The identification of the four principal triterpenoid components of Centella asiatica has been achieved by TLC on silica gel plates and mass spectrometry, as a modification of the method described in the European Pharmacopoeia (5th edn). A combination of ethyl acetate and methanol as the mobile phase was found to be successful in separating these compounds from the rest of the main components of the extract. The spots were detected with anisaldehyde solution. The separated compounds were confirmed by MALDI -TOF mass spectrometry.
Centella asiatica has been extensively studied but there has been no report to date that relates gene expression and centelloside production in non-differentiated tissues. We have determined the content of the four principal triterpenoid bioactive compounds of C. asiatica (asiaticoside, madecassoside, asiatic acid and madecassic acid) in calli grown in different media and checked the expression level of some of the genes in the centelloside biosynthetic pathway. The results when compared with data from in vitro plant cultures showed a significantly lower expression of the gene encoding beta-amyrin synthase in calli, which is consistent with the observed lower production of centellosides (less than 900 microg/g DW), while in the plants the production was around 1.5-2 mg/g DW. Moreover, we find an efficient housekeeping gene for this plant. The biosynthesis of phytosterols is also discussed.
Summary
Plant cell cultures constitute eco‐friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate‐elicited Taxus baccata cell cultures by complementary DNA‐amplified fragment length polymorphism (cDNA‐AFLP) indicated a correlation between an extensive elicitor‐induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate‐induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl‐CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl‐CoA ligase that localizes to the cytoplasm and is able to convert β‐phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β‐phenylalanyl‐CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.