基于血清药物化学的厚藤治疗急性痛风性关节炎质量标志物研究

Study on Q-Marker for treating acute gouty arthritis with Ipomoea pes-caprae based on serum pharmacochemistry

目的基于中药质量标志物(qualitymarker,Q-Marker)的概念,研究厚藤提取物对急性痛风性关节炎(acutegouty arthritis,AGA)大鼠的治疗作用,并分析厚藤提取物在正常大鼠及AGA大鼠的血中移行成分,为厚藤Q-Marker的确定提供依据。方法采用尿酸钠建立AGA大鼠模型,考察厚藤提取物对AGA大鼠关节肿胀指数、足垫肿胀程度和滑膜组织病理变化的影响,考察厚藤提取物对AGA大鼠血清中白细胞介素-1β(interleukin-1β,IL-1β)、巨噬细胞炎性蛋白-1α(macrophage inflammatory protein-1α,MIP-1α)和MIP-2水平的影响。采用液相色谱-四级杆-飞行时间串联质谱(LC-Q-TOF-MS/MS)技术对厚藤提取物进行定性分析,探寻正常大鼠和AGA大鼠ig厚藤提取物后的入血成分。结果与模型组比较,厚藤提取物组大鼠关节肿胀指数和足垫厚度均显著降低(P<0.05、0.01),厚藤提取物高、中剂量组大鼠血清中IL-1β、MIP-1α和MIP-2水平均显著降低(P<0.05),厚藤提取物高、中剂量组大鼠滑膜组织炎性细胞浸润减轻。从厚藤提取物中鉴定出树脂糖苷类、黄酮类、有机酸类、香豆素类、核苷类、木脂素类等53个成分,其中有机酸类和黄酮类为其主要成分,并推测了树脂糖苷类、黄酮类、有机酸类、香豆素类部分成分的裂解规律;正常大鼠含药血浆中鉴定出苹果酸、咖啡酸、咖啡酸甲酯和对羟基苯甲酸4个成分,AGA大鼠含药血浆中鉴定出莨菪亭、咖啡酸、咖啡酸甲酯和对羟基苯甲酸4个成分。结论厚藤提取物能够明显改善AGA大鼠关节肿胀,抑制血清中IL-1β、MIP-1α及MIP-2水平,并抑制炎症反应;采用LC-Q-TOF-MS/MS技术可快速鉴定厚藤水提物的主要成分为有机酸类和黄酮类,莨菪亭、咖啡酸、绿原酸及其异构体(新绿原酸、隐绿原酸、异绿原酸B、异绿原酸A、异绿原酸C)和原儿茶酸在AGA大鼠中以原型成分或代谢物形式入血,可作为厚藤治疗AGA的Q-Marker。

Objective To study the therapeutic effect of Ipomoea pes-caprae extract on acute gouty arthritis(AGA)rats and analyze the transitional components in blood of normal rats and AGA rats based on theory of quality marker(Q-Marker)of traditional Chinese medicine,and provide experimental basis for the determination of Q-Marker.Methods AGA model was induced by sodium urate.The effect of I.pes-caprae extract on joint swelling index,foot pad swelling and synovial tissue pathological changes in AGA rats were evaluated.The effect of I.pes-caprae extract on interleukin-1β(IL-1β),macrophage inflammatory protein-1α(MIP-1α),and MIP-2 levels in serum of AGA rats were observed.LC-Q-TOF-MS/MS was used to qualitatively analyze I.pes-caprae extract,and then the blood components of normal rats and AGA rats were identified after ig I.pes-caprae extract.Results Compared with model group,joint swelling index and foot pad thickness of rats in I.pes-caprae extract group were significantly decreased(P<0.05,0.01),IL-1β,MIP-1α,and MIP-2 levels in serum of high-and medium-dose I.pes-caprae extract groups were significantly decreased(P<0.05),infiltration of inflammatory cells in synovial tissue were reduced in high-and medium-dose I.pes-caprae extract groups.53 components were identified from I.pes-caprae extract,including resin glycosides,flavonoids,organic acids,coumarins,nucleosides,lignans,among which organic acids and flavonoids were the main components.Fragmentation rules of some compounds of resin glycosides,flavonoids,organic acids,and coumarins were also speculated.Four components including malic acid,caffeic acid,methyl caffeic acid and 4-hydroxybenzoic acid were identified in normal rat sample plasma.Four components including scopoletin,caffeic acid,methyl caffeic acid,and 4-hydroxybenzoic acid were identified in AGA rat sample plasma.Conclusion I.pes-caprae extract can significantly relieve joint swelling of AGA rats,inhibit IL-1β,MIP-1α,and MIP-2 levels in serum,and inhibit inflammatory response.Main chemical components from I.pes-caprae extract were rapidly identified as organic acids and flavonoids by using LC-Q-TOF-MS/MS.Scopoletin,caffeic acid,chlorogenic acid and its isomers(neochlorogenic acid,cryptochlorogenic acid,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C)and protocatechuic acid were injected into the blood in form of prototype components or their metabolites in AGA rats,which could be used as Q-Marker for the treatment of AGA with I.pes-caprae extract.