蓝靛果花色苷提取物对脂多糖诱导肝炎的抑制作用机制研究

发布时间：2018-01-01 16:12

本文关键词：蓝靛果花色苷提取物对脂多糖诱导肝炎的抑制作用机制研究　出处：《沈阳农业大学》2017年博士论文　论文类型：学位论文

【摘要】：蓝靛果(Lonicera caerulea L.)是忍冬科、忍冬属植物。蓝靛果果实中含有丰富的花色苷,具有较高的营养、保健和药用价值,有"第三代水果"的美称。研究表明,花色苷具有广泛的生物活性,如抗氧化、抗衰老等,机体长期摄入花色苷可以降低一些慢性疾病的发生率。脂多糖(lipopolysaccharide,LPS)是革兰氏阴性菌细胞壁外膜的主要组成成分,是一种内毒素,它可以通过刺激多种促炎因子表达来引发肝脏免疫反应,进而促进肝脏炎症的发生,然而肝脏炎症可以进一步引起肝硬化、肝癌等慢性肝脏疾病。因此,研究蓝靛果花色苷对LPS诱导肝脏炎症的抑制作用机制,为寻找潜在的保肝药物或功能性食品提供理论依据,具有重要意义。本文在对不同品种蓝靛果个体花色苷定性和定量分析的基础上,利用体外和体内实验,分别从氧化应激、能量代谢、肝脏功能、抗氧化能力、炎性因子表达、凋亡相关蛋白表达和丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路角度探讨蓝靛果花色苷提取物对LPS诱导肝脏炎症的抑制作用及其机理。其主要研究结果如下:采用0.10%HCl酸化的甲醇溶液超声波辅助提取蓝靛果花色苷,根据单因素实验结果,利用响应面分析方法优化提取工艺。经过实验稍作调整后,确定的最佳提取工艺为:料液比1:10、提取时间90 min、提取温度40℃C;D101大孔树脂纯化蓝靛果花色苷的静态吸附解吸优化条件为:吸附平衡时间3 h、解吸平衡时间2h、上样浓度2.52 mg/mL、甲醇浓度100%;动态吸附解吸优化条件为:上样流速2 BV/h、上样量490 mL、洗脱流速2 BV/h、蒸馏水用量2 BV。在此条件下纯化得到的蓝靛果花色苷提取物经冷冻干燥后为紫黑色粉末,色价为57.6,是未纯化时的11倍。通过计算得到花色苷含量为484.2 mgCYD-3-G/L。不同品种蓝靛果果实中花色苷组成及个体比例不同。实验在野生、'蓓蕾'、'NO.1'和'N0.2'四种蓝靛果中分别鉴定出14、11、11和12种个体花色苷。四个品种蓝靛果的主要花色苷均为矢车菊-3-葡萄糖苷,分别占各自总花色苷的89.8%、90.7%、89.6%和91.3%。矢车菊-3-乙酰己糖苷和芍药素-3-乙酰己糖苷首次在蓝靛果中发现,其中矢车菊-3-乙酰己糖苷在'蓓蕾'、'NO.1'和'NO.2'品种蓝靛果中被鉴定,芍药素-3-乙酰己糖苷仅在'NO.2'品种蓝靛果中被鉴定。四种蓝靛果的总花色苷含量无显著差异,野生和'蓓蕾'蓝靛果具有较高的抗氧化能力和总酚含量,'N0.2'品种蓝靛果的总可溶性固形物含量最高。实验通过比较分析不同品种蓝靛果花色苷组成和抗氧化能力,结合实际情况,选定适宜食用且抗氧化能力相对较高的'蓓蕾'品种蓝靛果作为后续实验的研究材料。通过测定体外模拟消化前、后样品中总花色苷含量和抗氧化能力相关指标发现:体外消化降低了总花色苷含量和抗氧化能力。体外消化后蓝靛果提取物的总花色苷含量降低67.6%,氧自由基清除能力(oxygen radical absorbance capacity,ORAC)值和总抗氧化能力(total antioxidant capacity,T-AOC)值分别下降了 80%和55.6%。此外,与未消化样品相比,体外消化后蓝靛果提取物中花色苷种类减少3种、酚酸减少1种。可见,体外模拟消化后样品的总花色苷含量及种类减少,抗氧化能力降低,由此可以推测经体外消化后的蓝靛果花色苷提取物的生物活性会下降。消化前、后的蓝靛果花色苷提取物可以有效地抑制LPS诱导的活性氧(reactive oxygen species,ROS)、8-羟基鸟核苷酸(8-hydroxydeoxyguanosine,8-OHdG)、蛋白羟基组、脂质过氧化产物的生成和谷胱甘肽(glutathione,GSH)的消耗;阻止ATP合成酶、细胞色素 c 氧化酶(cytochrome oxidase,COX)和琥珀酸脱氢酶(succinate dehydrogenase,SDH)活性降低和促炎因子表达(interleukin(IL)-1β和IL-6)上调;调节细胞内谷草转氨酶(aspartate aminotransferase,AST)、谷丙转氨酶(alanine aminotransferase,ALT)和胆碱酯酶活性;同时维持细胞结构。由此可知,蓝靛果花色苷提取物可以通过抑制细胞内氧化应激反应,维持细胞内能量代谢,调节肝细胞功能,来抑制LPS诱导的BRL-3A细胞炎症的发展。而且,未消化花色苷提取物的抑制效果好于消化后花色苷提取物的抑制效果。消化前、后蓝靛果花色苷提取物可以削减LPS诱导的细胞内过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)活性下降,进而维护细胞的抗氧化能力,表现为较高的T-AOC值;能够抑制细胞内核转录因子-κB(nuclear factor-kappa B,NF-κB)和肿瘤坏死因子(tumor necrosis factor-α,TNF-α)表达上调和IL-10表达下调,从而一定程度地阻止了炎症的发展;可以削减细胞内Caspase-3、cleaved Caspase-3、Bax、cleaved poly(ADP-ribose)polymerase(PARP)蛋白表达量增加和 PARP、B 淋巴细胞瘤-2(B-cell lymphoma,Bcl-2)蛋白表达量降低,进而阻止细胞异常凋亡。可见,蓝靛果花色苷提取物还可以通过抗氧化、抗炎和抗凋亡途径抑制LPS对肝细胞造成的损伤,对肝细胞具有一定的保护作用。此外,实验通过对大鼠肝脏组织及血清中相关指标分析,发现蓝靛果花色苷提取物可以通过抑制氧化应激反应(ROS、GSH)、肝功能失调(AST、ALT)、细胞周期循环异常、组织结构损伤、炎症因子表达(C反应蛋白(C-reactive protein,CRP)和IL-6),以及阻碍氧化应激反应和Toll样受体(toll-like receptors,TLR2和TLR4)相关联的MAPK信号通路转导,进而抑制肝脏炎症的发展,对肝脏具有一定的保护作用。并且,其对肝脏的保护效果在50-200 mg/kg bw范围内呈剂量依赖性。本实验结果补充了蓝靛果花色苷潜在的抗炎作用机制,为其作为预防慢性肝炎的功能性食品或补充药物提供理论依据。
[Abstract]:EELC (Lonicera caerulea L.) is Caprifoliaceae Lonicera edulis. Fruit contains rich anthocyanin, has high nutritional and medicinal value, health care, the "third generation fruit" reputation. The results show that anthocyanins have extensive biological activities, such as antioxidant, anti-aging, long body the intake of anthocyanins can reduce the incidence of some chronic diseases. Lipopolysaccharide (lipopolysaccharide, LPS) is a major component of the outer cell wall of Gram-negative bacteria, is a kind of endotoxin, it can promote the expression of inflammatory factor to trigger liver immune response by stimulating variety, and promote liver inflammation, liver inflammation can lead to cirrhosis, but further liver cancer, chronic liver disease. Therefore, inhibition mechanism of loniceraedulis anthocyanin induced inflammation of the liver on LPS, for finding potential liver protection medicine or functional food The theory basis, has important significance. Based on different varieties of Lonicera edulis anthocyanin individual qualitative and quantitative analysis on the use of in vitro and in vivo, respectively from oxidative stress, energy metabolism, liver function, antioxidant ability, expression of inflammatory factors and apoptosis related protein expression and mitogen activated protein kinase (mitogen-activated protein kinase. MAPK) to investigate the inhibitory effect and mechanism of loniceraedulis anthocyanin extract on LPS induced liver inflammation signal pathway angle. The main results are as follows: extraction of anthocyanins from Lonicera edulis Turcz by ultrasonic assisted acidified methanol solution of 0.10%HCl, according to the results of single factor experiments, the response surface analysis method to optimize the extraction process. After the experiment after a slight adjustment, the best the extraction process was determined as follow: solid-liquid ratio 1:10, extraction time 90 min, extraction temperature 40 C; purified sweetberry anthocyanin D101 macroporous resin The optimum conditions were: adsorption and desorption adsorption equilibrium time is 3 h, the desorption equilibrium time is 2h, the concentration of sample was 2.52 mg/mL, 100% methanol concentration; dynamic adsorption and desorption conditions were as follows: the optimal sampling rate 2 BV/h, sample volume 490 mL, elution flow rate of 2 BV/h, the volume of distilled water for 2 BV. in a purified extract the loniceraedulis anthocyanin obtained by freeze dried purple black powder, the color value is 57.6, is 11 times the purification can be computed. The anthocyanin content of different anthocyanins in 484.2 different varieties of mgCYD-3-G/L. indigo cosmetics. Composition and proportion of individuals. The experiment in the wild, 'Bud','NO.1'and'N0.2' four Lonicera edulis were identified in 14,11,11 and 12 kinds of individual anthocyanins. Four main varieties of loniceraedulis glycosides are cyanidin -3- glucoside, their total anthocyanins respectively 89.8%, 90.7%, 89.6% and 91.3%. -3- acetyl hexoside cornflower and Peony -3- acetyl hexoside first found in Lonicera edulis, including Centaurea -3- acetyl hexoside in 'Bud', identified'NO.1'and'NO.2' varieties of Lonicera edulis, paeonidin -3- acetyl hexoside was only identified in'NO.2'varieties of Lonicera edulis. There was no significant difference between the total anthocyanin content of four wild Lonicera edulis, and' Bud 'edulis has a higher antioxidant capacity and total phenol content, total soluble'N0.2' species of Lonicera edulis solids content is the highest. The experiment through the comparative analysis of composition and antioxidant capacity of different varieties of Lonicera edulis anthocyanin, combined with the actual situation, select appropriate edible and antioxidant capacity of the relatively high 'Bud' varieties of Lonicera edulis as research the material for the following experiments. Through the determination of in vitro digestion, find related indicators in the sample after the total anthocyanin content and antioxidant ability in vitro digestion reduces the total anthocyanin content and antioxidant can . the total anthocyanin content of Lonicera edulis extracts in vitro after digestion decreased 67.6%, oxygen free radical scavenging ability (oxygen radical absorbance capacity, ORAC) and total antioxidant capacity (total antioxidant, capacity, T-AOC) value dropped 80% and 55.6%. respectively. In addition, with no digestion samples compared to in vitro after digestion of anthocyanins indigo fruit extract 3 fewer species, 1 kinds of phenolic acids decreased. Obviously, in vitro digestion after total anthocyanin content and the types of samples decreased, decreased antioxidative ability, it can be speculated that the extract of Lonicera edulis anthocyanin after the in vitro digestion of biological activity will decline. Before digestion with extract of Lonicera edulis anthocyanin can be effective after inhibition of ROS induced by LPS (reactive oxygen species, ROS), 8- hydroxy guanine nucleotide (8-hydroxydeoxyguanosine, 8-OHdG), hydroxyl group protein, lipid peroxidation and glutathione oxidation products (generation Glutathione, GSH) to prevent consumption; ATP synthase, cytochrome c oxidase (cytochrome oxidase, COX) and succinate dehydrogenase (succinate dehydrogenase, SDH) activity decreased and proinflammatory cytokine expression (interleukin (IL) -1 beta and IL-6) up-regulated; regulation of intracellular aspartate aminotransferase (aspartate, aminotransferase, AST), alanine aminotransferase (alanine aminotransferase, ALT) and cholinesterase activity; while maintaining cell structure. Therefore, the loniceraedulis anthocyanin extract can inhibit the intracellular oxidative stress, maintain energy metabolism in cells, regulating the liver function, to inhibit BRL-3A cell inflammation induced by LPS. Moreover, the inhibitory effect of undigested anthocyanin extract good in the inhibitory effect of anthocyanin extracts after digestion. After digestion, the loniceraedulis anthocyanin extract can reduce LPS induced intracellular catalase (catalase, CA T), superoxide dismutase (superoxide dismutase, SOD) activity decreased, thereby maintaining the cellular antioxidant capacity showed a higher T-AOC value; can inhibit cell nuclear factor kappa B (nuclear factor-kappa B, NF- B) and tumor necrosis factor (tumor necrosis factor- TNF- up-regulated the expression of alpha, alpha) and IL-10 expression, thus to a certain extent prevented the development of inflammation; can reduce intracellular Caspase-3, cleaved, Caspase-3, Bax, cleaved poly (ADP-ribose) polymerase (PARP) protein expression was increased and PARP, B lymphocytes in -2 (B-cell lymphoma Bcl-2) protein expression decreased, thereby preventing abnormal cell apoptosis obviously, the loniceraedulis anthocyanin extract can antioxidant, anti-inflammatory and anti apoptosis inhibition of LPS on liver cell injury has protective effect on liver cells. In addition, through the experiment on rat liver Analysis of the relevant indicators in serum and found the loniceraedulis anthocyanin extract can inhibit oxidative stress (ROS, GSH), hepatic dysfunction (AST, ALT), cell cycle abnormalities, structure damage, inflammatory factor expression (C reactive protein (C-reactive protein, CRP) and IL-6), and prevents oxidative stress the reaction and Toll like receptor (Toll-like receptors, TLR2 and TLR4) associated with MAPK pathway, thereby inhibiting the development of inflammation of the liver, has a protective effect on the liver. And its protective effect on liver in a dose-dependent manner in the 50-200 mg/kg range of BW. The experimental results of the anti-inflammatory mechanism of indigo the anthocyanin in fruit of potential, and provide a theoretical basis for the prevention of chronic hepatitis as functional foods or supplements.

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS264.4;TS201.4

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