【作者】 张颖；

【导师】 王月华； 朱金艳；

【作者基本信息】 沈阳农业大学 ， 农业硕士（专业学位）， 2025， 硕士

【摘要】 笃斯越橘(Vaccinium uliginosumL.,VU)是杜鹃花科越橘属植物,富含多酚类活性物质,但其具体功能成分尚不明确,制约了其资源的高效利用。笃斯越橘多酚已被证实具有抗氧化、调节血脂等功能。近年来,去唾液酸糖蛋白受体1(Asialoglycoprotein receptor 1,ASGR1)因可调控胆固醇代谢被认为是新型降脂靶点,然而多酚类化合物是否能通过调控ASGR1表达发挥降脂作用尚待研究。本研究以笃斯越橘多酚(Vaccinium uliginosumL.polyphenol,VUP)为研究对象,系统分析其组成成分,通过体外和体内试验验证VUP对ASGR1的调控作用,并阐明其降脂机制。在此基础上,研发一款笃斯越橘降脂功能产品,为笃斯越橘资源在降脂方面的高值化开发提供科学依据,同时为天然多酚干预脂代谢紊乱提供新策略。主要研究结果如下:(1)全面挖掘笃斯越橘多酚类物质组成,分别提取笃斯越橘游离多酚(VUFP)和结合多酚(VUBP)。结果表明,VUFP和VUBP总酚含量分别为(519.95±4.56)mg/100g FW和(207.37±10.91)mg/100 g FW;VUFP中总花色苷含量为(68.77±0.96)mg/100mL FW,而VUBP中未检出。基于超高效液相色谱-电喷雾-串联质谱(UPLC-ESI-MS/MS)方法进行多酚组分鉴定,共鉴定出以黄酮类及酚酸类为主的885种多酚物质(VUFP 858种,VUBP 763种);同时鉴定出47种花色苷单体(VUFP 35种,VUBP 25种),其中矢车菊素-3-O-(6’’-O-乙酰)葡萄糖苷、矢车菊素-3,5-O-二半乳糖苷、矢车菊素-3-O-(酒石酰)鼠李糖苷-5-O-葡萄糖苷、矢车菊素-3-O-(6-O-丙二酰-β-D-葡萄糖苷)等30种花色苷单体物质是在笃斯越橘新检测到的。值得注意的是,在VU中检测到了天竺葵素及花青素与桑布双糖苷结合的花色苷。(2)利用油酸(OA)建立人肝癌细胞系2(Hep G2)细胞高脂模型,分别给予1、1.5、2 mg/mL浓度的VUP处理。结果表明,与OA组比,经过VUP处理的Hep G2细胞脂滴积累量显著减少20.03%-36.95%,TC、TG含量分别显著降低32.44%-65.82%、11.45%-62.43%,细胞中AST、ALT含量分别降低6.25%-16.39%、11.69%-27.44%,ASGR1含量降低5.1%-36.95%。表明VUP有降低脂质水平、改善肝损伤,降低Hep G2细胞中ASGR1含量的作用。(3)利用高脂饮食诱导C57BL/6小鼠建立高脂血症模型,评估VUP对高脂血症的影响。结果表明,VUP能够减轻高脂血症小鼠体重、降低TC、TG、低密度脂蛋白胆固醇(LDL-C)的含量,提高高密度脂蛋白胆固醇(HDL-C)的含量,并改善肝损伤。VUP抑制ASGR1 mRNA的表达水平,上调ATP结合盒转运蛋白G5(ABCG5)和G8(ABCG8)mRNA的表达水平;抑制ASGR1蛋白表达;上调磷酸化腺苷酸活化蛋白激酶(p-AMPK)的表达,激活腺苷酸活化蛋白激酶(AMPK)通路;抑制乳腺癌易感基因1号(BRCA1)和BRCA1相关RING域蛋白1(BARD1)的蛋白表达水平;上调肝X受体α(LXRα)蛋白表达水平,上调细胞色素P450家族成员7A1(CYP7A1)蛋白表达,降低胆汁胆固醇含量,增加胆汁酸含量;上调ABCG5和ABCG8蛋白表达,促进胆固醇的外排,增加粪便胆固醇含量。(4)以笃斯越橘为主要原料,药食同源原料葛根、玉竹和山楂为辅料,以感官评分、1,1-二苯基-2-苦基肼(DPPH)清除率为指标,设计一款降脂口服液。采用单因素试验及D-最优混料设计试验优化口服液配方,得到笃斯越橘降脂口服液的最优配方为:笃斯越橘汁50%,葛根提取液25%,玉竹提取液15%,山楂提取液10%。口服液感官评分为87.73分,DPPH清除率为92.388%,多酚含量为245.99 mg/100 mL。更多还原

【Abstract】 Vaccinium uliginosumL.(VU)is a bilberry plant in the Ericaceae family.It is rich in polyphenols,but its specific functional components are not clear,which restricts the efficient utilization of its resources.The polyphenols of Vaccinium uliginosumL.have been proved to have antioxidant and lipid regulation functions.In recent years,Asialoglycoprotein receptor 1(ASGR1)has been identified as a novel lipid lowering target because it can regulate cholesterol metabolism.However,whether polyphenols can play a role in lipid lowering by regulating the expression of ASGR1 remains to be studied.Based on this,this study took Vaccinium uliginosumL.polyphenol(VUP)as the research object,systematically analyzed its components,verified the regulatory effect of VUP on ASGR1 through in vitro and in vivo experiments,and clarified its lipid-lowering mechanism.On this basis,a lipid-lowering product of Vaccinium uliginosumL.was developed,which provided a scientific basis for the high-value development of the resource of Vaccinium uliginosumL.in lipid-lowering,and offered a new strategy for the intervention of natural polyphenols in lipid metabolism disorders.The main results are as follows:(1)The composition and functional properties of polyphenols of VU were comprehensively explored,and free polyphenols(VUFP)and bound polyphenols(VUBP)were extracted from VU.The results showed that the total phenol contents of VUFP and VUBP were(519.95±4.56)mg/100 g FW and(207.37±10.91)mg/100 g FW,respectively.The total anthocyanins content in VUFP was(68.77±0.96)mg/100 mL FW,but it was not detected in VUBP.Based on the Ultra performance liquid chromatography-electrospray tandem mass spectrometry(UPLC-ESI-MS/MS)method,a total of 885 polyphenols(VUFP858,VUBP 763)were identified,mainly flavonoids and phenolic acids.At the same time,47anthocyanin monomers(VUFP 35,VUBP 25)were identified,of which 30 anthocyanin monomers such as Cyanidin-3-O-(6’’-O-acetyl)glucoside,Cyanidin-3,5-O-Digalactoside,Cyanidin-3-O-(tartaryl)rhamnoside-5-O-glucoside,and Cyanidin-3-O-(6-O-malonyl-beta-D-glucoside)were newly detected in Vaccinium uliginosumL..Notably,pelargonidin and anthocyanins binding with sambulodisoside were detected in VU.(2)The hyperlipemia model of human hepatocellular carcinoma cell line 2(Hep G2)was established by oleic acid(OA)and treated with VUP at 1,1.5 and 2 mg/mL,respectively.The results showed that compared with the OA group,the accumulation of lipid droplets in Hep G2cells after VUP treatment was significantly reduced by 20.03%-36.95%,and the contents of total cholesterol(TC)and triglyceride(TG)were significantly decreased by 32.44%-65.82%and 11.45%-62.43%,respectively.Aspartate aminotransferase(AST)and alanine aminotransferase(ALT)contents decreased by 6.25%-16.39%and 11.69%-27.44%respectively,and ASGR1 contents decreased by 5.1%-36.95%.These results indicated that VUP could decrease lipid level,improve liver injury and ASGR1 content in Hep G2 cells.(3)Hyperlipidemia model was established in C57BL/6 mice by feeding a high fat diet to evaluate the effect of VUP on hyperlipidemia..The results showed that VUP could reduce the body weight of hyperlipidemia mice,reduce the content of TC,TG and low density lipoprotein cholesterol(LDL-C),increase the content of high density lipoprotein cholesterol(HDL-C),and improve the liver injury.VUP inhibited the expression level of ASGR1 mRNA,and up-regulated the expression levels of ATP-binding cassette transporter G5(ABCG5)and G8(ABCG8)mRNA.VUP inhibited the expression of ASGR1 protein,up-regulated the expression of phosphorylated adenylate activated protein kinase(p-AMPK)and activated the adenylate activated protein kinase(AMPK)pathway.It also inhibited the expression of BRCA1 and BRCA1-associated RING domain protein 1(BARD1),up-regulated the protein expression level of liver X receptorα(LXRα),up-regulated the protein expression of cytochrome P450 family member 7A1(CYP7A1),decreased bile cholesterol content,increased bile acid content.The protein expression of ABCG5 and ABCG8 was up-regulated,which promoted the efflux of cholesterol and increased the content of fecal cholesterol.(4)A lipid-lowering oral liquid was designed based on sensory score and clearance rate of 1,1-diphenyl-2-picrohydrazine(DPPH),using VU as the main raw material,pueraria,polygonatum odoratum and hawthorn as auxiliary materials.The single factor test and D-optimal mixing design test were used to optimize the formula of the oral liquid.The optimal formula of the oral liquid was as follows:50%of the VU juice,25%of the pueraria extract,15%of the polygonatum odoratum extract and 10%of the hawthorn extract.The sensory score of the oral liquid was 87.73 points,DPPH clearance rate was 92.388%,and the polyphenol content was 245.99 mg/100 mL.更多还原