Anti-hyperlipidemia Effect And Molecular Mechanism Of The Total Flavonoid Extract From Coreopsis Tinctoria

The flowers of Coreopsis tinctoria Nutt., so called Snow chrysanthemum, have been applied as a kind of popular tea flowers to prevent cardiovascular disease in Xinjiang district in China. It was recorded to possess detoxification, dampness and dysentery effects in Uygur folk medicine. Recent research convinced hypoglycemic, hypotensive,hypolipidemic and other functions of this herb, with flavonoids as the main bioactive ingredients. However, the true compounds responsible for its hypolipidemic effect remained to be clarified. Previously, the total flavonoid extract of Coreopsis tinctoria(CTE)has been processed from our group, which was proved to possess anti-hyperlipidemia effect in vivo. In the present thesis, separation and determination of four typical flavonoids of CTE was developed, followed by investigations of reducing triglyceride activities of CTE and some of these compounds using in vivo assays. Molecular mechanisms of hypolipidemic function of this herb will be partly elucidated accordingly.The dry flowers of Coreopsis tinctoria were extracted with 75% alcohol under reflux,the extract was condensed and then refined by an AB-8 macroporous resin column to give CTE as reporded before. Four flavonoids, Flavanomarein(Fla, 1), isookanin(2), Marein(3)and Okanin(4) were then separated with success using LX2000 resin, C18 silica gel and Sephadex LH-20 gel columns in proper order. Their structures were identified by UV, MS,1H- and13C- NMR and other spectroscopic techniques. A dual-wavelength HPLC method was then established for simultaneous determination of compounds 1-4 for the first time.An Agilent 1260 HPLC system was applied using Cosmosi L AR-II-C18(4.6mm×250mm,5μm) column; the mobile phase was acetonitrile(A)- 0.1% acetic acid(B), with graduate elution of 16% A(0-20 min) followed by 16-25% A(20-35 min) and followed by25%-90%A(35 ~ 45 min); a DAD was applied, with the detection wavelengths setting at 280(for flavanomarein) and 378 nm(for marein); the flow rate and column temperature was set at1.0 m L/min and 30 °C, respectively; and sample volume was 20 μL. Under this condition,the content of compounds 1-4 of CTE was determined to be 64.31, 20.54, 114.3 and16.64mg·g-1, respectively. Nine flavonoids in CTE were also identified by a LC/MS/MS analysis.The methods developed here were accurate and selective, which could be applied for the quality control of Coreopsis tinctoria and its extract accordingly.The cultured Human Hepatocellular Carcinoma Cell(Hep G2) were divided into normal, model, CTE(3.75、7.5、15 μg/m L), Marein(0.0125、0.025、0.05 μmol/m L), Fla(0.025、0.05、0.1 μmol/m L) and fenofibrate 200 μg/m L groups, respectively. To observe the influence of its lipid metabolism by different doses of CTE and the main compounds Marein, Flavanomarein, after Oleic acid induced steatosis in Hep G2 cells. After incubation with drugs for 24 hours, the triglyceride(TG),the Intracellular free fatty acid(FFA) and the cultured supernatant FFA were measured with Oil red staining and kits. In addition, the gene expressions of peroxisome proliferator-activated receptor(PPAR)α, diacylglycerol acyltransferase(DGAT) and carnitine palmitoyltransferase(CPT)1A were measured with Western-Blot. Results:(1) contrast the model group with the normal group, the intracellular TG and FFA levels and cultured supernatant FFA level were siginificantly increased after treatment with OA for 24h(P<0.01). Compared with the model group, the intracellular TG and FFA levels and cultured supernatant FFA level were siginificantly decreased after treatment with CTE(7.5 μg/m L、15 μg/m L) or Marein(0.025 μmol/m L equivalent to 22.45 μg/m L 、 0.05 μmol/m L equivalent to 11.25 μg/m L) for 24 hours(P<0.01 or P<0.05). But the Flavanomarein(0.025、0.05、0.1μmol/m L) did not have this effect(P>0.05). The results of Oil red staining is the same with it.(2) Western-Blot results:The CTE and marein could siginificantly increase the gene expressions of PPARα and CPT1A(P<0.01 or P<0.05), decrease the gene expressions of DGAT in Hep G2 cell(P<0.01 or P<0.05). It is showed that the mechanism of action may be related to the expression of DGAT and CPT1 A, which are regulated by the activation of PPAR.Conclusion:For the first time, the results showed that the CTE of the Coreopsis tinctoria.had significantly activity of reducing triglyceride. Marein is one of the effective components.