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

Avinash Kumar; Vijay Rani Rajpal; Ambika; Rachayya Mallikarjun Devarumath; Amita Kumari; Rakesh Thakur; Manju Chaudhary; Pradeep Pratap Singh; Shiv Murat Singh Chauhan; Soom Nath Raina

doi:10.1007/s12298-021-00972-w

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

Physiol Mol Biol Plants. 2021 Apr;27(4):727-746. doi: 10.1007/s12298-021-00972-w. Epub 2021 Mar 26.

Authors

Avinash Kumar^{1

2}, Vijay Rani Rajpal^{1

3}, Ambika^{4

5}, Rachayya Mallikarjun Devarumath^{1

6}, Amita Kumari², Rakesh Thakur⁷, Manju Chaudhary⁷, Pradeep Pratap Singh^{4

8}, Shiv Murat Singh Chauhan⁴, Soom Nath Raina^{1

7}

Affiliations

¹ Department of Botany, University of Delhi, 110007 New Delhi, Delhi India.
² Department of Botany, Vinoba Bhave University, Hazaribag, Jharkhand 825319 India.
³ Department of Botany, Hansraj College, Delhi University, New Delhi, Delhi 110007 India.
⁴ Department of Chemistry, University of Delhi, 110007 New Delhi, Delhi India.
⁵ Department of Chemistry, Hansraj College, Delhi University, Delhi, 110007 India.
⁶ Molecular Biology and Genetic Engineering Lab., Vasantdada Sugar Institute, Pune, Maharashtra India.
⁷ Amity Institute of Biotechnology, Amity University, Sector 125, 201303 Noida, Uttar Pradesh India.
⁸ Department of Chemistry, Swami Shraddhanand College, University of Delhi, New Delhi, Delhi 110036 India.

Abstract

Picrorhiza kurroa is a medicinally important, high altitude perennial herb, endemic to the Himalayas. It possesses strong hepato-protective bioactivity that is contributed by two iridoid picroside compounds viz Picroside-I (P-I) and Picroside-II (P-II). Commercially, many P. kurroa based hepato-stimulatory Ayurvedic drug brands that use different proportions of P-I and P-II are available in the market. To identify genetically heterozygous and high yielding genotypes for multiplication, sustained use and conservation, it is essential to assess genetic and phytochemical diversity and understand the population structure of P. kurroa. In the present study, isolation and HPLC based quantification of picrosides P-I and P-II and molecular DNA fingerprinting using RAPD, AFLP and ISSR markers have been undertaken in 124 and 91 genotypes, respectively. The analyzed samples were collected from 10 natural P. kurroa Himalayan populations spread across four states (Jammu & Kashmir, Sikkim, Uttarakhand and Himachal Pradesh) of India. Genotypes used in this study covered around 1000 km geographical area of the total Indian Himalayan habitat range of P. kurroa. Significant quantitative variation ranging from 0.01 per cent to 4.15% for P-I, and from 0.01% to 3.18% in P-II picroside was observed in the analyzed samples. Three molecular DNA markers, RAPD (22 primers), ISSR (15 primers) and AFLP (07 primer combinations) also revealed a high level of genetic variation. The percentage polymorphism and effective number of alleles for RAPD, ISSR and AFLP analysis varied from 83.5%, 80.6% and 72.1%; 1.5722, 1.5787 and 1.5665, respectively. Further, the rate of gene flow (Nm) between populations was moderate for RAPD (0.8434), and AFLP (0.9882) and comparatively higher for ISSR (1.6093). Fst values were observed to be 0.56, 0.33, and 0.51 for RAPD, ISSR and AFLP markers, respectively. These values suggest that most of the observed genetic variation resided within populations. Neighbour joining (NJ), principal coordinate analysis (PCoA) and Bayesian based STRUCTURE grouped all the analyzed accessions into largely region-wise clusters and showed some inter-mixing between the populations, indicating the existence of distinct gene pools with limited gene flow/exchange. The present study has revealed a high level of genetic diversity in the analyzed populations. The analysis has resulted in identification of genetically diverse and high picrosides containing P. kurroa genotypes from Sainj, Dayara, Tungnath, Furkia, Parsuthach, Arampatri, Manvarsar, Kedarnath, Thangu and Temza in the Indian Himalayan region. The inferences generated in this study can be used to devise future resource management and conservation strategies in P. kurroa.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-021-00972-w.

Keywords: Genetic diversity; HPLC; Molecular DNA markers; Phytochemical diversity; Picrorhiza kurroa; Picrosides.