| ObjectiveThe incidence of hyperlipidemia is increasing, threatening our physical health. Hyperlipidemia is primarily based on cholesterol of plasma, triglycerides, low-density lipoprotein, high density lipoprotein decreased to a state of blood lipid metabolism disorder state. Cholesterol metabolism disorder can bring serious adverse effects to the body, increased levels of plasma cholesterol is a major factor in causing atherosclerosis and containing a large amount of cholesterol in the atherosclerotic plaque, thus contributing to the accumulation of cholesterol in the blood vessel walls, which can be cause a series of cardiovascular diseases.Although a variety of western medicine are very significant lipid-lowering effect. But long-term use has different levels of side effects. Scholars based on the research of the traditional Chinese medicine, try to find out safe, reliable and effective herbal natural medicines of lipid-lowering. Many scholars study lipid-lowering of Botanicals, and at the same time long-term large number of experiments constantly have new advances and discoveries.This experiment, through in-vitro lipid-lowering experimental screening of the optimum combination of Composite Codonopsis Lanceolate (CCL), and according to the optimization of fermented Composite Codonopsis Lonceoloata (FCCL). Moreover, Fermented Composite Codonopsis Lonceoloata (FCCL) and non-fermented Composite Codonopsis Lonceoloata (NCCL) carry on the contrast experiment and found better lipid-lowering effect of FCCL. Finally, by hypolipidemic in-vivo experiments to explore the prevention effect of CCL for lipid-lowering and its mechanism.Medthods① Extracting separately Codonopsis lancelata, Lycium barbanma, and Onion skin. And in-vitro cytotoxicity experiment with the best concentration of screened three drugs. By L9 (34) orthogonal design, according to CCL extracts in-vitro, cell concentration affect the value of the internal and external TC, otherwise select the optimal formula combination of CCL.② According to the absorbing component of serum pharmacological experiments, taking nine rats, according to L9 (34) orthogonal design test configuration 9 groups CCL different set of liquid, in the light of a lavage 0.5 (g/kg · bw, in raw medicinal materials), the concentration of 5 times, continuous lavage 5 d,2 h after the last delivery, aseptic conditions eyes take blood, separation of collect serum in rats. Furthermore, it will be 9 group of rats serum, according to the categories in order to join training logarithmic phase of HepG2 cells (2 x 105/mL) culture, and set up blank control group (containing 10% fetal bovine serum), model control group (containing 50% fetal bovine serum).③ Lipid-lowering in-vivo experiments, randomly divided into 6 groups according to the total concentration of TC result, namely the normal control group, model control group, CCL high, medium and low dose groups (0.5,1,2 g/kg·bw in raw medicinal materials) and simvastatin group (10 mg/kg). Normal control group, model control group mice distilled water (1 mg/mL); CCL high, medium and low dose group mice amount of different concentration extract; Simvastatin group equal amounts of simvastatin lavage solution. Continuous lavage 4 w, weighing 2 times a week, and adjust the dosage according to the weight. Any time at the end of the fasting 12 h after the treatment, drinking water, ether anesthesia pick eyeball blood, cervical vertebra dislocation to death. Determination of serum TC, TG, LDL-C, HDL-C.④ CCL screening active dry yeast fermentation conditions, using L9 (34) orthogonal design method, with the content of total flavonoids as observation index, optimizing the best conditions for fermentation, extraction CCL. Respectively FCCL, NCCL ethanol extract, D101 macroporous adsorption resin column and the refined extract, respectively according to the concrete measurement steps for determination of total flavonoids in FCCL, NCCL ethanol extract and the content of total saponins.⑤) Oil Red O staining experiments were configured FCCL, NCCL ethanol extract of the experimental concentration, added to the zhang the logarithmic phase inside the liver cell culture, cultivate 48 h, observed intracellular lipid droplets.⑥ FCCL body lipid-lowering effect, the rats ether anesthesia, extract blood from tail, according to the TC results randomized into 6 groups, namely blank control group, model control group, normal FCCL low, medium and high dose group (0.0625,0.125, 0.5 g/kg·bw in raw medicinal materials) and simvastatin group (10 mg/kg). Normal control group, model control group mice distilled water (1 mg/mL); FCCL lavage, respectively, high, medium and low dose group, the same amount of different concentrations extract; Simvastatin group equal amounts of simvastatin lavage solution. Continuous lavage 6 w, weighing 2 times a week, and adjust the dosage according to the weight. Any time at the end of the fasting 12 h after the treatment, drinking water; Ether anesthesia, picked eye take blood, cervical vertebra dislocation executed, quickly removed the liver. Enzyme colorimetric determination of serum TC, TG, LDL-C, HDL-C, HMG-CoA, LCAT. Liver homogenate, take supernatant fluid under test. Determined according to kit of liver homogenate NO, NOS, iNOS content.Results① Combined with our previous findings, to determine the mass concentration of the three kinds of raw materials extract the best formulation screening the Codonopsis lanceolata at 0,100,200 μg/mL; onion skin 0,20,40 μg/mL; Lycium barbanma 0,40, 80μg/mL.② The best ratio of mass concentration is:Codonopsis lanceolata extract objects 200 μg/mL, Lycium barbanma extract 40 μg/mL and onion skin extract 40 μg/mL, added to the characteristics of culture cholesterol content is minimum. Thus the optimal formula combination of CCL for Codonopsis lanceolata extracts are 200 μg/mL, onion skin extracts 40 μg/mL and Lycium barbanma extract 40 μg/mL. Besides, Codonopsis lanceolata, onion skin and Lycium barbanma three extracts of mixing ratio is 5:1:1③ The CCL in vivo lipid-lowering experimental results show that the CCL is low, medium and high dose groups serum TC concentration was significantly lower than that in the model control group, the difference was statistically significant (P<0.05, P <0.01); CCL high、 medium and low-dose group of serum LDL-C concentration was significantly lower than that in the model control group, the difference was statistically significant (P<0.01). Clinical first-line lipid-lowering drug simvastatin-the positive control group of serum TC, LDL-C was significantly lower than the model group (P<0.01).④ The CCL active dry yeast best conditions for screening, the amount of active dry yeast was added to 8 g, glucose for 20 g, fermentation time is 3 days, extraction of total flavonoids was highest. FCCL with NCCL low, medium and high does groups were intracellular lipid drops significantly less than the model control group, and the FCCL with NCCL high dose group of cells within the lipid droplets to a minimum. The result shows that the FCCL high dose group is better reduce the content of intracellular lipid droplets than the NCCL high dose group.⑤ FCCL ethanol extract objects in rats, with experimental results show that, FCCL ethanol extract significantly reduced the role of lipids, otherwise based on experimental data indicates that the effect of the the FCCL high dose group lipids regulating and clinical first-line lipid-lowering drug simvastatin close.Conclusions(1) The concentration of the optimum combination of extracts derived from lipid-lowering of Codonopsis lancelata, Lycium barbanma, and Onion skin: Codonopsis Lanceolata extract 200 μg/mL, Onion Skin extracts 40 μg/mL, Lycium barbanma extract 40 μg/mL, besides Codonopsis lanceolata, onion skin and Lycium barbanma three extracts of mixing ratio is 5:1:1(2) The optimal dose of CCL lipid-lowering effect in vivo is 0.5 g/kg·bw.(3) CCL in active dry yeast was 2%, the amount of glucose was 5%, fermentation time for 3 d, total flavonoids and total saponins content was significantly increased and enhanced lipid-lowering effect.(4) FCCL extract can inhibit cholesterol synthesis rate-limiting enzyme. |
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