
运用多维在线高效液相色谱-生物化学检测方法筛选银杏叶提取物治疗老年痴呆疾病的药效成分
丁晓萍, 沈华旦, 方江济, 郭鹏程
中国药学杂志 ›› 2018, Vol. 53 ›› Issue (16) : 1407-1412.
运用多维在线高效液相色谱-生物化学检测方法筛选银杏叶提取物治疗老年痴呆疾病的药效成分
Multiple On-line HPLC Coupled with Biochemical Detection Methods for Screening Effective Components in Ginkgo biloba Extract
目的 建立高效液相色谱-生物化学检测(HPLC-BCD)多维在线联用分析方法,筛选银杏叶提取物治疗老年痴呆疾病(Alzheimer disease,AD)的药效成分。方法 运用HPLC-UV(紫外)-1,1-二苯基-2-三硝基苯肼/2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(DPPH/ABTS)、HPLC-UV-乙酰胆碱酯酶(AchE)方法快速筛选银杏叶提取物中的自由基清除剂和AchE抑制剂。以加兰他敏为阳性对照,考察在线HPLC-UV-AchE方法的稳定性和重现性。运用超高效液相色谱(UPLC)-LTQ/线性离子阱/静电场轨道阱高分辨质谱(orbitrap MS)(UPLC-LTQ/orbitrap MS)方法鉴定主要活性成分。结果 HPLC-UV-DPPH/ABTS方法检测到银杏叶提取物中的14个强抗氧化成分,而运用HPLC-UV-AchE方法发现了3个AchE抑制剂,通过UPLC-LTQ/orbitrap MS方法鉴定了3个酶抑制剂分别为4-O-beta-glucopyranosyl-cis-coumaric acid、ginkgolide A和3-O-[2-O-(β-D-glucosyl)-α-L-rhamnosyl] quercetin。结论 实验建立的在线HPLC-BCD方法具有良好的灵敏度、重现性和稳定性,能够快速筛选银杏叶提取物中的抗氧化剂和AchE抑制剂。为快速发现中药治疗相关疾病的有效成分提供一种有效、快速、便捷的分析手段。
OBJECTIVE To develop on-line high performance liquid chromatography-biochemical detection (HPLC-BCD) methods to screen effective components in Ginkgo biloba extract for treatment of Alzheimer's disease. METHODS Radical scavengers and AchE inhibitors in G.biloba leave extract were screened by HPLC-UV-DPPH/ABTS and HPLC-UV-AchE methods, respectively. The stability and repeatability of the on-line HPLC-UV-AchE method were investigated using galanthamine as a positive reference. The main active ingredients in the extract were identified by ultra high performance liquid chromatography linear ion trap/orbitrap high resolution mass spectrometry (UPLC-LTQ/orbitrap MS) method. RESULTS Fourteen antioxidants in G.biloba extract were detected by the HPLC-UV-DPPH/ABTS method, while three AchE inhibitors were found by the HPLC-UV-AchE method. The three AchE inhibitors were tentatively identified by UPLC-LTQ/orbitrap MS as 4-O-beta-glucopyranosyl-cis-coumaric acid、 ginkgolide A and 3-O-[2-O-(β-D-Glucosyl)-α-L-rhamnosyl]quercetin, respectively. CONCLUSION The proposed on-line HPLC-BCD method shows high sensitivity, good repeatability and stability. It can be used to screen antioxidants and AchE inhibitors in G. biloba extract. This will provide an effective, quick and convenient analysis tool to quickly find bioactive ingredients of traditional Chinese medicines for treating related diseases.
高效液相色谱-生物化学检测 / 乙酰胆碱酯酶 / 1, 1-二苯基-2-三硝基苯肼/2, 2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐自由基 / 银杏叶提取物 / 线性离子阱/静电场轨道阱高分辨质谱 {{custom_keyword}} /
on-line HPLC-BCD / acetylcholinesterase / DPPH/ABTS free radical / Ginkgo biloba extract / UPLC-LTQ/orbitrap MS {{custom_keyword}} /
国家自然科学基金项目资助(81403093);湖北省自然科学基金项目资助(2015CFB493)
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