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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Advances in Understanding the Metabolites and Metabolomics of Polygonum multiflorum Thunb: A Mini-review.

BACKGROUND: The roots of Polygonum multiflorum (PM) are a well-known traditional Chinese medicine, widely used to treat a variety of conditions in Southeast Asia, South Korea, Japan and other countries. It is known that Polygoni Multiflori Radix Praeparata (PMRP) may enhance the efficacy and reduce the toxicity of PM. However, reports of adverse reactions, such as hepatotoxicity, caused by PM or PMRP, have continuously appeared around the world, which increased the known risks of the medication and gradually gained the extensive attention of many researchers. The chemical constituents of PM that cause hepatotoxicity have not been distinctly elucidated using the traditional phytochemical screening. Recently, with the rapid development of metabolomics, there has been a growing need to explore the potential hepatotoxic components and mechanisms of PM. METHODS: The metabolites and metabolomics of PM were searched by the Web of Science, PubMed, Google scholar and some Chinese literature databases. RESULTS: A brief description of metabolites and metabolomics of PM is followed by a discussion on the metabolite- induced toxicity in this review. More than 100 metabolites were tentatively identified and this will contribute to further understanding of the potential hepatotoxic components of PM. Meanwhile, some toxic compounds were identified and could be used as potential toxic markers of PM. CONCLUSION: This review mainly outlines the metabolites and metabolomics of PM that have been identified in recent years. This study could help to clarify the potential hepatotoxic components and metabolic mechanisms of PM and provide a scientific reference for its safe clinical use in the future.

In vivo hepatotoxicity screening of different extracts, components, and constituents of Polygoni Multiflori Thunb. in zebrafish (Danio rerio) larvae.

Polygonum multiflorum Thunb. (PM) is a traditional Chinese medicine, commonly used to treat a variety of diseases. However, the hepatotoxicity associated with PM hampers its clinical application and development. In this study, we refined the zebrafish hepatotoxicity model with regard to the following endpoints: liver size, liver gray value, and the area of yolk sac. The levels of alanine aminotransferase, aspartate transaminase, albumin, and microRNAs-122 were evaluated to verify the model. Subsequently, this model was used to screen different extracts, components, and constituents of PM, including 70 % EtOH extracts of PM, four fractions from macroporous resin (components A, B, C, and D), and 19 compounds from component D. We found that emodin, chrysophanol, emodin-8-O-ß-D-glucopyranoside, (cis)-emodin-emodin dianthrones, and (trans)-emodin-emodin dianthrones showed higher hepatotoxicity compared to other components in PM, whereas polyphenols showed lower hepatotoxicity. To the best of our knowledge, this study is the first to identify that dianthrones may account for the hepatotoxicity of PM. We believe that these findings will be helpful in regulating the hepatotoxicity of PM.

Multiflorumisides HK, stilbene glucosides isolated from Polygonum multiflorum and their in vitro PTP1B inhibitory activities.

A phytochemical study on a 70% EtOH extract of dried roots of Polygonum multiflorum resulted in the isolation of four undescribed stilbene glucosides, namely multiflorumisides HK (1-4). The structures of the natural products were elucidated by 1D and 2D nuclear magnetic resonance (NMR) as well as mass spectroscopy analyses. Among them, multiflorumiside J (3) and multiflorumiside K (4) belong to rare tetramer stilbene glucosides. Moreover, the in vitro inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) were evaluated and the putative biosynthetic pathway was proposed. Notably, compounds 1-4 showed the inhibitory activity against PTP1B with the IC50 values of 1.2, 1.7, 1.5 and 4.6 µm, respectively. Based on the obtained results, stilbene glucosides could be the potential PTP1B inhibitors of P. multiflorum.

YiQiFuMai Powder Injection attenuates coronary artery ligation-induced myocardial remodeling and heart failure through modulating MAPKs signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: YiQiFuMai Powder Injection (YQFM), a traditional Chinese medicine prescription re-developed based on Sheng-Mai-San, is a classical and traditional therapeutic for clinical heart failure (HF) and angina. However, its potential mechanism against HF remains unclear. AIM OF THE STUDY: The present study observes the therapeutic role of YQFM and mechanisms underlying its effects on coronary artery ligation (CAL)-induced myocardial remodeling (MR) and HF. METHODS: MR and HF were induced by permanent CAL for 2 weeks in ICR mice. Then mice were treated with YQFM (0.13g/kg, 0.26g/kg and 0.53g/kg) once a day until 2 weeks later. Cardiac structure and function were evaluated by echocardiography. Serum lactate dehydrogenase (LDH), creatine kinase (CK) and malondialdehyde (MDA) were measured by biochemical kits and cardiomyocyte morphology was assessed by hematoxylin-eosin (HE) staining. Myocardial hydroxyproline (HYP), serum amino-terminal pro-peptide of pro-collagen type III (PIIINP), and Masson's trichrome staining were employed to evaluate cardiac fibrosis. Circulating level of N-terminal pro-B-type natriuretic peptide (NT-proBNP) was tested by ELISA kit to predict prognosis of CAL-induced HF. Effects of YQFM on the mitogen-activated protein kinases (MAPKs) pathway after CAL operation was evaluated by Western blotting and immunohistochemistry assay. RESULTS: YQFM (0.53g/kg) improved the left ventricular (LV) function and structure impairment after 2 weeks in CAL mice. YQFM administration also decreased LDH and CK activities, circulating levels of MDA, PIIINP, NT-proBNP, and HYP contents. Moreover, YQFM ameliorated cardiac injury and fibrosis. Furthermore, YQFM (0.53g/kg) inhibited the myocardial phosphorylation of MAPKs in HF mice. CONCLUSION: Our findings suggest that YQFM attenuates CAL-induced HF via improving cardiac function, attenuating structure damage, oxidative stress, necrosis, collagen deposition, and fibrosis. In addition, YQFM ameliorates cardiac remodeling and HF, partially through inhibiting the MAPKs signaling pathways. These data provide insights and mechanisms into the widely application of YQFM in patients with HF, MI and other ischemic heart diseases.