Phytochemical Analysis and Metabolite Diversity in Tinospora cordifolia
Plants produce a wide range of phytochemicals to attract pollinators, for selfdefense from multitude of biotic and abiotic stresses, and to interact with their environments for survival and spread (Akula et al., 2011). Environment influences the qualitative and quantitative composition of phytochemicals in plants. Phytochemical screening of plant is important in order to identify the new bioactive compounds, which could become new leads or new drugs (Rout et al., 2009). Ever since its inception, mass spectrometry (MS) has been extensively used in the analysis of plant metabolites (Jorge et al., 2016). The advantage of MS techniques has been utilized in the characterization of the phytoconstituents of Tinospora cordifolia stem (TCS) using LC-MS/MS in recent years (Bajpai et al., 2016; Bajpai et al.. 2017a, 2017b). Recent developments in the hyphenated liquid chromatography mass instrumentation (LC-MS) have led to quantum jump in the area of natural product chemistry. LC-MS has high selectivity and sensitivity for qualitative and quantitative analysis of bioactive compounds and their metabolites present in trace amount in plant extract (Reemtsma, 2003). With the considerable advancements in LC-MS and LC-MS/MS techniques, it is possible to obtain extremely precise chemical information such as UV chromatogram, exact/accurate mass, isotope distribution patterns, molecular formula, characteristic fragment pattern, structural elucidation, quantitation, and purity of a compound (De Villiers et al., 2016).
T. cordifolia (TC) is a dioecious plant and it is noteworthy that all the chemical analyses and biological activities carried out in the past somehow did not take this into account. In a recent study (Bajpai et al., 2016), stem samples from male and female plants were analyzed. This study also took into account the distribution and seasons to address edaphic factors and different growth phases regulated by seasons reflecting the metabolic status of the plants. The samples were obtained from different locations in India such as Lucknow, Jabalpur, and Kolkata during premonsoon (February to April) and postmonsoon (September to November) seasons. The plant material collected from wild is likely to have inherent chemical variations and/or an altogether different composition, which makes the quality management of this Ayurvedic drug (or any other plant drug) a challenge. Due to very wide distribution and extensive use of this plant in the traditional system of medicine, it is important to know the extent of variability, if any, in the metabolite profile due to geographical locations, seasons, and gender. Collection of plant material with the optimum concentration of bioactive compounds is of paramount importance for the preparation of effective herbal drugs. Therefore, it is important to determine the actual content(s) and variations in the bioactive compounds in the plant extract. Studies on this very important aspect however, are lacking. Recently, direct analysis in real time mass spectrométrie (DART MS) and LC-MS/MS studies have made some headways in this direction. High performance liquid chromatography-electrospray ionization-quadrupole time of flight tandem mass spectrometry (HPLC-ESI-QTOF-MS/MS) and ultra performance liquid chromatography-electrospray ionization-quadrupole linear ion trap tandem mass spectrometry (UPLC-ESI-QqQLI1-MS/MS) were also applied for the phytochemical profiling and quantitate bioactive phytochemical in TCS.
DESCRIPTION OF PLANT MATERIALS
Male and female plants with leaf, stem, flowers, and fruits were collected from three sites of naturally growing population from the bank of river Gomati, Lucknow, for consecutive years from 2009, 2010, 2011 and 2012. Five male and five female plant samples (stem) were collected from flowering male and female plants. In all 18 collections were made from three major geographical locations from Jabalpur (JBL), Madhya Pradesh; Kolkata (KOL), West Bengal; and Lucknow (LKO), Uttar Pradesh (UP), in year 2009 in post monsoon (September to November) and 2010 in premonsoon (February to April) and post monsoon (September to November) seasons and followed in 2011 and 2012. Collected plant samples were identified and deposited in the departmental herbarium of Botany division of Central Drug Research Institute (CSIR-CDRI), Lucknow, India. Voucher specimen numbers with geographical location and collection period are reported in Table 2.1. Botanical reference standard (BRS) of T. cordifolia stem (TCS) was obtained from Tulsi Amrit Pvt Limited, Indore, India (Batch No. 10TC 1438).