Screening Of Components For Activating Serum Aromatic Esterase From Ginger

Paraoxonase（PON）is an important hydrolysis enzyme in human.The activity of enzyme is related to many human diseases,such as cardiovascular,cerebrovascular diseases,atherosclerosis,cancer,type II diabetes and other diseases.Ginger has many pharmacological effects,like antioxidation,anti-atherosclerosis,decreasing cholesterol,enhance immune function and so on.It is usually used to treat the cardiovascular disease and increase the role of coronary blood flow.Therefore,the selection of PON1 agonist components from ginger has a very important research and practical significance for the prevention and treatment of some diseases.In the chemiluminescence（CL）method using 9-（4-methylphenoxycarbonyl）-10-methylacridinium trifluoromethanesulfonate as a substrate,the ginger crude extract of 5.00 × 10^-6 g/L improved K for the serum-mediated substrate hydrolysis from 0.0064 min^-1 to 0.0119 min^-1,being 1.86-fold as control value.At ginger crude extract of 4.00×10^-5 g/L,the agarose activity was found to be the highest by UV method with phenyl acetate as substrate.The two evaluation methods had the same conclusion was that ginger crude extracts can stimulate the serum PON1 hydrolysis substrate.The petroleum ether extract（PEE）,ethyl acetate extract（EAE）and n-butanol extract（n-BE）extracted from the ginger crude extract were obtained by CL method.PON1 has the stimulating effect when the three extracts were 8.00 × 10^-5 g/L.The K were 0.0099 min^-1,0.0081 min^-1 and 0.0068 min^-1,and the size of agonistic activity was: PEE > EAE > n-BE.When used UV method,there was the same conclusion.The agonist increased first and then decreased with the increase of concentration.When the extracts was 8.00×10^-5 g/L,the size of agonistic activity was: PEE > EAE > n-BE.Two samples（sample 1 #,sample 2 #）and components C（6-shagaols）,D,E（p-coumaric acid）,F（6-gingerol）were first separated from EAE by column chromatography and thin layer chromatography.Using CL method,it was found that sample 1 # was inhibitor for serum PON1,and the inhibitory effect was enhanced as the concentration was increasing.However,samples 2 # was agonist for serum PON1,and the agonism increased first and then decreased as the concentration was increasing.At the concentration of 1.00×10^-4 g/L,the agonism was up to the highest,K = 0.01519 min^-1,being 2.44-fold as control value.Component A and B（3-hydroxy-7-methoxyflavone）were further isolated from sample 2 #.But it was not possible to identify structural of component A and D due to the small amount.Component A,B and C had an agonistic effect on the serum PON1.The agonism increased first and then weakened with the increasing concentration.The K values were 0.0131min^-1,010104 min^-1 and 0.0205min^-1,and it was 2.05,1.63 and 3.20-fold as control value.When components were 1.00×10^-4 g/L,2.00×10^-6 g/L,1.00×10^-5 g/L,the size of the agonistic activity was: C > A> B.The components D,E and F had an inhibit effect on the serum PON1,and the inhibitory effects were enhanced with the increasing concentration.When using UV method,the component A,B and C were agonist for serum PON1.The agonism of the three components increased first and then decreased with the increasing concentration.At the optimum experimental concentration,the size of the agonistic activity was as followed: A(0.80×10^-5 g/L,70.60 U/L)> C(6.00×10^-5 g/L,64.79 U/L)≈ B(6.00×10^-5 g/L,64.59 U/L).The component E and F were inhibited on PON1.When the concentration was less than 4.00×10^-4 g/L,the inhibitory effect of component E was more large than component F;when the concentration was more than 4.00×10^-4 g/L,the inhibitory effect of component F on serum was more large than component E.It was found that the components E and F were competitive inhibition using the double reciprocal graph,and the average inhibitory constants were 8.98 mmol/L and 9.13 mmol/L,respectively.The size of the inhibitory activity was as followed: F> E.The component M and N obtained from PEE were evaluated by CL method.It was found that component M had an agonistic effect on serum PON1.The agonism increased with the concentration increasing.When beyond 1.00×10^-4 g/L,the agonism effect remained essentially unchanged,K = 0.0207 min^-1,being 3.23-fold as control value.While the component N was inhibitor for serum PON1,the inhibitory effect increases with the increasing concentration.However,the composition of the above components can not be identified due to the sample decomposition.