No abstract

Centella asiatica is a herbaceous plant used in medicine for its wound-healing and anti-inflammatory properties. Its bioactive compounds are ursane-type triterpene saponins known as centellosides. With the aim of increasing the biotechnological production of these compounds, C. asiatica cell suspensions were established and treated with two concentrations (100 and 200 lM) of methyl jasmonate (MeJA). The maximum centelloside production was observed in the stationary growth phase, reaching 0.16 mg g -1 dry weight (DW) at day 25 of the culture in the control and 1.11 mg -1 g DW at day 15 in the MeJA-elicited cultures. The elicitor did not change the centelloside pattern, with madecassoside being the main compound, followed by asiaticoside. Reverse transcription polymerase chain reaction (RT-PCR) analysis of the b-amyrin synthase gene (CabAS, the specific oxidosqualene cyclase that leads to centelloside formation) showed higher levels of expression in the elicited cultures than in the control. The maximum content of centellosides was obtained at day 15, with a time lag between gene activation and centelloside biosynthesis. In the cultures elicited with 200 lM MeJA, the centelloside production did not increase compared to the control. Both elicitor concentrations decreased the content of phytosterols. Thus, MeJa elicitation in this type of culture was dose-dependent and its inducing role was apparent at low concentrations.

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.

No abstract