). BA:N6-Benzyladenine Kn:Kinetin 2iP:N6-[2-Isopentenyl]adenine MS:Murashige and Skoog (1962) MSSM:Murashige and Skoog's basal medium + BA (1 mg l-1)

Production of withaferin A in shoot cultures of Withania somnifera.

We have developed an efficient transformation system for Bacopa monnieri, an important Indian medicinal plant, using Agrobacterium rhizogenes strains LBA 9402 and A4. Transformed roots induced by strain LBA 9402 spontaneously dedifferentiated to callus while excised roots induced by strain A4 spontaneously showed induction of shoot buds within 10 days. PCR and RT-PCR analysis revealed the presence and expression of the rolAB and rolC genes at the transcription level in pRi A4 transformed cultures indicating that the TL-DNA was integrated retained and expressed in the A4-Ri transformed shoots. Transformed calli showed the presence of rolAB or rol A, TR and ags genes. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes. Growth and biomass accumulation was significantly higher in the transformed shoots (twofold) and roots (fourfold) than in the non-transformed (WT) plants. In pRi A4-transformed plants, the content of bacopasaponin D, bacopasaponin F, bacopaside II and bacopaside V was enhanced significantly as compared to WT plants of similar age while bacoside A3 and bacopasaponin C content was comparable with that of WT plants. Significant increase in content of five bacopa saponins could be detected in pRi 9402-transformed callus cultures. There is an overall stimulatory effect on accumulation of bacopa saponins in transformed plants and cells of B. monnieri establishing the role of endogenous elicitation by Ri T-DNA of A. rhizogenes.

Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants

Sumita Jha

Papers

Elicitation: A biotechnological tool for enhanced production of secondary metabolites in hairy root cultures

Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera.

Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones

Production of withaferin A in shoot cultures of Withania somnifera.

Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants