Screening of some phytochemicals and bioactive properties of selected naturally grown and micropropagated medicinal plants of the Apocynaceae family

Abstract

Background of the research: Medicinal plants, their identification, preservation and phytochemical profiling have become extremely important due to excessive demand of plant derived bioactive secondary metabolites as natural products, which have potentiated present day herbal drug, pharmaceuticals and nutraceuticals based industries in an unprecedented manner. Medicinal plants are marketed for their therapeutic effect but those particular plants, where root part is more valuable than the other parts, have higher threat of eradication from the wild due to overexploitation. Under in-vitro culture system application of elicitor molecule reportedly increases total root biomass and content of secondary metabolites. In West Bengal, there is a gap in medicinal plant research especially in the southern part. So, after mindful consideration, three high-value, ethno-medicinally important medicinal plants have been selected from the Apocynaceae family, namely, Hemidesmus indicus, Tylophora indica and Cryptolepis buchanani, which, i. are marketed for the root part; ii. are found mainly in the southern plain of West Bengal; iv. have significant therapeutic efficacy. Application of micropropagation, must be accompanied with phytochemical screening (crucial for quality control and selection of elite chemotype) as well as pharmacological studies. Aim of the study: Establishment of fast, effective and innovative micro-propagation protocol, for selected medicinal plants, which can aid in their conservation, sustainable usage and could be useful for higher accumulation of plant biomass. Qualitative and quantitative assay of phytochemicals and marker compounds of selected medicinal plants as well as the comparative assessment, between wild and invitro counterparts, for the selection of elite chemotype. Screening of selected pharmacological activities with solvent extract/powder/fraction obtained from mother plant and regenerated plantlets to estimate therapeutic and commercial value of in-vitro culture of selected medicinal plants. Materials and methods: PGR plus elicitor fortified, MS media based micropropagation protocol of these medicinal plants was established where the regenerated plantlets were exposed to in-planta elicitation. The goal was to check its role on biomass generation and marker compound accumulation. Assessment of clonal fidelity was done; evaluation of various parameters and establishment of experimental model to estimate the role of different variables under in-vitro culture was performed. The mother plant (wild) and regenerated plantlets were tested cum compared for phytochemical content (TPC, TFC, TTC); photosynthetic pigment content; anti-oxidant efficacy (DPPH, FRAP, SOD, CAT, APX assay); anti-bacterial property and pancreatic lipase inhibitory activity. Moreover, quantification of marker compounds done via simple, robust and standardized HPTLC technique. Results: As root is the main part for the accumulation of marker compounds in all these three plants, the impact of different variables on the root length has been analyzed. MS- BAP (1.5 mg/l) combo has produced the highest (6.7) ‘number of shoots / explant’ and the ‘average shoot length’ is 11.7 cm in case of H. indicus. The values of these two parameters are 4.1 and 9.2 cm, respectively as observed in T. indica. In case of C. buchanani, Kin (1mg/l) has generated maximum shoot length (6.8 cm) despite of a trend of slow growth. In all these plants, IBA 1.5 mg/l have promoted rooting phenomenon more than the other combinations of auxin. Profuse base callus formation and increase in fresh weight of nodular meristemoids have been noted in T. indica which may be connected with lower multiplication rate in this plant. Methyl jasmonate (50 μM) based elicitation of healthy microshoots has yielded promising result in T. indica and H. indicus but this phenomenon is absent in C. buchanani. ISSR marker based genetic fidelity assessment has been done in support of the monomorphic plant production trend in tissue culture. Phytochemical screening has revealed the TPC, TFC, TTC, PPC content in mother plants and when the values were compared with the in-vitro ones, significant content wise compatibility has been observed in other two plants except C. buchanani. The activities of antioxidant enzymes (SOD, CAT, APX), DPPH, FRAP assays plus pancreatic lipase inhibitory activity of all these plants have confirmed the assumption that in-vitro raised plants can be a sustainable option against the wild plants in terms of basic bioactivity and phytochemical content. This idea was further supported by the rapid, validated, and sensitive densitometric HPTLC based quantification of marker compounds. In H. indicus and T. indica the values of the markers (MBAld and tylophorine respectively) are noteworthy in elicitor treated root samples and similar with naturally grown root sample. In C. buchanani, HPTLC was applied to assay the content of cryptolepine in leaf tissues of wild and micropropagated ones. The naturally harvested samples of C. buchanani have yielded better result in all phytochemical and pharmacological screening. In case of the anti-bacterial activity, the marker compounds have demonstrated significant result. Conclusions: The plants which have been selected for the present study; have traditional acceptance and commercial application. Considering their pharmaco-therapeutic importance plus overexploitation scenario the present work has recommended a fast and effective micropropagation technique, Multifaceted screening of all three medicinal plants; which have been collected from the southern part of West Bengal, is reported in the present work for the very first time. The results have also highlighted the prospective application of in-vitro micropropagation technique as a sustainable cum potential remedy against overexploitation of valuable medicinal plants.