1. An efficient combination of supercritical fluid extraction and high speed counter-current chromatograpgy to extract and purity homoisoflavonoids from Ophiopogon japonicus(thunb.) Ker-Gawler
Chengjun Ma Gang Li Juan Zhang Qiusheng Zheng Xiao Fan Zhenhua Wang
Department of Bioengineering ,College of Ocean , Yantai University, Yantai, China
Institute of Oceanology, Chinese Academy of Sciences, Qindao, China
College of Pharmacy, Shihezi University, Shihezi, China
Journal of separation science
March 31, 2009
Presenter : Kang MinSeok

2. Principle of analytical instrument Introduction
Experimental progress/result
Conclusion / Further study

3. Supercritical fluid Extractor
EXTRACTION

4. Supercritical fluid High temperature + High Pressure
Surface tension & density change
High diffusivity
Readily penetrates
porous & fibrous solids

5. Supercritical fluid extrator
CO2 SFE critical condition : 73.8bar , 31’C

6. Advantages of SFE
1. Higher diffusivities, lower viscosity, and lower surface tension.
2. Separation of analytes from solvent is fast and easy.
3. Having more selective separation power.
4. Does not have to worry about solvent residuals
5. Cheap, simple and many are safe.

8. Separation & Isolation
High Speed Count-Current Chromatography
Separation & Isolation

9. Counter-current distribution curve
K<1
K=1
K>1
if K=1 1 2 3 r
1000
500
250
125
375 n

10. Liquid-Liquid chromatography
Classification of CCC
Hydrostatic
CCC
CPC
CCC
Hydrodynamic
CCC
HSCCC
Liquid-Liquid chromatography

11. Separation zone
SETTLING
ZONE
MIXING ZONE
SETTLING
ZONE
SETTLING
ZONE

12. Characteristic of HSCCC
Versatility
Convenience and speed
Good resolution
Economical
Predictable and Reproducible
Total sample recovery

13. Introduction Ophiopogon japonicus Liliaceae / 소엽맥문동 Effect on
Cardiovascular system.
Steroidal glycosides
Steroid saponin
(ophiopogenin A~D)
Homoisoflavonoids
Ophiopogonone A ,B
Polysaccharides

14. Target componds Pharmacological Investigation of Homoisoflavonoids
Anti-inflammatory.
Anti-allergic
Anti-histamininic
Angioprotective
Insulin sensitizer activities
Potent-Phosphodiesterase inhibitor

15. Experiment outline Extraction Optimization of SFE
Scaling-up SFE & preparation crude extract
Separation
Selection two phase solvent system
HSCCC Working
Idenification
ESI-MS
1 H NMR
13C NMR

16. Orthogonal test design
Condition
Sample(powder) 40g
CO2 flow rate : 2.0L/min

17. Orthogonal test design
Sum of the Extraction yield at A1(15 pressure)
Mean value
Relation expression
Influence to the mean extraction : D > A > C > B

18. Orthogonal test design

19. Scale-up(100-fold) & HPLC analysis
87.5g crude extract/4kg
12.9%
3.4%
11.7%
Mobile phase
ACN : 0.3 % acetic acid=50:50v/v , flowrate 1.0ml/min , UV280 nm

20. Selection of suitable two-phase solvent system
Optimum range of partition coefficient 0.5<K<2.0

21. HSCCC separation Two phase solvent system
N-hexane / Ethyl acetate / methanol / acn/ water = 1.8 : 1.0 :1.0 : 1.2 : 1.0 v/v
Stationary(upper) Mobile(lower) Sf = 59.2% wavelength 280nm

22. HSCCC purification & HPLC idenification
13.5mg,97.3%
4.1mg, 98.3%
15.3mg,96.9%
Crude sample(140mg)

23. Identification
Fraction A: Colorless needles crystal. ESI-MS (m/z): 342
Fraction C: Colorless needles crystal. ESI-MS (m/z): 356
[M]+; 1H-NMR (CDCl3) d: 4.05 (1H, dd, J= Hz, H-2),
[M]+; 1H-NMR (CDCl3) d: 4.24 (1H, dd, J=11.7, 7.0 Hz, H-2),
4.35 (1H, dd, J= Hz, H-2), 2.66 l 3.01 (1H, m, H-3),
4.45 (1H, dd, J=11.6, 4.6 Hz, H-2), 2.79l3.06 (1H, m, H-3),
12.43 (1H, s, 5-OH), 5.29 (1H, s, 7-OH), 2.66 (1H, dd, J=10.2
12.95 (1H, s, 5-OH), (1H, s, 6-CHO), (1H, s, 7-
10.3 Hz, H-9), 3.20 (1H, d, J=10.2 Hz, H-9), 6.72 (3H, s, H-29,
OH), 1.98 (3H, s, 8-CH3), 2.65 (1H, dd, J=12.1, 9.0 Hz, H-9),
H-59, H-69), 5.96 (2H, s, -O-CH2-O-), 2.02 (3H, s, 6-CH3), 2.07
3.22 (1H, dd, J = 12.4, 3.6 Hz, H-9), 6.73 (3H, m, H-29, H-59,
(3H, s, 8-CH3); 13C-NMR (100 MHz, CDCl3), d: (C-4),
H-69), 5.91 (2H, s, -O-CH2-O-); 13C-NMR (100 MHz, CDCl3), d:
160.9 (C-5), (C-10), (C-7), (C-39), (C-
196.8 (C-4), (CHO-6), (C-10), (C-5), 164.1
49), (C-19), (C-69), (C-29), (C-59), 101.8
(C-7), (C-49), 146.2(C-39), (C-19), (C-69),
109.1 (C-29), (C-59), (C-11), (C-6), (C-
(C-6), (C-8), 99.8 (C-11), 99.3 (-O-CH2-O-), 69.1 (C-2),
8), (-O-CH2-O-), 69.8 (C-2), 45.6 (C-3), 31.6 (C-9), 5.8
44.3 (C-3), 31.3 (C-9), 7.4 (CH3-8), 6.8 (CH3-6).
(CH3-8
Fraction B: Orange needles crystal. ESI-MS (m/z): 354
[M]+; 1H-NMR (CDCl3) d: 7.54 (1H, s, H-2), (1H, s, 5-
OH), (1H, s, 7-OH), 3.73 (2H, s, H-9), (1H, s, 6-
CHO), 2.08 (3H, s, 8-CH3), 6.73 (3H, s, H-29, H-59, H-69), 5.91
(2H, s, -O-CH2-O-); 13C-NMR (100 MHz, CDCl3), d: 187.1
(OHC-6), (C-4), (C-5), (C-7), (C-10),
152.3 (C-2), (C-39), (C-49), (C-19), (C-
3), (C-69), (C-29), (C-59), (C-11), 104.8
(C-6), (C-8), (-O-CH2O-), 30.1 (C-9), 6.8 (CH3-8).

24. Conclusion & further study
1. Time waits for no one
>Investigation on the latest extraction system
2. Practical applications of HSCCC
3. Experimental design leads your effort to good result
4. Demand for statistics study & structure idenification.

25. Thank you for your attention