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

Abstract

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.

Quantitative and rapid quality assessment methods for the multi‐class bioactive constituents of Tinospora cordifolia using high‐performance liquid and thin layer chromatography analysis with tandem mass spectrometry characterization

“…Factors like soil, temperature, water, humidity, and species association have contributed substantially to these variations in TC. This quantification data showed a set of new contributing factors like the maturity of the stem, harvesting time with the influence of season, and association with this creeper [26, 27, 39, 40]. Further, this method and qualitative fingerprinting can provide an important clue in chemotaxonomy studies to understand this significant relationship lacking in previous reports.…”

“…Previous analytical studies on TC were focused on quantifying the isoquinoline alkaloids using simple TLC and LC-based techniques [19,24,25]. However, it is essential to focus on the compounds responsible for its protective effects with specific analytical or biomarkers from TC [26,27]. The recent LC-based MS/MS and Quadrupole-TOF studies have identified chemical markers from TC for quality control (QC).…”

“…Still, due to the usage of expensive hyphenated instrumental techniques and a large number of identifications, these reported methods lack practical adaptation in QC and research labs for quantification [19,25,28]. The literature focuses on ecological studies of TC but lacks data on the standardization of its bioactive and specific markers in aqueous extract matrix [26,28]. Thus, the present study used a simple RP approach for separating and standardizing chemical and biologically active compounds in TC extract (TCE) [10,29].…”

Isolation and HPLC assisted quantification of two iridoid glycoside compounds and molecular DNA fingerprinting in critically endangered medicinal Picrorhiza kurroa Royle ex Benth: implications for conservation

“…HPLC analysis has been used as a preferred method for isolation, chemical characterization, and quantification of phytochemicals in many medicinal plant species (Han et al 2008;Sultan et al 2008;Thapliyal et al 2012;Song et al 2015;Mehra et al 2017;Thakur et al 2020).The concentration of picrosides in dry rhizomes of different populations of P. kurroa, has been found to vary with altitude in different ecogeographical regions in Himalayas. In Himachal Pradesh, the highest concentration of P-I and P-II was observed between an altitude of 2733 m (Sainj) to 2750 m (Parsuthach), in Uttarakhand it was observed between 3580 m (Dayara) to 3680 m (Tungnath), while in Kashmir and Sikkim regions, it was observed at 3000 m altitude in Arampatri and Temza, respectively.…”