大黄及其主要成份的毒性毒理研究
摘要
大黄为我国四大最常用的中药之一，全国8000多种中药制剂中约有800多种含有大黄，是治疗多种疾病的重要中药，被称为中药“四大金刚”之一，在临床上有着广泛的应用。大黄药理有效成分主要为蒽醌类化合物，包括大黄酸、大黄素、大黄酚、芦荟大黄素、大黄素甲醚等。以往研究发现大黄可能具有肝肾毒性和致癌的潜力，但是对大黄中主要成份的毒性比较研究及毒性机制研究报道甚少，目前尚没有明确的定论。本课题采用体内、体外多种实验方法研究和探讨了大黄毒性反应的作用靶器官，大黄中主要成份的毒性比较研究，在此基础上利用代谢组学技术，探讨了大黄素等蒽醌的毒理学作用机制，初步证明了大黄毒性的客观真实性，为大黄的临床安全应用提供了参考依据。
研究发现大黄重复给药6个月产生毒性反应的剂量为10g／kg／d，无明显毒性反应的剂量为2.5g／kg／d(相当于临床常用剂量10g的15倍，换算成人的等效剂量约为0.4g／kg／d)，对SD大鼠的主要毒性表现为动物排稀便、软便、体重增长抑制，被毛脏乱、红染，精神不振等，血液生化检查发现BUN略有升高，病理检查发现大黄引起大鼠肾小管上皮细胞内色素沉积和水样变性、肝细胞轻度萎缩、肠粘膜轻度增厚。毒性反应主要靶器官为肾脏，毒性部位为肾小管上皮细胞，肝脏也可能是其毒性靶器官之一，上述毒性损害均可逆。
大黄中几种主要成份体外毒性比较试验发现，大黄素等对HK-2细胞增殖抑制的IC_(50)值分别为：大黄酸82.97μM，大黄素甲醚76.02μM，大黄素130.65μM，其毒性大小顺序为：大黄素甲醚＞大黄酸＞大黄素＞芦荟大黄素＞大黄酚；对于HepG2细胞的其IC_(50)值分别为：大黄酸67.71μM，大黄素125.30μM，毒性大小顺序为：大黄酸＞大黄素＞芦荟大黄素＞大黄酚和大黄素甲醚。大黄素等均能引起两种细胞增殖抑制，细胞萎缩和空泡化，LDH漏出率增加，线粒体膜电位降低，细胞出现明显的凋亡，大黄素使HK-2细胞周期阻滞在S期，而对HepG2细胞的周期没有明显的影响。Western印渍分析凋亡相关蛋白显示，大黄素和大黄酸处理HK-2细胞后，抑制细胞外信号调节激酶1／2(extracelluar signal regulated kinase 1／2，ERK1／2)的磷酸化，并且呈现明显的剂量和时间效应关系，细胞内Bcl-2表达降低，Bax没有明显的变化，致使促凋亡基因蛋白占优势，细胞向促调亡的方向发展，细胞色素c释放增加，从而使Caspase-8活化，激活下游caspase3，引起细胞的凋亡；对HK-2细胞的损伤效应可能是通过MAPK／ERK信号转导通路抑制ERK磷酸化，而导致了细胞周期阻滞和凋亡。Ames波动试验结果表明大黄素在加有S_9时具有弱的致突变性，是间接遗传毒性物质，大黄酸和大黄酚没有致突变性；体内试验发现大黄素对CYP450尤其是CYP1A1具有一定的诱导作用，表明大黄素还可能具有潜在的促癌作用。大黄素是大黄中含量最高的蒽醌成份，因此大黄素可能是大黄主要的毒性作用物质之一。
利用NMR技术研究大黄素处理后大鼠尿液、血浆和肾脏组织萃取物中内源性代谢产物谱的变化，研究发现血浆中肌氨酸、异丁酸盐和3-羟基丁氨酸(3-HB)升高，而乳酸、胆碱／磷酸卵磷酯、葡萄糖和多种氨基酸及VLDL／LDL等明显下降；尿液中肌酐、TMAO、醋酸盐、乳酸、葡萄糖和各种氨基酸明显升高，而柠檬酸盐、马尿酸和2-酮戊二酸含量下降，尿液中葡萄糖和氨基酸升高是肾小管损伤的典型标志；肾组织萃取物中醋酸盐、3-HB和肌酐明显增加，而乳酸和胆碱／磷酸卵磷脂下降；脂溶性成分中甘油三脂和细胞膜组成成份等脂类的比例发生改变；由此可以推测大黄素可能是通过改变脂类代谢，导致脂类成分的改变，破坏细胞膜性结构，致使肾小管上皮细胞受损而造成重吸收障碍。尿液中的氨基酸、葡萄糖、TMAO以及肌酐可以作为大黄素的主要生物标志物研究。
综上，大黄毒作用靶器官主要是肾脏，尤其是肾近曲小管上皮细胞，肝脏也可能是其毒性靶器官之一，大黄素是主要的毒性成分，损伤机制可能为大黄素通过MAPK／ERK信号转导通路抑制ERK磷酸化，肾脏脂类成分的改变，致使肾小管上皮细胞线粒体外膜受到损伤，细胞色素C从线粒体释放，进而引发细胞凋亡，导致肾小管重吸收障碍，从而形成氨基酸尿和葡萄糖尿，这种毒性作用是可逆的。
大黄的临床应用十分广泛，正常情况下使用大黄是安全的，但长期、大剂量使用会出现毒性反应，因此在临床上长期用药不要超过0.4g／kg／日，肝肾损伤患者应用时需要调整剂量，长期应用时要注意监测其肝肾功能。其他含蒽醌类药物如芦荟、决明子和虎杖等可能也有类似的毒性毒理机制，因此，应该建立质量标准，控制毒性成份的含量，加强对此类药物的不良反应监测。随着代谢组学技术的发展，此项新技术将会越来越多的用于其他中草药毒性毒理的研究，对于实现中药现代化具有重要的意义。
Rhubarb (Radix et Rhizoma Rhei) is widely used in traditional Chinese medicines, which is one of the four most frequently used herbs, and is formulated in more than 800 kinds of Chinese materia medica preparations. Emodin, rhein, chrysophanol, aloe-emodin and physcion are its essential active components.
Recently studies have demonstrated that rhubarb may have the potential of hepatotoxicity, nephrotoxicity and carcinogenicity. However, so far there is little studies concerning the toxic mechanisms and the comparative toxicologic assessment of its essential components. So, there are no identified conclusions about its toxicity. In this study, in vivo and in vitro experiments have been applied to explore the toxicity and the target organs of rhubarb, and compared the toxicity of its essential components. After then, the toxic mechanisms are elucidated based on metabonomic technologies in order to offer some toxicological information for its safely clinical usage.
The NOAEL for rhubarb in SD rats is 2.5g/kg/d, which is approximately equal to 15 times of clinical dosage. Clinical signs of toxicity of rhurbarb in SD rats at the dose level of (?) 10g/kg/d included: diarrhoea, retarded growth of body weight, dirty and red colored fur hair, depressed, et al. Serum biochemistry profiles displayed a trend of increase in BUN. Hydropic degeneration and pigment sediment in renal tubular epithelial cell, slight shrink in hepatocyte, and slightly thickening in mucous membrane of intestine were histopathologically found. From these results, the toxic target organ was estimated to be kidney (esp. the proximal tubules), and liver may be one of its target organs, too. All of those toxicity manifestations are reversible upon stopping dosing.
Comparative toxicity studies of its essential components indicated that emodin, rhein, and physcion markedly inhibited the proliferation of HK-2 cells after 48 hours culture, and their IC_(50) are 130.65μM ,82.97μM, 76.02μM respectively. And their relative toxicity was: physcion>rhein>emodin>aloe-ernodin>chrysophanol; For HepG2 cell, their IC50 are 67.71μM and 125.30μM for rhein and emodin respectively. And their relative toxicity was: rhein>emodin> aloe-emodin>physcion and chrysophanol. The toxic manifestations included: inhibition of the cells proliferation, cell shrinkage and vacuolation, LDH leakage increasement, and cell apoptosis followed by the decreasment of mitochondria membrane potential (MMP). Furhermore, emodin can block the cell cycle, and the percentage of cells in S phase was gradually increased and that in G_0/G_1 phase were decreased. Western blotting analysis of apoptotic related proteins revealed that after treatment with emodin and rhein, the phosphorylation of extracelluar signal regulated kinase 1/2 were inhibited, the expression level of Bcl-2 decreased significantly, the release of cytochrme C increased, caspase8 and caspase3 were activated in HK-2 cells, but the level of Bax hasn't changed. These results implied that emodin may lead to cell cycle blockage and apoptosis though suppressing MAPK/ERK signal pathway. Ames-fluctuation test showed that emodin has a weak mutagenic potential upon S9 metabolic activation, suggested that it is indirect genotoxicant. The rhein and chrysophanol have no mutagenic potential in this test system. Total CYP450 content and activities of CYP1A, CYP3A and CYP2B in rat liver were elevated after multiple induction with emodin. As emodin is the most abundant component of rhubarb, it may be its primary toxic substance.
An integrated metabonomic study with high-resolution ~1H NMR spectroscopy has been applied to investigate the biochemical composition of urine, serum, kidney tissue aqueous extracts (acetonitrile/water) and lipidic extracts (chloroform/methanol) obtained from emodin treated rats. It was found that the level of sarcosine isobutyrate and 3-HB was elevated, and the levels of VLDL/LDL, lactate, glucose and many kinds of amino acids, choline/phosphatidylcholine were decreased in serum. The level of creatinine, TMAO, acetate, lactate, glucose and many kinds of amino acids increased in urine, while citrate, hippurate and 2-oxoglutarate decreased significantly. The increased level of glucose and amino acids is the typical biomarker of impaired renal tubule. The predominant changes identified in kidney tissue aqueous extracts included an increase in the signal intensities of acetate, 3-HB, creatinine, and the reduced level of lactate, choline/phosphatidylcholine. In kidney tissue chloroform/methanol extracts, there was a remarkably increase in many of the lipid signals including CH_2CO, CH_2OPO_2, CH_2OCOR. And the level of the triglyceride terminal methyl and methylene groups reduced. From these results, we can infer that emodin may change the proportion of lipid moieties, destroyed the structure of cellular membrane, and damaged the renal tubular epithelial cell, which induced a decline in the reabsorption of low molecular weight compounds. Glucose, amino acids, TMAO and creatinine in urine should be identified as biomarkers of emodin.
In conclusion, the main toxic target organ of rhubarb is kidney, especially the proximal tubule epithelial cell, and liver may be one of its toxic target organs, too. Emodin is its primary toxic substance. The suppression of phosphorylation ERK through MAPK/ERK signal pathway, and alteration of the proportion of lipid moieties, may lead to the impairment of mitochondrial outer membrane in renal tubular epithelial cell, which caused the release of cytochrome c and apoptosis, and induced a decline in the reabsorption of low molecular weight compounds in renal tubule, amino-aciduria and glucosuria was finally observed. The toxic effects were reversible.
Rhubarb is widely and safely used in Chinese medicines usually, while the toxic effects will appear when it was used in large dose and for a long time. Therefor, when it's used for a long time, the dose won't exceed 0.4g/kg. When the patients have kidney or liver injury, the dose must be modified, and functions of kidney and liver should be monitored. Other herbs containing anthraquinone, such as aloes, Cassia seed, giant knotweed rhizome, may have similar toxic effects. Quality criteria should be established to control the content of toxic substance accordingly. With the development of metabonomics, this novel technology might provide additional discrimination in the toxicological effects of other herb in the future, which will contributes to the modernlization of traditional Chinese medicines.
研究发现大黄重复给药6个月产生毒性反应的剂量为10g／kg／d，无明显毒性反应的剂量为2.5g／kg／d(相当于临床常用剂量10g的15倍，换算成人的等效剂量约为0.4g／kg／d)，对SD大鼠的主要毒性表现为动物排稀便、软便、体重增长抑制，被毛脏乱、红染，精神不振等，血液生化检查发现BUN略有升高，病理检查发现大黄引起大鼠肾小管上皮细胞内色素沉积和水样变性、肝细胞轻度萎缩、肠粘膜轻度增厚。毒性反应主要靶器官为肾脏，毒性部位为肾小管上皮细胞，肝脏也可能是其毒性靶器官之一，上述毒性损害均可逆。
大黄中几种主要成份体外毒性比较试验发现，大黄素等对HK-2细胞增殖抑制的IC_(50)值分别为：大黄酸82.97μM，大黄素甲醚76.02μM，大黄素130.65μM，其毒性大小顺序为：大黄素甲醚＞大黄酸＞大黄素＞芦荟大黄素＞大黄酚；对于HepG2细胞的其IC_(50)值分别为：大黄酸67.71μM，大黄素125.30μM，毒性大小顺序为：大黄酸＞大黄素＞芦荟大黄素＞大黄酚和大黄素甲醚。大黄素等均能引起两种细胞增殖抑制，细胞萎缩和空泡化，LDH漏出率增加，线粒体膜电位降低，细胞出现明显的凋亡，大黄素使HK-2细胞周期阻滞在S期，而对HepG2细胞的周期没有明显的影响。Western印渍分析凋亡相关蛋白显示，大黄素和大黄酸处理HK-2细胞后，抑制细胞外信号调节激酶1／2(extracelluar signal regulated kinase 1／2，ERK1／2)的磷酸化，并且呈现明显的剂量和时间效应关系，细胞内Bcl-2表达降低，Bax没有明显的变化，致使促凋亡基因蛋白占优势，细胞向促调亡的方向发展，细胞色素c释放增加，从而使Caspase-8活化，激活下游caspase3，引起细胞的凋亡；对HK-2细胞的损伤效应可能是通过MAPK／ERK信号转导通路抑制ERK磷酸化，而导致了细胞周期阻滞和凋亡。Ames波动试验结果表明大黄素在加有S_9时具有弱的致突变性，是间接遗传毒性物质，大黄酸和大黄酚没有致突变性；体内试验发现大黄素对CYP450尤其是CYP1A1具有一定的诱导作用，表明大黄素还可能具有潜在的促癌作用。大黄素是大黄中含量最高的蒽醌成份，因此大黄素可能是大黄主要的毒性作用物质之一。
利用NMR技术研究大黄素处理后大鼠尿液、血浆和肾脏组织萃取物中内源性代谢产物谱的变化，研究发现血浆中肌氨酸、异丁酸盐和3-羟基丁氨酸(3-HB)升高，而乳酸、胆碱／磷酸卵磷酯、葡萄糖和多种氨基酸及VLDL／LDL等明显下降；尿液中肌酐、TMAO、醋酸盐、乳酸、葡萄糖和各种氨基酸明显升高，而柠檬酸盐、马尿酸和2-酮戊二酸含量下降，尿液中葡萄糖和氨基酸升高是肾小管损伤的典型标志；肾组织萃取物中醋酸盐、3-HB和肌酐明显增加，而乳酸和胆碱／磷酸卵磷脂下降；脂溶性成分中甘油三脂和细胞膜组成成份等脂类的比例发生改变；由此可以推测大黄素可能是通过改变脂类代谢，导致脂类成分的改变，破坏细胞膜性结构，致使肾小管上皮细胞受损而造成重吸收障碍。尿液中的氨基酸、葡萄糖、TMAO以及肌酐可以作为大黄素的主要生物标志物研究。
综上，大黄毒作用靶器官主要是肾脏，尤其是肾近曲小管上皮细胞，肝脏也可能是其毒性靶器官之一，大黄素是主要的毒性成分，损伤机制可能为大黄素通过MAPK／ERK信号转导通路抑制ERK磷酸化，肾脏脂类成分的改变，致使肾小管上皮细胞线粒体外膜受到损伤，细胞色素C从线粒体释放，进而引发细胞凋亡，导致肾小管重吸收障碍，从而形成氨基酸尿和葡萄糖尿，这种毒性作用是可逆的。
大黄的临床应用十分广泛，正常情况下使用大黄是安全的，但长期、大剂量使用会出现毒性反应，因此在临床上长期用药不要超过0.4g／kg／日，肝肾损伤患者应用时需要调整剂量，长期应用时要注意监测其肝肾功能。其他含蒽醌类药物如芦荟、决明子和虎杖等可能也有类似的毒性毒理机制，因此，应该建立质量标准，控制毒性成份的含量，加强对此类药物的不良反应监测。随着代谢组学技术的发展，此项新技术将会越来越多的用于其他中草药毒性毒理的研究，对于实现中药现代化具有重要的意义。
Rhubarb (Radix et Rhizoma Rhei) is widely used in traditional Chinese medicines, which is one of the four most frequently used herbs, and is formulated in more than 800 kinds of Chinese materia medica preparations. Emodin, rhein, chrysophanol, aloe-emodin and physcion are its essential active components.
Recently studies have demonstrated that rhubarb may have the potential of hepatotoxicity, nephrotoxicity and carcinogenicity. However, so far there is little studies concerning the toxic mechanisms and the comparative toxicologic assessment of its essential components. So, there are no identified conclusions about its toxicity. In this study, in vivo and in vitro experiments have been applied to explore the toxicity and the target organs of rhubarb, and compared the toxicity of its essential components. After then, the toxic mechanisms are elucidated based on metabonomic technologies in order to offer some toxicological information for its safely clinical usage.
The NOAEL for rhubarb in SD rats is 2.5g/kg/d, which is approximately equal to 15 times of clinical dosage. Clinical signs of toxicity of rhurbarb in SD rats at the dose level of (?) 10g/kg/d included: diarrhoea, retarded growth of body weight, dirty and red colored fur hair, depressed, et al. Serum biochemistry profiles displayed a trend of increase in BUN. Hydropic degeneration and pigment sediment in renal tubular epithelial cell, slight shrink in hepatocyte, and slightly thickening in mucous membrane of intestine were histopathologically found. From these results, the toxic target organ was estimated to be kidney (esp. the proximal tubules), and liver may be one of its target organs, too. All of those toxicity manifestations are reversible upon stopping dosing.
Comparative toxicity studies of its essential components indicated that emodin, rhein, and physcion markedly inhibited the proliferation of HK-2 cells after 48 hours culture, and their IC_(50) are 130.65μM ,82.97μM, 76.02μM respectively. And their relative toxicity was: physcion>rhein>emodin>aloe-ernodin>chrysophanol; For HepG2 cell, their IC50 are 67.71μM and 125.30μM for rhein and emodin respectively. And their relative toxicity was: rhein>emodin> aloe-emodin>physcion and chrysophanol. The toxic manifestations included: inhibition of the cells proliferation, cell shrinkage and vacuolation, LDH leakage increasement, and cell apoptosis followed by the decreasment of mitochondria membrane potential (MMP). Furhermore, emodin can block the cell cycle, and the percentage of cells in S phase was gradually increased and that in G_0/G_1 phase were decreased. Western blotting analysis of apoptotic related proteins revealed that after treatment with emodin and rhein, the phosphorylation of extracelluar signal regulated kinase 1/2 were inhibited, the expression level of Bcl-2 decreased significantly, the release of cytochrme C increased, caspase8 and caspase3 were activated in HK-2 cells, but the level of Bax hasn't changed. These results implied that emodin may lead to cell cycle blockage and apoptosis though suppressing MAPK/ERK signal pathway. Ames-fluctuation test showed that emodin has a weak mutagenic potential upon S9 metabolic activation, suggested that it is indirect genotoxicant. The rhein and chrysophanol have no mutagenic potential in this test system. Total CYP450 content and activities of CYP1A, CYP3A and CYP2B in rat liver were elevated after multiple induction with emodin. As emodin is the most abundant component of rhubarb, it may be its primary toxic substance.
An integrated metabonomic study with high-resolution ~1H NMR spectroscopy has been applied to investigate the biochemical composition of urine, serum, kidney tissue aqueous extracts (acetonitrile/water) and lipidic extracts (chloroform/methanol) obtained from emodin treated rats. It was found that the level of sarcosine isobutyrate and 3-HB was elevated, and the levels of VLDL/LDL, lactate, glucose and many kinds of amino acids, choline/phosphatidylcholine were decreased in serum. The level of creatinine, TMAO, acetate, lactate, glucose and many kinds of amino acids increased in urine, while citrate, hippurate and 2-oxoglutarate decreased significantly. The increased level of glucose and amino acids is the typical biomarker of impaired renal tubule. The predominant changes identified in kidney tissue aqueous extracts included an increase in the signal intensities of acetate, 3-HB, creatinine, and the reduced level of lactate, choline/phosphatidylcholine. In kidney tissue chloroform/methanol extracts, there was a remarkably increase in many of the lipid signals including CH_2CO, CH_2OPO_2, CH_2OCOR. And the level of the triglyceride terminal methyl and methylene groups reduced. From these results, we can infer that emodin may change the proportion of lipid moieties, destroyed the structure of cellular membrane, and damaged the renal tubular epithelial cell, which induced a decline in the reabsorption of low molecular weight compounds. Glucose, amino acids, TMAO and creatinine in urine should be identified as biomarkers of emodin.
In conclusion, the main toxic target organ of rhubarb is kidney, especially the proximal tubule epithelial cell, and liver may be one of its toxic target organs, too. Emodin is its primary toxic substance. The suppression of phosphorylation ERK through MAPK/ERK signal pathway, and alteration of the proportion of lipid moieties, may lead to the impairment of mitochondrial outer membrane in renal tubular epithelial cell, which caused the release of cytochrome c and apoptosis, and induced a decline in the reabsorption of low molecular weight compounds in renal tubule, amino-aciduria and glucosuria was finally observed. The toxic effects were reversible.
Rhubarb is widely and safely used in Chinese medicines usually, while the toxic effects will appear when it was used in large dose and for a long time. Therefor, when it's used for a long time, the dose won't exceed 0.4g/kg. When the patients have kidney or liver injury, the dose must be modified, and functions of kidney and liver should be monitored. Other herbs containing anthraquinone, such as aloes, Cassia seed, giant knotweed rhizome, may have similar toxic effects. Quality criteria should be established to control the content of toxic substance accordingly. With the development of metabonomics, this novel technology might provide additional discrimination in the toxicological effects of other herb in the future, which will contributes to the modernlization of traditional Chinese medicines.
引文
[1] 黄萍．中药不良反应的研究与监测[J]．中药新药与临床药理．1998；9(1)：54．
[2] 刘庆增．大黄的化学成分及药理作用[J]．中草药，1987，18(1)：41．
[3] 陈可冀．抗衰老中药学．北京：中医古籍出版社，1989：143～146．
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