摘要
黑米是优异的稻种资源,富含天然花色苷类化合物。花色苷是一类天然色素,属于黄酮类物质,具有抗氧化、无毒、无突变等特点。酒精滥用是全球范围内重要公共卫生问题,过量饮酒会在机体代谢过程中产大量的自由基与活性氧,导致肝脏及多器官损伤。本文研究了黑米中花色苷主要成分的提取工艺及对酒精性肝损伤的影响。
1.鉴定黑米花色苷的主要成分及确定其检测方法。以矢车菊素-3-葡萄糖苷为标准品,建立了黑米花色苷的高效液相色谱(HPLC-DAD)法。通过液质联用(HPLC-ESI-MS)鉴定出四种花色苷,分别为矢车菊素-3-葡萄糖苷(cyanidin-3-glucoside, 91.0%),矢车菊素-3,5-葡萄糖苷(cyanidin-3, 5-diglucoside, 0.92%),矢车菊素-3-芸香糖苷(cyanidin-3-rutinoside, 0.94%)和芍药素-3-葡萄糖苷(peonidin-3-glucoside, 7.13%)。
2.确定黑米花色苷最佳提取工艺,建立大孔吸附树脂纯化花色苷的方法。以矢车菊素-3-葡萄糖苷为跟踪指标,通过单因素和正交试验,对各因素进行研究,比较9种大孔吸附树脂对花色苷的静态吸附和解吸性能。黑米花色苷最佳提取条件,提取液乙醇/水/盐酸体积比为50/50/0.5,温度50℃,固液比为1:10 (g/mL),提取时间为1小时,提取次数为3次。确定了AB-8为最佳吸附树脂,最佳条件为:洗脱剂为80%乙醇,上样流速为1.0BV/h,解吸流速为2.0BV/h。结论:测定经树脂纯化后提取物中花色苷的含量达到22.5 %(粗提物含量为3.74%),树脂富集倍数为6.02。
3.研究了黑米四种花色苷在温度(80°C, 90°C和100°C;4°C, 20°C和37°C),pH1.0-6.0下的稳定性。结果表明四种花色苷降解遵循一级反应方程,随温度降解符合Arrhenius方程。四种花色苷的活化能(Ea),半衰期(t1/2 )和反应数率常数(k)不同且差异显著。花色苷的降解率均随温度和pH值得上升而增加,尤其是当温度达到100°C,pH值达到5.0时。贮藏温度和pH值对花色苷稳定性及色差有很大影响,花色苷溶液色差利用CIE L*a*b*参数对其进行评价。随温度增加在pH3.0时,L*, a*, b*值明显降低。
4.探讨了黑米花色苷提取物(AEBR)对大鼠慢性酒精肝损伤的影响。结果表明:雄性Wistar大鼠摄入酒精后,血清中谷草酰胺转氨酶(AST),丙氨酸转氨酶(ALT)和γ-谷氨酸转氨酶(GGT)以及肝组织中丙二醛(MDA)显著增加(P<0.05)。AEBR干预可显著缓解酒精伤害,尤其高剂量组(500mg/kg),AEBR(500mg/kg)处理可显著降低血清中酶(AST, ALT和GGT)活性(P<0.01),肝脏中MDA水平及甘油三酯(TG),总胆固醇的浓度(TCH)。花色苷干预组表现出正常的抗氧化体系,谷胱甘肽过氧化物酶(GSH-Px),超氧化物歧化酶(SOD)和谷胱甘肽-s-转移酶(GST)均在正常范围。以上结果均与组织学观察相一致。这表明AEBR能缓解酒精伤害,可明显减轻酒精对机体脂质代谢和抗氧化系统的不利影响。
Black rice is a good and important rice resource due to rich natural anthocyanins. As a group of flavonoid pigments, anthocyanins have not only their colorant potential but also significant health implications, because of their non-toxicity, non-mutagenic, antioxidant activity. As an important global issue on public health, alcohol abuse has been provoked widespread solicitude in the world because of its outcome including functional impairment of multiorgans or multisystems. Excessive free radical and reactive oxygen species (ROS) produced during ethanol metabolism result in liver damage. In this paper, the composition of anthocyanins extract from black rice (AEBR), the optimal extraction conditions of AEBR and effect of AEBR on alcohol-induced liver damage were studied.
1. A rapid determination method has been developed to elucidate the anthocyanin profiles in black rice by high performance liquid chromatography (HPLC)-electrospray ionization - mass spectrometry with diode array detection. Four anthocyanins were identified as cyandin-3-glucoside (91.01%), peonidin-3-glucoside (7.13%), cyanidin-3,5-diglucoside (0.92%) and cyanidin-3-rutinoside (0.94%). The main anthocyanins were cyandin-3-glucoside and peonidin-3-glucoside.
2. The optimal conditions for anthocyanins extraction from black rice was gotten, found the method of purifying anthocyanins with macroporous resins. Using cyanidin-3-glucoside as a monitoring parameter, the technologies and the conditions for extracting anthocyanins from black rice were optimized by the single factor experiments and orthogonal design, 9 different macroporous resins were studied. The results showed that the optimal parameters for extraction were ethanol / water / hydrochloric acid 50/50/0.5, temperature 50℃, solid-liquid ratio of 1:10, the extraction time is 1h, times of extraction is 3. By comparing static adsorption and desorption property of anthocyanins crude extract from black rice with 9 macroporous adsorbing resins, AB-8 was the most appropriate resin. The column was eluted by 80% ethanol at the flow rate of 2 BV per hour. Under this condition, the content of anthocyanins in the refined product was 22.5%, and AB-8 resin enrichment amount were 6.02 multiple. The processing of purifying anthocyanins was effective.
3. Thermal and storage stability of four anthocyanins from black rice in solution were investigated at temperatures (80°C, 90°C and 100°C) and (4°C, 20°C and 37°C) in the range of pH1.0-pH6.0. Reuslts indicated that degradation of anthocyanins followed the first-order reaction kinetics. The temperature-dependent degradation was adequately modeled on the Arrhenius equation. The calculated values of activation energies (Ea), temperature quotient (Q10), t1/2 (the time that 50% anthocyanin degradation would take) and k (degradation rate constant) were different for the four anthocyanins. The degradation rate of anthocyanin increased with increasing heating temperature and pH values. Especially, as heating temperature increasing to 100°C and pH value to 5.0. During storage, the storage temperatures and pHs had significant effect on characteristics, significant decrease in anthocyanin stability was observed with the increasing of pH and temperature. Colour characteristics was evaluated using colour indices such as CIE L*a*b* parameters. There was a decrease in L*, a*, b* value, especially at pH3.0 with the temperature increasing.
4. The anthocyanin-rich extract from black rice (AEBR) was evaluated for its protective effect against chronic ethanol-induced biochemical changes in male Wistar rats. Administration of ethanol to Wistar rats induced liver damage showed a significant increase (P<0.05) in the levels of serum aspartate transaminase (AST), alanine transaminase (ALT), gamma glutamyl transferase (GGT). The hepatic malondialdehyde (MDA) level, indicator of lipid peroxidation, was also increased (P<0.05) in ethanol-treated rats. In contrast, administration of AEBR along with alcohol significantly (P<0.01) decreased the activities of liver enzymes (AST, ALT and GGT) in serum towards near normal levels. In addition, administration of AEBR significantly (P<0.01) decreased the levels of lipid peroxidation markers (MDA) and the concentrations of serum and hepatic triglyceride (TG) and total cholesterol (TCH). Rats treated with AEBR showed a better profile of the antioxidant system with normal glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities while the levels of these enzymes were significantly (P<0.05) increased in alcohol-treated rats. All these results were accompanied by histological observations in liver. The results demonstrate that AEBR has a beneficial effect in reducing the adverse effect of alcohol.
引文
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