昆仑胎菊水溶性黄酮提取工艺优化及其对衰老小鼠肝组织SIRT1、p53表达的影响

Optimization of Extraction Process of Water-Soluble Flavonoids from Coreopsis tinctoria Buds and Its Effect on Expression of SIRT1 and p53 in Liver Tissues of Aging Mice

采用水溶剂法提取昆仑胎菊水溶性黄酮(water-solubleflavonoids from Coreopsis tinctoria buds,CBF)。在单因素试验的基础上,以提取液中总黄酮提取量为指标,响应面法优化得到CBF提取最优工艺:水提时间15 min、水提温度96℃、液料比71∶1(mL/g),在此条件下水溶性总黄酮提取量可达到145.45 mg/g。探讨CBF对D-半乳糖致衰老小鼠的抗衰老作用及其对衰老小鼠肝组织中沉默信息调节因子1(silent information regulator 1,SIRT1)、p53蛋白表达的影响。以长期皮下注射D-半乳糖(300 mg/(kg·d))建立衰老小鼠模型,连续造模45 d,建模的同时分别灌胃VE(50 mg/(kg·d))、CBF(180、700 mg/(kg·d)),观察各组小鼠体质量变化及行为学状态,HE染色法观测肝组织病理形态学变化,并采用免疫印迹法评价衰老小鼠肝组织中SIRT1、p53蛋白的表达。结果表明,CBF对小鼠生长发育无不良影响且可改善衰老小鼠的行为状况;显著减轻D-半乳糖引起的肝组织病理学损伤;显著提升衰老小鼠肝组织中SIRT1蛋白表达(P<0.05),有效抑制D-半乳糖引起的p53蛋白过量表达(P<0.05)。结论:响应面法建立的回归数据模型优化得到的CBF水提取工艺条件实际有效,同时发现CBF具有良好的抗衰老作用,其作用机理可能与激活SIRT1/p53信号通路有关。

Water-soluble flavonoids(CBF) were extracted from Coreopsis tinctoria buds using water as the extraction solvent. By combined used of one-factor-at-a-time method and response surface methodology, the optimal extraction conditions that provided the maximum yield of total flavonoids of 145.45 mg/g were determined as follows: extraction time15 min, temperature 96 ℃ and water-to-material ratio 71:1(mL/g). Thefinal aim of this study was to investigate the antiaging effect of CBF on D-galactose-induced aging mice and its impact on silent information regulator 1(SIRT1) and p53 protein expression in the liver of aging mice. The aging mouse model was established by long-term subcutaneous injection of D-galactose(300 mg/(kg·d) for 45 consecutive days, and the mice were also orally administered with either vitamin E(VE, 50 mg/(kg·d)) or CBF at 180 and 700 mg/(kg·d) during this period. The body mass and behavioral state of mice in each group were observed. Hepatic histopathological changes were evaluated by HE staining. The protein expression of SIRT1 and p53 in the liver of aging mice were evaluated by Western blotting. Results showed that CBF had no adverse effects on the growth of mice but significantly improved the bad behavior of aging mice. It alleviated the pathological damage to liver tissues caused by D-galactose, increased the expression of SIRT1 protein(P < 0.05), and inhibited the overexpression of p53 induced by D-galactose(P < 0.05). In conclusion, the water extraction method was applied successfully to extract CBF. It was also demonstrated that CBF possessed promising anti-aging effect, and the mechanism might be related to the activation of the SIRT1/p53 signaling pathway.