摘要
越桔(Vaccinium uliginosum L．)酒渣是果实酿酒初级利用后的工业废弃物，花色素是其中所含的生物活性成分之一。为探寻越桔酒渣综合利用的技术途径，实现资源深度开发，本文就越桔酒渣花色素的提取精制工艺、主要化学成分类型、抗氧化、抑制肿瘤及调节血压等方面展开研究。
采用单因素比较和正交试验法，首先探讨花色素提取和精制的技术条件，越桔酒渣花色素提取的最佳条件为：室温条件，80％pH3．0乙醇作浸提剂，料液比1：40，提取2．5h；用HPD100大孔树脂对色素进行精制，树脂柱径高比1：10，花色素浓度0．5g/L，流速1mL／min为最佳吸附条件，色素吸附量可达到0．0987g／mL湿树脂；60％pH3．0乙醇作洗脱液，流速1mL／min，4．5倍柱体积的洗脱液条件下解析效果最佳，色素回收率达到95．48％；制取的花色素呈紫红色，色价为91．5，得率为3．00％。
研究制取花色素理化性质表明，越桔酒渣花色素(Pigment from WildVaccinium uliginosum L．Wine Pomace，简称PVP)属花色苷类，为水溶性色素。采用高效液相法对PVP进行分析，初步确定PVP中含有槲皮素、杨梅素及异鼠李素等黄酮类化合物。
体外抗氧化活性试验表明，PVP表现出很强的清除DPPH·和·O_2~-活性，IC_(50)分别为10．69μg／mL和224．01μg／mL。同时，一定浓度的PVP可以抑制黄嘌呤氧化酶(xanthine oxidase，简称XO)的活性。10μg／mL的PVP能够抑制600μMH_2O_2对牛主动脉内皮细胞(bovine aortic endothelial cells，简称BAECs)的过氧损伤，清除细胞内活性氧(Reactive Oxygen Species，简称ROS)，改善内皮功能障碍。
采用MTT法观察PVP体外对人鼻咽癌(CNE)、肾癌(786-O)及结肠癌(HCT-8)细胞增殖的抑制作用，结果显示：PVP抑制人鼻咽癌细胞(CNE)增殖的IC_(50)为80．30 mg/L；对肾癌细胞(786-O)具有一定的抑制作用，300 mg/L为抑制细胞增殖的最高临界点，抑制率为68．43％；对人结肠癌细胞(HCT-8)增殖抑制的IC_(50)为250．29mg/L。据报道肠道具有酶解花色苷成花色素的作用，肠道对花色素苷的吸收具有重要意义，因此对人结肠癌细胞(HCT-8)展开进一步的研究。研究采用Wright染色法、吖啶橙染色法观察PVP作用HCT-8后的细胞形态，结果发现HCT-8经PVP作用后，出现凋亡特征，细胞变小皱缩，核凝固，细胞膜破裂等；流式细胞仪观察PVP对细胞周期的影响，表明PVP下调G_0／G_1期细胞比例，减少HCT-8细胞有丝分裂和细胞分化，从而促进细胞凋亡。
采用自发性高血压大鼠(spontaneously hypertensive rats，简称SHR)为模型，每日定时灌胃一次PVP，连续4周，结果发现，高剂量PVP(150mg／kg·dbw)SHR大鼠饮水量显著增高，体重增长缓慢，血压显著降低，血清甘油三酯(TG)含量显著减少，但PVP对血清葡萄糖(Glu)，总胆固醇(TC)，高密度脂蛋白总胆固醇(HDL-C)和总抗氧化能力(T-AOC)无显著影响，提示PVP除具一定的降压作用外还具有调节血脂的潜在作用。
蓝莓软胶囊(bluebeery soft capsulem，简称BSC)是以越桔酒渣提取物为主要成分的产品，搞清楚其生物学活性对于指导产业化生产具有重要意义。本文继续将SHR大鼠诱导成高血压高甘油三酯合并的动物模型，以研究BSC对血压和血脂的调节作用。研究参考文献方法，利用高脂高糖饲料喂养SHR大鼠，同时每日定时灌胃BSC一次，观察BSC对高甘油三酯SHR大鼠的影响。结果表明饲喂高脂高糖10周后，SHR大鼠血清TG水平显著升高，同时每日灌胃BSC不能预防SHR大鼠TG的升高，但低剂量的BSC(250mg／kg bw)可以使SHR大鼠血清TG的增长缓慢。BSC可以显著降低高甘油三酯SHR大鼠的血压，降幅接近10％，起到改善血压的作用；高剂量BSC(500g／kg bw)可以显著降低高甘油三酯SHR大鼠血清TC及游离脂肪酸(FFA)含量，提高血清HDL-C和肝脏肝酯酶(HL)含量；高低剂量的BSC都可以极显著减低肝脏丙二醛(MDA)含量，起到抗氧化的作用。综上所述，越桔酒渣提取物制成品具有一定的调节SHR大鼠血压和血脂功效。
Wild Vaccinium uliginosum L. wine pomaces were rubbishes of primary manufacture in the industry, which contained pigments, a bioactivity ingredient. The depth exploiture for resource of Vaccinium uliginosum L., this paper studied the extracting and redining technology of the pigment, primary component pigment type, antioxidant, anti-carcinogenic and regulating blood pressure.
We firstly focused the extracting and refining technology of the pigment of wild Vaccinium uliginosum L. wine pomaces(PVP).The results showed that the best extracting conditions was 80% ethanol used 40 fold volume of wine pomances, pH 3.0, 2.5 hours under room temperature;the HPD-100 macroporous resin was used to refine the pigment;and the best adsorption conditions was 0.5g/L pigment solution, 1 mL/min, diament vs height 1:1,and the adsorption capacity was 0.0987g/mL wet resin; the 60% ethanol was used eluting solvent, pH3.0, 1 mL/min and 3 fold eluting solvent volume were the best desorption conditions, the pigment can be gotten more than 95.48% of that all. The pigment produced by this methods was purple power, its color value was 91.5 and output ratio can get 3.00%。
Study on chemistry characteristic of the pigment showed that PVP was a type of anthocyanins, and had good water solubility. There were myricetin, quercetin and isorhamnetin in PVP by HPLC.
The results of antioxidant activity in vitro experiment demonstrated that PVP can scavenge DPPH and O_2~- strongly with IC_(50) of 10.69μg/mL and 224.01μg/mL. Meanwhile, in the experiment of xanthine oxidase (XO), PVP can inhabited the activity of XO(P<0.05).And 10μg/mL PVP can protect bovine aortic endothelial cells (BAECs) from H_2O_2 with the concentration of 600μM induced injury, decrease the contents of ROS and improve the endothelial dysfunction.
The anti-carcinogenic effect of PVP was to be evaluated by MTT. The results showed that PVP can inhibit the growth of human nasopharyngeal carcinoma cell (CNE) with IC_(50) 80.30 mg/L, PVP can inhibit the cytotoxic of human renal carcinoma cell (786-O) and 300 mg/L was the best concentration, and human colon carcinoma cell (HCT-8) with the IC_(50) was 250.29mg/L. some studies reported that anthocyanins also were effective presumably due to their deglycosylation to cyanidin by cecal bacteria and PVP induced-apoptosis on HCT-8 was investigated further. Several methods of morphological observation were used, such as Wright staining, acidine orange staining, the results showed the morphological changes were induced by PVP with apoptosis on HCT-8. And flow cytometry was used to study the cycle of HCT-8. The results were that the G_0/G_1 phase, cell mitosis and cell differentiation were decreased, so PVP can advance the apoptosis of HCT-8.
The blood pressure of SHR was lower than positive control (Pos) after taking the PVP for 4 weeks,the water taking was much more than Pos, the amplitude of weight was slower, (150mg/ kg·d·bw)PVP can decrease the plasmas TG of SHR, but there were no effect of Glu, TC, HDL-C and T-AOC in plasmas of SHR, which showed SHR can regulate the blood pressure and blood fats.
The main component of bluebeery soft capsulem (BSC) was the extraction of Vaccinium uliginosum L. wine pomaces, and it's important to make the bioactivity clear. So the model of spontaneously hypertensive rats (SHR) with hyperlipemia was used to study the bioactivity of BSC. Based on former research, the SHR consumed the food of high sugar and high fat, and intragastric administration every day for 10 weeks. The results demonstrated that the levels of TG in SHR increased significantly after taking food of high sugar and high fat, and BSC can prevent the increasing of TG. But (500g/kg·bw·d ) BSC can make the increasing slowly and decrease the blood pressure, the level of plasma TC and FFA ,increase the level plasma HDL-C and HL in liver of SHR with high TG. As well, BSC can decrease the level of MDA in liver significantly, and displayed the effect of antioxidant. In all, the products of Vaccinium uliginosum L. wine pomaces can regulate the blood pressure and blood lipids of SHR.
引文
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