目的: 研究经知母总黄酮干预后前列腺增生(benign prostatic hyperplasia,BPH)大鼠前列腺中花生四烯酸(arachidonic acid,AA)的代谢组学特征。方法: 采用Shim-PACK XR-ODS C18(75 mm×3.0 mm, 2.2 μm)色谱柱,以0.05%的甲酸-水溶液(浓氨试液调节pH至3.3)为流动相A,甲醇-乙腈(20∶80,v/v)为流动相B,梯度洗脱,流速0.3 mL·min-1,柱温40 ℃,进样量10 μL;质谱采用负离子扫描,多反应监测(MRM)模式。分别收集假手术组、模型组、知母总黄酮组和阳性对照组大鼠的前列腺组织,利用超高效液相色谱串联质谱(UPLC-MS/MS)法对其中的11个物质[包括花生四烯酸(AA)、5-羟二十碳四烯酸(5-HETE)、8-羟二十碳四烯酸(8-HETE)、11-羟二十碳四烯酸(11-HETE)、12-羟二十碳四烯酸(12-HETE)、15-羟二十碳四烯酸(15-HETE)、白三烯(LTB4)、前列腺素D2(PGD2)、前列腺素E2(PGE2)、前列腺素H2(PGH2)、血栓素B2(TXB2)]进行定量分析。结果: 待测物均能被很好地检出,在其线性范围内呈现良好的线性关系。组织样品中11个待测成分在高、中、低浓度下的提取回收率均＞75.7%,基质效应为85.5%~106.3%,准确度RSD＜11.2%,日内和日间精密度RSD分别＜11.0%和9.5%。方法的专属性、准确度、精密度、提取回收率、基质效应和稳定性方面均符合要求。研究表明,模型组大鼠前列腺内的AA及其代谢物浓度均显著高于假手术组;知母总黄酮可改变BPH发展过程,有效下调BPH大鼠前列腺中AA及其代谢物的浓度。结论: 知母总黄酮可以通过脂氧合酶(LOX)和环氧合酶(COX)的2条途径来调节AA代谢,抑制BPH中发生的炎症反应。
Objective: To investigate the metabonomic characteristics of arachidonic acid(AA) in the prostate of benign prostatic hyperplasia (BPH) rats after the intervention of the flavonoids extract of Anemarrhenae Rhizoma. Methods: Shim-PACK XR-ODS C18(75 mm×3.0 mm, 2.2 μm) column was performed in UPLC assay. The mobile phase A was 0.05% formic acid aqueous solution(concentrated ammonia test solution adjusted to pH 3.3)and mobile phase B was methanol-acetonitrile (20∶80, v/v). Gradient elution was performed at the flow rate of 0.3 mL·min-1, the column temperature was 40 ℃, and the injection volume was 10 μL. Negative ionscanning and multiple reaction monitoring(MRM) mode was employed formass spectrometry.The prostate tissues of rats in the sham group, BPH model group, flavonoids group and positive control group were collected and detected, and a total of eleven substances[including arachidonic acid (AA), 5-hydroxyeicosatetraenoic acid (5-HETE), 8-hydroxyeicosatetraenoic acid (8-HETE), 11-hydroxyeicosatetraenoic acid (11-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE), leukotriene B4 (LTB4), prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin H2 (PGH2), thromboxane B2 (TXB2)]were quantitatively analyzed by ultra-fast liquid chromatography tandem mass spectrometry(UFLC-MS/MS). Results: The objects to be measured could be well detected and its calibration curves were good linear in the linear range. The extraction recovery rates of 11 components to be tested in tissue samples at high, medium, and low concentrations were all greater than 75.7%, with matrix effects ranging from 85.5% to 106.3%, accuracy RSD<11.2%, and intra day and inter day precision RSD<11.0% and 9.5%, respectively.The selectivity, accuracy and precision, recovery, matrix effects, and stability of the method met the requirements. Studies had shown that the concentrations of AA and its metabolites in BPH model group rat prostate were significant higher than those in sham group rats.The flavonoids extract of Anemarrhenae Rhizoma could change the development process of BPH and effectively down-regulate the concentrations of AA and its metabolites in the BPH rat prostate.Conclusion: The flavonoids extract of Anemarrhenae Rhizoma can regulate AA metabolism through two pathway of lipoxygenase(LOX) and cyclooxygenase(COX), thereby inhibiting the inflammatory process occurring in BPH.
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