Alternative production of bilobalide and ginkgolides through callus induction of Ginkgo biloba leaves

Transcription

1 1/23/215 Alternative production of bilobalide and ginkgolides through callus induction of Ginkgo biloba leaves Agus Sukito, Sanro Tachibana and Asep Hidayat International Conference of Indonesia Forestry Researchers III October 215 Ginkgolide A Ginkgolide B Bilobalide Platelet-activating factor (PAF) antagonist PAF anaphylaxis 1

2 1/23/215 location Ginkgolides and bilobalide production from leaves climate seasonal variations Uneconomic Tissue culture of the ginkgo leaves Research objectives: To investigate the callus induction and production of bilobalide, ginkgolides A and B from callus cultures of G. biloba Fresh leaf Ethanol 7%, 1 minutes Anti-formin 1% 2 minutes Cut into small pieces (1x1 mm) MS medium Growth hormone Sucrose Gellan gum ph 5.6 Autoclave T=121 ºC t = 2 min Incubated at 25ºC in the dark 2

3 Biomass (g FW/flask) 1/23/ Kinetin.1 + NAA Kinetin.1 + NAA 2 2 Kinetin.1 + 2,4-D 1 1 Kinetin.1 + 2,4-D Incubation time (d) Fig. 1. Biomass of callus cultures in MS medium with addition of several combination of growth hormones Table. 1. Biomass of callus cultures in MS medium with addition of several combination of growth hormones Treatment Kinetin.1 + NAA 1 Kinetin.1 + NAA 2 Kinetin.1 + 2,4-D 1 Kinetin.1 + 2,4-D 2 Weight Cultivation time (days) Fresh 3.461± ± ±.211 Dry.472± ± ±.24 Fresh 3.99± ± ±.34 Dry.529± ± ±.1 Fresh 1.652± ± ±.181 Dry.83±.12.18±.15.99±.11 Fresh 1.69± ± ±1.166 Dry.57±.1.74±.15.67±.12 Extraction method: Extracted with 5% acetone Evaporated in vacuo Extracted with n-hexane Callus dry weight ( g) Water acetone Water soluble n-hexane soluble Adjusted to ph 2. Extracted with EtOAc Water soluble Water soluble EtOAc soluble Ginkgolide fraction TMS derivatization Fig. 2. Scheme of callus extraction method (Jeon et al., 1993) GC-MS 3

4 1/23/215 Standard samples TMS derivatization Bilobalide Ginkgolide A Ginkgolide B Fig. 3. Profile of Bilobalide, ginkgolides A and B in callus after TMS derivative by GC- MS Samples HPLC Bilobalide Shim-pak VP-ODS column Eluent : H 2 O/MeOH/Isopropanol = 72.5/17.5/1 Flow rate : 1 ml/min Temperature : 35ºC Detector : UV 22 nm Ginkgolide A Ginkgolide B Fig. 4. HPLC preparatives of bilobalide, ginkgolides A, B and C from callus of G. biloba 4

5 1/23/215 m/z [M-CH 3 ]=455 Fig. 5. Molecular ion of bilobalide in callus culture after TMS derivative by GC-MS m/z [M-CH 3 ]=537 Fig. 6. Molecular ion of ginkgolide A in callus culture after TMS derivative by GC-MS m/z [M-CH 3 ]=625 Fig. 7. Molecular ion of ginkgolide B standard sample after TMS derivative by GC-MS m/z [M-CH 3 ]=625 Fig. 8. Molecular ion of ginkgolide B in callus culture after TMS derivative by GC-MS 5

6 Content (mg/g DW) Content (mg/g DW) 1/23/ Bilobalide Ginkgolide A Ginkgolide B Fig. 9. Bilobalide and ginkgolides content in callus culture of G. biloba leaves with combination of kinetin and NAA hormone growth 2,5 2 Kinetin.1 + NAA 1 Kinetin.1 + NAA 2 Kinetin.1+2,4-D 1 Kinetin.1+2,4-D 2 1,5 1,5 Bilobalide Ginkgolide A Ginkgolide B Fig. 1. Bilobalide and ginkgolides content in callus culture of G. biloba leaves with combination of kinetin and 2,4-D hormone growth Method of cell suspension cultures: MS medium Sucrose Growth Hormone Callus Autoclave T=121 ºC t = 2 min shaken at 1 rpm, 25º C 3 d d 3 d Fig.11. Cell suspension culture method of G. biloba Table. 2. Combination of growth hormone concentration in cell suspension cultures Kinetin (mg/l) NAA (mg/l) 2,4-D (mg/l) TR1 TR2 TR3 TR4 1. TR5 TR6 TR7 TR8 6

7 Content (mg/g DW) PCV (%) 1/23/ TR1 TR2 TR3 TR4 TR5 TR6 TR7 TR8 Fig. 12. Biomass amount of cell suspension cultures with combination of growth hormone after incubation for 3 days TR1 TR2 TR3 TR4 TR5 TR6 TR7 TR8 TR1 TR2 TR3 TR4 TR5 TR6 TR7 TR8 TR1 TR2 TR3 TR4 TR5 TR6 TR7 TR8 Bilobalide Ginkgolide A Ginkgolide B Fig. 13. Production of bilobalide, ginkgolides A and B in cell suspension cultures with combination of growth hormone after incubation for 3 days Conclusions: The combination of.1 mg/l kinetin + 2 mg/l NAA was found to be the best for the induction of callus, which it resulted the highest biomass and amount of bilobalide (4.3 mg/g DW), ginkgolides A (3.1 mg/g DW) and B (3.5 mg/g DW). The highest biomass and production of bilobalide, ginkgolides A and B were observed in MS medium supplemented with.1 mg/l kinetin + 2 mg/l NAA in cell suspension cultures (6, 3.1, and 5.6 mg/g DW, respectively). It is a combination that suitable for both callus and cell suspension cultures for producing the bilobalide, ginkgolides A and B. 7

8 1/23/215 Bilobalide Ginkgolide A Ginkgolide B y = 72,518x ,4 R² =, Callibration curve of bilobalide y = 72,518x ,4 R² =, Shim-pak VP-ODS column Eluent : H 2 O/MeOH/Isopropanol = 72.5/17.5/1 Flow rate : 1 ml/min Temperature : 35ºC Detector : UV 22 nm Callibration curve of ginkgolide A y = 67,119x ,1 R² =, Callibration curve of ginkgolide B 8

9 1/23/215 Bilobalide 9

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