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马齿苋(Portulaca oleracea L.)是一种药食两用植物,味酸、性寒,具有清热解毒、凉血消肿之功效。现代药理研究证实其有抗衰老、降血糖、降血脂、抗动脉粥样硬化,以及保肝护肝等方面的药理活性[1-2]。目前对马齿苋保肝护肝的有效成分还不甚了解。本研究进一步明确马齿苋提取物对四氯化碳致小鼠急性肝损伤具有保护作用,并通过超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF/MS)技术分析马齿苋提取物的主要成分,采用电喷雾电离源正离子模式和负离子模式对色谱流出物进行检测,通过高分辨质谱分析结合相关文献鉴定出苹果酸、柠檬酸、亮氨酸、异亮氨酸、腺苷、琥珀酸、染料木素、酪氨酸、苯丙氨酸等15种主要成分。
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各组小鼠体重均呈上升趋势,各组体重差异无统计学意义(见图1),初步说明马齿苋提取物对小鼠生长没有明显的毒副作用。
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由图2可见,与对照组相比,CCl4急性化学性肝损伤模型组血清ALT、AST水平显著升高(P<0.001),说明造模成功。与模型组相比,全草高剂量组小鼠血清AST、ALT水平显著降低(P<0.05,P<0.01);沉淀高剂量组小鼠血清ALT显著降低(P<0.05),AST水平有降低趋势但不具有统计学意义。
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由图3可见,与对照组相比,CCl4急性化学性肝损伤模型组血清IL-6水平显著升高(P<0.01)。与模型组相比,全草高剂量组小鼠血清IL-6水平显著降低(P<0.05),沉淀高剂量组小鼠血清IL-6水平有降低趋势但不具有统计学意义。
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取马齿苋样品溶液,按照“1.4.3”项下进样分析,全扫描总离子流图如图4所示。
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将“Formula-Database-Generator”软件建立的马齿苋已知化学成分的数据库导入“Masshunter Qualitative Analysis”软件系统,依据数据库对马齿苋提取物总离子流质谱图进行鉴定和确证,将检索到的化学成分信息按照保留时间、质荷比、加合离子情况等进行分类整理。并将化合物按其质荷比在原质谱数据中进行反向提取,对提取到的质谱信息进行质量准确度的验证并根据同位素峰比例等信息进行元素组成分析。推测出15种化合物为马齿苋80%乙醇提取物的主要成分,按照保留时间顺序依次为:苹果酸、柠檬酸、亮氨酸、异亮氨酸、腺苷、琥珀酸、染料木素、酪氨酸、苯丙氨酸、佛手苷内酯、金莲花碱、6,7-二羟基香豆素、芳樟醇、金色酰胺醇/橙黄胡椒酰胺、金色酰胺醇酯/咸南藤酰胺乙酸酯(见表1),另外还有多糖、黄酮等化学成分。
表 1 马齿苋提取物UPLC-Q-TOF/MS分析结果
时间
(tR/min)分子式 离子峰归属 质荷比 偏差 化合物 理论值 实测值 0.847 C4H6O5 [M-H]- 133.0142 133.0147 −0.49 苹果酸[3-4] 1.019 C6H8O7 [M-H]- 191.0197 191.0198 −0.62 柠檬酸[3] 1.097 C6H13NO2 [M+H]+ 132.1019 132.1014 3.91 亮氨酸[5] 1.203 C6H13NO2 [M+H]+ 132.1019 132.1015 2.72 异亮氨酸[4] 1.293 C10H13N5O4 [M+H]+ 268.104 268.1047 −2.55 腺苷[6] 1.305 C4H6O4 [M-H]- 117.0193 117.0197 −3.23 琥珀酸[3] 1.555 C15H10O5 [M+NH4]+ 288.0866 288.0867 −0.21 染料木素[7] 2.053 C9H11NO3 [M+NH4]+ 199.1077 199.1074 1.69 酪氨酸[5] 2.168 C9H11NO2 [M+H]+ 166.0863 166.0859 2.03 苯丙氨酸[5] 3.567 C12H8O4 [M+NH4]+ 234.0761 234.0765 −1.81 佛手苷内酯[8] 4.638 C12H13NO3 [M+H]+ 220.0968 220.0967 0.61 金莲花碱[9] 4.929 C9H6O4 [M-H]- 177.0193 177.0192 0.98 6,7-二羟基香豆素[5] 7.301 C10H18O [M+COOH]- 199.034 199.1342 −1.21 芳樟醇[10] 8.646 C25H26N2O3 [M+H]+ 403.2016 403.2029 −3.12 金色酰胺醇/橙黄胡椒酰胺[9] 9.685 C27H28N2O4 [M+H]+ 445.2122 445.2128 −1.34 金色酰胺醇酯/咸南藤酰胺乙酸酯[9]
Protective effect of Portulaca oleracea L. extract on acute liver injury induced by carbon tetrachloride in mice and its chemical composition analysis
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摘要:
目的 探究马齿苋乙醇提取物对四氯化碳所致小鼠急性肝损伤的保护作用,并对其有效成分进行分析。 方法 将80%乙醇马齿苋提取物经离心、减压蒸馏、真空干燥为全草提取物、上清提取物和沉淀提取物。将80只ICR雄性小鼠随机分为8组:对照组、肝损伤模型组、马齿苋全草提取物低剂量组、高剂量组、马齿苋上清提取物低剂量组、高剂量组、马齿苋沉淀提取物低剂量组、高剂量组。经口灌胃去离子水或3种马齿苋提取物不同剂量混悬液1周后,分别皮下注射橄榄油或CCl4橄榄油溶液,16 h后收集血清,检测小鼠血清ALT、AST、IL-6水平,以评价马齿笕对肝损伤的保护作用。采用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF/MS)技术分析马齿苋的有效成分。 结果 与模型组相比,马齿苋全草提取物高剂量组小鼠血清AST、ALT、IL-6水平显著降低;沉淀高剂量组小鼠血清ALT显著降低(P<0.05),IL-6水平有所降低,但不具统计学意义;其他各干预组小鼠血清AST、ALT、IL-6水平没有显著性变化。应用UPLC-Q-TOF/MS法共鉴别出苹果酸、柠檬酸、亮氨酸、异亮氨酸、腺苷、琥珀酸、染料木素、酪氨酸、苯丙氨酸等15种主要成分。 结论 马齿苋乙醇全草提取物对CCl4急性肝损伤小鼠具有保肝抗炎作用,可能是15种有效成分发挥的联合作用。 -
关键词:
- 马齿苋 /
- 提取物 /
- CCl4急性化学性肝损伤 /
- 保护作用
Abstract:Objective To investigate the protective effect of the ethanol extract of Portulaca oleracea L. on acute liver injury induced by carbon tetrachloride in mice, and to analyze its effective components. Methods 80% ethanol purslane extract was centrifuged, vacuum distillated and vacuum dried into whole plant extract, supernatant extract and precipitated extract. Eighty ICR male mice were randomly divided into 8 groups: control group, liver injury model group, whole plant extract low-dose group, high-dose group, supernatant extract low-dose group, high-dose group, precipitation extract low-dose group, and high-dose group. After oral administration of distilled water or three kinds of purslane extract suspensions at different doses for 1 week, olive oil or CCl4 olive oil solution were injected subcutaneously respectively. After 16 hours, serum was collected to detect the levels of ALT, AST and IL-6 to evaluate the protective effect of purslane on acute liver injury. Ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) was used to analyze the effective components of purslane extract. Results Compared with the model group, the levels of serum AST, ALT and IL-6 in high-dose whole plant extract group were significantly reduced. The serum ALT level of mice in the high-dose precipitation extract group was significantly reduced (P<0.05). The serum IL-6 level was decreased, but there was no significant difference. There were no significant changes in the levels of serum AST, ALT and IL-6 in the other intervention groups. 15 main components such as malic acid, citric acid, leucine, isoleucine, adenosine, succinic acid, genistein, tyrosine and phenylalanine were identified by UPLC-Q-TOF/MS. Conclusion Purslane whole plant ethanol extract has hepatoprotective and anti-inflammatory effects on CCl4 acute liver injury mice, which may be a combined effect of 15 active components. -
Key words:
- purslane /
- extract /
- acute chemical liver injury of CCl4 /
- protection
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表 1 马齿苋提取物UPLC-Q-TOF/MS分析结果
时间
(tR/min)分子式 离子峰归属 质荷比 偏差 化合物 理论值 实测值 0.847 C4H6O5 [M-H]- 133.0142 133.0147 −0.49 苹果酸[3-4] 1.019 C6H8O7 [M-H]- 191.0197 191.0198 −0.62 柠檬酸[3] 1.097 C6H13NO2 [M+H]+ 132.1019 132.1014 3.91 亮氨酸[5] 1.203 C6H13NO2 [M+H]+ 132.1019 132.1015 2.72 异亮氨酸[4] 1.293 C10H13N5O4 [M+H]+ 268.104 268.1047 −2.55 腺苷[6] 1.305 C4H6O4 [M-H]- 117.0193 117.0197 −3.23 琥珀酸[3] 1.555 C15H10O5 [M+NH4]+ 288.0866 288.0867 −0.21 染料木素[7] 2.053 C9H11NO3 [M+NH4]+ 199.1077 199.1074 1.69 酪氨酸[5] 2.168 C9H11NO2 [M+H]+ 166.0863 166.0859 2.03 苯丙氨酸[5] 3.567 C12H8O4 [M+NH4]+ 234.0761 234.0765 −1.81 佛手苷内酯[8] 4.638 C12H13NO3 [M+H]+ 220.0968 220.0967 0.61 金莲花碱[9] 4.929 C9H6O4 [M-H]- 177.0193 177.0192 0.98 6,7-二羟基香豆素[5] 7.301 C10H18O [M+COOH]- 199.034 199.1342 −1.21 芳樟醇[10] 8.646 C25H26N2O3 [M+H]+ 403.2016 403.2029 −3.12 金色酰胺醇/橙黄胡椒酰胺[9] 9.685 C27H28N2O4 [M+H]+ 445.2122 445.2128 −1.34 金色酰胺醇酯/咸南藤酰胺乙酸酯[9] -
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