L-DOPA synthesis in Mucuna pruriens (L.) DC. is regulated by polyphenol oxidase and not CYP 450/tyrosine hydroxylase: An analysis of metabolic pathway using biochemical and molecular markers.

Mucuna pruriens L., commonly known as velvetbean or cow-itch, is a self-pollinated tropical legume of the family Fabaceae, known for its medicinal properties. The active principle L-DOPA extracted from the plant is a potent drug used in the treatment of Parkinson's disease. Although, it is hypothesized that a single step reaction can produce L-DOPA, the presence of optional routes makes the pathway more intricate. For instance, the catecholamine biosynthetic pathway, which leads to L-DOPA production, could occur by hydroxylation of tyrosine to L-DOPA either by polyphenol oxidase (PPO) or tyrosine hydroxylase (TH). Furthermore, Cytochrome P450 (CYP) enzymes can also cause hydroxylation of tyrosine, resulting in L-DOPA synthesis. Therefore, the present investigation was focused on validating the step, which catalyzes the synthesis of L-DOPA, at the biochemical and molecular levels. Enzyme inhibitor studies showed significant inhibition of PPO enzyme with corresponding decrease in L-DOPA synthesis while TH and CYP inhibition had no effect on L-DOPA synthesis. Activity staining of non-denaturing PAGE gel for PPO and TH showed activity only to PPO enzyme. Following in-gel assay and tryptic digestion of the excised stained gel portion, peptide recovery and LC-MS/MS analysis were performed. Degenerate primers based on peptide sequence resulted in an 800bp amplicon. The subsequent sub-cloning, RACE analysis and BLAST search resulted in the isolation of full-length PPO coding sequence of 1800 bp. Structure prediction and phylogenetic analysis of the obtained sequence revealed strong similarity to other plant PPO's like Glycine max, Vigna radiata and Vicia faba of the same family.

Corosolic acid content and SSR markers in Lagerstroemia speciosa (L.) Pers.: a comparative analysis among populations across the Southern Western Ghats of India.

Lagerstroemia speciosa commonly known as 'Banaba' is native of south-east Asia which exhibits both horticultural and therapeutic value. The anti-diabetic and anti-obese property of the tree is attributed to corosolic acid (CRA)-a pentacyclic triterpene seen predominantly in the mature leaves. Although there are studies on either chemical or genetic variation in L. speciosa from different regions, none have dealt with their association to discuss the formation of chemical diversity. For the first time, we have analyzed CRA content in 12 natural populations corresponding to 42 samples seen in the Southern Western Ghats (SWG) using chromatography techniques and genetic variation estimated using SSR markers. Significant variation in percentage distribution of CRA ranging from 0.005% to 0.868% dr.wt. was recorded wherein populations from the north SWG contain relatively more active principle (mean=0.321%) than their counterparts in the south (mean=0.064%). Similarly, SSR data showing relatively high rate of gene flow (Nm=2.72) and low genetic differentiation (FST=0.14) is indicative that populations from north are genetically more diverse than those in the south (Nm=0.48; FST=0.38). The scatter plot derived by Principle Component Analysis (PCA) of chemical and genetic data shows similar pattern of clustering that reveals strong association between the two sets of data. It is concluded that the observed variation in CRA content in natural populations of the species depends more on the genetic background and less on edaphic factors.