Magnetic Fe₃O₄ Immobilized β-glucosidase Hydrolysis For The Preparation Of Genipin And Evaluation Of Its Effect On The Relaxation Of Rat Thoracic Aorta

Eucommia ulmoides is a deciduous perennial tree.Its skin is a valuable tonic medicine in China and possesses hypotensive,hypoglycemic,anti-oxidation,anti-tumor and diuretic activities,etc.Geniposide is the main active ingredient in E.ulmoides,but it must be converted to genipin by the intestinal bacteria and then absorbed by the body thus playing its outstanding hypotensive and hypoglycemic activities.Therefore,the use of in vitro conversion process for the preparation of genipin can improve the therapeutic effect.In this paper,magnetic Fe3O4 immobilized P-glucosidase was first used to hydrolysis geniposide to genipin.In addition,we studied and compared the effect of geniposide and genipin on the relaxation of rat aorta for the first time,which provide a theoretical basis for the treatment of cardiovascular diseases by geniposide and genipin.The main studies and conclusions were as follows:1.Preparation and characterization studies of magnetic Fe3O4/CS nanoparticles.The preparation process of magnetic Fe304/CS nanoparticles were optimized first,and the optimal conditions were as follows:Fe2+:Fe3+=5:1,total iron concentration 0.05 moliL,pH value 11.At the same time,the prepared magnetic Fe3O4 and Fe3O4/CS nanoparticles were charactered from the aspects of X-ray diffraction,FT-IR and laser particle size analysis.The results showed that the Fe3O4/CS nanoparticles possessed homogeneous ingredients with the cubic spinel structure of nanocrystals.2.Hydrolysis of geniposide to genipin by magnetic Fe3O4/CS nanoparticles immobilizedβ-glucosidase.Firstly,we applied of magnetic Fe3O4/CS nanoparticles to immobilize β-glucosidase,and then the optimal conditions were determine as follows:temperature 50℃,pH 5.0 and time 3.5 h.Subsequently,the hydrolysis of geniposide to genipin by immobilized P-glucosidase was studied and the optimum conditions were determined as follows:hydrolysis time 6 h,pH value 5.0 and temperature 50℃.Under the optimized conditions,the hydrolysis and conversion rates of geniposide reached 90%and 96.80%,respectively.In addition,this immobilized P-glucosidase can be resued at least eight times and maintain more than 50%viability.The results show that the immobilized P-glucosidase method can increase the stability of enzyme and can be easy to control,easy separation enzyme from the reaction system,repeatedly used and reducing the cost.3.Study and compare of the effects of geniposide and genipin on the vasodilation of isolated rat thoracic aorta.Isolated vessels were applied to investigate the isotonic tension of thoracic aortic rings precontracted by norepinephrine(NE,1×10-6mol/L)or potassium chloride(KC1,30 mmol/L).The response action of geniposide and genipin on the vasodilation of isolated rat thoracic aorta were observed in the rings.The dependent contraction effects of geniposide and genipin on the extracellularand intracellular Ca2+-were also observed.Experimental results were exhibited as follows:(1)A concentration gradient(1×10-8 mol/L-1×10-4 mol/L)of geniposide and genipin had no effect on the resting tone.(2)A concentration gradient(1×10-8mol/L-1×10-4 mol/L)of geniposide and genipin treated on vasoconstriction induced by NE(1×10-6 mol/L)or KC1(30 mmol/L),which may produce concentration-dependent vasodilation.(3)A concentration gradient(1×10-8 mol/L-1×10-4 mol/L)of geniposide and genipin lead to the contraction of endothelium-denuded aortic rings induced by NE(1×10-6 mol/L)concentration-dependently,which was lower than relaxant of endothelium-intact aortic rings.(4)For NE-induced precontraction,the vasoconstriction of arterial rings treated with geniposide and genipin(1×10-8 mol/L-1×10-4 mol/L)changed weaker after adding L-NAME(1×10-44 mol/L).(5)For NE-induced precontraction,the vasoconstriction of arterial rings treated with geniposide and genipin(1×10-8 mol/L-1×10-4 mol/L)changed weaker after adding indomethacin(1×10-4 mol/L).(6)For NE-induced precontraction,the vasoconstriction of arterial rings treated with geniposide and genipin(1×10-8 mol/L-1 x 10-4 mol/L)didn’t change after adding K+channel blocker TEA(1×10-2 mol/L),4-AP(1×10-3 mol/L)and Gli(1×10-3 mol/L).(7)For the same consentration of geniposide and genipin(3×10-4 mol/L),NE(1×10-6 mol/L)can cause transient contraction,keeping on adding CaC12(2.5 mmol/L)can lead to vascular ring contraction.Compared with the control,geniposide and genipin can significantly reduce the vasoconstrictor effect induced by NE and CaC12 in Ca2+-free medium.The relaxant effect of geniposide and genipin was related with the VDC and ROC channels,which can control the extracelluar Ca2+influx and intracellular Ca2+release.Geniposide and genipin preferentially antagonized the contraction of rat thoracic artery in an endothelium-dependent manner and may be relevant to the production of NO and PGI2.Overall,the vasodilator effect of genipin was better than that of geniposide.