葛根芩连汤对实验性糖尿病动物模型降血糖机制的初步研究
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摘要
本研究在考察葛根芩连汤降血糖、降血脂作用的同时,通过复方及其各组成药物的总抗氧化活性(FRAP)和对有机自由基1-二苯基-2-苦基肼基(DPPH·)的清除作用对该方抗氧化活性进行了考察。采用液质联用方法分析正常动物、糖尿病动物模型及灌胃葛根芩连汤后的尿液中内源性代谢物的区别,采用主成分分析(PCA)和偏最小二乘法-判别分析(PLS-DA)的方法进行数据分析,寻找与糖尿病病发性潜在生物标记物,发展一种基于代谢组学的糖尿病早期诊断和药物筛选新方法。
首先研究葛根芩连汤对四氧嘧啶(ALX)诱导糖尿病小鼠和高脂高糖饲料加链脲佐菌素(STZ)诱导糖尿病大鼠的降糖作用,并通过考察该方对小鼠血脂的影响及其抗氧化活性,探讨其治疗糖尿病的作用机制。
以尾静脉注射ALX致糖尿病模型小鼠,以阿卡波糖为阳性对照,葛根芩连汤灌胃给药15d后,测定其空腹血糖和给药后1h血糖以及给药1h后血清中甘油三酯(TG)和高密度脂蛋白(HDL-C)的含量;分别用FRAP(Ferric reducing antioxidant power)法、DPPH·法测定了葛根芩连汤全方及组方单味药的抗氧化活性。同时以腹腔注射STZ致糖尿病模型大鼠,以阿卡波糖为阳性对照,葛根芩连汤灌胃给药20 d后,测定其空腹血糖和给药后1 h血糖。葛根芩连汤能显著降低ALX诱导的糖尿病模型小鼠和STZ诱导的糖尿病模型大鼠的血糖值,使小鼠血清中TG的含量降低和HDL-C的含量升高,且具有较好的抗氧化活性。葛根芩连汤对糖尿病大小鼠都有明显的降糖作用,并且通过降血脂和强抗氧化作用发挥对糖尿病动物的整体保护作用。方中黄芩的抗氧化作用中占有主导作用。
以高脂高糖饲料加STZ诱导的糖尿病大鼠为研究对象,通过高效液相色谱与质谱(LC-MS)联用技术对大鼠尿液分析。以流动相为乙腈(含0.1%甲酸)和超纯水(含0.1%甲酸)进行梯度洗脱,建立尿液代谢图谱。使用SIEVE软件对图谱数据处理,以SCORE大于0.6为标准,不同尿液质谱全扫描图为参照,空白组和模型组共检出393个化合物,模型与葛根芩连汤组共检出354个化合物。然后利用SIMCA-P软件对检出化合物进行PCA和PLS-DA分析,根据相关系数界值表,选择p﹤0.05的化合物,空白组和模型组得到104个化合物,模型与葛根芩连汤组得到164个化合物。以质荷比和保留时间为参照,从104和164个化合物中找到相同的31个潜在的生物标记物。通过HMDB和ChemSpider数据库中进行搜库比对,识别出分子量为197的化合物,使用标准品对保留时间、分子量以及串联质谱谱图进行对照,最终鉴定m/z197化合物为D-葡萄糖酸。在尿液中,它的趋势呈现出从空白组到模型组升高,模型组到葛根芩连汤组又降低的趋势。因此,通过代谢组学方法的分析也可以看出,葛根芩连汤对糖尿病有一定的防治作用。
This study is investigated the hypoglycemic and hypolipidemic of Gegenqinlian Decoction (GQD).The antioxidant activity of GQD is also measured through the total antioxidant activity and organic free radical (DPPH·) scavenging. By using LC-MS method, endogenous metabolites are analysised the difference in urine between normal animals and diabetic animals.And the data is analysised through the principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) methods, in order to look for the potential diabetes biomarkers, and develop diabetes early diagnosis methods based on metabonomics and new methods of drug screening.
The hypoglycemic effect of Gegen Qinlian Decoction (GQD) on alloxan induced diabetic model rats and STZ induced diabetic model mice was studied. The effect of hypolipidemic, antioxidant activity and the hypoglycemic mechanism of the decoction were evaluated. The diabetic mice were induced by the intravenous injection of alloxan (ALX). 15d after administration, the blood glucose, serum triglyceride (TG) and high density lipoprotein (HDL-C) were determined since 1 h after the last administration. The effect of antioxidation of GQD and each prescription herb was determined by Ferric reducing antioxidant power (FRAP) assay and 2,2-diphenyl-1-picryl-hydrazyl (DPPH·) free radical-scavenging assay, respectively. The diabetic rats were induced by the abdominal injection of STZ. 20 d after administration, the blood glucose was determined since 1 h after the last administration.
GQD could significantly reduce the blood glucose levels of alloxan-induced diabetic mice and STZ-induced diabetic rats, and decreases levels of serum TG and increases HDL-C levels. In addition, GQD has good antioxidant activity. GQD has significantly hypoglycemic effect. By the effect of hypolipidemic and antioxident, GQD plays a integral role in treatment of diabetes. In addition, the Scutellaria plays a leading role in the whole effect of antioxidation of GQD.
The urine of STZ-induced diabetic rats was detected by LC-MS analysis to establish metabolic graphy. The mobile phase was included acetonitrile (containing 0.1% formic acid) and ultra pure water (containing 0.1% formic acid) for gradient elution. 393 compounds of the blank group and model group, 354 compounds of model and Gegenqinlian Decoction group were detected through SIEVE software for SCORE greater than 0.6 standard. Then, detected compounds were analysised by using PCA and PLS-DA, control group and model group received 104 compounds, model and Gegenqinlian Decoction group got 164 compounds. Accrording to the m/z and retention time, the same 31 potential biomarkers were found from the 104 and 164 compounds. Compared to the HMDB and ChemSpider databases, the m/z197 compound was identified.And it was sured D-gluconic acid through the retention time, molecular weight and MS/MS spectra. In the urine, it showed a trend to the model group from the control group was increased, and a trend to the model group to Gegenqinlian Decoction group was decreased. Thus, Gegenqinlian Decoction has a certain role on diabetes prevention and treatment by the analysis of metabolomics approach.
首先研究葛根芩连汤对四氧嘧啶(ALX)诱导糖尿病小鼠和高脂高糖饲料加链脲佐菌素(STZ)诱导糖尿病大鼠的降糖作用,并通过考察该方对小鼠血脂的影响及其抗氧化活性,探讨其治疗糖尿病的作用机制。
以尾静脉注射ALX致糖尿病模型小鼠,以阿卡波糖为阳性对照,葛根芩连汤灌胃给药15d后,测定其空腹血糖和给药后1h血糖以及给药1h后血清中甘油三酯(TG)和高密度脂蛋白(HDL-C)的含量;分别用FRAP(Ferric reducing antioxidant power)法、DPPH·法测定了葛根芩连汤全方及组方单味药的抗氧化活性。同时以腹腔注射STZ致糖尿病模型大鼠,以阿卡波糖为阳性对照,葛根芩连汤灌胃给药20 d后,测定其空腹血糖和给药后1 h血糖。葛根芩连汤能显著降低ALX诱导的糖尿病模型小鼠和STZ诱导的糖尿病模型大鼠的血糖值,使小鼠血清中TG的含量降低和HDL-C的含量升高,且具有较好的抗氧化活性。葛根芩连汤对糖尿病大小鼠都有明显的降糖作用,并且通过降血脂和强抗氧化作用发挥对糖尿病动物的整体保护作用。方中黄芩的抗氧化作用中占有主导作用。
以高脂高糖饲料加STZ诱导的糖尿病大鼠为研究对象,通过高效液相色谱与质谱(LC-MS)联用技术对大鼠尿液分析。以流动相为乙腈(含0.1%甲酸)和超纯水(含0.1%甲酸)进行梯度洗脱,建立尿液代谢图谱。使用SIEVE软件对图谱数据处理,以SCORE大于0.6为标准,不同尿液质谱全扫描图为参照,空白组和模型组共检出393个化合物,模型与葛根芩连汤组共检出354个化合物。然后利用SIMCA-P软件对检出化合物进行PCA和PLS-DA分析,根据相关系数界值表,选择p﹤0.05的化合物,空白组和模型组得到104个化合物,模型与葛根芩连汤组得到164个化合物。以质荷比和保留时间为参照,从104和164个化合物中找到相同的31个潜在的生物标记物。通过HMDB和ChemSpider数据库中进行搜库比对,识别出分子量为197的化合物,使用标准品对保留时间、分子量以及串联质谱谱图进行对照,最终鉴定m/z197化合物为D-葡萄糖酸。在尿液中,它的趋势呈现出从空白组到模型组升高,模型组到葛根芩连汤组又降低的趋势。因此,通过代谢组学方法的分析也可以看出,葛根芩连汤对糖尿病有一定的防治作用。
This study is investigated the hypoglycemic and hypolipidemic of Gegenqinlian Decoction (GQD).The antioxidant activity of GQD is also measured through the total antioxidant activity and organic free radical (DPPH·) scavenging. By using LC-MS method, endogenous metabolites are analysised the difference in urine between normal animals and diabetic animals.And the data is analysised through the principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) methods, in order to look for the potential diabetes biomarkers, and develop diabetes early diagnosis methods based on metabonomics and new methods of drug screening.
The hypoglycemic effect of Gegen Qinlian Decoction (GQD) on alloxan induced diabetic model rats and STZ induced diabetic model mice was studied. The effect of hypolipidemic, antioxidant activity and the hypoglycemic mechanism of the decoction were evaluated. The diabetic mice were induced by the intravenous injection of alloxan (ALX). 15d after administration, the blood glucose, serum triglyceride (TG) and high density lipoprotein (HDL-C) were determined since 1 h after the last administration. The effect of antioxidation of GQD and each prescription herb was determined by Ferric reducing antioxidant power (FRAP) assay and 2,2-diphenyl-1-picryl-hydrazyl (DPPH·) free radical-scavenging assay, respectively. The diabetic rats were induced by the abdominal injection of STZ. 20 d after administration, the blood glucose was determined since 1 h after the last administration.
GQD could significantly reduce the blood glucose levels of alloxan-induced diabetic mice and STZ-induced diabetic rats, and decreases levels of serum TG and increases HDL-C levels. In addition, GQD has good antioxidant activity. GQD has significantly hypoglycemic effect. By the effect of hypolipidemic and antioxident, GQD plays a integral role in treatment of diabetes. In addition, the Scutellaria plays a leading role in the whole effect of antioxidation of GQD.
The urine of STZ-induced diabetic rats was detected by LC-MS analysis to establish metabolic graphy. The mobile phase was included acetonitrile (containing 0.1% formic acid) and ultra pure water (containing 0.1% formic acid) for gradient elution. 393 compounds of the blank group and model group, 354 compounds of model and Gegenqinlian Decoction group were detected through SIEVE software for SCORE greater than 0.6 standard. Then, detected compounds were analysised by using PCA and PLS-DA, control group and model group received 104 compounds, model and Gegenqinlian Decoction group got 164 compounds. Accrording to the m/z and retention time, the same 31 potential biomarkers were found from the 104 and 164 compounds. Compared to the HMDB and ChemSpider databases, the m/z197 compound was identified.And it was sured D-gluconic acid through the retention time, molecular weight and MS/MS spectra. In the urine, it showed a trend to the model group from the control group was increased, and a trend to the model group to Gegenqinlian Decoction group was decreased. Thus, Gegenqinlian Decoction has a certain role on diabetes prevention and treatment by the analysis of metabolomics approach.
引文
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