本文關(guān)鍵詞：藍(lán)靛果花色苷提取物對脂多糖誘導(dǎo)肝炎的抑制作用機(jī)制研究　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 藍(lán)靛果 花色苷 抑制 脂多糖 肝臟炎癥

【摘要】：藍(lán)靛果(Lonicera caerulea L.)是忍冬科、忍冬屬植物。藍(lán)靛果果實中含有豐富的花色苷,具有較高的營養(yǎng)、保健和藥用價值,有"第三代水果"的美稱。研究表明,花色苷具有廣泛的生物活性,如抗氧化、抗衰老等,機(jī)體長期攝入花色苷可以降低一些慢性疾病的發(fā)生率。脂多糖(lipopolysaccharide,LPS)是革蘭氏陰性菌細(xì)胞壁外膜的主要組成成分,是一種內(nèi)毒素,它可以通過刺激多種促炎因子表達(dá)來引發(fā)肝臟免疫反應(yīng),進(jìn)而促進(jìn)肝臟炎癥的發(fā)生,然而肝臟炎癥可以進(jìn)一步引起肝硬化、肝癌等慢性肝臟疾病。因此,研究藍(lán)靛果花色苷對LPS誘導(dǎo)肝臟炎癥的抑制作用機(jī)制,為尋找潛在的保肝藥物或功能性食品提供理論依據(jù),具有重要意義。本文在對不同品種藍(lán)靛果個體花色苷定性和定量分析的基礎(chǔ)上,利用體外和體內(nèi)實驗,分別從氧化應(yīng)激、能量代謝、肝臟功能、抗氧化能力、炎性因子表達(dá)、凋亡相關(guān)蛋白表達(dá)和絲裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信號通路角度探討藍(lán)靛果花色苷提取物對LPS誘導(dǎo)肝臟炎癥的抑制作用及其機(jī)理。其主要研究結(jié)果如下:采用0.10%HCl酸化的甲醇溶液超聲波輔助提取藍(lán)靛果花色苷,根據(jù)單因素實驗結(jié)果,利用響應(yīng)面分析方法優(yōu)化提取工藝。經(jīng)過實驗稍作調(diào)整后,確定的最佳提取工藝為:料液比1:10、提取時間90 min、提取溫度40℃C;D101大孔樹脂純化藍(lán)靛果花色苷的靜態(tài)吸附解吸優(yōu)化條件為:吸附平衡時間3 h、解吸平衡時間2h、上樣濃度2.52 mg/mL、甲醇濃度100%;動態(tài)吸附解吸優(yōu)化條件為:上樣流速2 BV/h、上樣量490 mL、洗脫流速2 BV/h、蒸餾水用量2 BV。在此條件下純化得到的藍(lán)靛果花色苷提取物經(jīng)冷凍干燥后為紫黑色粉末,色價為57.6,是未純化時的11倍。通過計算得到花色苷含量為484.2 mgCYD-3-G/L。不同品種藍(lán)靛果果實中花色苷組成及個體比例不同。實驗在野生、'蓓蕾'、'NO.1'和'N0.2'四種藍(lán)靛果中分別鑒定出14、11、11和12種個體花色苷。四個品種藍(lán)靛果的主要花色苷均為矢車菊-3-葡萄糖苷,分別占各自總花色苷的89.8%、90.7%、89.6%和91.3%。矢車菊-3-乙酰己糖苷和芍藥素-3-乙酰己糖苷首次在藍(lán)靛果中發(fā)現(xiàn),其中矢車菊-3-乙酰己糖苷在'蓓蕾'、'NO.1'和'NO.2'品種藍(lán)靛果中被鑒定,芍藥素-3-乙酰己糖苷僅在'NO.2'品種藍(lán)靛果中被鑒定。四種藍(lán)靛果的總花色苷含量無顯著差異,野生和'蓓蕾'藍(lán)靛果具有較高的抗氧化能力和總酚含量,'N0.2'品種藍(lán)靛果的總可溶性固形物含量最高。實驗通過比較分析不同品種藍(lán)靛果花色苷組成和抗氧化能力,結(jié)合實際情況,選定適宜食用且抗氧化能力相對較高的'蓓蕾'品種藍(lán)靛果作為后續(xù)實驗的研究材料。通過測定體外模擬消化前、后樣品中總花色苷含量和抗氧化能力相關(guān)指標(biāo)發(fā)現(xiàn):體外消化降低了總花色苷含量和抗氧化能力。體外消化后藍(lán)靛果提取物的總花色苷含量降低67.6%,氧自由基清除能力(oxygen radical absorbance capacity,ORAC)值和總抗氧化能力(total antioxidant capacity,T-AOC)值分別下降了 80%和55.6%。此外,與未消化樣品相比,體外消化后藍(lán)靛果提取物中花色苷種類減少3種、酚酸減少1種�？梢�,體外模擬消化后樣品的總花色苷含量及種類減少,抗氧化能力降低,由此可以推測經(jīng)體外消化后的藍(lán)靛果花色苷提取物的生物活性會下降。消化前、后的藍(lán)靛果花色苷提取物可以有效地抑制LPS誘導(dǎo)的活性氧(reactive oxygen species,ROS)、8-羥基鳥核苷酸(8-hydroxydeoxyguanosine,8-OHdG)、蛋白羥基組、脂質(zhì)過氧化產(chǎn)物的生成和谷胱甘肽(glutathione,GSH)的消耗;阻止ATP合成酶、細(xì)胞色素 c 氧化酶(cytochrome oxidase,COX)和琥珀酸脫氫酶(succinate dehydrogenase,SDH)活性降低和促炎因子表達(dá)(interleukin(IL)-1β和IL-6)上調(diào);調(diào)節(jié)細(xì)胞內(nèi)谷草轉(zhuǎn)氨酶(aspartate aminotransferase,AST)、谷丙轉(zhuǎn)氨酶(alanine aminotransferase,ALT)和膽堿酯酶活性;同時維持細(xì)胞結(jié)構(gòu)。由此可知,藍(lán)靛果花色苷提取物可以通過抑制細(xì)胞內(nèi)氧化應(yīng)激反應(yīng),維持細(xì)胞內(nèi)能量代謝,調(diào)節(jié)肝細(xì)胞功能,來抑制LPS誘導(dǎo)的BRL-3A細(xì)胞炎癥的發(fā)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、后藍(lán)靛果花色苷提取物可以削減LPS誘導(dǎo)的細(xì)胞內(nèi)過氧化氫酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)活性下降,進(jìn)而維護(hù)細(xì)胞的抗氧化能力,表現(xiàn)為較高的T-AOC值;能夠抑制細(xì)胞內(nèi)核轉(zhuǎn)錄因子-κB(nuclear factor-kappa B,NF-κB)和腫瘤壞死因子(tumor necrosis factor-α,TNF-α)表達(dá)上調(diào)和IL-10表達(dá)下調(diào),從而一定程度地阻止了炎癥的發(fā)展;可以削減細(xì)胞內(nèi)Caspase-3、cleaved Caspase-3、Bax、cleaved poly(ADP-ribose)polymerase(PARP)蛋白表達(dá)量增加和 PARP、B 淋巴細(xì)胞瘤-2(B-cell lymphoma,Bcl-2)蛋白表達(dá)量降低,進(jìn)而阻止細(xì)胞異常凋亡�？梢�,藍(lán)靛果花色苷提取物還可以通過抗氧化、抗炎和抗凋亡途徑抑制LPS對肝細(xì)胞造成的損傷,對肝細(xì)胞具有一定的保護(hù)作用。此外,實驗通過對大鼠肝臟組織及血清中相關(guān)指標(biāo)分析,發(fā)現(xiàn)藍(lán)靛果花色苷提取物可以通過抑制氧化應(yīng)激反應(yīng)(ROS、GSH)、肝功能失調(diào)(AST、ALT)、細(xì)胞周期循環(huán)異常、組織結(jié)構(gòu)損傷、炎癥因子表達(dá)(C反應(yīng)蛋白(C-reactive protein,CRP)和IL-6),以及阻礙氧化應(yīng)激反應(yīng)和Toll樣受體(toll-like receptors,TLR2和TLR4)相關(guān)聯(lián)的MAPK信號通路轉(zhuǎn)導(dǎo),進(jìn)而抑制肝臟炎癥的發(fā)展,對肝臟具有一定的保護(hù)作用。并且,其對肝臟的保護(hù)效果在50-200 mg/kg bw范圍內(nèi)呈劑量依賴性。本實驗結(jié)果補(bǔ)充了藍(lán)靛果花色苷潛在的抗炎作用機(jī)制,為其作為預(yù)防慢性肝炎的功能性食品或補(bǔ)充藥物提供理論依據(jù)。
[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.

【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TS264.4;TS201.4

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