委陵菜黄酮降血糖作用及其机制研究
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摘要
目的：通过糖尿病动物模型，筛选委陵菜抗糖尿病的有效部位及主要有效成分，并进一步探讨降血糖有效成分委陵菜黄酮降血糖作用的量效关系和时效关系。观察委陵菜黄酮对四氧嘧啶所致糖尿病小鼠以及链脲佐菌素诱导的糖尿病大鼠模型的降血糖作用，并从糖代谢、脂代谢、抗氧化等方面对其抗糖尿病机制进行初步分析。
方法：1．采用65mg/kg四氧嘧啶尾静脉注射建立四氧嘧啶糖尿病小鼠模型，挑选建模合格的小鼠，按血糖值和体重均匀分成10组。分别给予委陵菜不同极性部位溶液。各组动物每日灌胃1次，连续灌胃15天。眼静脉丛取血，以GOD法测血糖值，筛选降血糖有效部位。
2．委陵菜降血糖有效部位F部位，经色谱方法和制备型高效液相色谱分离得到黄酮类成分即委陵菜黄酮，以四氧嘧啶糖尿病小鼠模型，观察其降血糖效果，筛选降血糖有效成分。
3．按70mg／kg剂量四氧嘧啶尾静脉注射复制糖尿病小鼠模型，以正常小鼠及四氧嘧啶糖尿病小鼠为模型，将糖尿病小鼠按血糖值均匀分成7组，委陵菜黄酮按照不同剂量连续灌胃15天，采血测量空腹血糖，甘油三酯和总胆固醇水平。取胰腺做病理切片，供光镜观察各组小鼠胰腺病理学的改变。
4．采用高糖高脂饲料喂养8周，联合小剂量链脲佐菌素(STZ 30mg／kg)静脉注射的方法诱发实验性2型糖尿病大鼠模型，应用委陵菜黄酮连续灌胃10周，观察药物对实验性糖尿病大鼠糖代谢、脂代谢、糖耐量、胰腺病理形态学改变、SOD、MDA水平等的影响。
5．采用链脲佐菌素(STZ 60mg／kg)静脉注射的方法诱发实验性1型糖尿病大鼠模型，应用委陵菜黄酮连续灌胃4周，观察药物对实验性糖尿病大鼠糖代谢、脂代谢、抗氧化能力及胰腺病理形态学改变等的影响。
结果：1．实验结果表明，委陵菜F部位降低四氧嘧啶小鼠模型血糖的作用最强，与阳性对照组在给药后无显著性差异(P＞0．05)，血糖降低率分别为32．97%和33．30%，说明委陵菜F部位具有较强的抗糖尿病作用。为委陵菜抗糖尿病的有效部位。
2．委陵菜F部位经色谱分离后所得成分—委陵菜黄酮，药理实验表明高剂量组(0．4mg/kg)血糖降低率为52．19%，盐酸苯乙双胍阳性对照组血糖降低率为29．26%，说明该黄酮类化合物具有明显的降低四氧嘧啶小鼠模型血糖的作用，药效强于阳性对照药组，说明委陵菜黄酮为委陵菜降血糖的有效成分。
3．委陵菜黄酮对糖尿病小鼠具有显著的降血糖以及降低甘油三酯和总胆固醇水平的作用。说明委陵菜黄酮可降低血糖，同时也可改善血糖升高引起的脂质代谢异常，但对正常小鼠的血糖、血脂没有影响，顺反异构并不影响药效的因素。此外，病理学检查结果显示，委陵菜黄酮可明显对抗病因性糖尿病模型小鼠胰岛细胞萎缩，减轻胰岛细胞病理损伤，提示委陵菜黄酮对糖尿病模型的胰岛损伤有保护作用。
4．委陵菜黄酮对高糖高脂饲料结合小剂量链脲佐菌素所致糖尿病大鼠的血糖升高有明显降低作用，具有明显的降低糖尿病小鼠TC、TG水平，升高HDL-C水平的作用。委陵菜黄酮组能抑制OGTT后0min、30min、60min、120min和180min血糖的升高(P＜0．05)，改善葡萄糖致糖尿病大鼠的糖耐量减退。说明委陵菜黄酮可降低血糖，同时也可显著改善血糖升高引起的脂质代谢异常。在抗氧化指标的检测中，与糖尿病模型组相比较，委陵菜黄酮各剂量组，SOD水平明显升高，MDA水平明显降低(P＜0．05，P＜0．01)。说明委陵菜黄酮低剂量组能有效提高糖尿病大鼠的抗氧化能力。此外，病理学检查结果显示，委陵菜黄酮对糖尿病模型的胰岛损伤有保护作用。
5．委陵菜黄酮可以显著降低STZ诱导的1型糖尿病大鼠空腹血糖，促进肝糖原的合成，从而改善糖代谢。通过纠正糖尿病大鼠血清TC、LDL-C、FFA水平异常，升高HDL-C含量，纠正机体脂代谢紊乱。通过提高糖尿病大鼠血清SOD活力，降低NO含量，从而增强糖尿病大鼠的抗氧化能力。
结论：委陵菜降血糖具有显著的降血糖作用，改善糖尿病并发的脂代谢紊乱和抗氧化，保护胰岛β细胞的作用，从而治疗糖尿病及并发症。以上结果提示委陵菜黄酮将有望成为治疗糖尿病及其并发症的一种有前途的天然药物。
Objective: The present study was to investigate the anti-diabetic effect ofpotentilla flavone on alloxan-induced diabetic mice and streptozotocin-induceddiabetic rats, and to explore the anti-diabetic effect and its mechanisms of potentillaflavone.
Methods: 1 .Mice were initated with intervenous injection 65mg/kg alloxan toestablish diabetic model mice, the alloxan-diabetic mice and normal mice wereequally divided into 10 groups according to body weight and FBG. Different polarityfraction were given intragastrically for successive 15 days,once a time. Fast bloodsugar was measured using GOD method to screen anti-diabetic active component.
2. A main flavonoid constituent, named potentilla flavone of the anti-diabeticactive fraction F were isolated by the methods of silica gel column chromatographyand preparative high performance(HPLC). Anti-diabetic effects were observed byusing alloxan-induced diabeticmice, screen anti-diabetic active constituent.
3. Diabetic animal models were induced by the intervenous injection of alloxan(70mg·kg~(-1)) in mice, the alloxan-diabetic mice and normal mice were equally dividedinto 7 groups. The above groups were given intragastrically the potentilla flavone forsuccessive 15 days and then blood samples were taken to determine blood glucose,TG and TC. Made the pancreatic gland pathological section to observe pancreasorganization through optics microscope. Pathologic changes of pancreatic tissues ineach group were observed.
4. Male type 2 diabetic model rats were induced by feeding with high glucose andfat diet together with intervenous injection of 35mg/kg STZ and then intragastricallythe potentilla flavone for 10 weeks. Then we observed the effects of the testedcomponent on glycometabolism, lipid metabolism, oral glucose tolerance, pancreaspathomorpholgoy change, serum SOD, MDA level in diabetic mice.
5. According to the purpose of the experiment, type 1 diabetic model was inducedby STZ(60mg/kg)in rats and then intragastrically given the potentilla flavone for 4weeks. Then the effects of the tested component on glycometabolism, lipidmetabolism, antioxidation capability, pancreas pathomorpholgoy change in diabeticrats were observed.
Results: 1 .In the anti-diabetic experiment, fraction F revealed significantlypoglycemic effect comparing with the phenformin group(P＜0.01),and their reduction rates were 32.97% for the fraction F group and 33.30% for the phenformin group.This observation suggests that potentilla flavone is an anti-diabetic active fraction.
2. A main constituent was isolated from the fraction F. By using spectroscopicmethod, the strueture was elucidated as a flavonoid, named poteniilla flavone. Inthe anti- diabetic experiment, potentilla flavone revealed the significanthypoglycemic effect in alloxan-induced diabetic mice, its reduction rate of bloodglucose was 52.19%, and reduction rate of phenformin group was 29.26%. The aboveobservation suggests that potentilla flavone is an anti-diabetic active constituent.
3. The results showed that potentilla flavone can decrease significantly the levelof the blood sugar, TG and TC. The observation indicated potentilla flavone candecrease blood sugar and improve the lipid decompensation in mice. Pathologic resultof pancreas islet indicated that potentilla flavone has repair effects. potentilla flavonehas the effect of decreasing blood glucose and serum lipid of the diabetic mice butshows no effects on blood glucose and serum lipid of normal mice. The FBG andTC,TG level in trans-potentilla flavone and cis-potentilla flavone-treated group withsame dosage show no difference. The result of morphyology pancreas isletβcellshow that it can restore the histology structure of damaged pancreas isletβcell.
4. Diabetic model rats were induced by combination of high-fat-sugar diet for 8weeks and low dose streptozotocin (STZ, 30mg/kg iv) injection in SD rats. Diabeticrats were randomly divided into 5 groups and normal group. After being given drugsor distilled water for 10 weeks, the levels of FBG, serum lipid, serum MDA and theactivity of SOD were measured. The blood sugar in rat at 0, 30, 60, 120 and 180minute after taking glucose were measured. Furthermore, pathologic changes ofpancreatic tissues in each group were observed. Experimental results indicate thatpotentilla flavone can lower blood glucose levels and significantly lower the level ofserum cholesterol, triglyceride and increase high-density lipoprotein level. Thepotentilla flavone group is able to enhance the SOD activity, reduce the production ofMDA(P＜0.05, P＜0.01) and lower 30, 60, 120 and 180min BG after OGTT ascompared with the diabetic model group, the difference is significant(P＜0.05). HEstaining of pancreas shows that potentilla flavone can improve pancreatic injury indiabetic mice.
5. The results showed that potentilla flavone can improve glycometabolism bydecreasing the level of FBG and increasing hepatic glycogen content. The content oftotal cholesterol, LDL-C, FFA level was rectified and HDL-C content in serum increased, so lipid metabolism was improved. Promote the antioxidant capability byincreasing the activity of SOD and low the content of MDA in serum of diabetic rats.
The research results suggest that potentilla flavone can improve glycometabolismand lipid metabolism, promote antioxidant capability, protect the islet cell injury thatwas induced by streptozotocin and improve the impaired glucose tolerance. Therefore,potentilla flavone will be expected to develop a promising natural product to treatdiabetic mellitus and its complications.
方法：1．采用65mg/kg四氧嘧啶尾静脉注射建立四氧嘧啶糖尿病小鼠模型，挑选建模合格的小鼠，按血糖值和体重均匀分成10组。分别给予委陵菜不同极性部位溶液。各组动物每日灌胃1次，连续灌胃15天。眼静脉丛取血，以GOD法测血糖值，筛选降血糖有效部位。
2．委陵菜降血糖有效部位F部位，经色谱方法和制备型高效液相色谱分离得到黄酮类成分即委陵菜黄酮，以四氧嘧啶糖尿病小鼠模型，观察其降血糖效果，筛选降血糖有效成分。
3．按70mg／kg剂量四氧嘧啶尾静脉注射复制糖尿病小鼠模型，以正常小鼠及四氧嘧啶糖尿病小鼠为模型，将糖尿病小鼠按血糖值均匀分成7组，委陵菜黄酮按照不同剂量连续灌胃15天，采血测量空腹血糖，甘油三酯和总胆固醇水平。取胰腺做病理切片，供光镜观察各组小鼠胰腺病理学的改变。
4．采用高糖高脂饲料喂养8周，联合小剂量链脲佐菌素(STZ 30mg／kg)静脉注射的方法诱发实验性2型糖尿病大鼠模型，应用委陵菜黄酮连续灌胃10周，观察药物对实验性糖尿病大鼠糖代谢、脂代谢、糖耐量、胰腺病理形态学改变、SOD、MDA水平等的影响。
5．采用链脲佐菌素(STZ 60mg／kg)静脉注射的方法诱发实验性1型糖尿病大鼠模型，应用委陵菜黄酮连续灌胃4周，观察药物对实验性糖尿病大鼠糖代谢、脂代谢、抗氧化能力及胰腺病理形态学改变等的影响。
结果：1．实验结果表明，委陵菜F部位降低四氧嘧啶小鼠模型血糖的作用最强，与阳性对照组在给药后无显著性差异(P＞0．05)，血糖降低率分别为32．97%和33．30%，说明委陵菜F部位具有较强的抗糖尿病作用。为委陵菜抗糖尿病的有效部位。
2．委陵菜F部位经色谱分离后所得成分—委陵菜黄酮，药理实验表明高剂量组(0．4mg/kg)血糖降低率为52．19%，盐酸苯乙双胍阳性对照组血糖降低率为29．26%，说明该黄酮类化合物具有明显的降低四氧嘧啶小鼠模型血糖的作用，药效强于阳性对照药组，说明委陵菜黄酮为委陵菜降血糖的有效成分。
3．委陵菜黄酮对糖尿病小鼠具有显著的降血糖以及降低甘油三酯和总胆固醇水平的作用。说明委陵菜黄酮可降低血糖，同时也可改善血糖升高引起的脂质代谢异常，但对正常小鼠的血糖、血脂没有影响，顺反异构并不影响药效的因素。此外，病理学检查结果显示，委陵菜黄酮可明显对抗病因性糖尿病模型小鼠胰岛细胞萎缩，减轻胰岛细胞病理损伤，提示委陵菜黄酮对糖尿病模型的胰岛损伤有保护作用。
4．委陵菜黄酮对高糖高脂饲料结合小剂量链脲佐菌素所致糖尿病大鼠的血糖升高有明显降低作用，具有明显的降低糖尿病小鼠TC、TG水平，升高HDL-C水平的作用。委陵菜黄酮组能抑制OGTT后0min、30min、60min、120min和180min血糖的升高(P＜0．05)，改善葡萄糖致糖尿病大鼠的糖耐量减退。说明委陵菜黄酮可降低血糖，同时也可显著改善血糖升高引起的脂质代谢异常。在抗氧化指标的检测中，与糖尿病模型组相比较，委陵菜黄酮各剂量组，SOD水平明显升高，MDA水平明显降低(P＜0．05，P＜0．01)。说明委陵菜黄酮低剂量组能有效提高糖尿病大鼠的抗氧化能力。此外，病理学检查结果显示，委陵菜黄酮对糖尿病模型的胰岛损伤有保护作用。
5．委陵菜黄酮可以显著降低STZ诱导的1型糖尿病大鼠空腹血糖，促进肝糖原的合成，从而改善糖代谢。通过纠正糖尿病大鼠血清TC、LDL-C、FFA水平异常，升高HDL-C含量，纠正机体脂代谢紊乱。通过提高糖尿病大鼠血清SOD活力，降低NO含量，从而增强糖尿病大鼠的抗氧化能力。
结论：委陵菜降血糖具有显著的降血糖作用，改善糖尿病并发的脂代谢紊乱和抗氧化，保护胰岛β细胞的作用，从而治疗糖尿病及并发症。以上结果提示委陵菜黄酮将有望成为治疗糖尿病及其并发症的一种有前途的天然药物。
Objective: The present study was to investigate the anti-diabetic effect ofpotentilla flavone on alloxan-induced diabetic mice and streptozotocin-induceddiabetic rats, and to explore the anti-diabetic effect and its mechanisms of potentillaflavone.
Methods: 1 .Mice were initated with intervenous injection 65mg/kg alloxan toestablish diabetic model mice, the alloxan-diabetic mice and normal mice wereequally divided into 10 groups according to body weight and FBG. Different polarityfraction were given intragastrically for successive 15 days,once a time. Fast bloodsugar was measured using GOD method to screen anti-diabetic active component.
2. A main flavonoid constituent, named potentilla flavone of the anti-diabeticactive fraction F were isolated by the methods of silica gel column chromatographyand preparative high performance(HPLC). Anti-diabetic effects were observed byusing alloxan-induced diabeticmice, screen anti-diabetic active constituent.
3. Diabetic animal models were induced by the intervenous injection of alloxan(70mg·kg~(-1)) in mice, the alloxan-diabetic mice and normal mice were equally dividedinto 7 groups. The above groups were given intragastrically the potentilla flavone forsuccessive 15 days and then blood samples were taken to determine blood glucose,TG and TC. Made the pancreatic gland pathological section to observe pancreasorganization through optics microscope. Pathologic changes of pancreatic tissues ineach group were observed.
4. Male type 2 diabetic model rats were induced by feeding with high glucose andfat diet together with intervenous injection of 35mg/kg STZ and then intragastricallythe potentilla flavone for 10 weeks. Then we observed the effects of the testedcomponent on glycometabolism, lipid metabolism, oral glucose tolerance, pancreaspathomorpholgoy change, serum SOD, MDA level in diabetic mice.
5. According to the purpose of the experiment, type 1 diabetic model was inducedby STZ(60mg/kg)in rats and then intragastrically given the potentilla flavone for 4weeks. Then the effects of the tested component on glycometabolism, lipidmetabolism, antioxidation capability, pancreas pathomorpholgoy change in diabeticrats were observed.
Results: 1 .In the anti-diabetic experiment, fraction F revealed significantlypoglycemic effect comparing with the phenformin group(P＜0.01),and their reduction rates were 32.97% for the fraction F group and 33.30% for the phenformin group.This observation suggests that potentilla flavone is an anti-diabetic active fraction.
2. A main constituent was isolated from the fraction F. By using spectroscopicmethod, the strueture was elucidated as a flavonoid, named poteniilla flavone. Inthe anti- diabetic experiment, potentilla flavone revealed the significanthypoglycemic effect in alloxan-induced diabetic mice, its reduction rate of bloodglucose was 52.19%, and reduction rate of phenformin group was 29.26%. The aboveobservation suggests that potentilla flavone is an anti-diabetic active constituent.
3. The results showed that potentilla flavone can decrease significantly the levelof the blood sugar, TG and TC. The observation indicated potentilla flavone candecrease blood sugar and improve the lipid decompensation in mice. Pathologic resultof pancreas islet indicated that potentilla flavone has repair effects. potentilla flavonehas the effect of decreasing blood glucose and serum lipid of the diabetic mice butshows no effects on blood glucose and serum lipid of normal mice. The FBG andTC,TG level in trans-potentilla flavone and cis-potentilla flavone-treated group withsame dosage show no difference. The result of morphyology pancreas isletβcellshow that it can restore the histology structure of damaged pancreas isletβcell.
4. Diabetic model rats were induced by combination of high-fat-sugar diet for 8weeks and low dose streptozotocin (STZ, 30mg/kg iv) injection in SD rats. Diabeticrats were randomly divided into 5 groups and normal group. After being given drugsor distilled water for 10 weeks, the levels of FBG, serum lipid, serum MDA and theactivity of SOD were measured. The blood sugar in rat at 0, 30, 60, 120 and 180minute after taking glucose were measured. Furthermore, pathologic changes ofpancreatic tissues in each group were observed. Experimental results indicate thatpotentilla flavone can lower blood glucose levels and significantly lower the level ofserum cholesterol, triglyceride and increase high-density lipoprotein level. Thepotentilla flavone group is able to enhance the SOD activity, reduce the production ofMDA(P＜0.05, P＜0.01) and lower 30, 60, 120 and 180min BG after OGTT ascompared with the diabetic model group, the difference is significant(P＜0.05). HEstaining of pancreas shows that potentilla flavone can improve pancreatic injury indiabetic mice.
5. The results showed that potentilla flavone can improve glycometabolism bydecreasing the level of FBG and increasing hepatic glycogen content. The content oftotal cholesterol, LDL-C, FFA level was rectified and HDL-C content in serum increased, so lipid metabolism was improved. Promote the antioxidant capability byincreasing the activity of SOD and low the content of MDA in serum of diabetic rats.
The research results suggest that potentilla flavone can improve glycometabolismand lipid metabolism, promote antioxidant capability, protect the islet cell injury thatwas induced by streptozotocin and improve the impaired glucose tolerance. Therefore,potentilla flavone will be expected to develop a promising natural product to treatdiabetic mellitus and its complications.
引文
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