Metabolomic analysis of methyl jasmonate-induced triterpenoid production in the medicinal herb Centella asiatica (L.) urban

Abstract

Centella asiatica is an important source of biologically active pentacyclic triterpenoids. The enhancement of the biosynthesis of the centellosides by manipulation of associated metabolic pathways is receiving much attention. Jasmonates play critical roles in plant metabolism by up-regulating the expression of genes related to secondary metabolites. Here, we investigated the effect of methyl jasmonate (MeJa) in C. asiatica through targeted metabolomic profiling of asiaticoside and madecassoside as well as their aglycones, asiatic acid and madecassic acid. Cell suspensions were treated with 0.2 mM MeJa for 2, 4 and 6 days. Liquid chromatography coupled to mass spectrometry (LC-MS) was used to explore induced changes in metabolite profiles, both qualitatively and quantitatively. Principal component analysis (PCA)-derived scores plots revealed clusters of sample replicates for control and treated samples at 2, 4 and 6 days while loading plots aided in identifying signatory biomarkers (asiatic acid and madecassic acid, as well as asiaticoside and madecassoside) that clearly demonstrate the variability between samples. In addition to increased biosynthesis of the targeted centelloids, other differential changes in the intracellular metabolite profiles reflected the response of the C. asiatica cells to the MeJa-treatment as a reprogramming of the metabolome.

Figure 1

UHPLC-MS (BPI) chromatograms showing treatment-related variations. Ten day old cell suspensions were treated with 0.2 mM MeJa for 2, 4 and 6 days and intracellular metabolites extracted with ethanol. Four ion peaks (m/z 973.5, 957.5, 503.3 and 487.3) were more predominantly observed in the 4 days samples than in other conditions (2 and 6 days). These ion peaks were further identified as the targeted triterpenoids/centelloids (Table 1).

Figure 2

(A) PCA scores plot of a representative experiment. A 4-component model explains 87.2% variation. The plot was computed using the first two components (PC1 and PC2) which explain 60.5% of the variation (B) PCA loading plot for all time points (2, 4 and 6 days). The mass ions m/z = 487.3426, 957.5069, 503.3382 and 973.5014, identified as asiatic acid, asiaticoside, madecassic acid and madecassoside respectively, contribute to the clustering of the samples.

Figure 3

A variable importance in projection (VIP) plot of day 4 samples. The VIP plot indicates that the mass ions m/z 487.3426, 503.3382, 957.5069 and 973.5014 were accountable for the significant separation in the model as their VIP scores were significantly greater than 1.0.