Preparative isolation and purification of antioxidative stilbene oligomers from Vitis chunganeniss using high-speed counter-current chromatography in stepwise elution mode MinSeok Kang Department of Chemistry, Zhejiang University, Hangzhou, China College of Food Science and Biological Engineering, Zhejiang Gengshang University, Hangzhou, China Shan He Yanbin Lu Liyan Jiang Bin Wu Feiying Zhang Yuanjiang Pan Journal of separation science April

Introduction HSCCC in Stepwise elution mode Sample profile (Vitis chunganeniss) Experiment procedure & result HSCCC DPPH & Electron paramagnetic resonance Further study (Application)

KD=KD= KD=KD= C upper C lower K K 0.5 ≤ ≤ 1.0(2.0) Proper range of K value The distribution constant

Journal of Chromatographic Science, Vol. 47, May/June 2009 Judging K value is not easy work. Inevitable selection

What is the stepwise elution mode? Add up two systems If you are suffer from finding proper solvent system at one step These peaks are suitable for 5:5:6:4 These peaks are suitable for 1:1:1:1

Sample profile 东南葡萄 Vitaceae 东南葡萄 Vitaceae Traditionally using for… 1.Infectious hepatitis 2.Physical injury Traditionally using for… 1.Infectious hepatitis 2.Physical injury No phytochemical or pharmacological investigation has been reported.

Sample Preparation Dried powder of V. chunganeniss (2.5kg) MeOH extraction Crude sample (270g) -> Aqueous solution Crude sample (270g) -> Aqueous solution Solvent Fractionation with EA ( 5 times ] Crude sample (120g) Evaporated under reduced pressure at 40’C HSCCC separation

Preparation of two-phase solvent system Methanol : water gradient [ MeOH 0-40min / 30-70% ] Add up Add up

HSCCC Procedure 1. Entirely filled with the upper phase 2. lower(mobile) phase was pumped into the column 3. Flow rate : 5.0mL/min, revolution speed : 450rpm 4. Injection after hydrodynamic equilibrium (800mg) HSCCC type TBE-1000A HSCCC type TBE-1000A 5. Detection using UV (280nm)

HSCCC result Stepwise elution point All target was isolated Compound 3 wasn’t isolated

Structure Identification Negative ESI-MS Negative ESI-MS 1 H-NMR 1 H-NMR 13 C-NMR 13 C-NMR Hopeaphenol Hopeaphenol Amurensin G Amurensin G Vitisin A Vitisin A

Activity DPPH Assay 1.180uL (O.15mM DPPH in EtOH) + Sample 20uL -> 96 well plate 2.Incubated at 37’C for 30min nm absorbance DPPH Assay 1.180uL (O.15mM DPPH in EtOH) + Sample 20uL -> 96 well plate 2.Incubated at 37’C for 30min nm absorbance Electron paramagnetic resonance DPPH result

electron paramagnetic resonance a spinning electron behaves like a tiny magnet absorption of electromagnetic radiation (in the form of microwaves) absorption of electromagnetic radiation (in the form of microwaves) Getting signal

Activity Electron paramagnetic resonance Scavenging effect on reactive oxygen species(ROS) Hydroxyl Radicals OH· ROS Superoxide anions O 2 −. Singlet oxygen 1 O 2 DMPO-OH adduct DMPO-OOH adduct TEMP- 1 O 2 adduct TEMP- 1 O 2 adduct Fenton type reaction Photo-irradiated riboflavin/EDTA Rose bengal in combination with photo-irradiation Species Sourse Detection Spin-Trap DMPO TEMP Modulation frequency : 100kHz Microwave frequency : 9.86GHz Microwave power : mW Temperature : 298K Modulation amplitude : 1G Scan Width : 200G Time constant : ms Modulation frequency : 100kHz Microwave frequency : 9.86GHz Microwave power : mW Temperature : 298K Modulation amplitude : 1G Scan Width : 200G Time constant : ms

Activity of Vitisin A Electron paramagnetic resonance It was not effective scavenger of hydroxyl radical It was not effective scavenger of Superoxide anion IC 50 of 1 O 2 = 6.9umol > EGCG(14.5 umol) IC 50 of 1 O 2 = 6.9umol > EGCG(14.5 umol) Reported Activity of Vitisin A : anti-obesity, anti-inflammatory, hepatotoxic activity

Further study 1. The more you think, the more you get 2. Application of our HSCCC procedure Various solvent systems Two or three combined systems Stepwise elution & obtain target compounds

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