【作者】 袁晓晴；

【导师】 汤坚；

【作者基本信息】 江南大学 ， 食品科学， 2007， 博士

【摘要】 癞葡萄（Momordica charantia L.Var.abbreviata Ser.,MCV）是葫芦科（Cucurbitaceae）苦瓜属的一个变种,原产于印度东部。由江苏省优质产品开发服务中心驯化改良而成,无锡富仁生物技术有限公司大面积栽培获得成功。现代药理研究证明苦瓜具有降血糖、降血脂、抗癌、抗艾滋病病毒、抑菌、抗氧化等功效。本论文以癞葡萄鲜果为原料,对癞葡萄水提物的降血糖活性进行了研究,并从中筛选出降血糖活性肽MC2-1-5。同时通过碱性蛋白酶Alcalase酶法水解癞葡萄蛋白制备具有降血糖活性的酶解物（MCPHs）,并对MC2-1-5和MCPHs的降糖机理进行了研究,研究结果如下:原料癞葡萄鲜果的化学组成为:水分92.28%,灰分0.48%,粗蛋白1.42%,粗脂肪0.93%,还原糖1.48%,多糖1.55%,粗纤维0.78%。以料水比、盐浓度、提取时间和提取温度为因素,以蛋白提取率为指标,通过正交实验确定癞葡萄水提物的最佳提取工艺条件。综合考虑成本、后续工作量等因素,确定提取条件为料液比1:2,盐浓度0.05 mol/L,提取时间4 h,提取温度40℃。对在此工艺条件下得到的水提物进行氨基酸组成分析,结果表明:水提物中含量最高的氨基酸为精氨酸,其次为谷氨酸和天门冬氨酸,含量分别为22.06%、9.75%、9.54%。三者约占水提物氨基酸总含量的41.35%。新鲜癞葡萄精氨酸、谷氨酸和天门冬氨酸的含量依次为20.13%、12.51%和9.63%,三者约占癞葡萄水提物氨基酸总含量的42.27%。通过四氧嘧啶型糖尿病小鼠研究了水提物的降血糖作用。结果表明不同剂量（250、500和750 mg/kg）的癞葡萄水提物可显著降低糖尿病小鼠的血糖浓度;水提物的降血糖作用在灌胃后2 h就已开始,并在4 h后达到最大。本实验中的最佳有效剂量为500 mg/kg,该剂量水提物比20 mg/kg剂量的格列本脲片产生的降糖效果更佳。然而,水提物对正常小鼠的血糖浓度不会产生显著影响。此外,水提物还可显著改善糖尿病小鼠的糖耐量。以500 mg/kg剂量连续治疗糖尿病小鼠15 d显著降低小鼠空腹血糖浓度,且和时间成正相关。到第15 d,血糖值较治疗前下降59.88%。水提物还显著增加糖尿病小鼠的体重、降低小鼠的采食量和饮水量。以小鼠血糖浓度为指标,通过超滤、Sephadex G-25凝胶柱和半制备RP-HPLC从水提物中筛选降血糖肽。研究了进料流速、操作压力、操作温度和料液浓度对膜通量的影响。根据结果选择超滤条件:进料流速200 L/h、温度20℃、压力0.10 MPa、料液浓度20 g/L。超滤将癞葡萄水提物分成浓缩液（MC1）和透过液（MC2）两个组分,动物实验表明MC2对糖尿病小鼠血糖浓度有显著影响,在灌胃2 h和4 h后使小鼠血糖浓度降低46.15%和52.59%。而MC1对血糖浓度影响不显著。Sephadex G-25凝胶色谱柱将MC2分成六个组分,动物实验表明MC2-1在以20 mg/kg剂量灌胃时即可显著降低四氧嘧啶型糖尿病小鼠的血糖浓度,在灌胃2 h和4 h后使小鼠血糖浓度降低47.43%和51.87%。其他组分对小鼠血糖浓度无显著影响。用半制备RP-HPLC柱将MC2-1分成十个组分,动物实验表明组分MC2-1-1和MC2-1-5在以2 mg/kg剂量灌胃时即可显著降低四氧嘧啶型糖尿病小鼠的血糖浓度。与相应的初始血糖值相比,MC2-1-1在灌胃2 h和4 h后分别使小鼠血糖降低43.84%和54.57%;MC2-1-5在灌胃2 h和4 h后分别使小鼠血糖降低61.70%和69.18%。其他组分对小鼠血糖浓度无显著影响。MC2-1-5还可显著改善小鼠的糖耐量。红外光谱图显示多肽MC2-1-5在500～4000 cm^-1范围具有肽的特征吸收峰。串联飞行时间质谱仪测得MC2-1-5的相对分子质量为3405.5174。蛋白测序仪测得MC2-1-5的N端前10个氨基酸残基排列次序为GHPYYSIKKS。癞葡萄水溶性蛋白不具备降血糖功效,为充分利用这部分蛋白,酶解癞葡萄蛋白制备降血糖酶解物。以水解度、蛋白回收率和MCPHs的降血糖活性为指标,从六种蛋白酶中选出碱性蛋白酶Alcalase 2.4L水解癞葡萄蛋白。通过单因素试验和响应面分析确定Alcalase水解癞葡萄蛋白的最佳工艺条件:[E]/[S]2.37、pH9.2,温度57℃。在此工艺条件下制备了三种不同DH的MCPHs（8.5%、11%和13.5%）,动物实验表明DH为8.5%的MCPHs不具备降血糖活性,DH为11%和13.5%的MCPHs都具有显著的降血糖活性,而DH为11%的MCPHs的降血糖活性高于DH为13.5%的降血糖活性。根据吸附和解吸效果选择DA201-C大孔吸附树脂对MCPHs进行脱盐。脱盐后MCPHs的含量从脱盐前的76.67%提高至脱盐后的85.69%;MCPHs的回收率为78.38%。脱盐率为92.49%。脱盐后的MCPHs仍有显著的降血糖作用,且降血糖作用较脱盐前稍有提高。研究了MC2-1和MCPHs的降血糖机理。经MC2-1和MCPHs治疗28 d后,与模型对照组相比,糖尿病小鼠体重显著上升;采食量和饮水量显著下降;脏器/体重比恢复至接近正常水平;空腹血糖值下降;糖耐量显著改善;肝糖原含量显著增加。经MC2-1治疗的糖尿病小鼠的血清胰岛素水平显著提高,但经MCPHs治疗的糖尿病小鼠的血清胰岛素水平未见显著改善。经MC2-1和MCPHs治疗28 d后,小鼠血清FFA、TC、TG和LDL-C水平下降;HDL-C水平上升,说明MC2-1对糖尿病所引起的高血脂症有一定的治疗作用;小鼠MDA含量下降,SOD活性和GSH-Px活性上升,说明MC2-1和MCPHs能够改善糖尿病小鼠体内的氧化应激机制和提高其抗氧化能力。胰腺、肝脏和肾脏组织形态观察表明MC2-1和MCPHs能改善四氧嘧啶对这些器官细胞造成的损伤。上述结果表明MC2-1的降糖机理是修复糖尿病小鼠胰岛β细胞、促进胰岛素分泌、增加肝糖原含量及提高小鼠抗氧化能力;MCPHs的降糖机理是修复糖尿病小鼠胰岛β细胞,增加肝糖原含量及提高小鼠抗氧化能力。由于MC2-1-5是MC2-1中的主要降血糖组分,因而推测MC2-1-5的降糖机理与MC2-1类似。更多还原

【Abstract】 Momordica charantia L.Var.abbreviata Ser.（MCV） is a member of Cucurbitaceae family. Momordica charantia variety was originally growing wild in eastern India.It was domesticated and improved by Jiangsu High-quality Farm Product Development Center,China and has been successfully planted over large areas by Wuxi Furen Biological Science and Technology Co., Ltd.It is reported that Momordica Charantia L.has many pharmacological benefits such as antidiabetic,hypocholesterolemic,hypo-triglyceridemic,antitumor,antibacterial,anti-HIV, anti-inflammatory,antioxidant,etc.In this dissertation,the hypoglycemic effect of an aqueous extract from MCV and a purified peptide called MC2-1-5 with hypoglycemic effect from aqueous extract was stuied.At the same time,MCV protein hydrolysates（MCPHs） with hypoglycemic effect were prepared by Alcalase.The hypoglycemic mechanism of MC2-1-5 and MCPHs was studied in alloxan-induced diabetic mice.The chemical composition of the fresh MCV fruit were analyzed and found to be as follows:92.28%moisture,0.48%ash,1.42%protein,0.93%fat,1.48%reducing sugar,1.55% of polysaccharide and 0.78%crude fiber.The effect of MCV fruit to water ratio,salt concentration,extraction temperature and extraction time on extraction rate of MCV protein were studied.The parameters affecting protein extraction were optimized by an orthogonal experimental design,that is to say,MCV fruit to water ratio of 1:2,salt concentration of 0.05 mol/L,extraction temperature of 40℃and extraction time of 4 h.Amino acid（AA） composition profile of MCV aqueous extract showed that the content of Arginine was highest, followed by Glutamic acid and Aspartic acid of 22.06%,9.75%and 9.54%respectively.The total percentage composition of the three AA was 41.35%.The AA composition profile of fresh MCV showed that the content of Arginine,Glutamic acid and Aspartic acid was 20.13%, 12.51%and 9.63%respectively.The total percentage composition of the three AA was 42.27%.The acute（a single dose） and chronic（15 d） hypoglycemic effects of aqueous extract were studied in alloxan-induced diabetic mice.The results showed that an aqueous extract from MCV fruit at different doses（250,500 and 750 mg/kg） produced a significant hypoglycemic effect in alloxan-induced diabetic mice within 2 h after oral administration,which was maintained for 4 h.As far as the most effective dose under the experimental conditions stuied is concerned,it was found to be 500 mg/kg in all groups.The hypoglycemic effect of the aqueous extract administered at a dose of 500 mg/kg was more effective than that of glibenclamide administered at a dose of 100 mg/kg.However,the aqueous extract didn’t have significant effect on blood glucose level（BGL） in normal mice.In addition,the hypoglycemic effect of aqueous extract was further confirmed by the oral glucose tolerance test（OGTT）.A 15-day treatment of the aqueous extract at a dose of 500 mg/kg in diabetic mice continuously lowered the fasting blood glucose（FBG） level.Treatment of alloxan-induced mice for 15 days with the aqueous extract at a dose of 500 mg/kg produced a 59.88%fall in FBG level.During the treatment period,the aqueous extract significantly increased the body weight（BW）,lowered the food and water intake in diabetic mice. Using BGL in diabetic mice as an index,the peptide with hypoglycemic effect was purified by ultrafiltration（UF）,Sephadex G-25 and semi-RP-HPLC.The influence of input flow rate,operating pressure,operating temperature and sample concentration on flux was studied.The UF conditions used were:input flow rate 200 L/h,temperature 20℃,pressure 0.1 MPa and sample concentration 20 g/L.On separating MCV aqueous extract into two fractions （MC1 and MC2） by UF,MC2 showed significant hypoglycemic effect in alloxan-induced mice. It produced a reduction of 46.15%and 52.59%in BGL after 2 and 4 h respectively of oral administration.MC1 didn’t show hypoglycemic effect in diabetic mice.Six different fractions of MC2 were obtained by Sephadex G-25 gel filtration chromatography.The in vivo test showed MC 2-1 at a dose of 20 mg/kg could lower the BGL in diabetic mice.It produced a reduction of 47.43%and 51.87%in BGL after 2 and 4 h respectively of oral administration.Other fractions had no significant effect on BGL in diabetic mice.A further ten different fractions of MC 2-1 were obtained through Semi-HPLC.The in vivo test showed MC 2-1-1 and MC 2-1-5 at a dose of 2 mg/kg could lower the BGL in diabetic mice,while other fractions had no significant effect on BGL in diabetic mice.MC2-1-1 produced a reduction of 43.84%and 54.57%in BGL after 2 and 4 h respectively of oral administration.MC2-1-5 produced a reduction of 61.70%and 69.18%in BGL after 2 and 4 h respectively of oral administration.In addition,MC2-1-5 improved the oral glucose tolerance in diabetic mice.An Infrared spectrograph showed MC2-1-5 to have characteristic IR absorption peak of peptide in the spectral region 500～4000 cm^-1.MALDI TOF-MS showed the molecular weight of MC2-1-5 was 3405.5174 Da.Protein Sequencer showed the N terminal frist ten AAs of MC2-1-5 were GHPYYSIKKS.MCV souble protein did not have hypoglycemic effect.In order to make use of the MCV soluble protein,the protein was hydrolyzed to produce MCPHs with hypoglycemic effect.The MCV protein was hydrolyzed using six different proteases.The results showed Alcalase 2.4L to have the best hydrolysing capacity.The protein recovery was also highest with Alcalase.The hydrolysis parameters using Alcalase were optimized as follows:enzyme to substrate ratio 2.37%,pH 9.2 and hydrolysis temperature 57℃.Three hydrolysates with different DH（8.5%, 11%and 13.5%） were obtained using Alcalase under the optimized conditions.The hypoglycemic effect of the three hydrolysates was stuied in alloxan-induced diabetic mice.The results showed that the MCPHs at DH 11%had the best hypoglycemic effect,followed by MCPHs at DH 13.5%.MCPHs at DH 8.5%didn’t have any hypoglycemic effect.DA201-C Macroporous resin was chosen to desalt MCPHs by comparing the adsorbing and desorbing capacity.The optimal conditions for desalting using DA201-C macroporous resin were determined.Under the optimal conditions,the desalting rate and recovery of MCPHs was 92.49%and 82.38%respectively.The content of MCPHs increased from 76.67%to 85.69%.The hypoglycemic effect of desalted MCPHs was better than that of MCPHs before desalting.The hypoglycemic mechanism of MC2-1 and MCPHs was studied.Compared with the diabetic control,a 28-d treatment with MC2-1 and MCPHs increased the BW and hepatic glycogen content,lowered food intake and water intake,made the organ/body weight near to normal and improved the oral glucose test in diabetic mice.In addition,MC2-1 in particular, increased the serum insulin level in diabetic mice.MC2-1 and MCPHs could improve the lipid profile in diabetic mice.After a 28-d treatment with MC2-1 and MCPHs,free amino acid（FFA）,total cholesterol（TC）,triglyceride （TG） and low density lipoprotein cholesterol（LDL-C） lowered significantly,while high density lipoprotein cholesterol（HDL-C） increased significantly.MC2-1 and MCPHs could improve the mechanisms of oxidative stress and antioxidant capacity in diabetic mice.After a 28-d treatment with MC2-1 and MCPHs,malondialdehyde（MDA） content lowered while SOD activity and GSH-Px activity increased.Tissue morphology showed that MC2-1 and MCPHs could improve the cell injury in islet,liver and kidney induced by alloxan in diabetic mice.In summary,the hypoglycemic mechanism of MC2-1 is to recover the isletβcell,so as to stimulate insulin secretion,to increase the hepatic glycogen content and improve antioxidant capacity in diabetic mice.The hypoglycemic mechanism of MCPHs is similar to that of MC2-1, except that it couldn’t stimulate insulin secretion.Since MC2-1-5 is the main hypoglycemic constituent in MC2-1,it is speculated that the hypoglycemic effect of MC2-1-5 was similar to that of MC2-1.更多还原