Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China; Tianjin Key Laboratory of Phytochemistry and Pharmaceutical Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China.
Tianjin Key Laboratory of Phytochemistry and Pharmaceutical Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China.
J Ethnopharmacol. 2015 Apr 22;164:210-22. doi: 10.1016/j.jep.2015.02.018. Epub 2015 Feb 16.
Jinqi Jiangtang Tablet is a traditional Chinese anti-diabetic formula containing three ingredients: Coptis chinensis Franch. (dried rhizome of C. chinensis Franch., Coptis deltoidea C. Y. Cheng et Hsiao and Coptis teeta Wall.), Astragalus membranaceus (Fisch.) Bunge. (dried root of A. membranaceus (Fisch.) Bge. var. mongholicus (Bge. ) Hsiao and A. membranaceus (Fisch.) Bge. ) and Lonicera japonica Thunb. (dried alabastrum or with nascent flowers of L. japonica Thunb. ). Free radicals, α-glucosidase, α-amylase, aldose reductase and lipase are different targets related with diabetes. However, there are no chromatographic methods employed in screening the anti-diabetic compounds from natural products basing on these targets simultaneously. The present study was aimed at the establishment of a multi-targets integrated fingerprinting to clarify the possible mechanism of the action of Traditional Chinese Medicines which simultaneously contained multiple chemical characteristics and effects of constitutions.
The multi-targets integrated fingerprinting was developed and validated to screen anti-diabetic compounds from natural products by using ultra-high-performance liquid chromatography/quadruple-time-of-flight mass spectrometry, fraction collector and microplate reader. Ultra performance liquid chromatography was employed to separate the components in Jinqi Jiangtang Tablet, which were identified by quadruple-time-of-flight mass spectrometry to acquire their structural information and collected by the fraction collector. Finally the active fractions were tested for scavenging 1, 1-diphenyl-2-picrylhydrazyl radical and inhibition of α-glucosidase, α-amylase, aldose reductase, and lipase activities in vitro by microplate reader.
Our tests revealed that the Jinqi Jiangtang Tablet showed inhibitory activity against α-glucosidase, α-amylase, aldose reductase and lipase with IC50 values of 0.80 ± 0.02 mg/mL, 1.28 ± 0.13 mg/mL, 0.80 ± 0.02 mg/mL, 1.90 ± 0.18 mg/mL respectively and the scavenging activity with IC50 value of 1.71 ± 0.178 mg/mL. The bioactive fractions were identified to be alkaloids, flavonoids and phenolic acids. The phenolic acids possessed antioxidant activities, namely the scavenging effect on 1, 1-diphenyl-2-picrylhydrazyl rull;). The alkaloids exhibited inhibitory activity against α-glucosidase, aldose reductase, α-amylase, and lipase. The flavonoids also showed mild inhibition on α-glucosidase, aldose reductase, α-amylase and lipase.
The results demonstrate that Jinqi Jiangtang Tablet can scavenge free radicals and inhibit α-glucosidase, aldose reductase, α-amylase and lipase, which may be the possible mechanism of action of Jinqi Jiangtang Tablet for the treatment of diabetes and associated complications. Compared with conventional chromatographic separation and activity assays, the multi-targets integrated fingerprinting, which simultaneously contains the chemical characteristics and multiple effects of constitutions could comprehensively and properly reveal the activity of Jinqi Jiangtang Tablet. The results also show that the multi-targets integrated fingerprinting is a novel and powerful tool for screening and identifying active ingredients from Traditional Chinese Medicines.
金芪降糖片是一种中药降糖配方，包含三种成分：黄连（黄连、三角叶黄连和云连的干燥根茎）、黄芪（膜荚黄芪和蒙古黄芪的干燥根）和金银花（金银花的干燥花蕾或带新生花的蜜腺）。自由基、α-葡萄糖苷酶、α-淀粉酶、醛糖还原酶和脂肪酶是与糖尿病相关的不同靶点。然而，目前还没有基于这些靶点同时筛选天然产物中降糖化合物的色谱方法。本研究旨在建立一种多靶点综合指纹图谱，以阐明同时含有多种化学特征和成分作用的中药的作用机制。
采用超高效液相色谱/四极杆飞行时间质谱、馏分收集器和微孔板读数仪，建立并验证了多靶点综合指纹图谱，以筛选天然产物中的降糖化合物。超高效液相色谱用于分离金芪降糖片中的成分，通过四极杆飞行时间质谱对其进行鉴定，获取其结构信息，并通过馏分收集器进行收集。最后，通过微孔板读数仪测试活性馏分对 1,1-二苯基-2-苦基肼自由基的清除作用以及对α-葡萄糖苷酶、α-淀粉酶、醛糖还原酶和脂肪酶活性的抑制作用。
研究表明，金芪降糖片对α-葡萄糖苷酶、α-淀粉酶、醛糖还原酶和脂肪酶具有抑制活性，IC50 值分别为 0.80 ± 0.02 mg/mL、1.28 ± 0.13 mg/mL、0.80 ± 0.02 mg/mL 和 1.90 ± 0.18 mg/mL，其清除自由基的活性 IC50 值为 1.71 ± 0.178 mg/mL。生物活性馏分被鉴定为生物碱、黄酮类和酚酸。酚酸具有抗氧化活性，即对 1,1-二苯基-2-苦基肼自由基的清除作用。生物碱对α-葡萄糖苷酶、醛糖还原酶、α-淀粉酶和脂肪酶具有抑制作用。黄酮类化合物也对α-葡萄糖苷酶、醛糖还原酶、α-淀粉酶和脂肪酶有轻微抑制作用。
研究结果表明，金芪降糖片可清除自由基并抑制α-葡萄糖苷酶、醛糖还原酶、α-淀粉酶和脂肪酶，这可能是金芪降糖片治疗糖尿病及其相关并发症的作用机制。与传统的色谱分离和活性测定相比，同时包含化学成分和多种成分作用的多靶点综合指纹图谱能够全面、恰当地反映金芪降糖片的活性。结果还表明，多靶点综合指纹图谱是筛选和鉴定中药活性成分的一种新颖而强大的工具。
