Mercedes Bonfill

Steroidal Lactones from Withania somnifera, an Ancient Plant for Novel Medicine

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 Alzheimers disease. The most recent attempts in biotechnological production of withanolides are also discussed.

Biotechnological production of taxol and related taxoids: current state and prospects.

Taxol is one of the most effective anti-cancer drugs ever developed. The natural source of taxol is the inner bark of several Taxus species, but it accumulates at a very low concentration and with a prohibitively high cost of extraction. Another problem is that the use of inner bark for taxol production implies the destruction of yew trees. For all these reasons, the growing demand for taxol greatly exceeds the supply that can be sustained by isolation from its natural source and alternative sources of the drug are being sought. Although taxol has been prepared by total synthesis, the process is not commercially viable. Taxol can also be semisynthetically produced via the conversion of baccatin III or 10-deacethylbaccatinIII found in Taxus needles but the cost and difficulty of the extraction process of the semisynthetic precursors are also very high. The most promising approach for the sustainable production of taxol and related taxoids is provided by plant cell cultures at an industrial level. Taxol is currently being clinically used against different tumour processes but due to the difficulty of its extraction and formulation, as well as the growing demand for the compound, new taxol analogues with improved properties are being studied. In this revision we discuss current research in the design of new taxol-related compounds, the chemical structure/anti-cancer activity relationship and new formulations of the drug. We also consider the optimizing strategies to improve taxol and related taxoid production in cell cultures, as well as the current knowledge of taxol metabolism, all of which are illustrated with examples, some of them from our own research.

Relation between the amount of rolC gene product and indole alkaloid accumulation in Catharanthus roseus transformed root cultures

Summary Transformed root lines of Catharanthus roseus were established after the inoculation of aseptic stem segments with Agrobacterium rhizogenes strain A4, and cultured in a hormone-free B5/2 solid medium. In these root cultures, in addition to the changes in the ethylene accumulated, the amount of rol C gene product was determined using specific antibodies in order to investigate their relation to growth rate, morphology and the indole alkaloid production of the cultured roots. Our results showed the presence, in all transformed root lines considered, of vindoline and catharanthine, the two monomer precursors for the enzymatic synthesis of the antineoplastic drugs vinblastine and vincristine. In the culture conditions of our experiment, roots with thin morphology that reached the highest rol C gene product amount showed lower growth rate and higher alkaloid production when compared with those with thick morphology and lowest rol C gene product amount. The effect of the protein encoded by this gene on ethylene production is also discussed.

Improved Paclitaxel and Baccatin III Production in Suspension Cultures of Taxus media

A cell suspension culture of Taxus media was established from a stable callus line of this species. The growth rate and production of paclitaxel and baccatin III of this cell suspension were significantly increased during the shake flask culture in its respective optimum media for cell growth and product formation, which were selected after assaying 24 different culture media. The highest yields of paclitaxel (2.09 mg L−1) and baccatin III (2.56 mg L−1) in the production medium rose (factors of 7.0 and 3.0, respectively) in the presence of methyljasmonate (220 μg g−1 FW). When the elicitor was added together with mevalonate (0.38 mM) and N‐benzoylglycine (0.2 mM), the increase in the yields of paclitaxel and baccatin III was even higher (factors of 8.3 and 4.0, respectively). Thereafter, a two‐stage culture for cell suspension was carried out using a 5–l stirred bioreactor running for 36 days, the first stage being in the cell growth medium until cells entered their stationary growth phase (12 days) and the second stage being in the production medium supplemented with the elicitor and two putative precursors in the concentrations indicated above. Under these conditions, 21.12 mg L−1 of paclitaxel and 56.03 mg L−1 of baccatin III were obtained after 8 days of culture in the production medium.

Production of Taxol ® and baccatin III by a selected Taxus baccata callus line and its derived cell suspension culture

Abstract Small callus pieces excised from a selected 1-year-old stable callus line of Taxus baccata were grown on B5 solid medium supplemented with either phenylalanine (1 mM) or the elicitor compound VSO4 (0.05 or 0.1 mM). Under these conditions, callus growth and the production of Taxol® and baccatin III were tested during 8 weeks of culture. Growth was enhanced by the presence of both phenylalanine and VSO4. The elicitor compound VSO4 (0.05 and 0.1 mM) moreover clearly increased Taxol® and baccatin III production, especially at the end of the culture. The specific activity of PAL in callus pieces cultured under the different conditions studied, is also considered. In addition, liquid cultures were prepared from the selected callus line in order to study the effect of the VSO4 addition to the culture medium on Taxol® and baccatin III production. The presence of this elicitor (VSO4; 0.05 mM), increased Taxol® and baccatin III production (by a factor of 2.5 and 3.6, respectively) from 5.2 μg g−1 DW to 13.1 μg g−1 DW for Taxol® and from 4.4 μg g−1 DW to 16.0 μg g−1 DW for baccatin III in the T. baccata cell suspension cultures. It also increased their release into the medium by a factor of 3.2 and 2.2, respectively

Effect of Agrobacterium rhizogenes T-DNA on alkaloid production in Solanaceae plants

Abstract Inoculation of leaf sections of tobacco, Duboisia hybrid and Datura metel Solanaceae plants with A4 strain of Agrobacterium rhizogenes , induced transformed roots with the capacity to produce putrescine-derived alkaloids. In general, the hairy roots obtained showed two morphologies: typical hairy roots with high capacity to produce alkaloids and callus-like roots with faster growth capacity and lower alkaloid production. The aux 1 gene of A. rhizogenes was detected by PCR analyses in all roots showing callus-like morphology. However, this gene was only detected in 25–60% of the root cultures established showing typical hairy morphology. This fact suggests a significant role of aux genes in the morphology of transformed roots. Inoculation of leaf sections with A. tumefaciens strain C58 C1 carrying the pRiA4TR- (deletion of aux genes) did not produce roots with callus-like morphology.

Inhibition of paclitaxel and baccatin III accumulation by mevinolin and fosmidomycin in suspension cultures of Taxus baccata

To achieve a better understanding of the metabolism and accumulation of paclitaxel and baccatin III in cell cultures of Taxus, inhibitors of the early steps in the terpenoid pathway were applied to a cell suspension culture of Taxus baccata: fosmidomycin as an inhibitor of the non-mevalonate branch of the pathway, and mevinolin as an inhibitor of the mevalonate branch. Synthesis of both taxanes in the cell suspension was first increased when cultured in the product formation medium supplemented with methyljasmonate (100 microM). The product formation medium was selected after assaying 24 different culture media. When fosmidomycin (200 microM) was added to the product formation medium together with the elicitor, the accumulation of paclitaxel and baccatin III was reduced by up to 3.0 and 1.5 times, respectively, whereas the inhibitory effect of mevinolin (1 microM) was only clearly exerted in the case of paclitaxel. Under the conditions of our experiment, we conclude that in the synthesis of both taxanes, the non-mevalonate pathway is the main source of the universal terpenoid precursor isopentenyl diphosphate (IPP).

A rational approach to improving the biotechnological production of taxanes in plant cell cultures of Taxus spp.

Taxol is a complex diterpene alkaloid scarcely produced in nature and with a high anticancer activity. Biotechnological systems for taxol production based on cell cultures of Taxus spp. have been developed, but the growing commercial demand for taxol and its precursors requires the optimization of these procedures. In order to increase the biotechnological production of taxol and related taxanes in Taxus spp. cell cultures, it is necessary not only to take an empirical approach that strives to optimize in-put factors (cell line selection, culture conditions, elicitation, up-scaling, etc.) and out-put factors (growth, production, yields, etc.), but also to carry out molecular biological studies. The latter can provide valuable insight into how the enhancement of taxane biosynthesis and accumulation affects metabolic profiles and gene expression in Taxus spp. cell cultures. Several rational approaches have focused on studying the transcriptomic profiles of key genes in the taxol biosynthetic pathway in Taxus spp. cell cultures treated with elicitors such as methyl jasmonate, coronatine and cyclodextrins in relation with the taxane pattern, production and excretion to the culture medium. These studies have provided new insights into the taxol biosynthetic pathway and its regulation. Additionally, identifying genes with low levels of expression even in the presence of elicitors, together with metabolomics studies, has shed light on the limiting steps in taxol biosynthesis and could help define suitable metabolic targets for engineering with the main aim of obtaining highly productive Taxus cultured cells. In this review, we have summarized the latest endeavors to enhance the molecular understanding of the action mechanism of elicitors in Taxus spp. cell cultures. Developments in the ongoing search for new and more effective elicitation treatments and the application of metabolic engineering to design new transgenic cell lines of Taxus with an improved capacity for taxane production are described.

Influence of plant growth regulators on the growth and essential oil content of cultured Lavandula dentata plantlets

Micropropagation of L. dentata L. through shoot-tips (ca. 5 mm) was achieved successfully. Micropropagated plantlets were cultured without plant regulators in the culture medium (control) or in media containing 0.1 mg l−1 of 6-benzyladenine (BA) and/or indole-3-butyric acid (IBA). These plantlets were examined for their essential oil content and composition in relation to growth rate and density and secretory or postsecretory stage of glandular hairs at five weeks of culture. The growth rates of these plantlets were not always correlated with their essential oil content (0.26%–0.65% v/w). However, all cultured plantlets showed a positive correlation between oil accumulation and the percentage of glandular hairs in secretory stage. Quantitative changes in the major monoterpene components (1,8-cineole, fenchol, borneol and camphor) and sesquiterpene content of plantlet oil, were also observed in response to the effect of varying growth regulator concentration in the culture medium. The influence of this effect on HMG-CoA reductase activity of cultured plantlets is also discussed.

Coronatine, a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate.

Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received much attention recently for its potential to act as a plant growth regulator and elicitor of plant secondary metabolism. To gain more insight into the mechanism by which elicitors can affect the biosynthesis of paclitaxel (Px) and related taxanes, the effect of coronatine (Cor) and methyl jasmonate (MeJA) on Taxus media cell cultures has been studied. For this study, a two-stage cell culture was established, in which cells were first cultured for 14 days in a medium optimised for growth, after which the cells were transferred to medium optimised for secondary metabolite production. The two elicitors were added to the medium at the beginning of the second stage. Total taxane production in the cell suspension was significantly enhanced by both elicitors, increasing from a maximum level of 8.14mg/L in control conditions to 21.48mg/L (day 12) with MeJA and 77.46mg/L (day 16) with Cor. Expression analysis indicated that the txs, t13oh, t2oh, t7oh, dbat, pam, bata and dbtnbt genes were variably induced by the presence of the elicitors. Genes encoding enzymes involved in the formation of the polihydroxylated hypothetical intermediate (TXS, T13OH, T2OH, T7OH) and the phenylalanoil CoA chain (PAM) were stronger induced than those encoding enzymes catalysing the last steps of the Px biosynthetic pathway (DBAT, BAPT and DBTNBT). Notably, although taxane accumulation differed qualitatively and quantitatively following MeJA- or Cor-elicitation, gene expression induction patterns were similar, inferring that both elicitors may involve distinct but yet uncharacterised regulatory mechanisms.