1. Two-stage culture of Hypericum perforatum L. adventitious roots : effect of elicitation on production of biomass and secondary metabolites Cui XiHua Supervised by Professor Kee-Yoeup Paek Research Center for The Development of Advanced Horticultural Technology

2. Hypericum perforatum L. - Hypericum perforatum L.: Known as St. John’s wort, widely grown in various parts of the world for commercial purposes. -Current market : More than 40 drugs from 28 pharmaceutical companies were manufactured by Germany as antidepressant. 2 - The main active compounds: 1. Naphthodianthrones (Hypericin, 0.03~0.3% ) 2. Phloroglucinols (Hyperforin) 3. Flavonoids (Quercetin, hyperin) 4. Phenolic acid (Chlorogenic acid) -Efficacy: Treatment of depression, Dietary supplement phyto-medicine, Anxiolytic effect, Antibacterial activity Anti-inflammatory activity

3. Two stage culture of H. perforatum adventitious roots Stage 1: Biomass increase Adventitious root culture for 6 weeks in a bulb-type bubble bioreactor containing 1/2 MS liquid medium(NH 4 + :NO 3 - = 5:25 mM) supplemented with 1 mg l -1 IBA, 0.1 mg l -1 kinetin, and 30 g l -1 sucrose Stage 2: Secondary metabolites accumulation 3 1.Methyl jasmonate elicitation 2.Salicylic acid elicitation 3.Lactalbumin hydrolysate elicitation

4. Fig. 1. Production process of H. perforatum adventitious roots Fig. 1. Production process of H. perforatum adventitious roots In vitro plant Adventitious root induction from leaf explant Selection of root line Bioreactor culture Scaled up production HarvestDryFinal product Stage 1: Biomass increase

5. 배수체 검증 Number of nuclei Fluorescence intensity Fig.2. Typical DNA histograms of stained nuclei from mother plants and the adventitious root 5

6. Table1. Bioactive compounds in the adventitious root and mother plant of H. perforatum. Plant source Chlorogenic acid (mg g -1 DW) Total phenolics (mg g -1 DW) Total flavonoids (mg g -1 DW) Total target compounds (mg g -1 DW ) In–vitro b Adventitious root 0.55 ± 0.02 a 57.88 ± 2.9238.38 ± 2.1396.81 Ex–vitro c Leaves 13.98 ± 0.0439.13 ± 1.2823.02 ± 0.6776.13 Stems 0.57 ± 0.014.13 ± 0.383.20 ± 0.077.9 Roots 0.46 ± 0.0210.75 ± 0.526.60 ± 0.0317.81 a Values are mean ± S.E. for three replicates. b Data was taken after 6 weeks of bioreactor culture. c Data was taken in fields. 6 주요 약효성분 (1)

7. Fig.3. MS/MS spectrum of the precursor ion at m/z 503 in negative mode Hypericin LC-MS/MS analysis of hypericin, quercetin and hyperin 주요 약효성분 (2)

8. Fig.4. MS/MS spectrum of the precursor ion at m/z 463 in negative mode Hyperin

9. Fig.5. MS/MS spectrum of the precursor ion at m/z 301 in negative mode Quercetin

10. Identification of bioactive compounds by LC-MS/MS A: StandardB: Adventitious root Quercetin Chlorogenic acid Hyperin Fig.6. LC-MS/MS chromatogram of hypericin, quercetin, chlorogenic acid, hyperin standards (A) and adventtitious root extract (B) of H. perforatum using ESI negative mode Hypericin A A B B

11. Table 2. Bioactive compounds in adventitious roots and in vitro and ex vitro mother plants. Plants Leaves Stems Roots In - vitro ex - vitro Plant source Hypericin (μg g -1 DW) Quercetin (μg g -1 DW) Hyperin (μg g -1 DW) In-vitro Adventitious roots a 24.920.979.50 Plant b 263.141.6497.64 Ex-vitro c Leaves 247.883.45139.94 Stems 12.960.6160.11 Roots -0.095.24 a Data was taken after 6 weeks of bioreactor culture. b Data was taken after 4 weeks of solid culture of plant. c Data was taken in fields. Adventitious roots Quantitation of bioactive compounds by LC-MS/MS

12. 12 Log phase Exponential growth phase Stationary phase Declining phase Elicitation Stage 2: Enhancement of bioactive compound production

13. 1 wk2 wks4 wks 1 wk 2 wks4 wks 5 wks Elicitation 5 wks 6 wks 13 4w 5w Elicitation -1 Effect of methyl jasmonate (MJ) concentration and day of addition Elicitation

14. Fig.7.Biomass accumulation in the adventitious roots as affected by MJ concentration and day of addition for 1 week. 4w: MJ addition after 4 weeks of culture 5w: MJ addition after 5 weeks of culture 14

15. Fig. 10. Chlorogenic acid content in the adventitious roots as affected by MJ concentration and day of addition for 1 week. Fig. 8. Total phenolics content in the adventitious roots as affected by MJ concentration and day of addition for 1 week. Fig. 9. Total flavonoids content in the adventitious roots as affected by MJ concentration and day of addition for 1 week. 4w: MJ addition after 4 weeks of culture 5w: MJ addition after 5 weeks of culture 15

16. Fig. 13. Total polysacharides content in the adventitious roots as affected by MJ concentration and day of addition for 1 week. Fig. 11. Vitamin C content in the adventitious roots as affected by MJ concentration and day of addition for 1 week. Fig. 12. Vitamin E content in the adventitious roots as affected by MJ concentration and day of addition for 1 week. 4w: MJ addition after 4 weeks of culture 5w: MJ addition after 5 weeks of culture 16

17. MJ conc. (μmol) Phenolic compounds Total polysaccharides Chlorogenic acid Total phenolics Total flavonoids Total target compounds mg l -1 MJ addition-after 4 weeks of culture Cont. 8.78762.00456.861227.641235.70 50 21.69812.45519.441353.581127.27 100 24.70812.44529.371366.51980.43 150 24.92776.62506.421307.96925.44 200 26.92806.44503.041336.39937.73 MJ addition-after 5 weeks of culture Cont. 10.71861.39545.941418.051297.90 50 21.68944.88610.531577.091195.25 100 24.63964.79633.931623.351027.20 150 23.22942.13612.531577.891085.78 200 25.26960.84622.091608.191142.95 Table 3. Total production of bioactive compounds in the adventitious root (per 1 L medium) as affected by MJ elicitation after 1 week a. a Adventitious roots were cultured for 5 weeks before MJ elicitation. Total production = Mean of dry weight x Mean of each bioactive compound content 17

18. MJ conc. (μmol) Hypericin (μg g -1 DW) Quercetin (μg g -1 DW) Hyperin (μg g -1 DW) MJ addition-after 4 weeks of culture Cont. 5.860.2115.72 50 2.040.4113.32 100 1.810.2411.37 150 2.000.3412.14 200 1.210.3912.57 MJ addition-after 5 weeks of culture Cont. 24.920.979.50 50 13.680.339.18 100 10.270.459.74 150 6.130.4711.45 200 3.850.2610.25 Table 4. Quantification of hypericin, quercetin and hyperin by LC- ESI-MS/MS in the adventitious roots as affected by MJ concentration for 1 week in 3 L bioreactor culture.

19. Optimal concentration: 4 weeks old or 5 weeks old adventitious roots were treated with 100 µmol methyl jasmonate in 3 L bioreactors for 1 week. : When 100 µmol methyl jasmonate was added 4 weeks or 5 weeks after inoculation, total production of phenol compounds (per 1 L medium) were 1.1 fold higher than in the adventitious root without elicitation. Time of elicitation: when 5 weeks old adventitious roots were added 100 µmol methyl jasmonate, total production of phenol compounds (per 1 L medium) was 1.2 fold higher than in 4 weeks old adventitious roots added with 100 µmol methyl jasmonate. MJ can not increased the contents of hypericin, quercetin and hyperin. The highest accumulation of hypericin and quercetin were observed after 6 weeks of culture, and the highest contents of hyperin were observed after 5 weeks of culture. CONCLUSION 1. 19

20. SA conc. (μmol) Fresh wt. (g l -1 ) Dry wt. (g l -1 ) Growth ratio Cont. 135.99 azaz 14.58 a 57.32 50 128.01 b 14.64 a 57.54 100 126.74 b 14.39 a 56.56 150 126.09 b 14.27 a 56.06 200 117.60 c 14.24 a 55.96 Table 5. Effect of salicylic acid concentration on biomass accumulation for 1 week. Z Mean separation within columns by Duncan’s multiple range test at 5% level. Cont.50100 150200μmol 20 Elicitation-2 Salicylic acid elicitation (addition: 5weeks after inoculation)

21. Table 6. Accumulation of bioactive compounds in the adventitious root as affected by salicylic acid concentration for 1 week. a Data was taken after 6 weeks of culture. b Mean separation within columns by Duncan’s multiple range test at 5% level. SA conc. (μmol) Chlorogenic acid a (mg g -1 ) Total phenolics (mg g -1 DW) Total flavonoids (mg g -1 DW) Vitamin E (mg g -1 FW) Vitamin C (mg g -1 FW) Total polysacharides (mg g- 1 DW) Cont. 0.56 ab b 65.95 a 48.21 a 4.50 ab 0.56 d76.68b 50 0.56 ab 61.38 a 42.42 b 4.25 b 0.63 b91.01a 100 0.62 a 61.31 a 43.30 b 4.58 ab 0.68 a91.55a 150 0.61 a 57.90 ab 41.46 b 4.63 a 0.61 bc81.15ab 200 0.62 a 50.65 b 34.84 c 3.77 c 0.58 cd79.24b Cont.50100150200μmol 21

22. LH conc. (μmol) Fresh wt. (g l -1 ) Dry wt. (g l -1 ) Growth ratio Cont. 125.11 azaz 14.48 a 56.93 25 125.99 a 14.34 a 56.36 50 123.60 a 14.28 a 56.12 100 116.08 b 14.19 a 55.77 200 116.44 b 14.03 a 55.12 Table 7. Effect of lactalbumin hydrolysate concentration on biomass accumulation for 1 week. Z Mean separation within columns by Duncan’s multiple range test at 5% level. Cont.2550100200μmol 22 Elicitation-3 Lactalbumin hydrolysate elicitation (addition: 5weeks after inoculation)

23. Table 8. Accumulation of bioactive compounds in the adventitious root as affected by lactalbumin hydrolysate concentration for 1 week. a Data was taken after 6 weeks of culture. b Mean separation within columns by Duncan’s multiple range test at 5% level. LH conc. (μmol) Chlorogenic acid (mg g -1 ) Total phenolics (mg g -1 DW) Total flavonoids (mg g -1 DW) Vitamin EVitamin C Total polysaccharides (mg g- 1 DW) Cont. 0.53 ab 59.04 ab 42.87 ab 4.71 a 0.61 b 67.95 b 25 0.49 b 56.01 bc 39.53 bc 4.14 c 0.59 bc 81.40 a 50 0.49 b 53.19 c 38.20 c 4.64 ab 0.59 bc 79.83 a 100 0.50 b 54.26 c 38.05 c 4.34 bc 0.59 bc 84.17 a 200 0.55 a 62.35 a 45.53 a 4.43 abc 0.69 a 83.02 a Cont. 2550100200μmol 23

24. CONCLUSION 2. Optimal elicitors for both intensive biomass and secondary metabolite accumulation in adventitious root of H. perforatum : Methyl jasmonate elicitation significantly increased the contents of total phenolics, total flavonoids, chlorogenic acid, and Vitamin C. Salicylic acid and lactabumin hydrolysate elicitation significantly increased total polysaccharide contents and Vitamin C. 24

25. 25 연구실적 생물반응기를 이용한 H. perforatum 부정근의 배양조건 확립

26. 26 생물반응기를 이용한 H. perforatum 부정근의 배양조건 확립

27. 감사합니다. 27