胶原蛋白铬螯合物的制备及降血糖功效的研究
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摘要
本文用脱脂猪皮的盐酸消化液与0.2M无机铬溶液以一定比例混合，使铬离子与消化液中的小肽或者氨基酸螯合，化学滴定法测定其中铬含量(3.34％)，红外光谱法测定螯合程度。将此螯合物作为降血糖功能因子，检测其对糖尿病模型鼠的血液指标及肝糖原水平的影响。
实验动物采用昆明种小鼠，糖尿病模型用化学试剂四氧嘧啶按照200mg／kg腹腔注射，正常鼠以同样剂量注射生理盐水，空腹血糖超过11.1mmol／L的小鼠纳为糖尿病模型。实验小鼠分为5组：正常组，正常喂铬组，模型组，模型喂铬组，模型米糠铬组。造模组以饮水方式按照1ug／d只的剂量喂以Cr~(3+)，每周处死一批，试剂盒测定血液指标。饲养6周后，正常鼠喂铬不会使血糖浓度和血脂指标发生显著变化(P＞0.05)；模型喂铬组的血糖浓度和甘油三酯含量与模型组相比，下降显著(P＜0.05)；喂养米糠铬小鼠血糖浓度起伏很大，与模型组相比没有差异(P＞0.05)。各组小鼠随机取肝脏匀浆，用生理盐水提取可溶性蛋白乙醇沉淀后以样品缓冲液溶解，采用聚丙烯凝胶电泳分离，各组的蛋白带没有区别。
取70只正常小鼠分为两组，其中一组喂铬，方法同上，另一组作为对照，每隔一天处死一批，血液测定法同上。饲养12天后，喂铬组最终的血液指标与正常组比较没有差异，刚喂铬后几天差异明显。同样作肝脏蛋白电泳，差别明显。
热应激实验：另取24只小鼠，雌雄各半，分为四组，雌雄各有一组喂以铬，剂量同上，四周后将这些小鼠置于43℃，相对湿度30％的烘箱中，记录各小鼠的死亡时间，1小时死亡率。耐热时间雌鼠比雄鼠略长，但没有统计学意义，喂铬后雌鼠耐热时间显著增长(P＜0.05)，雄鼠相差不显著(P＞0.05)。取肝、肾、心、腿提取蛋白电泳，没有差异。
In this thesis, the hydrochloric acid assimilation solution and 0.2M CrCl3 were mixed in a certain proportion, which could make Cr3+ and peptide or amino acid in the assimilation solution chelate. The chromium content was determined in chemical titration and the chelating extent was determined in infrared spectrum. The substance chelating chromium was treated as a functional factor lowering blood sugar, whose influence to the blood parameter and liver of diabetes rats starch was determined.
Experimental animals are KM rats, by injecting alloxan in the dose of 200mg/kg into the abdomen, diabetes animal model were established, in comparison, normal rats were injected in the same dose of 0.9% sodium chloride solution. After this, those were treated as diabetes model rats whose fasting blood sugar content was above 11.1mol/L. Experimental rats were divided into 5 groups: normal rats, normal rats fed chromium, diabetes rats, diabetes rats fed chromium, diabetes rats fed chromium and rice bran. Diabetes were raised in the dose of 1ug/d Cr3+ by drinking, 6 subject rats in each group were killed every week, their blood parameters were determined by biochemical reagent kit. After 6 weeks administration, no significant difference can be observed between normal rats blood parameters and those of rats fed chromium(P>0.05); there was an obvious decrease between the blood sugar content and triglyceride of diabetes rats and those of diabetes raised chromium(P<0.05); rice bran and chromium did have no effect on the blood sugar and other parameters, the former changed violently and had no difference in comparison with diabetes rats(P>0.05). The livers were taken from each group randomly to homogenize them, the soluble protein was extracted in the 0.9% sodium chloride solution and deposited in alcohol. After centrifugation, the protein was dissolved in the sample buffer solution and separated in SDS-PAGE, there was no difference between the protein strips.
70 rats were divided into 2 groups, one was fed chromium in the same method as above in comparison with the other, 6 rats each group were killed and blood parameters were determined in the same method.. After 12 days administration, there was no difference between normal rats and those fed chromium, though emerging significant difference at the beginning, their livers were disposed in the same method as above, the result showed obvious difference.
Heat stress experiment: 24 rats were divided into 4 groups, of which the half is male and the other female. One male group and female group were fed chromium in the dose of lug/d, after 4 weeks administration, all rats were placed in the oven at 43 C with relative humidity, the heat tolerance time and the death rate after 1hour of each rat was noted. The heat tolerance time of female rat was longer than that of male rat, but there was no significant difference between those. The heat tolerance time of female rat fed chromium was more longer than the other female group(P<0.05), and there was no difference between the male groups(P<0.05), and the soluble protein extracted in the 0.9% sodium chloride solution was separated in SDS-PAGE.
实验动物采用昆明种小鼠，糖尿病模型用化学试剂四氧嘧啶按照200mg／kg腹腔注射，正常鼠以同样剂量注射生理盐水，空腹血糖超过11.1mmol／L的小鼠纳为糖尿病模型。实验小鼠分为5组：正常组，正常喂铬组，模型组，模型喂铬组，模型米糠铬组。造模组以饮水方式按照1ug／d只的剂量喂以Cr~(3+)，每周处死一批，试剂盒测定血液指标。饲养6周后，正常鼠喂铬不会使血糖浓度和血脂指标发生显著变化(P＞0.05)；模型喂铬组的血糖浓度和甘油三酯含量与模型组相比，下降显著(P＜0.05)；喂养米糠铬小鼠血糖浓度起伏很大，与模型组相比没有差异(P＞0.05)。各组小鼠随机取肝脏匀浆，用生理盐水提取可溶性蛋白乙醇沉淀后以样品缓冲液溶解，采用聚丙烯凝胶电泳分离，各组的蛋白带没有区别。
取70只正常小鼠分为两组，其中一组喂铬，方法同上，另一组作为对照，每隔一天处死一批，血液测定法同上。饲养12天后，喂铬组最终的血液指标与正常组比较没有差异，刚喂铬后几天差异明显。同样作肝脏蛋白电泳，差别明显。
热应激实验：另取24只小鼠，雌雄各半，分为四组，雌雄各有一组喂以铬，剂量同上，四周后将这些小鼠置于43℃，相对湿度30％的烘箱中，记录各小鼠的死亡时间，1小时死亡率。耐热时间雌鼠比雄鼠略长，但没有统计学意义，喂铬后雌鼠耐热时间显著增长(P＜0.05)，雄鼠相差不显著(P＞0.05)。取肝、肾、心、腿提取蛋白电泳，没有差异。
In this thesis, the hydrochloric acid assimilation solution and 0.2M CrCl3 were mixed in a certain proportion, which could make Cr3+ and peptide or amino acid in the assimilation solution chelate. The chromium content was determined in chemical titration and the chelating extent was determined in infrared spectrum. The substance chelating chromium was treated as a functional factor lowering blood sugar, whose influence to the blood parameter and liver of diabetes rats starch was determined.
Experimental animals are KM rats, by injecting alloxan in the dose of 200mg/kg into the abdomen, diabetes animal model were established, in comparison, normal rats were injected in the same dose of 0.9% sodium chloride solution. After this, those were treated as diabetes model rats whose fasting blood sugar content was above 11.1mol/L. Experimental rats were divided into 5 groups: normal rats, normal rats fed chromium, diabetes rats, diabetes rats fed chromium, diabetes rats fed chromium and rice bran. Diabetes were raised in the dose of 1ug/d Cr3+ by drinking, 6 subject rats in each group were killed every week, their blood parameters were determined by biochemical reagent kit. After 6 weeks administration, no significant difference can be observed between normal rats blood parameters and those of rats fed chromium(P>0.05); there was an obvious decrease between the blood sugar content and triglyceride of diabetes rats and those of diabetes raised chromium(P<0.05); rice bran and chromium did have no effect on the blood sugar and other parameters, the former changed violently and had no difference in comparison with diabetes rats(P>0.05). The livers were taken from each group randomly to homogenize them, the soluble protein was extracted in the 0.9% sodium chloride solution and deposited in alcohol. After centrifugation, the protein was dissolved in the sample buffer solution and separated in SDS-PAGE, there was no difference between the protein strips.
70 rats were divided into 2 groups, one was fed chromium in the same method as above in comparison with the other, 6 rats each group were killed and blood parameters were determined in the same method.. After 12 days administration, there was no difference between normal rats and those fed chromium, though emerging significant difference at the beginning, their livers were disposed in the same method as above, the result showed obvious difference.
Heat stress experiment: 24 rats were divided into 4 groups, of which the half is male and the other female. One male group and female group were fed chromium in the dose of lug/d, after 4 weeks administration, all rats were placed in the oven at 43 C with relative humidity, the heat tolerance time and the death rate after 1hour of each rat was noted. The heat tolerance time of female rat was longer than that of male rat, but there was no significant difference between those. The heat tolerance time of female rat fed chromium was more longer than the other female group(P<0.05), and there was no difference between the male groups(P<0.05), and the soluble protein extracted in the 0.9% sodium chloride solution was separated in SDS-PAGE.
引文
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[4] Edward S．Horton Rattaele Napoli现代营养学
[5] 马中书，邱明才，对1型糖尿病和2型糖尿病的再认识，天津医药，2001，29(6)：380～382
[6] 周爱儒等主编，生物化学，人民卫生出版社
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[9] 陈婉华等，螺旋藻对糖尿病大鼠的疗效观察，微生物学杂志，1998 18(4)：21～24
[10] Barbara J．Stoecker，现代营养学，第34章
[11] 张林等．微量元素铬在治疗糖尿病中的临床应用．国外医学地理学分册．1996 17(4)：161～162
[12] 王文君 欧阳克蕙等 铬的生物学功能及其应用效果．饲料博览．2000，10：12～13
[13] Mertz W Chromium occurrence and function in biological systems. Physiol Rev (1969)49: 163～239
[14] 程忠刚 畜禽铬的营养研究及应用 饲料博览．1998，10(6)：10～15
[15] Schwarz K, Mertz W, A glucose tolerance factor and its differentiation from factor 3. Arch Biochem biophys 1957 72: 515～518
[16] 颜世铭等，微量元素导论，上海：同济大学出版社，1992，309
[17] Sayato Y, Nakamuro K, Matsui S, Ando M,Metabolic fate of chromium compounds. 1.Comparative behavior of chromium in rat administered with Na_2~(51)CrO_4 and ~(51)CrCl_3,J Pharmacobiodyn 1980 3 : 17～23
[18] David ML, Seaborn CD, Stoecker B J, Effects of over_the_counter drugs on chromium retention and urinary excretion in rats, Nutr Res 1995 15 : 202～210
[19] Offenbacher EG, Promorion of chromium absorption by ascorbic acid,
Trace Elem Electrolytes 1994 11:178～181
[20] Dowling HJ, Offenbacher EG, Pi-Sunyer FX, Effects of plasma transferring and albumin on the absorption of trivalent chromium. Nutr Res 1990 10 : 1251～1260
[21] Moshtagine AA,Ani M, Bazrafshan MR, Comparative binding study of alumium and chromium to human transferring : effect of iron.Biol Trace Elem Res 32 : 39～46
[22] Lim TH, Sargent T Ⅲ, Kusubov N, Kinetics of trace element chromium(Ⅲ) in the human body.Am J Physiol 244 : R445～R454
[23] Kozolovsky AS, Moser PB, Reiser S, Anderson RA, Effects of diets high in simple sugars on urinary chromium losses.Metabolism 1986 35: 515～518
[24] Seaborn CD, Cheng NZ, Adeleye B, et al, Chromium and chronic ascorbic acid depletion effects on tissue ascorbate,managanese and ~(14)C retention from ~(14)C-ascorbate in guinea pigs.Biol Trace Elem Res 1994 41 : 279～294
[25] Freund H, Atamian S, Fischer JE, Chromium deficiency during total parenteral nutrition.JAMA 1979 241 : 496～498
[26] 王振来 钟艳玲等．微量元素铬的研究进展．中国饲料．2001，4：16～17
[27] Glismann W.Effect of trivalent Chromium on glucose, Tolerance Metab, 1996, 15 : 510～520
[28] Mertz W, Chromium in human nutrition:a review, J Nutr 1993 123 : 626～633
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