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首页> 外文期刊>Chinese Medicine >UHPLC-QQQ-MS/MS assay for the quantification of dianthrones as potential toxic markers of Polygonum multiflorum Thunb: applications for the standardization of traditional Chinese medicines (TCMs) with endogenous toxicity
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UHPLC-QQQ-MS/MS assay for the quantification of dianthrones as potential toxic markers of Polygonum multiflorum Thunb: applications for the standardization of traditional Chinese medicines (TCMs) with endogenous toxicity

机译：UHPLC-QQQ-MS / MS测定用于定量促性蒽醌的潜在有毒标志物：具有内源性毒性的中药（TCMS）标准化的应用

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摘要

The raw and processed roots of Polygonum multiflorum Thunb (PM) are commonly used in clinical practice to treat diverse diseases; however, reports of hepatotoxicity induced by Polygoni Multiflori Radix (PMR) and Polygoni Multiflori Radix Praeparata (PMRP) have emerged worldwide. Thus, it is necessary for researchers to explore methods to improve quality standards to ensure their quality and treatment effects. In the present study, an ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method was optimized and validated for the determination of dianthrones in PMR and PMRP using bianthronyl as the internal standard. Chromatographic separation with a gradient mobile phase [A: acetonitrile and B: water containing 0.1% formic acid (v/v)] at a flow rate of 0.25?mL/min was achieved on an Agilent ZORBAX SB-C18 column (2.1?mm?×?50?mm, 1.8?μm). The triple quadrupole mass spectrometer (TQMS) was operated in negative ionization mode with multiple reaction monitoring for the quantitative analysis of six dianthrones. Moreover, compounds 5 and 6 were further evaluated for their cytotoxicity in HepaRG cells by CCK-8 assay. The UHPLC-QQQ-MS/MS method was first developed to simultaneously determine six dianthrones in PMR and PMRP, namely, polygonumnolides C1–C4 (1–4), trans-emodin dianthrones (5), and cis-emodin dianthrones (6). The contents of 1–6 in 90 batches of PMR were in the ranges of 0.027–19.04, 0.022–13.86, 0.073–15.53, 0.034–23.35, 0.38–83.67 and 0.29–67.00?μg/g, respectively. The contents of 1–6 in 86 batches of commercial PMRP were in the ranges of 0.020–13.03, 0.051–8.94, 0.022–7.23, 0.030–12.75, 0.098–28.54 and 0.14–27.79?μg/g, respectively. Compounds 1–4 were almost completely eliminated after reasonable processing for 24?h and the contents of compounds 5 and 6 significantly decreased. Additionally, compounds 5 and 6 showed inhibitory activity in HepaRG cells with IC50 values of 10.98 and 15.45?μM, respectively. Furthermore, a systematic five-step strategy to standardize TCMs with endogenous toxicity was proposed for the first time, which involved the establishment of determination methods, the identification of potentially toxic markers, the standardization of processing methods, the development of limit standards and a risk–benefit assessment. The results of the cytotoxicity evaluation of the dianthrones indicated that trans-emodin dianthrones (5) and cis-emodin dianthrones (6) could be selected as toxic markers of PMRP. Taking PMR and PMRP as examples, we hope this study provides insight into the standardization and internationalization of endogenous toxic TCMs, with the main purpose of improving public health by scientifically using TCMs to treat diverse complex diseases in the future.
机译：Polygonum Multiflorum thunb（PM）的原始和加工根部通常用于临床实践以治疗多种疾病;然而，全世界都出现了由Polygoni Multiflori基数（PMR）和Polygoni Multiflori radix praparata（PMRP）诱导的肝毒性的报道。因此，研究人员需要探索提高质量标准以确保其质量和治疗效果的方法。在本研究中，优化了超高效液相色谱三重四极杆质谱（UHPLC-QQQ-MS / MS）方法并验证了使用Bianthronyl作为内标测定PMR和PMRP中的临时杆菌。用梯度流动相[A：乙腈和B：在Agilent Zorbax SB-C18柱上以0.25Ωml/ min的流速（2.1Ωmm ？×？50？mm，1.8？μm）。在负电离模式下操作三重四极杆质谱仪（TQMS），具有多重反应监测，用于六个展领的定量分析。此外，通过CCK-8测定法进一步评价化合物5和6的肝细胞中的细胞毒性。首先开发UHPLC-QQQ-MS / MS方法以同时测定PMR和PMRP中的六个临时杆，即PolyGonolides C1-C4（1-4），转发蛋白Dianthrones（5）和CIS-eModin Dianthrones（6） 。分别为0.027-19.04，0.022-13.86,0.073-15.53,0.034-23.35,0.38-83.67和0.29-67.00？μg/ g的范围。 86批的商业PMRP中1-6中的含量分别为0.020-13.03,0.051-8.94,0.022-7.23,0.030-12.75,0.098-28.54和0.14-27.79？μg/ g。在合理加工后几乎完全消除化合物1-4，并且化合物5和6的含量显着降低。另外，化合物5和6在Heparg细胞中显示出IC 50值为10.98和15.45Ωμm的抑制活性。此外，首次提出了一种系统化的五步策略，以使具有内源性毒性的TCM标准化，涉及确定方法的建立，识别潜在有毒标志物，加工方法的标准化，限制标准的发展和风险 - 菲尼特评估。促性蒽酮的细胞毒性评价结果表明，转铁蛋白Dianthrones（5）和顺式 - 大黄素促进剂（6）可选成PMRP的有毒标志物。采取PMR和PMRP作为示例，我们希望本研究能够深入了解内源性有毒TCMS的标准化和国际化，主要目的是通过科学使用TCMS在未来进行科学来治疗不同的复杂疾病。

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