Immobilized ?-glucosidase Hydrolysis For The Preparation Of Pinoresinol Diglucoside Aglycone And Evaluation Of Its Effect On The Relaxation Of Rat Thoracic Aorta

As a natural product,Eucommia ulmoides is a unique traditional Chinese herbal medicine,can regulate blood pressure,regulate blood lipids,antioxidant,anti-cancer,anti-bacterial anti-inflammatory and so on.Pinoresinol diglucoside is the main active ingredient of buck in Eucommia ulmoides,it must be converted into aglycone structure by intestinal flora and enzymes so that its medicinal activity effective.Therefore we use magnetic Fe3O4/CS composite immobilized ?-glucosidase innovatively,provide a suitable environment in vitro to make pinoresinol diglucoside converted into aglycone structure.Then,we studied and compared the effect of pinoresinol diglucoside and pinoresinol diglucoside aglycone on the relaxation of rat aorta for the first time,which provide a theoretical basis for the treatment of cardiovascular diseases by pinoresinol diglucoside and pinoresinol diglucoside aglycone.The main studies and conclusions were as follows:1.Hydrolysis pinoresinol diglucoside by magnetic Fe3O4/CS nanoparticles immobilized P-glucosidase.Firstly,we applied of magnetic Fe3O4/CS nanoparticles to immobilize ?-glucosidase,got the immobilized enzyme of energy recovery rate 89.44%.Subsequently,the hydrolysis of pinoresinol diglucoside by immobilized ?-glucosidase was studied and the optimum conditions were determined as follows:hydrolysis time 6.17 h,pH value 5.03 and temperature 47.8?.Under the optimized conditions,the hydrolysis rates of pinoresinol diglucoside reached 87.69%,In addition,this immobilized ?-glucosidase can be resued at least eight times and maintain more than 51%viability.better stability and easier to recycle,reduce costs effectively.2.Analysis the hydrolyzate of pinoresinol diglucoside by enzyme.Analysis and identification Pinoresinol diglucoside hydrolyzate composition and structure using HPLC-DAD-MS,discovery feature of this hydrolyzate has less polar,single component.It is the aglycone structure(M=358)that pinoresinol diglucoside(M=682)lost two glucose structure(M=162).detection and identification the structure of the hydrolysis products using nuclear magnetic resonance proton spectroscopy(1H-NMR)and carbon spectroscopy(13C-NMR),with the results of mass spectrometry to confirm each other,eventually identified the structure as pinoresinol diglucoside aglycone.3.Study and compare of the effects of Pinoresinol diglucoside and its aglycone on the vasodilation of isolated rat thoracic aorta.To compare the affect of Pinoresinol diglucoside and its aglycone in different concentrations on thoracic aortic rings precontracted by potassium chloride(KCl).To compare the affect of pinoresinol diglucoside and its aglycone in different concentrations on the thoracic aortic rings has been pretreated by different functional drugs.Experimental results were exhibited as follows:(1)Different concentration of pinoresinol diglucoside and its aglycone had no effect on the resting tone.(2)PDG and PDG-A both have effect of concentration-dependent relaxation on the pre vasoconstrictor by KCl,which PDG-A significantly higher than PDG.The relaxation(%)was 35.2%for PDG,80.36%for PDG-A,PDG-A 50.16%higher than PDG.(3)PDG and PDG-A both have effect of endothelium-dependent relaxation on the pre vasoconstrictor by KC1.The relaxation(%)of PDG was 33.51%and 3.06%for endothelium+and endothelium-,the relaxation(%)of PDG-A was 81.12%and 5.26%for endothelium+ and endothelium-,significant different(P<0.05).(4)The relaxation effect of PDG and PDG-A might be related to the release of nitric oxide(NO,vasodilators).Take L-nitro-arginine methyl ester(L-NAME)pre-incubating vascular rings,blocking the release of NO,The relaxation(%)was 14.29%for PDG,21.82%for PDG-A,Compared with the control group was significantly(P<0.05).(5)The relaxation effect of PDG and PDG-A might be related to the release of epoprostenol(PGi2,vasodilators).Take indomethacin(Indo)pre-incubating vascular rings,blocking the release of PGI2,The relaxation(%)was 15.67%for PDG,17.86%for PDG-A,Compared with the control group was significantly(P<0.05).(6)The relaxation effect of PDG and PDG-A might be unrelated to the potassium channels.Pre-incubating vascular rings with TEA,4-AP and Gli(K+ channel blocker)respectively,The relaxation(%)of PDG was 35.07%for TEA,31.62%for 4-AP,40.74%for Gli;relaxation(%)of PDG-A was 73.42%for TEA,78.75%for 4-AP,76.25%for Gli.Compared with the control group was not significantly(P>0.05)(7)Both of PDG and PDG-A could inhibit vasoconstriction caused by calcium release and internal flow of calcium.The norepinephrine(NE)could caused transient contraction in Ca2+ free medium,keeping on adding CaCl2 could lead to vascular ring contraction.Compared with the control,pinoresinol diglucoside and its aglycone can significantly reduce the vasoconstrictor effect induced by NE and CaCl2 in Ca2+-free medium.In summary,relaxant effect of blood vessels caused by pinoresinol diglucoside aglycone was significantly higher than Pinoresinol diglucoside;Vasodilation both are concentration-dependent and endothelium-dependent,The mechanism may be associated with endothelial factor NO,PGI2 related,also could inhibit vasoconstriction caused by calcium release and internal flow of calcium,...