The Studies On Monomer Abstraction And Pharmacodynamics Of Yulangsan Flavone Components

Yulangsan (YLS) is a folk herb widely used in Guangxi province. Its major active components of YLS include flavones, saponins and polycoses. Early researches showed that the extractives from YLS had the positive effects on hypertension, scavenging free radical, relieving myocardial ischemical reperfusion injury, increasing coronary artery flow rate , antiinflammatory and anticancer. Unfortunately, the prior researches were mainly focused on above- mentioned investigations, the active monomers and their mechanisms of action of YLS are not fully known.This study was designed to separate and purify the YLS flavonoid monom- ers and identify their molecular structure. Anti-oxidation in vitro, toleration to hypoxia and anticoagulation in vivo of YLS flavonoid monomers were investi- gated. At the same time, the hypoxia/ reoxygenation(H/R) injury and the H2O2- inducing cadiocyte apoptotic model of cultured neonatal rat cardiomyocytes were produced to inspect the possible mechanisms of YLS flavonoid monomers in vivo and in vitro.Objective:1.To study the technology for extracting total flavonoids from YLS.2.To separate and purify the YLS flavonoid monomers and identify their molecular structures. 3.To evaluate the effects of YLS flavonoid monomers on anti-oxidation in vitro, toleration to hypoxia and anticoagulation in vivo.4.To observe the effects of two flavone morphons on H/R injury in myo- cardial cells and explore its mechanisms.5.To investigate the effects of two flavone morphons on H2O2-induced cadiocyte apoptosis and its mechanisms.Methods:1.The technology for extracting total flavonoids from YLS1.1 Strong aqua reaction , AlCl3 reaction , MgAC reaction and HCl-Mg reaction were used to identify the total flavonoids of YLS.1.2 Rutin was used as control article, the OD275nm of YLS was determined and it was substed to the standard curvilinear equation.The contents of total fla- vonoids were calculated by the following formulas: Content %=C total flavonoids/CYLS×100% Extraction rate %= Content×WT extrac (g)/Crude drug (g)×100%1.3 To optimize the extraction of the total flavonoids from YLS A comparison of extract methods was conducted among the extractions of traditional heat refluxing, microwave and cold diffusion with the extraction yield of total flavonoids from YLS as major targets, meanwhile, the extraction time, extraction frequence, reagent dosage, extraction cost, and personnel and environmental protections were investigated .2.To separate and purify the YLS flavonoid monomers and identify their molecular structures.2.1 Separation:Using petroleum for getting rid of pigment and esters , acetoacetate as a abstracted solvent, the EtoAC extractant from YLS was extracted by the process of condense and cryodesiccation.Reciped a glass chromatographic column(10cm×120cm) aidding pressurize igniter,and gel silica H for TLC was used as separating fixed phase(sample:gel silica H equal 1:30 ). PE / EtOAc mixed solven was used for gradient elution according PE→EtOAc polarity order . The identical portions were merged by TLC.Antioxidative activity of the prefractionational fraction was sieved by using the system of proceeding oxyradical (·O2-) and hydroxy radical(·OH) were established in vitro. The optimal fraction was segregated in multilevel by gel silica column chromatography repeatedly, while different ratio PE/EtOAc was used as an eluant.2.2 Purification:Recryst,PTLC and PHPLC were used to purify the YLS flavonoid mono- mers.2.3 Identification:TLC and HPLC were used to determine the purity of two monomers.UV and IR were applyed to analysis the mother nucleus, substituent and functional groups. ESI-MS was taken to evaluate the molecular mass. NMR was adopted to characterize the molecular structure, while the steric configuration was assay- ed by x-ray monocrystal diffraction technology.3. To study the anti-oxidation of YLS flavonoid monomers in vitro, tolera- tion to hypoxia and anticoagulation in vivo.3.1 The anti-oxidation of YLS flavonoid monomers in vitroYLS A～B were dissolved in 1%DMSO as 0.5, 1.0, 2.0 mg.ml-1 respecti- vely. The cleaning and depressant effects of two flavone monomers from YLS on free radicals were studied by using the system of proceeding·O2- and·OH in vitro while 8 groups were established randomly as follows: normal, positive drug, and the low, middle, high-dose of YLS A～B group.3.2 The effects on anticoagulation in vivo90 mice were randomly divided into 9 groups with 10 mice in each group: normal, solvent,positive drug, and the low, middle, high-dose of YLS A～B group. Capillary tube method of thrombotest was used to investigate the anti- coagulated blood effects.3.3 The effects on toleration to hypoxiaHypoxia model of mice at normal pressure were used to evaluate the anti- hypoxia effects of two flavone monomers.4.The effects of two flavone morphons and the drug-containing sera on H/R injury in myocardial cellsThe hypoxia/reoxygenation injury model of cultured neonatal rat cardio- myocytes was developed in vitro. Active cardiocytes were picked out and divid- ed into following 17 groups : normal, H/R model, positive drug, blank serum, solvent, the low, middle, high-dose of YLS A～B morphons and the drug-con- taining serum.The cell morphous, the survival rate, the contents of MDA , T- SOD, GSH , LDH , NOS, TNF-аand the ctivities of Na+-K+-ATP, Ca2+-Mg2+- ATP enzymes were determined in the cultured neonatal rat cardiomyocytes inju- ried by H/R.5.The effects of two flavone morphons on cadiocyte apoptosis induced by H2O2 and its mechanisms.The cadiocyte apoptosis model was established by adding 100μmol·L-1 H2O2 as final concentration into cell culture fluid for 16h. The grouping and medication were same as above, while the apoptosis rate of the cadiocytes was detected by AnnexV-FITC/PI double staining.The immunohistochemical method was used to detect the protein expressions of NF-κBp65, Bcl-2 and Bax.Results:1.The technology on extracting total flavonoids from YLSThe microwave extraction was superior to the traditional heat refluxing extraction and cold diffusion. The specific technology was just as follows: an ethanol concentration of 60%; an extraction temperature below 60°C; a material to solvent ratio of 1:8; a microwave power of 240W; an extraction time of 20 min with the extraction carried out for one times. According to this technology, the extraction rate of total flavonoids was 3.16 % (vs crude drug), while the pur- ity was 22.5 % (vs rutin).2.The purification and identification of the YLS flavonoid monomersTwo flavonoid monomers were separated from YLS first time. YLS A: C17H16O3 , jasmineneedle crystal ,mp. 120.0-121.0℃;UV(MeOH) ?λmax(nm): 260 ,300; ESI-MS m/z: 291.17 [M+Na]+, 307.13 [M+K]+; IR(KBr)(cm-1):3501 , 3123 , 2929 , 2852 , 1789, 1623, 1604, 1568, 1528, 1462, 1372, 1285, 1228, 1165, 1133; 1HNMR(DMSO-d6 , 500MHz)δ:3.86(6H , s, H-OCH3 X2, H-7, 8), 7.60(1H, brd, J=6.9 Hz, H-4),7.60(5H, m, H-2′,3′,4′,5′,6′), 7.72(1H, J=8.8Hz, H-α),8.00(1H, d, J=8.8Hz, H-?),8.13(2H, brd, J=6.5Hz, H-3, 5); 13CNMR(DMSO-d6 , 125 MHz)δ: 174.2(C-7′),110.6(C-α),121.5(C- ?),119.6(C-1),147.8(C-2, 6),128.6(C-3, 5),130.9(C-4),130.9(C-1′),129.2(C-2′,3′,4′,5′,6′), 60.2(C-7), 60.2(C-8)。Chemical name: Cis-2,6- di-methoxyl chalcone. According to the results retrieving by SciFinder data base, it was separated and reported NMR data primary.YLS B: C18H14O4 , orange formula-crystal, mp. 135.0-136.0℃;UV(MeOH)λmax(nm): 238, 350; ESI-MS m/z: 295.00[M+H]+, 610.99 [2M+ Na]+, 263.35 [M?OMe]+; IR(KBr)(cm-1):3501, 3133, 2929, 2831, 1739, 1600, 1562, 1474, 1380, 1262, 1219, 1137, 1102, 1062, 1025; 1HNMR(CDCl3 , 500MHz)δ3.95(3H, s, -OCH3), 7.2(1H, d, H-5'), 8.2(1H, d, H-4'), 8.17(2H, s, H-2', 6'), 7.77(1H, s, H-α),7.57(5H, m, H-2, 3, 4, 5, 6); 13CNMR(CDCl3 , 125MHz)δ60.2(C-9'), 104.2(C-α),110.0(C-5'), 117.0(C-3'), 119.8(C-8'), 122.0(C-1'), 128.4(C-2'), 128.6(C-2, 4, 6),130.6(C-3, 5),141.8(C-1),145.7(C-4'), 150.0(C-6'), 154.8(C-7'), 158.2(C-?),175.0(C=O);Crystal data: Monoclinic, P2(1)/c, a=10.8813(16)nm, b= 9.7954(14) nm, c= 13.3473(19) nm,α= 90°, ?=103.697(2)°,γ= 90°, V=1382.2(3)nm3 ,Z=4, Dc=1.414 mg/m3, F(000)=616 ,μ=0.100 mm-1.Chemical name : Cis-6'-methoxyl- ?- hydroxy- benzene-furan chalcone. It was a new compound because no identical structure was reported in SciFinder data base up to now. 3.The effects of YLS flavonoid monomers on anti-oxidation in vitro, toleration to hypoxia and anticoagulation in vivo.Two flavone monomers of low, middle and high concentrations (final concentrations were 0.5, 1.0, 2.0mg/ml respectively) could scavenge·O2- and·OH (P<0.05 vs control). Meanwhile, they inhibited the production of·O2- significantly (P<0.05 vs control). The effects were concentration-dependent. Two flavone monomers of middle and high dose could significantly prolong the bleeding time in mice and increased the survival time of hypoxia mice( P<0.05 vs control ). 4.The effects of two flavone morphons on H/R injury in myocardial cells Cadiocyte fused to flakiness and aligned radiatly under optical microscope, in which cell parapodium interlaced reticulation, cell beat synchronization could be observed . The endochylema vacuolization , cell parapodium contracted or disappeared, refractive index decreased, obform cadiocyte increased as well as cell detachment , float , dissolve, necrosis, cell beat extentand frequency inhibi- tion, and rhythm irregularity were displayed in H/R model group.These changes were attenuated significantly in two flavone morphons and their drug-containing sera of middle and high dosage groups(P<0.05 vs model). Compared with the model group, preconditioning by two flavone morphons and drug-containing sera of middle and high dosage could enhance the survival rate of myocardial cells after H/R. Meanwhile, it increased the activities of GSH, cNOS, tNOS, T-SOD, Na+-K+-ATP and Ca2+- Mg2+-ATP enzymes and decreased the releases of MDA , LDH , iNOS , and TNF-а(P<0.05) with dosage depend- ent.5.The effects of two flavone morphons on cadiocyte apoptosis induced by H2O2 and its mechanismsFCM tests showed that the apoptosis rate of cadiocytes in low , middle, high dose of two flavone morphons and their drug-containing sera groups decr- eased significantly compared with those of model group(P<0.01).In addition, compared with the model group, two flavone morphons and drug-containing sera groups at middle , high dose could inhibit significantly the protein expressions of NF-κBp65 and Bax, and it could increase the protein expressions of Bcl-2 and the ratio of Bcl-2/Bax(P<0.01).Conclusions:1.Two flavonoids monomers from YLS are separated initially.YLS A is separated and reported NMR data primary , while YLS B is a new compound according to the data base in SciFinder.2.Two flavone monomers have potent effects on scavenging free radicals, anti-hypoxia, anticoagulated blood, protecting H/R injury in myocardial cells and relieving cadiocytes apoptosis, meanwhile, it shows certain dose-effect relationship. Its protective mechanisms may be related to its anti-oxidative stress injury, increasing the activities of GSH, cNOS, tNOS, T-SOD, Na+-K+-ATP , Ca2+- Mg2+-ATP enzymes , decreasing the releases of MDA, LDH, iNOS, TNF-а, and relieving cadiocyte apoptosis by down-regulating the protein expressions of NF-κBp65 and Bax, up-regulating the protein expressions of Bcl-2 and the ratio of Bcl-2/Bax.