On-line identification of the chemical constituents of Polygoni Multiflori Radix by UHPLC-Q-ToF MS/MS.

Introduction: Polygoni Multiflori Radix (PMR) is a type of Chinese herbal medicine with rich chemical composition and pharmacological activity used widely in medicine and food. However, in recent years, there have been increasing numbers of negative reports about its hepatotoxicity. Identification of its chemical constituents for quality control and safe use is very important. Methods: Three solvents of different polarities (water, 70% ethanol, and 95% ethanol solution) were used to extract the compounds from PMR. Extracts were analyzed and characterized by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-ToF MS/MS) in the negative-ion mode. Results: 152 compounds were detected and identified: 50 anthraquinones, 33 stilbene derivatives, 21 flavonoids, seven naphthalene compounds, and 41 other compounds. Eight other compounds were reported for the first time in the PMR-related literature, and eight other compounds were potentially new compounds. Discussion: This study lays a solid foundation for the screening of toxicity and quality-control indicators of PMR.

Exploratory Quality Control Study for Polygonum multiflorum Thunb. Using Dinuclear Anthraquinones with Potential Hepatotoxicity.

In recent years, the hepatotoxicity of Polygoni Multiflora Radix (PMR) has attracted increased research interest. Some studies suggest that anthraquinone may be the main hepatotoxic component. Most of the relevant studies have focused on the mononuclear anthraquinone component rather than binuclear anthraquinones. The hepatotoxicity of dinuclear anthraquinone (dianthrone) was investigated in a cell-based model. Next, a method for the determination of six free and total dianthonones in PMR and PMR Praeparata (PMRP) was established using ultra-high-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS), which was then used to analyze the collected samples. The data show that four binuclear anthraquinone compounds were hepatotoxic and may be potential toxicity indicators for the safety evaluation of PMR and PMRP. Herein, we provide a theoretical basis for the improvement of PMRP quality standards.

A stepwise strategy integrating metabolomics and pseudotargeted spectrum-effect relationship to elucidate the potential hepatotoxic components in Polygonum multiflorum.

Polygonum multiflorum (PM) Thunb., a typical Chinese herbal medicine with different therapeutic effect in raw and processed forms, has been used worldwide for thousands of years. However, hepatotoxicity caused by PM has raised considerable concern in recent decades. The exploration of toxic components in PM has been a great challenge for a long time. In this study, we developed a stepwise strategy integrating metabolomics and pseudotargeted spectrum-effect relationship to illuminate the potential hepatotoxic components in PM. First, 112 components were tentatively identified using ultraperformance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-Q-TOF-MS). Second, based on the theory of toxicity attenuation after processing, we combined the UPLC-Q-TOF-MS method and plant metabolomics to screen out the reduced differential components in PM between raw and processed PM. Third, the proposed pseudotargeted MS of 16 differential components was established and applied to 50 batches of PM for quantitative analysis. Fourth, the hepatocytotoxicity of 50 batches of PM was investigated on two hepatocytes, LO2 and HepG2. Last, three mathematical models, gray relational analysis, orthogonal partial least squares analysis, and back propagation artificial neural network, were established to further identify the key variables affecting hepatotoxicity in PM by combining quantitative spectral information with toxicity to hepatocytes of 50 batches of PM. The results suggested that 16 components may have different degrees of hepatotoxicity, which may lead to hepatotoxicity through synergistic effects. Three components (emodin dianthrones, emodin-8-O-ß-D-glucopyranoside, PM 14-17) were screened to have significant hepatotoxicity and could be used as toxicity markers in PM as well as for further studies on the mechanism of toxicity. Above all, the study established an effective strategy to explore the hepatotoxic material basis in PM but also provides reference information for in-depth investigations on the hepatotoxicity of PM.

Integrated spatially resolved metabolomics and network toxicology to investigate the hepatotoxicity mechanisms of component D of Polygonum multiflorum Thunb.

ETHNOPHARMACOLOGICAL RELEVANCE: The liver toxicity of Reynoutria multiflora (Thunb.) Moldenke. (Polygonaceae) (Polygonum multiflorum Thunb, PM) has always attracted much attention, but the related toxicity materials and mechanisms have not been elucidated due to multi-component and multi-target characteristics. In previous hepatotoxicity screening, different components of PM were first evaluated and the hepatotoxicity of component D [95% ethanol (EtOH) elution] in a 70% EtOH extract of PM (PM-D) showed the highest hepatotoxicity. Furthermore, the main components of PM-D were identified and their hepatotoxicity was evaluated based on a zebrafish embryo model. However, the hepatotoxicity mechanism of PM-D is unknown. AIM OF THE STUDY: This work is to explore the hepatotoxicity mechanisms of PM-D by integrating network toxicology and spatially resolved metabolomics strategy. MATERIALS AND METHODS: A hepatotoxicity interaction network of PM-D was constructed based on toxicity target prediction for eight key toxic ingredients and a hepatotoxicity target collection. Then the key signaling pathways were enriched, and molecular docking verification was implemented to evaluate the ability of toxic ingredients to bind to the core targets. The pathological changes of liver tissues and serum biochemical assays of mice were used to evaluate the liver injury effect of mice with oral administration of PM-D. Furthermore, spatially resolved metabolomics was used to visualize significant differences in metabolic profiles in mice after drug administration, to screen hepatotoxicity-related biomarkers and analyze metabolic pathways. RESULTS: The contents of four key toxic compounds in PM-D were detected. Network toxicology identified 30 potential targets of liver toxicity of PM-D. GO and KEGG enrichment analyses indicated that the hepatotoxicity of PM-D involved multiple biological activities, including cellular response to endogenous stimulus, organonitrogen compound metabolic process, regulation of the apoptotic process, regulation of kinase, regulation of reactive oxygen species metabolic process and signaling pathways including PI3K-Akt, AMPK, MAPK, mTOR, Ras and HIF-1. The molecular docking confirmed the high binding activity of 8 key toxic ingredients with 10 core targets, including mTOR, PIK3CA, AKT1, and EGFR. The high distribution of metabolites of PM-D in the liver of administrated mice was recognized by mass spectrometry imaging. Spatially resolved metabolomics results revealed significant changes in metabolic profiles after PM-D administration, and metabolites such as taurine, taurocholic acid, adenosine, and acyl-carnitines were associated with PM-D-induced liver injury. Enrichment analyses of metabolic pathways revealed tht linolenic acid and linoleic acid metabolism, carnitine synthesis, oxidation of branched-chain fatty acids, and six other metabolic pathways were significantly changed. Comprehensive analysis revealed that the hepatotoxicity caused by PM-D was closely related to cholestasis, mitochondrial damage, oxidative stress and energy metabolism, and lipid metabolism disorders. CONCLUSIONS: In this study, the hepatotoxicity mechanisms of PM-D were comprehensively identified through an integrated spatially resolved metabolomics and network toxicology strategy, providing a theoretical foundation for the toxicity mechanisms of PM and its safe clinical application.

Pharmacokinetics and metabolism of trans-emodin dianthrones in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum multiflorum Thunb. (PM) is a common traditional Chinese medicine with diverse biological activities of resolving toxins, nourishing livers and promoting hairs. Nevertheless, in recent years hepatotoxic adverse reactions caused by the administration of PM have raised worldwide concerns. In our previous study, we found that emodin dianthrones showed hepatotoxicity and may be potential toxicity markers. However, the metabolic transformation and pharmacokinetic behavior of emodin dianthrones in vivo have still not been elucidated. AIM OF THE STUDY: Taking trans-emodin dianthrones (TED) as an example, the present study was conducted to investigate the pharmacokinetics and bioavailability of TED in rats and characterized its metabolic transformation in the plasma, urine and feces of rats. MATERIALS AND METHODS: A rapid and sensitive UPLC-qqq-MS/MS method was developed for accurate quantification of TED in plasma and successfully applied to the pharmacokinetic evaluation of TED in rats after intravenous and oral administration. A reliable UFLC-Q-TOF-MS high resolution mass spectrometry combined with a scientific metabolite identification strategy was used to comprehensively characterize the metabolic transformation of TED in plasma, urine and feces in rats. RESULTS: The established UPLC-qqq-MS/MS method had a linear range of 1-500 ng/mL, and the method was accurate and reliable to meet the quantitative requirements. When 20 mg/kg TED was given by gavage rats, it was rapidly absorbed into the circulatory system and had a long half-life time of 6.44 h and wide tissue distribution in vivo. While intravenous injection of 0.4 mg/kg TED in rats, it was rapidly metabolized and eliminated with a half-life time of 1.82 h. The oral absorption bioavailability of TED was only 2.83%. Furthermore with a sensitive UFLC-Q-TOF-MS technique and metabolite identification strategy, 21 metabolites were successfully identified, including 11 in plasma, 12 in urine and 18 in feces. The main Ⅰ and Ⅱ phase metabolic processes involved glucuronidation, oxidation, carbonylation, (de)methylation, sulfation and hydrogenation. CONCLUSION: TED could be rapidly absorbed into the blood circulation and widely distributed and slowly metabolized in the body and underwent extensive cleavage and metabolic transformation in vivo. The study provided a basis for in-depth elucidation of the toxicology and mechanism research of TED, but also laid the foundation for further research on the material basis of hepatotoxicity of PM.

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.

Effects of fish oil supplementation on glucose control and lipid levels among patients with type 2 diabetes mellitus: a Meta-analysis of randomized controlled trials.

BACKGROUND: Previous studies have yielded inconsistent findings on the role of fish oil in type 2 diabetes mellitus (T2DM). We systematically summarized the available evidence from randomized controlled trials (RCT) and aimed to investigate the effects of fish oil supplementation on glucose control and lipid levels among patients with T2DM. METHODS: A comprehensive literature search was performed in electronic databases (PubMed, ProQuest, Cochrane Library, CNKI, VIP, and Wanfang) to identify all relevant RCTs which were published up to May 31st, 2019. We used Modified Jadad Score system to evaluate the quality of each included RCT. The pooled effects were estimated using random-effects model and presented as standardized mean differences with 95% confidence intervals. RESULTS: A total of 12 RCTs were included in this meta-analysis. There was no significant difference in glucose control outcomes comparing fish oil supplementation to placebo. The effect size of fasting plasma glucose (FPG) was 0.13 (95% CI: - 0.03 to 0.28, p > 0.05). No marked change was observed in fasting insulin (FINS), glycosylated hemoglobin (HbA1c), and HOMA of insulin resistance (HOMA-IR) levels. Fish oil supplementation was associated with a decrease of triglyceride (TG) level by - 0.40 (95%CI: - 0.53 to - 0.28, p < 0.05), and an increase of high density lipoprotein (HDL) cholesterol level by 0.21 (95%CI: 0.05 to 0.37, p < 0.05). In subgroup analysis, HDL cholesterol level was higher among Asian and low-dose(< 2 g/d n-3 PUFA) subgroups compared to their counterparts (p < 0.05). TG level was lower in mid and long duration groups, along with an inconspicuous difference in short duration group. CONCLUSIONS: This meta-analysis shows that among patients with T2DM, fish oil supplementation leads to a favorable blood lipids profile but does not improve glucose control.