UPLC-DAD Assisted Phytochemical Quantitation Reveals a Sex, Ploidy and Ecogeography Specificity in the Expression Levels of Selected Secondary Metabolites in Medicinal Tinospora cordifolia: Implications for Elites’ Identification Program
Authors: Thakur Rakesh Kr. , Rajpal Vijay Rani , Raina S.N. , Kumar Pawan , Sonkar Anand , Joshi Lata 
Published: January 2020

Background:
Medicinal phytochemistry involving UPLC-DAD in an exhaustive analysis
involving quantification of eight commercially important phytochemicals viz. syringin, cordifolioside
A, magnoflorine, tinocordiside, palmatine, 20β-hydroxyecdysone, L-tetrahydropalmatine and berberine
has been done in 143 accessions from eight states and the union territories of Delhi and Jammu &
Kashmir of India representing three different ploidy levels viz. diploid (2x), triploid (3x) and synthetic
tetraploid (4x). The study was done to assess the effect of sex, ploidy level and ecogeography on the
expression level of secondary metabolites in stems of dioecious, medicinally important shrub Tinospora
cordifolia.

Methods:
Two different UPLC-DAD methods were used for the quantification of eight selected phytochemicals
from the alcoholic stem extracts of T. cordifolia accessions. The Waters Acquity UPLC system
hyphenated to the QTOF micromass system, equipped with PDA and ESI-Q-TOF detectors was
utilized for the quantitative analysis, Mass Lynx v 4.0 software was used for data analysis.

Results:
Significant quantitative changes were observed in the analysed secondary metabolites among
different accessions of T. cordifolia. The triploid (3x) cytotypes revealed higher amounts of seven out
of eight analysed secondary metabolites than diploids and only 20β-hydroxyecdysone was observed to
be present in significantly higher amount in diploid cytotypes. Further, at the tetraploid level, novel induced
colchiploid (synthetic 4x) genotypes revealed increase in the yield of all of the analysed eight
phytochemicals than their respective diploid counterparts. The quantity of active principles in tetraploid
cytotypes were also higher than the average triploid levels at multiple locations in five out of eight
tested phytochemicals, indicating the influence of ploidy on expression levels of secondary metabolites
in T. cordifolia. Additionally, at each of the three ploidy levels (2x, 3x and synthetic 4x), a significant
sex specificity could be observed in the expression levels of active principles, with female sex outperforming
the male in the content of some phytochemicals, while others getting overexpressed in the
male sex. The manifestation of diverse ecogeographies on secondary metabolism was observed in the
form of identification of high yielding accessions from the states of Madhya Pradesh, Delhi and Himachal
Pradesh and the Union territory of Jammu & Kashmir. Two triploid female accessions that contained
approximately two- to eight fold higher amounts of five out of the eight analysed phytochemicals
have been identified as superior elites from the wild from the states of Delhi and Madhya Pradesh.

Conclusion:
The paper shows the first observations of ploidy specificity along with subtle sex and ecogeography
influence on the expression levels of secondary metabolome in T. cordifolia.