Biosynthesis and enrichment of targeted bioactive secondary metabolites through in-vitro and in-planta system and elucidation of their putative biosynthetic pathway

In-vitro biosynthesis and enrichment of targeted cardiac glycosides from Calotropis sp. and Nerium oleander. The emerging role of cardiac glycosides (CGs) is the prevention and/or treatment of proliferative diseases like cancer [Molecular Interventions 8: 36-49 (2008)]. During last few years several papers have reported promising in-vitro and in-vivo anticancer activity of CGs. Various CGs viz. calactin, calotoxin, calotropin, frugoside, proceroside, uscharidin, uscharin, uzarigenin, voruscharin, 2”-oxovoruscharin, etc. from Calotropis sp. and oleandrin, cardenolide N-1, oleander, cardenolide N-4, etc. from N. oleander were isolated in recent past. Most of these compounds have prominent cytotoxic effect and UNBS-1450, a semisynthetic cardenolide derivate of 2″-oxovuscharin in Phase I clinical trial [BiochemPharmacol. 81(1):13-23 (2011)]. Various crude extracts and fractions of different parts of these plants have also shown marked anti-cancer and anti-tumor activities. CGs are basically steroid like compounds with two distinct moieties i.e. glycon and aglycon (genin). Chemical synthesis of such compounds is very challenging because of their complex structure. On the other hand, quantity of these compounds in the plant is very less and hence their isolation from plant sources is not cost effective and detrimental to the biodiversity. So, the alternative option for getting these compounds is their production through plant tissue culture techniques. Our group is actively involved to establish the in-vitro biosynthesis and enrichment protocol of some selected bioactive CGs and determination of their putative biosynthetic pathway from C. gigantea and C. procera (DOI: 10.4103/pm.pm_507_18; DOI: 10.1038/srep34464; DOI: 10.1007/s 11 627-0 12-948 1-9). Same type of work is also going on from the plant N. oleander.

In-vitro biosynthesis and enrichment of targeted indole alkaloids from Alstonia scholaris. The enormous therapeutic importance and biological activities of A. scholaris is mainly due to great diversity of alkaloids. However, indole alkaloids (IAs) are particularly responsible for anticancer and antitumor activities. Out of many IAs present in this species, echitamine and its derivatives are the major ones. Antitumor and anticancer activities of echitamine chloride are well established [Cancer BiochemBiophys 17(1–2):79–88 (1999)]. However, echitamine and its derivatives are mainly concentrated in stem bark and root of the plant and these parts are not recommended for bulk collection. Hence, in-vitro biosynthesis of such IAs through plant tissue culture technique may play a significant role

Our team has standardized the callus induction and proliferation protocol from leaf explants of A. scholaris along with in-vitro biosynthesis and enrichment of four bioactive IAs viz. echitamine, acetylechitamine, tubotaiwine and picrinine. Quantitative estimation of these IAs was performed by using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS). (https://doi.org/10.1007/s10725-019-00570-7; DOI 10.1007/s11240-014-0579-0]. At present, efforts are going on to elucidate the putative biosynthetic pathway of these IAs.

In-planta enrichment of targeted pyranocarbazoles in Murraya koenigii
Pyranocarbazoles or carbazole alkaloids (CAs) are the major constituents of M.koenigii plant and these are reported to have many important biological activities such as anti-proliferative, anti-obesity, lipid lowering, etc. According to literatures, at least eight CAs of this plant viz. mahanimbine, girinimbine, mahanine, mahanimbicine, 9- carbethoxy-3-methyl carbazole, koenoline, pyrayafoline-D and murrayafoline –I are presently known for their cytotoxic properties. However, bio-availability of such CAs in the plant is very less and hence isolation of these compounds is not cost effective. On the other hand, chemical synthesis of these compounds is a very challenging aspect due to their complex structure. So, the alternative option for producing these compounds is their in-vitro and in-planta biosynthesis. Our group has accepted the challenge to biosynthesize some targeted CAs in potted plants by exposing them in different biotic/abiotic stresses, altering edaphic factors and manipulating micro/macro nutrients, etc. Our study has also been designed to assess the effect of various seasons and growth stages of plants on the accumulation of CAs. Biological activities of selected M. koenigii leaf extracts will be done and the results will be correlated it with CAs accumulation. This work will be the first of its kind as there is no report on enhanced production and accumulation of CAs in any plant.

Morpho-taxonomical identification and authentication of medicinal plants, their collection, characterization and IUCN red list status determination
Proper identification, authentication and IUCN red list status (RET status) of plants are the prerequisites for starting any plant based research. Knowledge on red list status of plant is very important because one should not start drug development research on any threatened plant until it is under captive cultivation. Out of different identification approach, morpho-taxonomical one is the fastest and most reliable. Our group is actively involved in survey of medicinal plants, their identification, documentation of ethnobotanical knowledge, collection of plant samples/materials, authentication, characterization and their IUCN red list status determination.

Nomenclature update of herbarium specimens deposited at CDRI’s internationally recognized medicinal plant Herbarium
Plant nomenclature is an integral part of plant taxonomy. However, scientific names of plants are not permanent and can be changed due to different taxonomical reasons from time to time as per the recommendations of the International Code of Nomenclature for algae, fungi, and plants (ICN). Therefore, the final determination of correct name of any plant is a very tricky taxonomical task and hardcore taxonomist is required for this purpose. In addition, it is very important to every Herbarium administrator to review the latest nomenclature of all the deposited specimens and update it accordingly. As the specimens of all Herbaria are arranged according to an accepted system of plant classification, they may require rearrangements after nomenclature update.

Our institute is harboring a very important internationally recognized medicinal plant Herbarium with its acronym ‘CDRI’. More than 31,000 herbarium specimens are stored in this Herbarium and these are arranged according to Bentham & Hooker’s system of classification. The species deposited in this Herbarium represent the angiosperm flora from all over India. Our group is actively involved to review the nomenclature of these specimens and update it as and when required. The specimens with updated nomenclature are properly placed at their new position.

Establishment of virtual Herbarium
A virtual Herbarium is a digitized form of traditional Herbarium. Virtual Herbaria are established to improve the availability of specimens to a wider range. At the same time, it is very helpful to increase the longevity of herbarium specimens by avoiding their frequent handling.

Our group is involved to digitize our traditional Herbarium and to make a specimen database. For this purpose, all the specimens of our Herbarium are being scanned and their digital images along with all the field information mentioned on each sheet are incorporated in a specially designed software prepared by our computer division. After completion of this work, the database along with digital images of herbarium specimens will be available on the internet.