Study On Preparation Of Flavone Aglycone From Ginkgo Biloba L Extract

The Ginkgo biloba L is one of the oldest plants on the planet in which the flavone compounds is the main active ingredient. In this paper, acid, free and immobilized enzyme were used to hydrolysis flavonoids preparing flavonoids aglycones with higher biological activity.And macroporous resin was used to separate and enrich the hydrolysate to prepare crude flavonoids aglycones.Firstly, the extract of leaves of Ginkgo biloba L (GBE) was hydrolyzed by hydrochloric acid andβ-glucosidase respectively to prepare the flavone aglycone. The orthogonal design test was adopted to find the optimal processing conditions of both methods. It was showed that the optimal acidic hydrolysis conditions are as follows: 80 % methanol as media in which acid concentration 4 mol/L,ratio of GBE weight to solvent volume (mg/mL) 2∶1, hydroly- zing 4 h at 70℃.Under those conditions, the transformation rate of flavone glycosides reached 93.8 % and the yield of flavonoids aglycones reached 9.23 %;The optimal enzymatic hydrolysis conditions are as follows: in the hydrolyzed fluid system with the pH 5.0, enzyme concentration 5×10-3 mg/mL,that is 8 international units of enzyme activity per milliliter, hydrolyzing 6 h at 40℃. Under those conditions, the transformation rate of flavone glycosides reached 88.2 % and the yield of flavonoids aglycones reached 9.08 %.Through comparing their HPLC spectrum of the hydrolysates, it was known that there are only flavone aglycones which can be observed in the acidic hydrolysate, while in the enzymatic hydrolysate there are still many other functional ingredients.So the method of enzymatic hydrolysis is helpful to the comprehensive bio-functional of GBE.Secondly, theβ-glucosidase was immobilized efficiently and easily by entrapping into the calcium alginate. The immobilizedβ-glucosidase transformed flavone glycosides to flavone aglycone in order to decrease the cost of process.The effects of temperature,pH value,treatment time andβ-glycosidase concentration on the transformation reaction of immobilizedβ-glucosidase were studied. The storing and operation stability of immobilizedβ-glucosidase were also discussed. The optimal conditions were as follows: in the hydrolyzed fluid system with the pH 5.0, the immobilizedβ-glycosidase concentration 0.1 g/mL, that is 9.6 international units of enzyme activity per milliliter, hydrolyzing 4 h at 40℃.Under this conditions the yield of transformation was 86.6 %.In addition,the storing and operation stability of immobilizedβ-glucosidase were greatly improved.Finally, to study the separation effect of macroporous resin for the purification of flavone aglycone, one type of macroporous resin was selected from three kinds of resins, and their adsorption dynamics were studied. The results showed that YWD07a resin was the best one. The optimal separation conditions were as following: flavone aglycone concentration of sample solution was 3.26 mg/mL, at pH 5.0, flow rate 2 bed volume (BV) per hour. Using 6 BV 70 % (v/v) ethanol as the desorption solvent. The content of flavone aglycone increased from 18.5g/100g to 80.5 g/100 g in the crude product, and the recovery of flavone aglycone reached 73.6 %.