Preparative isolation and purification ofantioxidative stilbene oligomers from Vitis

chunganeniss using high-speed counter-currentchromatography in stepwise elution mode

2009 10.22 MinSeok Kang

Department of Chemistry, Zhejiang University, Hangzhou, ChinaCollege 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 30 2009

Introduction• HSCCC in Stepwise elution mode• Sample profile (Vitis chunganeniss)

Experiment procedure & result• HSCCC• DPPH & Electron paramagnetic resonance

Further study (Application)

KD

=

C upper

C lower

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

Traditionally using for…1. Infectious hepatitis2. Physical injury

No phytochemical or pharmacological investigation has been reported.

Sample Preparation

Dried powder of V. chunganeniss (2.5kg)

MeOH extractionCrude sample

(270g)-> Aqueous

solutionSolvent 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

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 : 450rpm4. Injection after hydrodynamic equilibrium (800mg)

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

1H-NMR

13C-NMR Hopeaphenol

Amurensin GVitisin A

Activity

DPPH Assay 1. 180uL (O.15mM DPPH in EtOH) + Sample 20uL ->

96 well plate2. Incubated at 37’C for 30min 3. 517 nm absorbance

Electron param-agnetic reso-

nance

DPPH result

electron paramagnetic resonance

a spinning electron behaves like a tiny magnet

absorption of electromagnetic radiation (in the form of microwaves)

Getting signal

Activity Electron paramagnetic res-onance

Scavenging effect on reactive oxygen species(ROS)

Hydroxyl Radicals

OH·

ROS

Superoxide anions

O2−.

Singlet oxygen 1O2

DMPO-OH adduct

DMPO-OOH adduct

TEMP- 1O2

adduct

Fenton type reaction

Photo-irradiated riboflavin/EDTA

Rose bengal in combination with photo-irradiation

Species

Sourse

Detec-tion

Spin-Trap

DMPO TEMP

Modulation frequency : 100kHzMicrowave frequency : 9.86GHzMicrowave power : 20.11 mWTemperature : 298K

Modulation amplitude : 1GScan Width : 200GTime constant : 81.92 ms

Activity of Vitisin AElectron paramagnetic res-onance

It was not effective scavenger of hydroxyl radical

It was not effective scavenger of Superoxide anion

IC50 of 1O2 = 6.9umol> EGCG(14.5 umol)

Reported Activity of Vitisin A : anti-obesity , anti-inflammatory , hepa-totoxic 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

Thank you for your attention