Optimization of salicylic acid and chitosan treatment for bitter secoiridoid and xanthone glycosides production in shoot cultures of Swertia paniculata using response surface methodology and artificial neural network.

BACKGROUND: In this study, response surface methodology (RSM) and artificial neural network (ANN) was used to construct the predicted models of linear, quadratic and interactive effects of two independent variables viz. salicylic acid (SA) and chitosan (CS) for the production of amarogentin (I), swertiamarin (II) and mangiferin (III) from shoot cultures of Swertia paniculata Wall. These compounds are the major therapeutic metabolites in the Swertia plant, which have significant role and demand in the pharmaceutical industries. RESULTS: Present study highlighted that different concentrations of SA and CS elicitors substantially influenced the % yield of (I), (II) and (III) compounds in the shoot culture established on modified ½ MS medium (supplemented with 2.22 mM each of BA and KN and 2.54 mM NAA). In RSM, different response variables with linear, quadratic and 2 way interaction model were computed with five-factor-three level full factorial CCD. In ANN modelling, 13 runs of CCD matrix was divided into 3 subsets, with approximate 8:1:1 ratios to train, validate and test. The optimal enhancement of (I) (0.435%), (II) (4.987%) and (III) (4.357%) production was achieved in 14 days treatment in shoot cultures of S. paniculata elicited by 9 mM and 12 mg L- 1 concentrations (SA) and (CS). CONCLUSIONS: In optimization study, (I) show 0.170-0.435%; (II) display 1.020-4.987% and (III) upto 2.550-4.357% disparity with varied range of SA (1-20 mM) and CS (1-20 mg L- 1). Overall, optimization of elicitors to promote secoiridoid and xanthone glycoside production with ANN modeling (r2 = 100%) offered more significant results as compared to RSM (r2 = 99.8%).

Cloning and functional analysis of geraniol 10-hydroxylase, a cytochrome P450 from Swertia mussotii Franch.

Swertia mussotii Franch has anti-hepatitis activity and contains a high level of iridoid monoterpenoids. The cytochrome P450 monooxygenase (CYP) geraniol 10-hydroxylase (G10H) is thought to play an important role in iridoid monoterpenoid and indole alkaloid biosynthesis. Here we report the isolation of a full-length cDNA clone of S. mussotii G10H (SmG10H). The predicted gene product was a 496 residue protein designated CYP76B10, the sequence of which was highly similar to that of the CYP76 family, particularly to Catharanthus roseus G10H (80.2% homology). SmG10H transcripts were much more abundant in the leaves than in either the root or the stem, and were derived from a single copy gene. SmG10H expression was upregulated by treatment with methyljasmonate (MeJA) over a period from 6 h to 36 h after treatment. Accumulation of swertiamarin increased after elicitation by MeJA. SmG10H was heterologously expressed in both Escherichia coli and Pichia pastoris (yeast), forming a 55.5-kDa protein. Based on analysis in vitro, SmG10H was found to have catalytic activity hydroxylating geraniol. In the SmG10H overexpression plants, the level of SmG10H transcript and the contents of 10-hydroxygeraniol and swertiamarin increased simultaneously.