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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

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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.

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Acknowledgements

This work was supported by grant for scientific research from the Department of Biotechnology, Government of India. Avinash Kumar acknowledges SRF from CSIR during his Ph.D. The authors are thankful to Prof. (Retd.) Ravinder Raina, Department of Forest Products, Dr. Y. S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India, for easing collection of germplasm from Himachal. The authors extend their sincere gratitude to anonymous reviewers for their constructive comments that helped in the improvement of the manuscript.

Funding

This work was supported by grant for scientific research from the Department of Biotechnology, Government of India.

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Authors and Affiliations

  1. Department of Botany, University of Delhi, 110007, New Delhi, Delhi, India

    Avinash Kumar, Vijay Rani Rajpal, Rachayya Mallikarjun Devarumath & Soom Nath Raina

  2. Department of Botany, Hansraj College, Delhi University, New Delhi, Delhi, 110007, India

    Vijay Rani Rajpal

  3. Department of Chemistry, University of Delhi, 110007, New Delhi, Delhi, India

    Ambika, Pradeep Pratap Singh & Shiv Murat Singh Chauhan

  4. Department of Botany, Vinoba Bhave University, Hazaribag, Jharkhand, 825319, India

    Avinash Kumar & Amita Kumari

  5. Amity Institute of Biotechnology, Amity University, Sector 125, 201303, Noida, Uttar Pradesh, India

    Rakesh Thakur, Manju Chaudhary & Soom Nath Raina

  6. Department of Chemistry, Hansraj College, Delhi University, Delhi, 110007, India

    Ambika

  7. Molecular Biology and Genetic Engineering Lab., Vasantdada Sugar Institute, Pune, Maharashtra, India

    Rachayya Mallikarjun Devarumath

  8. Department of Chemistry, Swami Shraddhanand College, University of Delhi, New Delhi, Delhi, 110036, India

    Pradeep Pratap Singh

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Contributions

Contributors Role Taxonomy (CRediT): Avinash Kumar: Sample Collection, Methodology, Investigation (molecular profiling), Validation, Formal analysis, Writing—Original Draft preparation. Ambika: Methodology, Investigation (Chemical profiling), Formal analysis, Validation, (chemoprofiling). Amita Kumari: Analysis. Rachhaya.Mallikarjun Devarumath: Sample Collection. Rakesh Thakur: Analysis. Manju Chaudhary: Analysis. Pradeep Pratap Singh: Analysis (Chemical profiling). Shiv Murat Singh Chauhan: Conceptualization, Supervision (Chemical profiling work). SN Raina: Conceptualization, Resources, Overall Supervision, Project administration, Funding acquisition. Vijay Rani Rajpal: Supervision, Visualization, Validation, Writing and editing.

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Kumar, A., Rajpal, V.R., Ambika et al. 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 27, 727–746 (2021). https://doi.org/10.1007/s12298-021-00972-w

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