In vitro propagation and sustention of hypericin rich

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

Introduction ØHypericum perforatum is an important medicinal herb of Hypericaceae family ØIt is mainly used as an antidepressant and possesses broad pharmacological activities such as anti-microbial, anti-viral, cytotoxic, anti-inflammatory, antitumor etc. (Palmer and Keller 2010) ØMedicinal properties are attributed to the presence of various classes of biochemical compounds: Classes Phytochemicals Naphthodianthrones Hypericin and Pseudohypericin Flavonoids Rutin, Hyperoside, Isoquercitrin, Quercitrin and Quercetin Phloroglucinols Hyperforin and Adhyperforin Tannins Cathechin and Epicatechin Proanthocyanidins Procyanidin B 2 Bioflavonoids Biapigenin and Amentoflavone

ØHypericin is a potential photosensitizing anticancer agent ØHypericum preparations are generally standardized based on defined hypericin concentrations of 0. 3 -0. 5% ØHerbal preparations containing hypericin are worth of an estimated value of US$ 210 and 570 million in the USA and worldwide, respectively ØHypericin accumulates predominantly in foliar parts of the plant ØIn natural habitat H. perforatum propagates by means of runners or from seeds ØTissue culture techniques can be used as an alternative option for rapid multiplication of this medicinally important plant species for the hypericin production

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

Effect of different concentrations and combinations of growth hormones on shoot multiplication MS + growth hormones (mg/L) BAP IBA KN 0 1 2 0 0 1 1 0 3 0 0. 5 1 0 0 2 3 0 0 0 1 3 3 4 4 2 Parameters of shoot multiplication Days to multiple shoot formation Shoots per explant (Mean ± S. E. ) 18 -20 9 -10 8 -9 5 -6 9 -10 6 -8 5 -6 8 -9 10. 3 ± 0. 5 15. 3 ± 0. 5 17. 6 ± 0. 3 22. 2 ± 0. 6 36. 3 ± 0. 3 21. 3 ± 0. 3 30. 6 ± 0. 3 35. 3 ± 0. 3 21. 6 ± 0. 3 Shoot length (cm) (Mean ± S. E. ) 6. 6 ± 0. 5 8. 9 ± 0. 5 8. 8 ± 0. 5 10. 4 ± 0. 5 12. 2 ± 0. 5 9. 7 ± 0. 3 8. 5 ± 0. 6 10. 6 ± 0. 5 11. 1 ± 0. 3

Effect of different concentrations and combinations of growth hormones on root multiplication MS + growth hormones (mg/L) NAA IBA KN 0 1 1 0 3 1 2 0 1 0 0. 5 2 3 0 3 4 4 2 0 0 1 1 2 2 0 Parameters of root formation Days to root initiation 20 -22 10 -12 7 -8 9 -10 10 -12 7 -8 10 -12 Number of roots Root length per shoot explant (cm) (Mean ± S. E. ) 3. 3 ± 0. 3 1. 8 ± 0. 5 3. 5 ± 0. 6 2. 6 ± 0. 5 4. 8 ± 0. 6 3. 5 ± 0. 5 5. 8 ± 0. 3 3. 8 ± 0. 5 4. 8 ± 0. 5 2. 2± 0. 5 5. 0 ± 0. 5 2. 5± 0. 3 5. 2 ± 0. 5 2. 8 ± 0. 6 4. 7 ± 0. 6 3. 1 ± 0. 5 4. 9 ± 0. 6 2. 9 ± 0. 3

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

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

Effect of different growth hormones on hypericin production in shoot cultures of H. perforatum MS + growth hormones (mg/L) BAP IBA KN 0 0 0 1 1 0 0 1 2 1 0 0 0 2 3 3 4 2 0 1 3 3 4 4 1 1 2 0 Hypericin content (µg/mg) 0. 000 ± 0. 000 0. 062 ± 0. 004 0. 108 ± 0. 003 0. 063 ± 0. 003 0. 085 ± 0. 001 0. 096 ± 0. 001 0. 119 ± 0. 001 0. 091 ± 0. 004 0. 054 ± 0. 002 0. 037 ± 0. 002

Effect of temperature, light condition on hypericin (µg/mg) production in shoots growing on MS medium Type of condition MS + sucrose + IBA + KN + agar 25 0 C 15 0 C MS + sucrose + IBA + KN 25 0 C 15 0 C Light 0. 119 ± 0. 001 0. 009 ± 0. 001 0. 098 ± 0. 002 0. 006 ± 0. 003 Dark 0. 003 ± 0. 002 0. 001 ± 0. 001 0. 002 ± 0. 002 0. 001 ± 0. 001

-- Effect of time duration on sustention of hypericin rich shoots in glasshouse conditions S. No. Tissue Type Hypericin ( μg/mg ) 1 2 3 4 In vitro grown(1. 5 month) Hardened (1 st year) Hardened (2 nd year) Field grown (1 st year) 0. 11 0. 32 0. 37 0. 41 5 Field grown (2 nd year) 0. 44 Hardened plants (1 -2 yrs)

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

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