First report of triterpenes pathway in Calotropis procera revealed to accumulate beta-amyrin

Abstract

The major reports on Calotropis procera (C. procera) indicated the importance of this plant as a resource of pharmaceutically active ingredients as well as its medical advantages. β-amyrin (BA) is a significant substance in this plant and has a pharmacological effects in some frameworks, like focal and fringe sensory system, digestive and immune systems. In this study, the impact of sunlight before and after irrigation on the BA production in C. procera is studied its pathway with involved eight key enzymes. The eight enzymes' genes were characterized and successfully submitted to NCBI; AAS (acc.no. KU997645) for α-amyrin synthase, BAS (acc.no. MW976955) for β-amyrin synthase, SE (acc.no. MW976956) for squalene epoxidase, SS (acc.no. MW976957) for squalene synthase, GPPS, (acc.no. MW976958) for geranyl pyrophosphate synthase, FPPS (acc.no. MW976959) for farnasyl pyrophosphate synthase, CAS1, (acc.no. MZ00598) for cycloartenol synthase1 and LS (acc.no. MZ005982) for lupeol synthase. qRT-PCR analysis revealed high expression levels of GPPS, FPPS, SS, SE, and BAS genes at all times specially midday. Otherwise, CAS1, LS and BAS expression levels were very low at all daylight periods. The UPLC β-amyrin data are in accordance with qRT-PCR results. This indicates that triterpenes biosynthetic pathway in C. procera is going to β-amyrin accumulation with the highest level at midday.

Keywords: Beta-amyrin biosynthesis; Calotropis procera; Hydration; Light.

Fig. 1

Triterpenoids biosynthetic pathway. Low and no regulated enzymes in red color, high and regulated enzymes in blue color. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

The relative expression of C. procera GPPS, FPPS, SS, SE, BAS, AAS, CAS1 and LS genes before and after irrigation under various light periods (D, iD, M, iM, PD and iPD). Data are stated as means ± SD (black bars) for three different replicates. ^**Indicate for the significant difference between different treatments P < 0.01.

Fig. 3

The triterpenoid total ion chromatogram (TIC) evaluated by the scan mode of LC/MS/MS (a) represents two groups triterpenoid and MRM (b) represents alpha amyrin (Rt. 18.6, precursor 589, daughter ion 427).

Fig. 4

The β-amyrin as percentages from the total glycosides before and after irrigation under various light periods (D, iD, M, iM, PD and iPD).

Fig. 5

The α-amyrene (Rt. 18.6, precursor 589, daughter ion 427) before irrigation at Dawn (a), Midday (b), pre-dusk (c) and after irrigation at Dawn (d), Midday (e) and Pre-dusk (f).