基于非酶糖基化反应的槐花抑制AGEs形成的活性组分筛选
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摘要
糖毒引起的血热是消渴病诸多并发症产生的病理基础。晚期糖基化终产物(advanced glycation end products,AGEs)被认为是一个潜在的糖毒因子,可引起血热。中药槐花具有清热凉血功效。该实验主要通过构建体外非酶糖基化反应模型——牛血清白蛋白/丙酮醛与槐花孵育前后的色谱反应体系(BSA/MGO reaction system),筛选出槐花抑制AGEs的活性组分。建立HPLC分析方法,发现反应后有8个色谱峰发生变化,推测槐花中的活性组分与MGO反应生成新的物质,且活性组分可能为黄酮类组分芦丁。进一步考察不同比例的芦丁与MGO反应6 d后(1∶1,1∶3,3∶1)对AGEs生成的反应规律,结果显示以1∶3比例反应时抑制AGEs生成活性最明显,且24 h内抑制作用最大,3 d后趋于稳定。采用LC-ESI-MS/MS对芦丁与MGO反应的生成物进行鉴定,表明新生成的7个物质分别为芦丁与MGO反应后的单/双加成产物。该研究表明,芦丁是槐花清热凉血功效的活性组分,其作用在于形成新的物质抑制糖毒产物的生成,这为合理应用槐花提供参考依据。
Sugar-poison caused blood-heat is the pathological basis of many complications of diabetes. Advanced glycation end products( AGEs) are considered as the potential glycotoxic factor that can cause blood-heat. Sophorae Flos hold the effect of removing pathogenic heat from blood. In this study,chromatographic non-enzymatic glycation reaction system of bovine serum albumin( BSA)/methylglyoxal( MGO) and Sophorae Flos was established to identify active components in Sophorae Flos inhibiting AGEs formation. The HPLC was used to analyze chromatograms before and after the incubation of Sophorae Flos and methylglyoxal. Changes of chromatographic peaks of eight compounds was found. It is speculated that this change may be due to new substance produced by the reaction of active components in Sophorae Flos and methylglyoxal,and these active components may be flavonoid component rutin. Further investigation for the effects of rutin and MGO reaction( 1 ∶ 1,1 ∶ 3,3 ∶ 1) for 6 days on the formation of AGEs was performed. The results showed that the inhibition activity of rutin on AGEs production was most obvious when the reaction ratio was 1 ∶3,and the most inhibition was in 24 h and stabilized after 3 d. The product of the reaction of rutin with MGO was identified by LC-ESI-MS/MS,which indicated that the newly formed seven substances were the mono-and di-MGO adducts of rutin. This study showed that rutin is the active component on Sophorae Flos for removing pathogenic heat from blood by forming new compounds to inhibit the formation of sugar poison products,which provides reference for rational application of Sophorae Flos.
Sugar-poison caused blood-heat is the pathological basis of many complications of diabetes. Advanced glycation end products( AGEs) are considered as the potential glycotoxic factor that can cause blood-heat. Sophorae Flos hold the effect of removing pathogenic heat from blood. In this study,chromatographic non-enzymatic glycation reaction system of bovine serum albumin( BSA)/methylglyoxal( MGO) and Sophorae Flos was established to identify active components in Sophorae Flos inhibiting AGEs formation. The HPLC was used to analyze chromatograms before and after the incubation of Sophorae Flos and methylglyoxal. Changes of chromatographic peaks of eight compounds was found. It is speculated that this change may be due to new substance produced by the reaction of active components in Sophorae Flos and methylglyoxal,and these active components may be flavonoid component rutin. Further investigation for the effects of rutin and MGO reaction( 1 ∶ 1,1 ∶ 3,3 ∶ 1) for 6 days on the formation of AGEs was performed. The results showed that the inhibition activity of rutin on AGEs production was most obvious when the reaction ratio was 1 ∶3,and the most inhibition was in 24 h and stabilized after 3 d. The product of the reaction of rutin with MGO was identified by LC-ESI-MS/MS,which indicated that the newly formed seven substances were the mono-and di-MGO adducts of rutin. This study showed that rutin is the active component on Sophorae Flos for removing pathogenic heat from blood by forming new compounds to inhibit the formation of sugar poison products,which provides reference for rational application of Sophorae Flos.
引文
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[13] Lv L,Shao X,Chen H,et al. Genistein inhibits advanced glycation end product formation by trapping methylglyoxal[J]. Chem Res Toxicol,2011,24(4):579.
[14] Wang W,Yagiz Y,Burant T J,et al. Phytochemicals from berries and grapes inhibited the formation of advanced glycation end-products by scavenging reactive carbonyls[J]. Food Res Int,2011,44(9):2666.
[15] Dorota S N,Georgios K,Wieslaw W,et al. Evaluation of the in vitro inhibitory effects of buckwheat enhanced wheat bread extracts on the formation of advanced glycation end-products(AGEs)[J]. LWT-Food Sci Technol,2014,58(2):327.
[16]依秋霞,李敬,林生生,等.浅析“糖毒”与糖尿病肾病发病机制[J].辽宁中医杂志,2014,41(6):1139.
[17]王春梅,郭冀萍,汤利红.论“糖毒”与糖尿病[J].湖北中医杂志,2006(10):16.
[18]田春雨,薄海美,林飞武,等.双益方改善2型糖尿病模型大鼠胰岛素抵抗的药效及作用机制[J].中国实验方剂学杂志,2017,23(22):137.
[19]彭孟凡,刘保松,苗明三.补肾法治疗多囊卵巢综合征的理论探讨[J].中国实验方剂学杂志,2019,25(6):204.
[20]吕兴,许惠琴,刘斌,等.莫诺苷对晚期糖基化终末产物加重链脲佐菌素诱导糖尿病肾病保护作用及其机制[J].中草药,2014,45(21):3109.
[21]陈娟,章丽,张明华,等.牡丹皮体外抑制AGEs生成及活性成分筛选研究[J].中国中药杂志,2016,41(5):891.
[22] Park S H,Do M H,Lee J H,et al. Inhibitory effect of arachishypogaea(peanut)and its phenolics against methylglyoxal-derived advanced glycation end product toxicity[J]. Nutrients,2017,9(11):1214.
[2]岳仁宋,曹立虎,齐方洲,等.小议“糖毒”[J].辽宁中医杂志,2012,39(7):1286.
[3] Rhee S Y,Kim Y S. The role of advanced glycation end products in diabetic vascular complications[J]. Diabetes Metab J,2018,42(3):188.
[4] Byun K,Yoo Y,Son M,et al. Advancedglycation end-products produced systemically and by macrophages:a commoncontributor to inflammation and degenerative diseases[J]. Pharmacol Ther,2017,177:44.
[5] Szkudlarek A,Poz·ycka J,Maciz·ek-Jurczyk M. Influence of piracetam on gliclazide-glycated human serum albumin interaction.a spectrofluorometric study[J]. Molecules,2018, doi:10. 3390/molecules24010111.
[6]李媛,曾克武,王学美.糖尿病相关脑微血管病变研究进展[J].中国中药杂志,2017,42(12):2247.
[7] Schalkwijk C G. Vascular AGE-ing by methylglyoxal:the past,the present and the future[J]. Diabetologia,2015,58(8):1715.
[8]王亚娇,苏珂,龙艳,等.2型糖尿病视网膜病变患者血清超敏C反应蛋白与胰岛素抵抗的相关性[J].中国老年学杂志,2014(10):2874.
[9] Webster J,Urban C,Berbaum K,et al. The carbonyl scavengersaminoguanidine and tenilsetam protect against the neurotoxic effects of methylglyoxal[J]. Neurotox Res,2005,7:95.
[10]中国药典.一部[S]. 2015:354.
[11]韩玲玲,樊海瑞,林明珠,等.槐花醇提物对2型糖尿病合并高尿酸血症小鼠疗效研究[J].中药材,2017,40(11):2697.
[12] Chen Q C,Zhang W Y,Jin W,et al. Flavonoids and isoflavonoids from Sophorae Flos improve glucose uptake in vitro[J].Planta Med,2010,76(1):79.
[13] Lv L,Shao X,Chen H,et al. Genistein inhibits advanced glycation end product formation by trapping methylglyoxal[J]. Chem Res Toxicol,2011,24(4):579.
[14] Wang W,Yagiz Y,Burant T J,et al. Phytochemicals from berries and grapes inhibited the formation of advanced glycation end-products by scavenging reactive carbonyls[J]. Food Res Int,2011,44(9):2666.
[15] Dorota S N,Georgios K,Wieslaw W,et al. Evaluation of the in vitro inhibitory effects of buckwheat enhanced wheat bread extracts on the formation of advanced glycation end-products(AGEs)[J]. LWT-Food Sci Technol,2014,58(2):327.
[16]依秋霞,李敬,林生生,等.浅析“糖毒”与糖尿病肾病发病机制[J].辽宁中医杂志,2014,41(6):1139.
[17]王春梅,郭冀萍,汤利红.论“糖毒”与糖尿病[J].湖北中医杂志,2006(10):16.
[18]田春雨,薄海美,林飞武,等.双益方改善2型糖尿病模型大鼠胰岛素抵抗的药效及作用机制[J].中国实验方剂学杂志,2017,23(22):137.
[19]彭孟凡,刘保松,苗明三.补肾法治疗多囊卵巢综合征的理论探讨[J].中国实验方剂学杂志,2019,25(6):204.
[20]吕兴,许惠琴,刘斌,等.莫诺苷对晚期糖基化终末产物加重链脲佐菌素诱导糖尿病肾病保护作用及其机制[J].中草药,2014,45(21):3109.
[21]陈娟,章丽,张明华,等.牡丹皮体外抑制AGEs生成及活性成分筛选研究[J].中国中药杂志,2016,41(5):891.
[22] Park S H,Do M H,Lee J H,et al. Inhibitory effect of arachishypogaea(peanut)and its phenolics against methylglyoxal-derived advanced glycation end product toxicity[J]. Nutrients,2017,9(11):1214.