B. T. Ramesha

Endophytic fungal strains of Fusarium solani, from Apodytes dimidiata E. Mey. ex Arn (Icacinaceae) produce camptothecin, 10-hydroxycamptothecin and 9-methoxycamptothecin

Camptothecin and 10-hydroxycamptothecin are two important precursors for the synthesis of the clinically useful anticancer drugs, topotecan and irinotecan. In recent years, efforts have been made to identify novel plant and endophytic fungal sources of camptothecin and 10-hydroxycamptothecin. In this study we have isolated endophytic fungi strains from Apodytes dimidiata (Icacinaceae), a medium sized tree from the Western Ghats, India. The fungi were identified as Fusarium solani using both ITS rDNA sequencing and spore morphology. Two strains, MTCC 9667 and MTCC 9668 were isolated, both of which produced camptothecin and 9-methoxycamptothecin in their mycelia; one of the strains, MTCC 9668 also produced 10-hydroxycamptothecin, though in small amounts. The yields of camptothecin in MTCC 9667 and MTCC 9668 were 37 and 53 microg/100g, respectively, after 4 days of incubation in broth culture. The yields of 10-hydroxycamptothecin and 9-methoxycamptothecin in MTCC 9668 were 8.2 and 44.9 microg/100g, respectively. Further research in optimizing the culture conditions of these fungal strains might permit their application for the production of camptothecin and 10-hydroxycamptothecin.

Dysoxylum binectariferum Hook.f (Meliaceae), a rich source of rohitukine

Rohitukine, a chromane alkaloid, is a precursor of flavopiridol, a promising anti-cancer compound. Currently in Phase III clinical trials, flavopiridol is a potent inhibitor of several cyclin-dependent kinases (CDKs). Rohitukine was first reported from Amoora rohituka (0.083% dry weight) followed by that in Dysoxylum binectariferum (0.9% dry weight), both belonging to the family Meliaceae. Here, we report incredibly high yields of rohitukine (7% dry weight) in trees of D. binectariferum from the Western Ghats, India. Crude extracts of the tree were found to be highly effective against ovarian and breast cancer lines tested.

New plant sources of the anti-cancer alkaloid, camptothecine from the Icacinaceae taxa, India

In this study, the production of camptothecine and its derivatives, in thirteen species of the family Icacinaceae, namely, Apodytes dimidiata, Codiocarpus andamanicus, Gomphandra comosa, Gomphandra coriacea, Gomphandra polymorpha, Gomphandra tetrandra, Iodes cirrhosa, Iodes hookeriana, Miquelia dentata, Miquelia kleinii, Natsiatum herpeticum, Pyrenacantha volubilis and Sarcostigma kleinii is reported. Seeds of M. dentata were found to produce the highest content of camptothecine (1.0-1.4% by dry weight of seeds). Full scan LC-MS and ESI-MS/MS analysis of M. dentata revealed, besides camptothecine, a number of other derivatives, namely, 10-hydroxycamptothecine, 9-methoxycamptothecine, 20-deoxycamptothecine. Crude extract preparations of the seeds of M. dentata were effective against a breast cancer cell line (IC50=3.82 μg/ml for MDA MB273 cell lines) and two ovarian cancer cell lines (IC50=2.8 μg/ml for NCI/ADR-RES and 4.5 μg/ml for SKOV). These results are the first reports of camptothecine and its derivatives in these species and offer rich alternative plant sources for the anticancer compound, camptothecine.

Chemical Profiling of Nothapodytes nimmoniana for Camptothecin, an Important Anticancer Alkaloid: Towards the Development of a Sustainable Production System

Camptothecin (CPT), a pyrrolo quinoline alkaloid, is one of the most promising anticancer drugs of the 21st century. The compound was first isolated from the Chinese deciduous tree, Camptotheca acuminata. CPT exhibits a broad spectrum of antitumor activity both under in vitro and in vivo conditions. Irinotecan (CPT11) and Topotecan (TPT), two water-soluble derivatives of CPT, have been approved by the United States Food and Drug Administration for treating colorectal and ovarian cancers as well as against several types of brain tumor in children. Although CPT has been reported to exist in several species, the highest concentration (about 0.3 %) to date has been realized from Nothapodytes nimmoniana. The tree commonly referred to as “stinking tree” is native to warmer regions of South India. In the last few decades, driven by the enormous demand for CPT, there has been a decline of at least 20 % in the population, leading to red listing of the species. In recent years, efforts have been initiated in India to identify high-yielding individuals and populations of N. nimmoniana in its natural distribution range with the ultimate aim of using these lines to develop clonal orchards, as well as in developing in vitro production systems.In this chapter, we briefly review the overall status of N. nimmoniana as a source of CPT. Drawing upon existing literature as well as ongoing work at our laboratory, we discuss the basic patterns of accumulation of CPT in N. nimmoniana. We review the population variability for CPT accumulation along the distributional range of the species in the Western Ghats, India. Using a relatively new tool, namely the ecological niche model, we predict the chemical hot-spots of the species in the Western Ghats and offer a test of this prediction. Finally, we discuss strategies for a sustainable model of extraction of CPT from N. nimmoniana.

Sequestration of Camptothecin, an Anticancer Alkaloid, by Chrysomelid Beetles

Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of eukaryotic toposiomerase-I. Several derivatives of CPT are in clinical use against ovarian and lung cancers. CPT has been reported from several plant species belonging to the order Asterids, with the highest concentration in Nothapodytes nimmoniana (family Icacinaceae). In this paper, we report an intriguing observation of chrysomelid beetles (Kanarella unicolor Jacobby) feeding on the leaves of N. nimmoniana without any apparent adverse effect. LC-MS/MS analysis of the beetles indicated that 54.9% of the ingested CPT’s was recovered from the wings, followed by lesser amounts in the head and abdomen. LC-HRMS analysis revealed that most of the CPT in the insect body was in the parental form available in the plants without any major metabolizable products, including sulfated and glucuronilated forms. The mechanism by which the beetles are able to tolerate substantially high levels of CPT in their body tissue is under investigation.

Biodiversity and Chemodiversity: Future Perspectives in Bioprospecting

Biological diversity and its constituent chemical diversity have served as one of the richest sources of bioprospecting leading to the discovery of some of the most important bioactive molecules for mankind. Despite this excellent record, in the recent past, however, bioprospecting of biological resources has met with little success; there has been a perceptible decline in the discovery of novel bioactive compounds. Several arguments have been proposed to explain the current poor success in bioprospecting. Among them, it has been argued that to bioprospect more biodiversity may not necessarily be productive, considering that chemical and functional diversity might not scale with biological diversity. In this paper, we offer a critique on the current perception of biodiversity and chemodiversity and ask to what extent it is relevant in the context of bioprospecting. First, using simple models, we analyze the relation among biodiversity, chemodiversity and functional redundancies in chemical plans of plants and argue that the biological space for exploration might still be wide open. Second, in the context of future bioprospecting, we argue that brute-force high throughput screening approaches alone are insufficient and cost ineffective in realizing bioprospecting success. Therefore, intelligent or non-random approaches to bioprospecting need to be adopted. We review here few examples of such approaches and show how these could be further developed and used in the future to accelerate the pace of discovery.

Genetic structure of the rattan Calamus thwaitesii in core, buffer and peripheral regions of three protected areas in central Western Ghats, India: do protected areas serve as refugia for genetic resources of economically important plants?

Given the increasing anthropogenic pressures on forests, the various protected areas—national parks, sanctuaries, and biosphere reserves—serve as the last footholds for conserving biological diversity. However, because protected areas are often targeted for the conservation of selected species, particularly charismatic animals, concerns have been raised about their effectiveness in conserving nontarget taxa and their genetic resources. In this paper, we evaluate whether protected areas can serve as refugia for genetic resources of economically important plants that are threatened due to extraction pressures. We examine the population structure and genetic diversity of an economically important rattan, Calamus thwaitesii, in the core, buffer and peripheral regions of three protected areas in the central Western Ghats, southern India. Our results indicate that in all the three protected areas, the core and buffer regions maintain a better population structure, as well as higher genetic diversity, than the peripheral regions of the protected area. Thus, despite the escalating pressures of extraction, the protected areas are effective in conserving the genetic resources of rattan. These results underscore the importance of protected areas in conservation of nontarget species and emphasize the need to further strengthen the protected-area network to offer refugia for economically important plant species.

Cross-species amplification of coconut microsatellite markers in rattans

Summary Cross-species amplification of microsatellite loci is considered to be a time-saving as well as a cost-effective approach for developing locus specific markers for new species. In an attempt to identify molecular markers to determine gene flow events and genetic differentiation of populations in rattans, microsatellite primer pairs developed for Cocos nucifera (Arecaceae) were examined for cross-species amplification in rattans. Of the six microsatellite primer pairs screened, two (CNZ34 and CN2A5) yielded good cross-species amplification across different rattan genera and species from South and Southeast Asia. Based on microsatellite-specific PCR amplification products, cluster analysis was performed. The dendrogram indicated a clear grouping of rattans based on their genera and species. The coconut locus specific primer pairs could be successfully used to construct molecular differentiation of rattan genera across different geographical locations.

Stylish lengths: mate choice in flowers.

The styles of flowers may represent an arena for pollen competition in the race to fertilize ovules. Accordingly, selection should favour a longer ‘race’ to better discriminate among variable pollen by increasing style length. Sampling across a taxonomically diverse range of wild and outcrossed species, we found that the distribution of style lengths within plants were skewed towards longer styles, as predicted. In self-pollinated domesticated species, where discrimination among pollen is less important, we found no such pattern. We conclude that style length is under directional selection towards longer styles as a mechanism for mate choice among pollen of variable quality.

Development of polymorphic microsatellite loci in Nothapodytes nimmoniana, a medicinally important tree from the Western Ghats, India.

Nothapodytes nimmoniana is a medicinally important tree species that occur in the Western Ghats, a megadiversity hotspot in southern India. Inner stem bark of the tree contains an important anti‐cancer alkaloid, camptothecin for which the natural population of the tree is heavily harvested. In this paper, we report the isolation and characterization of eight polymorphic microsatellite loci using enrichment hybridization protocol. Analysis of 36 individuals representing two populations revealed three to 12 alleles per locus. Observed heterozygosity ranged from 0.21 to 0.94 for the two populations. None of the loci tested showed linkage disequilibrium. These markers are invaluable for evaluating the genetic structure and assessing the genetic impacts of harvesting of N. nimmoniana in the Western Ghats to formulate strategies for conservation of the species.