In vitro propagation and sustention of hypericin rich shoots of Hypericum perforatum

1. In vitro propagation and sustention of hypericin rich shoots of Hypericumperforatum Department of Biotechnology and Bioinformatics Jaypee University of Information Technology Waknaghat-173234, Solan (HP) Presented by: Dr. Hemant Sood

2. Introduction • Hypericum perforatum is an important medicinal herb of Hypericaceae family • It is mainly used as an antidepressant and possesses broad pharmacological activities such as anti-microbial, anti-viral, cytotoxic, anti-inflammatory, anti-tumor etc. (Palmer and Keller 2010) • Medicinal properties are attributed to the presence of various classes of biochemical compounds:

3. Hypericin is a potential photosensitizing anticancer agent • Hypericum preparations are generally standardized based on defined hypericin concentrations of 0.3-0.5% • Herbal preparations containing hypericin are worth of an estimated value of US$ 210 and 570 million in the USA and worldwide, respectively • Hypericin accumulates predominantly in foliar parts of the plant • In natural habitat H. perforatum propagates by means of runners or from seeds • Tissue culture techniques can be used as an alternative option for rapid multiplication of this medicinally important plant species for the hypericin production

4. Methodology H. perforatum plants were procured from the National Bureau of Plant Genetic Resources (NBPGR), Shimla, H.P., India Shoot apices were surface sterilized Cultured on MS media supplemented with 3% sucrose and different concentrations of IBA, BAP and KN Optimization of growth hormones, temperature, light condition and solid/liquid media for hypericin production Root induction Hardening

5. Effect of different concentrations and combinations of growth hormones on shoot multiplication

6. Effect of different concentrations and combinations of growth hormones on root multiplication

7. Micropropagation of H. perforatum Explant inoculated Shoot initiation Shoot multiplication Root induction Hardening

8. Quantification of hypericin Shoot tissues were ground to a fine powder in liquid nitrogen 100 mg of powdered material percolated in 10 ml methanol and sonicated for 1 hour Centrifuged at 6000 rpm for 15 minutes and filtered through 0.22 µm membrane filter Subjected to RP-HPLC Hypericin was identified on the basis of their retention time and comparison of UV spectra with the authentic standard

9. Effect of different growth hormones on hypericin production in shoot cultures of H. perforatum

10. Effect of temperature, light condition on hypericin (µg/mg) production in shoots growing on MS medium

11. -- Effect of time duration on sustention of hypericin rich shoots in glasshouse conditions Hardened plants (1-2 yrs)

12. Conclusion • A rapid protocol for in vitro multiplication and hypericin production in H. perforatum • Maintenance of hypericin rich hardened shoots under glass house conditions • This study can propose future avenues of commercialization of hypericin rich shoots to meet industrial demands for developing formulations from H. perforatum

13. Thank you