民族药准噶尔乌头乙醇提取物化学成分及其对大鼠内源性代谢物影响的研究

发布时间：2018-07-28 06:35

【摘要】：目的:分析准噶尔乌头乙醇提取物的化学成分,观察其单次灌胃后24h内对大鼠血液生化指标及其内源性代谢物的影响,为进一步研究该药的毒性机理、临床中毒诊断及新药开发提供科学依据。方法:采用高效液相色谱(HPLC)技术对准噶尔乌头乙醇提取物的化学成分进行定性定量分析;于大鼠单次灌胃准噶尔乌头乙醇提取物(7.8mg/kg)前及灌胃后0.5、1、2、6、12、24h分别从大鼠眼眶后静脉采集血液样本,通过全自动生化分析仪检测AST、ALT、TP、ALB、TC、TG、TBIL、Crea、GLU、LDH含量,用1H-NMR技术检测大鼠血清内源性代谢物的变化情况,分析给药前后各参数值的变化;结合SIMCA软件对以上数据进行模式识别(主成分分析法(PCA)、偏最小二乘法(PLS-DA)和正交偏最小二乘法-判别分析法(OPLS-DA)),寻找差异代谢物。结果:(1)准噶尔乌头乙醇提取物HPLC谱图的色谱条件为:乙腈-水(0.3%磷酸)(B:A)作为流动相,梯度洗脱:10~16%B(0~12 min)、16~35%B(12~30 min)、35~45%B(30~40 min)、45%B(40~45 min),检测波长为235 nm,温度35℃,流速1.0 mL/min,进样量20μL。本方法在分离度、精密度、稳定性、加样回收率等方法学考察中均符合要求。(2)准噶尔乌头乙醇提取物中含有宋果灵(54.9±4.5 mg)、苯甲酰乌头原碱(7.3±1.6 mg)、乌头碱(3.7±0.8 mg)、草乌甲素(1.2±0.3 mg)、3-脱氧乌头碱(3.6±0.5 mg)共5种生物碱成分。(3)大鼠单次灌胃准噶尔乌头乙醇提取物(7.8mg/kg)后起AST、TG、GLU、LDH升高(P0.05),ALB、TP、TC、TBIL降低(P0.05))。其中多数生化指标值在灌胃后1~2h内出现变化高峰(升高或下降),对LDH和AST的影响最为显著。(4)核磁共振氢谱(1H-NMR)代谢组学分析表明准噶尔乌头乙醇提取物灌胃前后大鼠血清的1H-NMR谱存在明显差异,主要差异性标记物有:α-葡萄糖、β-葡萄糖、肌酸水平升高(p0.05),氨基酸(丙氨酸、谷氨酸、甘氨酸、酪氨酸、亮氨酸、苏氨酸、赖氨酸、苯丙氨酸)、脂类水平呈先降后升趋势;丙酮酸和乳酸水平降低(p0.05)。结论:(1)建立的HPLC检测方法稳定可靠;(2)准噶尔乌头乙醇提取物中含有宋果灵、苯甲酰乌头原碱、乌头碱、草乌甲素、3-脱氧乌头碱5种生物碱成分。(3)准噶尔乌头乙醇提取物可能对心肌、肝脏有不同程度的损害,并对大鼠的糖代谢、脂类代谢和氨基酸代谢产生影响。(4)大鼠内源性代谢物的差异性变化有可能作为该药中毒检测的毒性标记物。
[Abstract]:Objective: to analyze the chemical constituents of the ethanol extract from Junggar aconitum and to observe the effect of the extract on the blood biochemical index and its endogenous metabolites in rats within 24 hours after single oral administration, in order to further study the toxic mechanism of the drug. The diagnosis of clinical poisoning and the development of new drugs provide scientific basis. Methods: the chemical constituents of ethanol extract of Junggar aconitum were qualitatively and quantitatively analyzed by high performance liquid chromatography (HPLC). Blood samples were collected from the posterior orbital vein of rats before and after single gastric administration of ethanol extract of Junggar aconitum (7.8mg/kg) and 0.5g / 2h after oral administration for 24 hours. The content of TBILTILTBILTILTBILLLU LDH was measured by automatic biochemical analyzer, and the results showed that the blood samples were collected from the posterior orbital vein of the rats before and after oral administration of the ethanol extract of Junggar aconitum (7.8mg/kg), and the contents of the blood samples were measured by automatic biochemical analyzer. The changes of serum endogenous metabolites in rats were detected by 1H-NMR technique, and the changes of parameters before and after administration were analyzed. The above data were identified with SIMCA software (principal component analysis, (PCA), partial least squares (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA),) to search for different metabolites. Results: (1) the chromatographic conditions of HPLC spectra of ethanol extract of Zhunggar aconitum were as follows: acetonitrile-water (0.3% phosphoric acid) (B: a) as mobile phase, gradient elution of 10: 1016 and B (0 ~ 12 min) and B (12 ~ 30 min) ~ 35 ~ (35) B (3040 min) ~ 45B (4045 min), the detection wavelength was 235 nm, the temperature was 35 鈩，

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