Effect Of Dendranthema Indicum Effective Parts And Peanut Shell Effective Parts On The Antihypertension And Blood Vessel Protection

Objects:As a killer of the healthy of people,Hypertension has a major cause of death and disability.At present,hypertension is only could be controlled but not be cured.Thus,attention is beggining to be focused in the Chinese traditional medicine which has little side effect.In this study,we prepared the effective part of Dendranthema Indicum(DEP)which is a kind of Chinese traditional medicine for anti-hypertension and the effective part of peanut shell(PEP)which is used to control blood pressure among the people through some methods of purification.Then we studied the effect of DEP and PEP on the blood pressure and blood lipid of SHR,and the mechsnism of anti-hypertension of DEP and PEP through some respects containing RAAS,endothelial function,immunoglobulin,gastrointestinal hormone and biologic peptide.At the same time,we further rearched the mechanism of antihypertension through protecting blood vessel with the experiments in vivo and vitro.Methods 1.The method of preparing PEP:The peanut shell was crushed and extracted by ethanol,then concentrated to extract.The extract was dissolved by high concentration ethaonl.The Luteolin solution was purified by the method of alcohol extracting-water precipitating.Through the selection and optimization of Static-dynamic adsorption parameters,the best macroporous resin and the optimal method of purification was obtained.The method of alcohol extracting-water precipitating was designed to obtain the products which had high purity of Luteolin.Duplicated test was performed to detect the stability of method.2.The method of preparing PEP:The Dendranthema Indicum extracted by ethanol,then concentrated to extract.According to the solubility of BUD,solid-liquid extraction was chose;The solvents which could dissolve the Buddleoside as little as possible while the impurity as much as possible was chose with the comprehensive consideration of the solubility of Buddeloside and impurity.A method,which had simple operations,small loss of Buddleoside and high efficiency of improving the purity was designed to purify the crude extracts by solid-liquid extraction with the selected solvents,and the influence of the temperature(4℃,30℃)on the reliability of this method was examined.Duplicated test was performed to detect the stability of method.3.The effect evaluation of PEP、DEP and P&DEP:The effect of DEP,PEP andP&DEP with different ratios and dosages on the blood pressure were examined by CODA Mouse&Rat Tail-Cuff Blood Pressure System during the successive administration of six weeks;The blood flow of tails were tested by the instrument of Moor FLPI speckle full frame real-time scanning imaging system in the sixth week;The blood lipid and liver/kidney function were analyzed by fully automatic biochemical analyser.The blood indexes about RAAS,inflammation,immunity,gastrointestinal hormone and relax/shrink blood vessel were detected by ELISA kit;The effect.and mechanism of PEP and DEP on the RAAS of the kidney was analyzed by western-blot.4.The protection effect of PEP and DEP on the blood vessel:The oxidative stress HUVEC model was established to simulate the progress that oxidative stress harm the endothelial cells.It was used to examine the antioxidation of DEP and PEP.The ROS in cells,proliferation activity and apoptosis of cells were determined by fluorescently labeled probe,MTT and flow cytometry,respectively.The mechanism was analyzed by western-blot.At the same time,the proliferation and migration VSMCs model achieved by the stimulation of AngⅡ was established to examine the effect of BUD and LUT.The ROS in cells,proliferation and migration of cells were examined by fluorescently labeled probe,MTT and transwell assay.The mechanism was analyzed by western-blot.The endothelium function was evaluated by examine the EDRF and EDCF in the blood of SHR by ELISA kit.The structure of artery vessel was to observe pathological sectionResults 1.The method of preparing PEP was as follows:(1)Primary purification of recrystallization:30%ethanol-water solution of Luteolin for loading was obtained through dilution of 80%ethanol-water solution of Luteolin;(2)DI01 was selected to purify the Luteolin.According to the date of static adsorption tests,the first-order kinetics model and Langmuir isotherm model were found to fit best.The optimal condition for the purification through macroporous resin was as follows:loading temperature:25℃,loading concentration of Luteolin solution:0.341 mg/ml,flow rate for loading:2 BV/h,loading volume:5 BV,ethanol-water concentration for elution:40%ethanol-water solution,flow rate for elution:2 BV/h,volume for elution:4BV.(3)The final purification of recrystallization::the products from the purification of D101 macroporous resin was dissolved by 80%ethanol-water solution,then the ethanol concentration was diluted slowly to 20%by water and the precipitation was centrifuged to collect after incubation for 0.5 hours in normal temperature.46.5±0.8g LUT could be collected and the purity of Luteolin in the product was above 50%in the duplicated test.2.The method of preparing PEP was as follows:the crude extracts from 2.5 kg flowers of Dendranthema Indicum was extracted sufficiently by 125ml ethyl acetate one time,125 ml ethanol one time,125 ml 40%ethanol-aqueous solution two times and 125 ml 20%ethanol-aqueous solution one time,respectively.The purity of Buddleoside in DEP was above 50%,the recovery rate was about 64.8±1.2%;It was proved that the temperature had no influence on the stability of method.The products obtained from the duplicated test were stable.3.(1)The effect evaluation of PEP、DEP and P&DEP:①The blood pressure containing SBP,DBP and MAP in the treated groups except PEP(150 mg/kg)and P&DEP(60&75mg/kg)were reduced obviously compared with SHR control group.And the blood pressure of P&DEP(45&30 mg/kg)was lower significantly than any other single drug treated group.②Microcirculation:The microcirculation of rat’s tail of all treated groups had no obvious difference with those of SHR control group;③Blood lipid:All treated groups could lower TG significantly but only G12 could lower TC significantly in the treated groups,and no treated groups had effect on the HDL-c;④Liver and kidney function:In the examination of effect of BUD and LUT on the liver/kidney,we found that only P&DEP(45&30mg/kg)and P&DEP(75&15mg/kg)could lower Cre significantly and only DEP(75mg/kg)could lower UA obviously;(2)In the examination of blood indexes:① RAAS in the treated groups(except valsartan group)were suppressed significantly compared with in the SHR model group,valsartan group was only effective to suppress the ALD significantly;In the treated groupsPEP(75 mg/kg)、DEP(75 mg/kg)、P&DEP(30:90 mg/kg)、P&DEP(45:30 mg/kg),ACE/Ang Ⅱ in the kidney was lowered while ACE2 was updated;In addition,the signaling pathway of Erkl/2 was inhibited obviously.②Endothelium function:inflammation factor,IL-1βin the treated groups except P&DEP(90&60mg/kg)was lowered significantly than in the valsartan group,and TNF-αin part treated groups(Valsartan、PEP(75 mg/kg)、DEP(75 mg/kg)、P&DEP(30:90 mg/kg)、P&DEP(45:30 mg/kg))was reduced obviously(P<0.01);Vasoconstrictor and vasodilator:EDCF in the treated groups(except)was lowered significantly than in the model group,and TXA2 in part treated groups(Valsartan、PEP(75 mg/kg)、DEP(75 mg/kg)、P&DEP(30:90 mg/kg)、P&DEP(45:30 mg/kg))was reduced obviously,but the EDRF、EDHF and ET-1 in all treated groups had no obvious difference with in the model group;③Immune globulin:IgG in all treated groups had obvious difference with in the model groupand the same with IgM(except valsartan and PEP75mg/kg);④Gastrointestinal hormone:Resistin and SP in all treated groups were lowered significantly,but leptin was not lowered obviously;⑤Bioactive peptide:NPY in all treated groups had significant difference with in the model group,but NT had not;4.The blood vessel protection effect of PEP and DEP:(1)The inhibition effect of endothelium cell damage:In the DEP and PEP treated groups,the expression of ACE/AngⅡwas suppressed obviously while ACE2 was enhanced significantly;In the stimulation of H2O2,the ROS and apoptosis rate of HUVEC were enhanced while the proliferation activity was reduced obviously.These variations were inhibited by the treatment of DEP(PEP had no effect).The expression of SOD was enhanced to improve the tolerance of HUVEC against ROS with the improvement of phosphorylation of Nrf-2 mediated by the activation of PI3K/Akt.(2)The inhibition of proliferation and migration of VSMCs induced by AngⅡ:At the same time,in the stimulation of AngⅡ,the ROS in the VSMCs was enhanced significantly,and whatsmore the proliferation rate and migration rate were also enhanced obviously.But the treatment of DEP and PEP both could block these variations.The mechanism was related with the activation of Erk1/2 and the reduction of MMP-2/MMP-9;(3)The inhibition of vessel remodeling:In vivo assays,the ratio between thichness and lumen was reduced significantly in the treatment of DEP and PEP compared with in SHR control group.(4)The endothelial function protection:In addition,the endothelium-derived contracting factors were reduced obviously in the BUD and LUT treated groups.Conclusion 1.The developed method for purifying the peanut shell effective parts was reliable,the purity of Luteolin in the products was above 50%.2.The developed method for purifying the Dendranthema Indicum effective parts was reliable,it could be used to prepare products with hectogram level.3.Effect evaluation:(1)The administration of DEP,PEP and P&DEP with different ratios and dosages was effective to lowering blood pressure for SHR,and P&DEP(45:30mg/kg)had the optimum efficiency;(2)PEP,DEP and P&DEP all have part of hypolipidemic effects to lower TG;PEP and DEP had no obvious influence on the function of liver/kidney;The mechanism of antihypertension:(1)The antihypertensive effect of PEP and DEP had a direct relation with the inhibition of circulatory RAAS and local RAAS in the kidney,and the adjustment of local RAAS in the kidney might be medicated by the inhibition of ERK 1/2 signaling pathway.(2)The protection of endothelial function:It could inhibit the index of inflammation and relax/shrink blood vessel.In addition,the antihypertensive effect might be related with the indexes adjustment of Ig,gastrointestinal hormone and bioactive peptide.4.One of the main mechanisms of antihypertensive effect of DEP and PEP was to reduce the damage of blood vessel and protect the endothelial function caused by ROS induced by RAAS.PEP and DEP could protect the endothelial function and inhibit the vascular remodeling,the mechanisms contained several aspects:(1)In vivo assays,it also showed that PEP and DEP could reduce the oxdative stress through inhibiting the expression of AngⅡ in the aorta mediated by ACE and ACE2.(2)Through the improvement of phosphorylation of Nrf-2 mediated by the signaling pathway of PI3K/Akt,DEP could promote the expression of SOD to improve the tolerance of endothelial cells to the ROS;(3)LUT(the main effective components of PEP)and BUD(the main effective components of PEP)could reduce the ROS induced by AngⅡ,and also could inhibitthe proliferation and migration of VSMCs through the regulation of Erk1/2 and MMP-2/9;...