Enhanced absorption and inhibited metabolism of emodin by 2,3,5,4＇-tetrahydroxystilbene-2-O-β-D-glucopyranoside： Possible mechanisms for Polygoni Multiflori Radix-induced liver injury

Polygoni Multiflori Radix(PMR) has been commonly used as a tonic in China for centuries. However, PMR-associated hepatotoxicity is becoming a safety issue. In our previous in vivo study, an interaction between stilbenes and anthraquinones has been discovered and a hypothesis is proposed that the interaction between stilbene glucoside-enriching fraction and emodin may contribute to the side effects of PMR. To further support our previous in vivo results in rats, the present in vitro study was designed to evaluate the effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside(TSG) on the cellular absorption and human liver microsome metabolism of emodin. The obtained results indicated that the absorption of emodin in Caco-2 cells was enhanced and the metabolism of emodin in human liver microsomes was inhibited after TSG treatment. The effects of the transport inhibitors on the cellular emodin accumulation were also examined. Western blot assay suggested that the depressed metabolism of emodin could be attributed to the down-regulation of UDP-glucuronosyltransferases(UGTs) 1A8, 1A10, and 2B7. These findings definitively demonstrated the existence of interaction between TSG and emodin, which provide a basis for a better understanding of the underlying mechanism for PMR-induced liver injury.

Spectrum-Toxicity Correlation Study Revealed the Influence of the Nine-Time Steaming and Sun Drying Method on Hepatotoxic Components of Polygoni Multiflori Radix

Objective:Polygoni Multiflori Radix(PM)is a traditional herbal medicine with repeated reports of liver injury events in recent years.We wondered whether the classical processing method,namely,nine-time steaming and sun drying(NSSD),had toxicity-attenuating effects on PM and the relationships between toxicity and times of processing,as well as with the alteration trends of its compounds.Materials and Methods:The chemical fingerprints of different PM extracts were developed using ultra-high-performance liquid chromatography.The spectrum-toxicity correlation between the chemical fingerprints and hepatocellular toxicity was analyzed with multiple correlation analysis.Results:The results suggested that the hepatotoxicity of NSSD processing products markedly decreased with the repeated steaming and sun drying,which was obviously superior to the product processed by the modern method.Comprehensive analysis revealed that the contents of cis-stilbene glycoside and emodin-8-O-β-D-glucoside related to liver injury susceptibility were reduced with the times of NSSD processing,which was consistent with the decreased trend of hepatocellular toxicity.After the five times of NSSD,the contents of them as well as the hepatotoxicity of PM were steady.Moreover,we found that the contents of catechin and physcion declined rapidly after the one time of NSSD and then remained stable until the nine times of NSSD.Based on the fact,they could be utilized to indicate whether PM products were processed by steaming and sun drying.Conclusions:This paper confirmed that the NSSD had a good influence on the toxicity attenuating to PM and found four compounds which could apply for the quality control of PM.

Simultaneous Determination of Four Anthraquinones in Polygoni Multiflori Radix with Single Reference Standard by High Performance Liquid Chromatography

Objective: To establish a rapid, accurate and reliable analytical method for the simultaneous determination of four major anthraquinones in Polygoni Multiflori Radix(PMR) using single reference standard.Methods: The four components including emodin-8-O-β-D-(EMG), physcion-8-O-β-D-glucoside, emodin and physcion were separated on an ODS C18 column within 13 min and detected at 280 nm. Emodin was selected as the reference standard, and the response factor for each analyte with respect to emodin were calculated. Robustness were also tested including different columns, equipments, temperatures, detection wavelengths, and other chromatographic conditions which might influence stability of response factors.Results: The method was validated in terms of linearity(r^2> 0.9995), LOQs(0.820–3.05 ng/m L), LODs(0.180–0.920 ng/m L), precision,accuracy(95.8–103.6%, RSD < 2.80%) and stability. A total of 40 batches of PMR were analyzed and the results were found to have no statistically significant differences compared with those obtained using the external standard method.Conclusion: This work provided a single standard to determine multi-components method for quantitation of four anthraquinones in PMR,which could be applied in the quality control of this herbal drug.

Chemical profiles and metabolite study of raw and processed Polygoni Multiflori Radix in rats by UPLC-LTQ-Orbitrap MS^n spectrometry

The raw and processed roots of Plygonum multiflorum Thunb(PM) are used to treat different diseases in clinical practice. In order to clarify the influence of processing, a comparative study of chemical substance analysis was carried out. As the xenobiotics with a high enough exposure in target organs being considered as the potential effective or toxicity components, an in vivo study was also implemented to characterize the constitutes and metabolites, and meanwhile, the factor of compatibility with black bean were also considered. As a result, a total of 148 compounds were detected in PM extracts and more than 40 compounds were only detected in the processed products, which were probably new components produced during the steaming process. In in vivo study, 7 prototype components and 66 metabolites were detected or tentatively identified, 24 of which were reported for the first time. Our results indicated that processing greatly changed the chemical composition of PM and influenced the disposition of the compounds in vivo. To the best of our knowledge, this was the first global comparative study of raw and processed PM. These results expanded our knowledge about the influence of processing of PM and provided the essential data for further efficacy or toxicity studies.

Two New Glucosides from Radix Polygoni multiflori Preparata

2,3,5,4'-tetrahydroxystilbene-2-O-(2'-O-feruloyl)-beta -D-glucopyranoside, 2,3,5,4'-tetra hydroxystilbene-2-O-(2'-O-p-coumaroyl)-beta -D-glucopyranoside were isolated from Radix Polygoni multiflori Preparata. Structures were elucidated by chemical and spectral evidences.

Hematopoietic effect of small molecular fraction of Polygoni multiflori Radix Praeparata in cyclophosphamide-induced anemia mice

The aim of this study is to investigate the protective effects of a small molecular fraction(SMF) of Polygoni multiflori Radix Praeparata(PMRP) in a cyclophosphamide(CTX) induced anemia mouse model. Small molecular fraction of PMRP was prepared and identified by high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(HPLC-Q-TOF-MS). In pharmacology, we examined the peripheral hemogram and thymus and spleen index. The content of granulocyte-macrophage colony-stimulating factor(GM-CSF) in serum was mensurated by enzyme-linked immunosorbent assay(ELISA);The level of superoxide dismutase(SOD), catalase(CAT), total antioxidant capacity(T-AOC), and malondialdehyde(MDA) in serum and spleen tissue homogenate were detected, and glutathione peroxidase(GSH-PX) was assayed in spleen. The results show that SMF can significantly accelerate the recovery of peripheral hemogram, increase the activity of antioxidant enzymes and GM-CSF in serum and spleen. SMF also increases the number of spleen cells, improves bone marrow pathology. In conclusion, the SMF of PMRP promoted the recovery of hematopoietic function in a CTX-induced anemia mouse, which can support SMF to be used as an adjunct to chemotherapy to counteract its side effects.

Screening and Identifying Hepatotoxic Components in Polygoni multiflori Radix and Polygoni multiflori Radix Praeparata

Objective: In this study, the hepatotoxic components of Polygoni multiflori Radix and Polygoni multiflori Radix Praeparata(known as Heshouwu [HSW] and Zhiheshouwu [ZHSW] in China, respectively) were screened, isolated, and identified. Materials and Methods: The ethanol extracts of HSW and ZHSW were separated into 80 fractions according to their polarity in the preparation liquid phase. Chang liver cell line was used to screen the toxic components of HSW and ZHSW in vitro. The obtained toxic mixture was further collected, isolated, and identified to confirm the hepatotoxic compounds of HSW and ZHSW. Results: The identifid hepatotoxic compounds include 2,3,5,4’-tetrah ydroxystilbene-2-O-β-D-glucoside, emodin, physcion-8-O-β-d-glucoside, physcion, and citreorosein, the fist two among them were the main components of HSW and ZHSW. After processing of HSW, the contents of 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside and emodin in ZHSW were signifiantly decreased. Conclusions: The traditional processing with herb has signifiant effects on the components, especially the toxic components, in the extract of HSW and is an effective method to reduce its toxicity.

Radix Polygoni Multiflori Praeparata and Dioscorea Bulbifera Rhizome Decoctions Display Combined Effects Detected by a Three-Probe Drug Cocktail with Substrates of Rat Hepatic Cytochrome P450 Enzymes

Objectives: Radix Polygoni Multiflori Praeparata (RPMP) and Dioscorea Bulbifera Rhizomes (DBR) are used in Chinese herbal medicine and have been frequently reported for adverse reactions on liver. In this research, we aimed to evaluate in vivo effects of RPMP and DBR on rat cytochrome P450 enzymes (CYP1A2, CYP2E1 and CYP3A2) with their respective substrates as probes. Methods: Rats were orally administered RPMP, DBR and RPMP/DBR combination at 12, 10 and (12 + 10) g/kg, respectively, or saline as a control, once daily for 7 days. Thereafter, a cocktail containing 10 mg/kg caffeine, 20 mg/kg chlorzoxazone and 10 mg/kg dapsone was tail vein injected to rats. At defined time points, plasma drug concentrations were simultaneously evaluated by HPLC. Pharmacokinetic parameters simulated by DAS software were used to assess RPMP and/or DBR effects on cytochrome P450 enzymes activity. ANOVA and Dunnett’s test were used for data analysis. Results: Caffeine metabolism was enhanced in RPMP animals and reduced after pretreatment with DBR, but no effect was observed in RPMP/DBR combination group. Chlorzoxazone and dapsone metabolism was enhanced in both RPMP and DBR groups and consequently in combination group. The data suggested that RPMP independently induces rat CYP1A2, CYP2E1 and CYP3A2 activity, while DBR independently inhibits activity of rat CYP1A2 and induces that of CYP2E1 and CYP3A2. RPMP/DBR combination showed no significant benefit compared with the two drugs alone and even showed a neutralized effect in CYP1A2 activity. Conclusions: Caution is needed when RPMP and/or DBR are co-administered with drugs metabolized by human CYP1A2, CYP2E1 and CYP3A4.

天麻首乌片致严重肝损伤1例分析

目的 探讨严重肝损伤与天麻首乌片的相关性。方法 通过1例服用天麻首乌片导致严重肝损伤的不良反应,梳理其组方中药肝毒性的文献研究结果,采用《中国药物性肝损伤诊治指南(2023年版)》相关标准及更新后的RUCAM量表对肝损伤分型及与天麻首乌片的因果关系进行评估,探讨治疗策略的合理性。结果 天麻首乌片中制何首乌和炒蒺藜可能造成肝损伤,本例患者肝损伤为肝细胞损伤型,RUCAM评分7分(很可能),治疗策略通常包括保肝、利胆、抗炎等。文献分析发现,人类白细胞抗原(HLA)-B*35:01等位基因是何首乌肝损伤的风险因素,可作为预测何首乌所致肝损伤的潜在生物标志物。结论 本例患者严重肝损伤与天麻首乌片存在相关性;药品上市许可持有人应主动监测相关品种的不良反应,并及时更新说明书,减少患者用药风险。

何首乌-牛膝药对抗维甲酸诱导骨质疏松作用及活性成分筛选研究

目的研究何首乌-牛膝药对的抗骨质疏松作用及筛选活性成分。方法采取液相色谱-质谱(HPLC-MS)联用法分析何首乌-牛膝药对指纹共有峰并进行成分确认;采用维甲酸复制大鼠骨质疏松模型及酶联免疫吸附(ELISA)方法,探讨何首乌-牛膝药对的抗骨质疏松作用;应用SPSS分析色谱结果与肝、肾湿重、骨干湿比数据和ELISA测定结果的相关性筛选何首乌-牛膝药对抗骨质疏松活性成分。结果HPLC-MS联用法分析结果共确定10个共有指纹峰成分,分别为:没食子酸、杯苋甾酮、β-蜕皮甾酮、25-R-牛膝甾酮、二苯乙烯苷、25-S-牛膝甾酮、大黄素、大黄素甲醚、齐墩果酸-3-O-β-D-葡萄糖醛酸苷、牛膝皂苷D。生物学指标测定结果表明何首乌-牛膝药对能使维甲酸所致骨质疏松大鼠血清骨碱性磷酸酶(BALP)和骨钙素(BGP-OCN)指标升高,抗酒石酸酸性磷酸酶5b(TRACP-5b)和血清I型胶原C端肽(CTX-1)降低;何首乌-牛膝药对组肝、肾指数明显下降,骨干湿比上升,体质量变化明显。结论何首乌-牛膝药对具有抗骨质疏松作用,筛选出作用最显著活性成分为没食子酸、β-蜕皮甾酮、大黄素和齐墩果酸-3-O-β-D-葡萄糖醛酸苷。

炆何首乌乌发的药效评价及其网络药理学机制研究

目的:对炆何首乌乌发的药效进行评价,并对其作用机制进行研究。方法:构建小鼠白发模型,通过白发面积在新生毛发区域占比、苏木精-伊红(HE)染色及血浆中酪氨酸酶(TYR)的含量来评价炆何首乌的乌发作用;并利用网络药理学及免疫荧光探讨其作用机制。结果:与假手术组比较,模型组小鼠背部白发面积明显升高,皮肤组织中含黑色素的毛囊数量明显降低,血浆中TYR含量明显降低。与模型组比较,阳性药、制何首乌(中剂量、高剂量)及炆何首乌(低剂量、中剂量、高剂量)均能明显降低小鼠白发面积;制/炆何首乌低剂量、中剂量、高剂量能够明显增加小鼠皮肤组织中黑色素毛囊数量;制/炆何首乌高剂量可以明显提高小鼠血浆中TYR含量。在生药量相同的条件下,与制何首乌比较,炆何首乌中剂量能明显增加黑色素毛囊数量;炆何首乌低剂量、高剂量能明显增加小鼠血浆中TYR含量。网络药理学分析发现,炆何首乌的乌发作用涉及黑色素生成和Wnt信号通路等。免疫荧光结果表明,与假手术组比较,模型组小鼠皮肤组织中淋巴增强结合因子1(LEF1)及TYR表达水平明显降低;炆何首乌组小鼠皮肤组织中LEF1及TYR表达水平均明显增高。结论:炆何首乌对小鼠白发模型具有一定的改善作用,其作用机制可能与上调LEF1及TYR的表达水平有关。

何首乌化学成分、药理作用及肝毒性的研究进展

从化学成分、药理作用及肝毒性3个方面综述何首乌的研究进展。何首乌含有二苯乙烯苷类、蒽醌类、磷脂类、黄酮类等多种化学成分,具有延缓衰老、抗癌、抗疲劳、抗炎镇痛、保护心血管等药理作用。目前,何首乌引起肝毒性的化学成分探讨主要集中于3类,即蒽醌类、二苯乙烯苷类及鞣质类。加大何首乌肝毒性成分、肝毒性机制研究的力度,加强中药安全用药知识宣教,规范其炮制及使用,可以在一定程度上降低何首乌肝毒性。

何首乌及其中成药制剂导致的中草药相关肝损伤的回顾性研究

目的:观察何首乌、润燥止痒胶囊和百乐眠胶囊引起的中草药相关肝损伤(HILI)在接受中医药治疗人群中的可能发生率,探索其危险因素。方法:以2020年1月至2022年6月在上海中医药大学附属曙光医院门诊接受中医药治疗时曾运用何首乌、润燥止痒胶囊、百乐眠胶囊的患者为研究对象,分析HILI发生的可能性。结果:运用何首乌导致HILI的可能发生率为2.64‰,运用润燥止痒胶囊导致HILI的可能发生率为2.40‰,运用百乐眠胶囊导致HILI的可能发生率为2.06‰。甲状腺疾病是影响服用百乐眠胶囊患者HILI发生的危险因素(P<0.001)。结论:何首乌及其中成药制剂导致的中草药相关肝损伤发生率较低,应当正确评价中草药相关肝损伤,安全合理应用中药。

指纹图谱技术结合化学计量学方法鉴别制首乌产地的研究

目的建立一种指纹图谱技术结合化学计量学方法的制首乌产地快速鉴别方法。方法建立33批不同产地制首乌的指纹图谱并对其进行相似度评价,对色谱峰信息进行偏最小二乘判别法分析(PLS-DA)和线性判别分析。结果以对照指纹图谱为参照,不同批次药材间化学成分及含量差异较小,采用指纹图谱结合相似度的评价方法无法对其产地进行区分;选取PLS-DA分析法,基于变量权重重要性排序(VIP)值确定其中的差异标志物,由此建立不同产地制首乌的线性判别函数模型并对不同产地制首乌进行预测,模型对测试集的验证结果良好。结论指纹图谱技术结合化学计量学方法可作为一种有效鉴别不同产地制首乌的方法。

中成药相关肝损伤研究进展

药物性肝损伤是指由化学药品、生物制品、中草药及其制剂等所导致的肝损伤,是临床常见的药物不良反应之一。近年来,含何首乌、补骨脂、淫羊藿的中成药导致肝损伤的临床报道逐渐增多,其导致肝损伤的原因及机制受到广泛关注,并取得一定进展。本文主要对含此类组分的中成药及其肝损伤的原因、发病机制等进行综述。

何首乌安全用药指南

何首乌为常用中药,在临床治疗和日常保健中使用广泛,但近年来屡见有肝损伤报道,引起国内外广泛关注,部分含何首乌和首乌藤制剂肝损伤风险已被国家食品药品监督管理局多次通报。近年来相关研究已取得实质性进展,揭示了其特异质肝损伤的基本属性、主要成因、物质基础和分子机制,发现其易感人群的基本特征和生物标志物,表明何首乌仅对极少数特定人群有肝损伤风险,对绝大多数人群是安全的。研究成果为正确客观地认识何首乌致肝损伤、合理制定何首乌及相关制剂安全用药措施提供了科学依据。为此,中华中医药学会组织全国相关领域专家,起草制定了《何首乌安全用药指南》,旨在帮助国内外公众和相关机构,科学认识、评估和规避何首乌肝损伤风险,指导何首乌及相关制剂的合理使用,保障广大消费者的健康权益,同时促进何首乌及相关产业的健康持续发展。该指南已通过中华中医药学会的审核,并进行了发布,编号T/CACM 1328-2019。

何首乌致肝损伤的信号通路及其作用机制

何首乌是一种临床常用的补益类中草药,相关肝损伤事件近年来被频繁报道,其安全性问题逐渐引起国内外关注。本文梳理近年来何首乌致药物性肝损伤信号通路及作用机制的研究进展,基于信号通路角度,为临床正确合理应用何首乌提供新思路。现有研究证据表明,何首乌参与调控多条信号通路,通过破坏线粒体功能、加重胆汁酸淤积、诱发免疫应激、氧化应激、内质网应激等多种途径导致肝细胞死亡,多靶点、多途径、多层次诱导药物性肝损伤的发生发展。

何首乌肝损伤风险的发现、评价与防控策略研究

近年来,中草药相关的不良反应/事件频繁被报道,尤其针对新发现“有毒”中药的安全性问题,往往难以做出科学的回答并制定有效解决方案。中药由于自身的复杂性及缺乏系统性研究,使得我们对中草药相关肝损伤的成因机制了解有限。因此,破解中草药安全性难题亟需在中药毒性认知及安全风险防控方面实现突破。笔者团队以何首乌肝损伤作为典型范例,基于首创中药药源性肝损伤因果关系评价“整合证据链法”,证实了何首乌致肝损伤的客观真实性。进一步基于团队创新性建立的病证毒理学方法,科学揭示了何首乌肝损伤的物质基础及成因机制,也提出并证实了中药免疫特异质肝损伤“三因致毒”机制假说。在此基础上,阐明了何首乌特异质肝损伤易感人群的病证特征,并筛选发现了多种生物标志物,包括基因标志物HLA-B*35:01、免疫学及代谢组标志物。最后,探索创建了基于易感人群识别、易感物质控制以及个性化用药的“人-药-用”三维风险防控的安全用药策略和方法,为全面揭示“有毒”中药毒副反应科学内涵并建立科学有效的风险防控对策提供了基础。

制首乌不同配伍方式对黑色素及酪氨酸酶活性的影响

目的观察制首乌不同配伍方式的水提物对黑色素上调率及酪氨酸酶活性的影响,优选制首乌“乌须发”最佳的配伍方式。方法分别采用蘑菇酪氨酸酶NaOH裂解法及多巴胺速率氧化法研究制首乌不同配伍方式对黑色素生物合成及酪氨酸酶活性的影响。结果制首乌不同配伍方式均具有黑色素上调的作用,整体表现为制首乌-黑芝麻>制首乌-侧柏叶>制首乌-桑椹>制首乌-女贞子,且在一定范围内呈一定的剂量依赖性;制首乌不同配伍方式对酪氨酸酶的激活率均不同,其中制首乌-侧柏叶和制首乌-桑椹对酪氨酸酶具有抑制作用,但是随着浓度的增加,抑制作用逐渐减弱;制首乌-黑芝麻和制首乌-女贞子对酪氨酸酶具有激活作用,制首乌-黑芝麻较制首乌-女贞子激活作用明显。结论制首乌-黑芝麻为制首乌“乌须发”的最佳配伍,制首乌-黑芝麻配伍能够激活酪氨酸酶的活性,上调黑色素含量。

Efficacy of Heshouwu(Radix Polygoni Multiflori)on gut mircobiota in mice with autoimmune encephalomyelitis

OBJECTIVE:To learn the mechanisms between gut microbiome and the autoimmunity benefits on Traditional Chinese Medicine(TCM)in central nervous system(CNS),we investigated the neuro-protection effects and gut mircobiota changes of Heshouwu(Radix Polygoni Multiflori)on experimental autoimmune encephalomyelitis(EAE),an animal model of multiple sclerosis(MS).METHODS:Mice were randomly divided into four groups:EAE mice(control phosphate-buffered saline group),50 mg·kg^(-1)·d^(-1)Heshouwu(Radix Polygoni Multiflori)-treated EAE mice,100 mg·kg^(-1)·d^(-1)Heshouwu(Radix Polygoni Multiflori)-treated EAE mice,and 200 mg·kg^(-1)·d^(-1)Heshouwu(Radix Polygoni Multiflori)-treated EAE mice.The spinal cords were stained with hematoxylin and eosin(HE)and luxol fast blue for evaluating inflammatory infiltration and demyelination.The percentages of granulocyte macrophage-colony stimulating factor(GM-CSF)+CD4+,interleukin 17(IL-17)+CD4+,Foxp3 CD4+,and interferon-γ(IFN-γ)+CD4+T cells in the inguinal lymph nodes(LNs)and brain were determined by flow cytometry analysis.16S rRNA gene sequencing was employed to analyze the changes in gut microbiota.RESULTS:We found that Heshouwu(Radix Polygoni Multiflori)alleviated the disease severity and neuropathology of EAE as evaluated by clinical and histopathologyical scores.Heshouwu(Radix Polygoni Multiflori)increased the diversity and abundance of the gut microbiota,and decreased Firmicutes/Bacteroidetes ratio(F/B ratio).Heshouwu(Radix Polygoni Multiflori)also decreased the concentrations of IL-10,and IL-21 and increase the levels of GM-CSF,IL-17A,IL-17F and IL-22 in serum of EAE mice.Moreover,Heshouwu(Radix Polygoni Multiflori)modulated the T cell responses by inhibiting Th17 cells and restoring Treg cells in the small intestine lymphoid tissues and inguinal lymph nodes.Microbiota-depleted mice receiving Heshouwu(Radix Polygoni Multiflori)-treated fecal microbiota transplantation had lower disease severity,neuropathology scores and alleviation of Th17/Treg imbalance compared to ad libitum group.CONCLUSIONS:Our findings suggested that the vital neuro-protection role of Heshouwu(Radix Polygoni Multiflori)(TCM)in immunomodulation effects partly by regulations of gut microbiome.