Isolation And Preparation Of Bioactive Constituents In Silybum Marianum And Their Antioxidant Activity

Silymarin seeds,the seeds of Milk thistle[Silybum marianum L. Gaertn.(Asteraceae)]have been used for the treatment of the liver diseases for hundreds of years.This hepatoprotective activity is attributed to the flavonolignans including silybin,isosilybin,silychristin and silydianin in the seeds.A recent review of the bioactivity of silymarin discussed its potential applications in the treatment of cancer,neurological,gastrointestinal, nephropathic,cardiological and skin disorders.In the present study,the six flavonolignans were extracted,separated and identified for the systematic research of the main bioactive constituents of silymarin.Silymarin seeds were ultrasound-assisted extracted with ethyl acetate.The extraction solutions were evaporated in vaccum to remove organic solvents. Silymarin was separated by high speed counter-current chromatography(HSCCC) using a biphasic solvent system A composed of petroleum ether-ethyl acetate-methanol-water(1:4:3:4,V/V) to yield silychristin(â… ) and Fraction 2. Fraction 2 was separated by HSCCC using a biphasic solvent system E composed of TMBE-methanol-water(2:1:1,V/V) to yield silydianin(â…¡),silybin and isosilybin.This method has advantages of larger sample load and high recovery.Silybin and isosilybin obtained from HSCCC separation were isolated using preparative high performance liquid chromatography to yield silybin A(â…¢), silybin B(â…£),isosilybin A(â…¤) and isosilybin B(â…¥),respectively.The chemical structure of the obtained silychristin,silydianin,silybin A, silybin B,isosilybin A and isosilybin B were identified by their spectra of ESI-MS,¹H-NMR and ¹³C-NMR.The antioxidant activity of silymarin and six compounds(â… -â…¥) was investigated by DPPH radical scavenging method and via pyrogallol auto-oxidation method.The results showed that the antioxidant activity silybin A, silybin B,isosilybin A and isosilybin B was low comparing with vitamin C.But the antioxidant activity of silychristin and silydianin at concentration more than 100.0μg/mL could reach to about 30-50%of vitamin C while the antioxidant activity of silymarin was 40-50%of vitmin C.Therefore,the antioxidant activity of silymarin is mainly contributed by silychristin and silydianin.The above results will provide technical support and basic reference for the study of bioactivities of silymarin.