【作者】 黄坚；

【导师】 杨凌；

【作者基本信息】 上海中医药大学 ， 中药学， 2022， 博士

【摘要】 背景中药的毒副作用仍然是影响中药更广泛应用的关键性挑战。现有83种“有毒中药”药材被列入药典,但其中明确标明其主要效应成分上下限的只有13种,剩余的药材中,没有物质基础的明确定性与定量的上限表述。也即至少在已经被视为“有毒中药”的药材仍存在着毒副作用相关物质基础不清晰,而同时,这些药物又在临床实践者进行推广应用,两者之间存在着管理上的冲突,这一冲突性挑战让具体应用者无法规避毒副作用风险或缺乏应用可遵循的基本准则。上述药材主要涉及三个方面的物质基础不清晰:1、效应物质包括有效成分与毒副作用产生相关成分不清晰;2、相关成分的ADME属性不清晰;3、多成分间的组合产生的药药相互作用（DDI）及其毒副作用不清晰。以中药山豆根为例,山豆根为2020年版《中国药典》收载的83味“有毒”中药之一,临床使用常引起胃肠道反应、肝毒性和神经毒性等多种不良反应,对其毒性物质基础的认识尚存在争议,药典质量标准中,仅对苦参碱（MT）和氧化苦参碱（OMT）两个指标成分的总量规定了含量检测下限,也即山豆根属于没有毒性物质基础表述的药材范例。因此,有必要对山豆根的主要毒性成分及其致毒因素进行系统研究。目标:本论文针对山豆根毒性物质基础不明,将开展以下研究工作:1、主要成分中哪些是毒性成分;2、主要毒性成分与代谢的相关关系;3、主要毒性成分与哪些药材或中药单体成分配伍后会产生药药相互作用（DDI）或减缓毒性作用;4、主要毒性成分的相关作用机制。方法:面对以上挑战,本研究利用以下三种研究策略与方法开展研究:1、对18批不同来源山豆根主成分进行比较,鉴定出山豆根高含量活性成分并进行含量测定。制备山豆根提取物和细分组分,利用组分敲除（Fraction Knockout Extract,FKE）策略,即FKE结合体外毒性的筛选,对山豆根主毒性成分进行筛选和鉴定。2、对其中经过确认的主毒性成分的整体药物代谢动力学进行了监测。3、研究马卡因伴随性代谢过程与山豆根及马卡因毒性作用间的关系,确认代谢对毒性作用的影响,并明确马卡因致毒机制。结果:1、各不同批次山豆根中主要成分的比较通过指纹图谱比对,共鉴定出14种主要成分,对不同批次山豆根中占据前10位的主要单体成分进行定量测定,接近千分之一排前列的生物碱成分为:OMT、MT;黄酮类成分为山豆根酮（SPN）、马卡因（MKN）和三叶豆紫檀苷（TF）;其中因槐果碱（SC）、氧化槐果碱（OSC）的含量差异巨大（变异系数CV>50%）,故选用了含量较稳定的三种黄酮类单体成分（SPN、MKN及TF）与两种生物碱指标成分（MT和OMT）作为候选目标成分。2、敲减法的提取物和组分制备1)提取物制备:分别制备山豆根总水提物（SDG-W-1）和山豆根总醇提物（SDG-E-1）。指纹图谱和成分分析显示SDG-W-1含大量生物碱成分（MT和OMT）及少量黄酮类成分（TF和MKN）;而SDG-E-1除含有生物碱成分（MT和OMT）外,还含有较多的黄酮类成分（SPN、MKN、和TF）。2)细分组分制备:以SDG-E-1为基础,经柱层析按极性由大到小分段收集制备成四种组分分别命名为水洗脱组分（SDG-Fr.1）,40%甲醇洗脱组分（SDG-Fr.2）,70%甲醇洗脱组分（SDG-Fr.3）和100%甲醇洗脱组分（SDG-Fr.4）。指纹图谱和成分分析显示SDG-Fr.1仅含少量生物碱MT;SDG-Fr.2为生物碱成分富集部位,含有大量的MT和OMT;SDG-Fr.3即含有生物碱成分MT和OMT,也含有黄酮类成分TF;SDG-Fr.4为黄酮类成分富集部位,主要含MKN和SPN,4个细分组分形成了不同目标成分和含量差异的成分敲除组合。3、主要单体毒性成分的确认以体外正常肝细胞系的细胞毒性与线粒体毒性分析测试为监测标准,对不同组分中各主要成分单体的毒性贡献进行了相关性分析,发现黄酮类成分SPN、MKN、TF的细胞毒性相关性高且有统计学意义,而生物碱成分MT和OMT的毒性相关性小且无统计学意义。由于水溶性组分中,以MKN为主,TF是MKN的糖苷衍生物;而脂溶性组分中,主要以SPN为主。两种山豆根黄酮成分均可显著降低LO2细胞的增殖活力,并改变线粒体膜电位造成线粒体毒性。4、MKN的UGT代谢及小鼠体内整体动力学变化在前期同组研究人员已证实MKN可被UGT代谢的基础上,我们进而发现UGT广谱抑制剂穗花杉双黄酮（AMF）在体外可抑制MKN的葡萄糖醛酸化结合作用;整体动力学研究发现,AMF也可抑制MKN在小鼠体内的葡萄糖醛酸化,其动力学变化是使MKN的血浆半衰期(t_1/2)和药时曲线下面积AUC（0-t）均增加了1.4倍,MKN体内暴露量增加。5、SPN的UGT体外抑制作用与SPN对小鼠的直接毒性作用前期同组研究人员已确认SPN是强效的UGT广谱抑制剂。我们进而发现SPN在体外也可抑制MKN的UGT代谢。此外,小鼠经灌胃给予SPN后可引起血清ALT、AST活性升高,还可引起血清总胆红素（TBIL）,主要是间接胆红素（IBIL）含量的显著升高,呈现出一定的肝毒性以及与MKN协同增毒作用。6、UGT代谢抑制剂与MKN的联合给药对小鼠毒性的影响正常小鼠连续灌胃给予MKN 15天,未引发小鼠肝毒性;但利用AMF抑制UGT模型可导致小鼠肝脏毒性,AMF与MKN联合用药,可明显提升小鼠体内MKN血药浓度、升高血清ALT、AST活性,增加肝脏重量和肝脏指数,并使小鼠肝细胞出现点状坏死和脂肪变性等病理性改变。7、MKN对GSH耗竭小鼠的肝毒性作用MKN及其反应性代谢产物在体外可直接与GSH结合。MKN与BSO联合给药可导致小鼠严重的肝毒性,表现出血清ALT和AST活性升高,肝内GSH含量下降,肝细胞脂肪变性和广泛坏死。结论:本研究利用了组分敲除策略证明了山豆根中主要成分中的黄酮类成分是肝毒性成分;但MKN通过UGT代谢解毒,在正常大小鼠难以引发肝脏毒性;当使用UGT代谢阻断剂时,MKN可造成肝毒性,也即UGT代谢能力的差异或配伍药物中的UGT抑制剂会影响MKN的毒性作用,是MKN产生DDI、或增强MKN毒副作用的主要易感机制;山豆根药材中的脂溶性成分SPN是强效的UGT抑制剂、并可引起肝损伤。如果在使用山豆根药材时未祛除脂溶性组分,或添加了其它含有UGT广谱抑制剂AMF的药材（如连翘等）,其肝毒性会明显增强;GSH是体内重要的解毒机制,GSH下降也可导致MKN毒性明显增强。上述研究是对中药毒性物质基础的有益探索,相关体内作用过程和作用机制的逻辑递进型研究将为阐明人体代谢介导的中药毒性提供新思路。更多还原

【Abstract】 BackgroundToxic side effects of Traditional Chinese Medicines（TCM）remain a key challenge that affects the wider application of them.There are 83"toxic TCM"listed in the Pharmacopoeia,but only 13 of them have been clearly stated the limits of their main effect ingredients,and the remaining TCM have no clear qualitative and quantitative limits of their ingredients.In other words,at least for the TCM that have been regarded as"toxic TCM",there is still a serious challenge of unclear ingredients of toxic side effects which have been already applied in clinical practices.This makes it impossible for applicators to avoid the risk of toxic side effects and turns out a lack of basic guidelines for application.Specifically,the ingredients of these TCM is unclear in three aspects:1)the effect ingredients including active ingredients and related ingredients of toxic side effects are unclear;2)the ADME characteristics of related ingredients are unclear;3)which multi-component combinations will produce Drug-Drug Interactions（DDI）and thus lead to toxic side effects?Taking the TCM Shandougen（SDG）as example,it is one of the 83 toxic TCM listed in the 2020 edition of the Chinese Pharmacopoeia.SDG is mainly used for the treatment of sore throat,but a variety of adverse reactions have shown in clinical application,such as gastrointestinal reaction,hepatotoxicity and neurotoxicity.However,the understanding of the toxic ingredients of SDG remains controversial.In its quality standard,there are only lower limit of the total amount of Matrine（MT）and Oxymatrine（OMT）.In other words,SDG is an example of a TCM with no base expression of toxic substances.Therefore,it is necessary to systematically research the main toxic ingredients of SDG and their toxic factors.PurposeThis dissertation will address the following specific questions about the toxic ingredients of SDG:1)Which of the major ingredients are toxic;2)Does the main toxic ingredients related with human metabolism and how does it relate to metabolism;3)What herbs or herbal ingredients would have DDI when the SDG are combined with them;4)What are the mechanisms associated with the major toxic ingredients?MethodsTowards these challenges,this study was conducted by using the following three research strategies and methods.1.To comparing the main ingredients of 18 batches of SDG from different sources,and identify the active ingredients with high content.To prepare extracts and fractions of SDG and identify of the main toxic ingredients of SDG using Fraction Knockout Extract（FKE）strategy by screening vitro toxicity.2.To monitor the in vivo pharmacokinetics of the identified main toxic ingredients and their metabolites.3.To study the animal hepatotoxicity of the main toxic ingredients of SDG under the clearance mechanism also known as metabolic mechanism.Result1.Comparison of the main ingredients in different batches of SDGA total of 14 main ingredients were identified through fingerprint matching.Among them,the ten ingredients with the highest content were quantitatively determined by liquid chromatography,among which the contents of Sophocarpine and Oxysophocarpine varied greatly in different batches of SDG（coefficient of variation>50%）.Therefore,these two alkaloids（MT and OMT）together with three flavonoid ingredients（SPN,MKN and TF）of SDG with relatively stable contents were selected as candidate target ingredients.2.Preparation of FKE1)Extract preparation:taking water or 70%ethanol as the solvent,the aqueous extract and the alcoholic extract of SDG were prepared by heating extraction method;2)The alcoholic extract of SDG was combined with four main ingredients by column chromatography to collect four fractions:A,water eluted fraction（containing MT）;B,40%methanol eluted fraction（containing MT and OMT）;C,70%methanol eluted fraction（containing MT,OMT and TF）;and D,100%methanol eluted fraction（containing MKN and SPN）.3.Confirmation of the main toxic ingredientsThe toxicity of different ingredients was screened and tested by in vitro LO2 cell lines combined with cytotoxicity test and high content analysis of mitochondrial toxicity,and it was found that the cytotoxicity of SPN,MKN and TF of flavonoid ingredients were higher among the five ingredients with IC₅₀ of 13.6μM,105.1μM and 160.4μM.However,while the cytotoxicity of alkaloid ingredients MT and OMT was less,with IC₅₀>1000μM.On the basis of each different knockdown fraction,by the correlation comparison between the addition of the knocked-down fraction and its toxic alteration,it was confirmed that in the water-soluble fraction,its main toxic ingredient was MKN,while in the fat-soluble fraction,the main toxic ingredient was SPN.The major toxicity changes were not related to the increase or decrease in the other three main ingredients.In addition,both flavonoid ingredients significantly reduced the viability of LO2 cells and caused mitochondrial toxicity by altering the mitochondrial membrane potential（MMP）,while the related effects of the main alkaloid ingredients of SDG were not significant.4.UGT metabolism of MKN and in vivo pharmacokinetic changes in miceBased on the previous study on in vitro UGT metabolism of MKN,it was found that the broad-spectrum inhibitor of UGT,AMF,could inhibit MKN glucuronidation reaction in vitro;compared with untreated mice,AMF,UGT inhibitor,could affect the MKN pharmacokinetic behavior of mice;The half-life(t_1/2)and Area Under Curve（AUC）of MKN were increased by 1.4-fold,MKN exposure was increased in vivo.5.In vitro UGT inhibition of SPN and direct toxic effects of SPN on miceIn a previous study by the same group,SPN was also shown to be a broad-spectrum inhibitor of UGT,with significant inhibitory effects on all 10 UGT subenzymes.In this study,Mice was administrated SPN by P.O.,the serum ALT,AST activity and Serum Total Bilirubin（TBIL）levels were significant increased of mice;but Alkaline Phosphatase（ALP）activity and Total Bile Acid（TBA）levels were not increased.6.Toxic effects of co-administration of UGT metabolic inhibitors and MKN in miceNo liver injury observed in mice which were treated MKN by ig for 15 days;however,the use of AMF,type of UGT inhibition,led to hepatotoxicity in mice.The combination of AMF and MKN significantly elevated MKN blood concentration,increased serum ALT and AST activities,increased liver weight and liver index,and caused pathological changes such as local necrosis and steatosis in mouse hepatocytes.7.Hepatotoxicity induced by MKN on GSH-depleted miceMKN and its reactive metabolite（s）in vitro was trapped by GSH.The administration of MKN in combination with BSO to mice resulted in serious hepatotoxicity characterized by the elevation of plasma ALT and AST activity;decreased the hepatic GSH level;steatosis and extensive necrosis of hepatocytes.ConclusionIn this study,FKE strategy was used to find flavonoids of SDG which are hepotatoxic ingredients.However,MKN is mainly detoxified through UGT metabolism,when blocking UGT metabolism,MKN can cause liver toxicity,that is,differences in UGT metabolism ability or UGT inhibitors will affect MKN toxicity.UGT inhibition is the main susceptibility mechanism for MKN to produce DDI or to enhance MKN toxicity effects.The SPN,which is a lipid-soluble ingredient in SDG is a potent UGT inhibitor,and it can directly cause hepatocytes injury.If the fat-soluble components are not removed or added AMF or other UGTs inhibitors（such as Forsythia suspensa）when using SDG,the hepatotoxicity of SDG will be significantly enhanced.In addition,GSH is an important detoxification mechanism in the body,and decreased levels of GSH can lead to hepatotoxicity of MKN.This study is a useful exploration of the toxic ingredients of toxic TCM,and the study of the relevant in vivo process and mechanism provides new ideas for the elucidation of the ingredients of toxic TCM from human metabolism-induced toxicity and its DDI.更多还原