苹果根皮苷氧化物POP2的制备及其抑制3T3-L1前脂肪细胞增殖与分化的研究

【作者】 王娟；

【导师】 孟永宏；

【作者基本信息】 陕西师范大学 ， 工程硕士（专业学位）， 2018， 硕士

【摘要】 类黄酮及其氧化产物,如儿茶素、茶多酚、茶黄素、茶红素等,均具有非凡的降脂功能。苹果多酚及其氧化产物也是一类重要的类黄酮,并且在苹果酵素发酵过程中大量产生。目前,苹果多酚的降脂作用已有报道,但是苹果多酚的氧化产物,尤其其代表性成分根皮苷氧化产物及其降脂作用未见报道。为了拓展苹果多酚的应用领域,本研究以根皮苷为原料,在体外用多酚氧化酶大量制备根皮苷氧化物POP2,研究根皮苷酶促氧化的反应历程,分离纯化后用HPLC、ESI-MS、IR对其结构进行表征,检测对比根皮苷与POP2的自由基清除活性,使用3T3-L1前脂肪细胞模型评价POP2的降脂功能,并通过RT-PCR和蛋白免疫印迹检测,研究POP2抑制脂肪细胞分化的分子机制。取得的主要结果如下:(1)根皮苷酶促氧化的最佳条件为:pH=6.5、温度为35℃、底物浓度为3 mg/mL,乙醇添加浓度为20%。当酶促氧化时间为48 h时,反应体系中仅存在POP2—种氧化产物,此时根皮苷的转化率为90.2%,分离纯化后得到纯度为94.7%的氧化物POP2,用质谱分析得到POP2的m/z为467,IR分析得到官能团的特征吸收峰,与推测的POP2的结构式吻合。(2)利用DPPH和ABTS试剂检测了根皮苷和POP2清除自由基的能力,DPPH自由基清除浓度(IC50)分别为:POP2为102.56±0.09μg/mL,根皮苷为278.79±0.34μg/mL,ABTS 自由基清除浓度(IC50)分别为:POP2 为 93.99±0.19μg/mL,根皮苷为153.18±0.24μg/mL,证明了 POP2清除自由基的能力远远强于根皮苷。以POP2为处理药物作用于3T3-L1前脂肪细胞,MTT法检测到POP2可以抑制3T3-L1前脂肪细胞的增殖,IC50分别为:24 h时为1214.342±0.445 μg/mL,48 h时为991.765±0.854μg/mL,当POP2浓度大于300μg/mL就会对细胞产生毒性,细胞的形态发生改变,数目减少,所以分化时选取POP2的浓度梯度均小于300 μg/mL。通过油红O染色观察到POP2处理后的细胞脂滴数量明显减少,定量分化结果显示当POP2浓度为250 μg/mL时可以抑制47.87%前脂肪细胞的分化,细胞内TG的含量随着POP2浓度的增加也逐渐降低,说明POP2能显著抑制前脂肪细胞的分化,抑制细胞内TG的积累。(3)通过RT-PCR检测细胞分化的关键转录因子,当POP2的浓度在50～250μg/mL 时,可以显著下调 PPARγ、C/EBPα、FAS、GLUT-4 的表达;通过 Western blot检测了分化过程中关键蛋白PPARγ和C/EBPα蛋白的表达,发现POP2可以抑制这两种蛋白的表达,并且具有剂量依赖性。本课题利用3T3-L1前脂肪细胞评价了 POP2的降脂功能,推测POP2抑制脂肪细胞分化的机制可能是通过抑制PPAR γ和C/EBP a基因的表达,从而下调其下游基因FAS和GLUT-4,抑制脂肪细胞中脂质的合成,同时证明了根皮苷氧化物POP2有作为抗氧化剂和降脂类成分的潜力,为其应用奠定了理论基础。更多还原

【Abstract】 Flavonoids and their oxidation products,such as catechin,tea polyphenol,theaflavins,and thearubigins,all have the extraordinary lipid-lowering function.Apple polyphenols and their oxidation products are also important flavonoids and produced in large quantities during the fermentation of apple enzymes.At present,the lipid-lowering effect of apple polyphenols has been reported,but the oxidation products of apple polyphenols,especially its representative component phloridzin oxidation products and its lipid-lowering effect has not been reported.In order to expand the application of apple polyphenols,in this study,the phloridzin oxide POP2 was prepared by polyphenol oxidase in vitro and to study the reaction course of enzymatic oxidation of phloridzin.The structure of POP2 was characterized by HPLC,ESI-MS and IR after separation and purification.The free radical scavenging activity of phloridzin and POP2 were detected,the anti-obesity function of POP2 was evaluated by 3T3-L1 preadipocyte model.The molecular mechanism of POP2 inhibiting adipocyte differentiation was detected by RT-PCR and Western blot.The main results are as follows.(1)Phloridzin enzymatic oxidation of the optimal conditions for:pH=6.5,temperature of 35 ℃,substrate concentration of 3 mg/mL,ethanol addition concentration was 20%.When the enzymatic oxidation time was 48 h,only one oxidation product of POP2 was present in the reaction system.At this time,the conversion rate of phloridzin was 90.2%,and the purity of 94.7%of POP2 was obtained after isolation and purification.The m/z of POP2 was 467 by the ESI-MS positive mode,the molecular weight was 466.The characteristic absorption of the functional group obtained by IR analysis was in agreement with the structural formula of POP2.(2)The free radical scavenging ability of phloridzin and POP2 were detected by DPPH and ABTS reagents.The IC50 concentration of DPPH free radical scavenging was 102.56 ±0.09 μg/mL for POP2 and 278.79 ±0.34 μg/mL for phloridzin,respectively.The IC50 of ABTS free radical scavenging was 93.99 ±0.19μg/mL for POP2 and 153.18 ±0.24 μg/mL,respectively.We proved the ability of scavenging free radicals is much stronger than phloridzin.The 3T3-L1 preadipocyte model was used as the experimental object,POP2 was used as the treatment drug,and MTT assay was detected POP2 can inhibit the proliferation of 3T3-L1 preadipocytes.The IC50 of 24 h and 48 h was:1214.342±0.445 μg/mL and 991.765±0.854 μg/mL,respectively.When the concentration of POP2 was greater than 300 μ/mL,it would be toxic to cells,and the morphology of cells would change and the number would decrease.Therefore,the concentration gradient of POP2 was less than 300 μg/mL in cell differentiation.The number of lipid droplets after POP2 treatment was significantly reduced by oil red O staining.The results of quantitative differentiation showed that when POP2 concentration was 250 μg/mL,47.87%of preadipocyte differentiation could be inhibited to differentiation.The content of intracellular TG gradually decreases with the increase of POP2 concentration.It indicated that POP2 can significantly inhibit the differentiation of preadipocytes and the accumulation of intracellular TG.(3)The key transcription factors for cell differentiation were detected by RT-PCR.When the concentration of POP2 was 50-250 μg/mL,the expressions of PPARγ,C/EBP α,FAS,and GLUT-4 were significantly down-regulated.The expression of key proteins PPAR γ and C/EBP α were detected by Western blot.It revealed that POP2 can inhibit the expression of these two proteins in a dose-dependent manner during differentiation.In this study,3T3-L1 preadipocytes were used to evaluate the lipid-lowering function of POP2.It was speculated that the mechanism of POP2 inhibiting adipocyte differentiation may be through down-regulating the expression of PPARγ and C/EBPαgenes,thereby down-regulating the downstream genes FAS and GLUT-4 and inhibit the synthesis of cellular lipids.At the same time,it was proved that the phloridzin oxides had the potential as an antioxidant and lipid reducing component,laying a theoretical foundation for its application.更多还原