BACKGROUND: Polygonum multiflorum (PM), a widely used traditional Chinese medicine herb, is divided into two forms, namely raw polygonum multiflorum (RPM) and polygonum multiflorum praeparata (PMP), according to the processing procedure. Emerging data has revealed the differential hepatotoxicity of RPM and PMP, however, its potential mechanism is still unclear.
METHODS: In our study, we investigated the differential hepatotoxicity of RPM and PMP exerted in C57BL/6 mice. First, sera were collected for biochemical analysis and HE staining was applied to examine the morphological alternation of the liver. Then we treated L02 cells with 5 mg / mL of RPM or PMP. The CCK8 and EdU assays were utilized to observe the viability and proliferation of L02 cells. RNA sequencing was performed to explore the expression profile of L02 cells. Western blotting was performed to detect the expression level of ferroptosis-related protein. Flow cytometry was used to evaluate ROS accumulation.
RESULTS: In our study, a significant elevation in serum ALT, AST and TBIL levels was investigated in the RMP group, while no significant differences were observed in the PMP group, compared to that of the CON group. HE staining showed punctate necrosis, inflammatory cell infiltration and structural destruction can be observed in the RPM group, which can be significantly attenuated after processing. In addition, we also found RPM could decrease the viability and proliferation capacity of L02 cells, which can be reversed by ferroptosis inhibitor. RNA sequencing data revealed the adverse effect of PM exerted on the liver is closely associated with ferroptosis. Western blotting assay uncovered the protein level of GPX4, HO-1 and FTL was sharply decreased, while the ROS content was dramatically elevated in L02 cells treated with RPM, which can be partially restored after processing.
CONCLUSIONS: The hepatotoxicity induced by RPM was significantly lower than the PMP, and its potential mechanism is associated with ferroptosis.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease (MAFLD) is a severe threat to human health. Polygonum multiflorum (PM) has been proven to remedy mitochondria and relieve MAFLD, but the main pharmacodynamic ingredients for mitigating MAFLD remain unclear.
OBJECTIVE: To research the active ingredients of PM adjusting mitochondria to relieve high-fat diet (HFD)-induced MAFLD in rats.
METHODS: Fat emulsion-induced L02 adipocyte model and HFD-induced MAFLD rat model were used to investigate the anti-MAFLD ability of PM and explore their action mechanisms. The adipocyte model was also applied to evaluate the activities of PM-derived constituents in liver mitochondria from HFD-fed rats (mitochondrial pharmacology). PM-derived constituents in liver mitochondria were confirmed by ultra-high-performance liquid chromatography/mass spectrometry (mitochondrial pharmacochemistry). The abilities of PM-derived monomer and monomer groups were evaluated by the adipocyte model and MAFLD mouse model, respectively.
RESULTS: PM repaired mitochondrial ultrastructure and prevented oxidative stress and energy production disorder of liver mitochondria to mitigate fat emulsion-induced cellular steatosis and HFD-induced MAFLD. PM-derived constituents that entered the liver mitochondria inhibited oxidative stress damage and improved energy production against cellular steatosis. Eight chemicals were found in the liver mitochondria of PM-administrated rats. The anti-steatosis ability of one monomer and the anti-MAFLD activity of the monomer group were validated.
CONCLUSIONS: PM restored mitochondrial structure and function and alleviated MAFLD, which may be associated with the remedy of oxidative stress and energy production. The identified eight chemicals may be the main bioactive ingredients in PM that adjusted mitochondria to prevent MAFLD. Thus, PM provides a new approach to prevent MAFLD-related mitochondrial dysfunction. Mitochondrial pharmacology and pharmacochemistry further showed efficient strategies for determining the bioactive ingredients of Chinese medicines that adjust mitochondria to prevent diseases.

In this study, natural deep eutectic solvents (NADES) formed by choline chloride (ChCl), sucrose, fructose, glucose, and xylose, were used to extract antioxidants from the halophyte Polygonum maritimum L. (sea knotgrass) and compared with conventional solvents (ethanol and acetone). NADES and conventional extracts were made by an ultrasound-assisted procedure and evaluated for in vitro antioxidant properties by the radical scavenging activity (RSA) on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, oxygen radical absorbance capacity (ORAC), and copper chelating activity (CCA). Samples were profiled by liquid chromatography (LC)-electrospray ionization (ESI)-QTOF-MS analysis. ChCl:fructose was more efficient in the DPPH assay, than the acetone extract. ChCl:sucrose and ChCl:fructose extracts had the highest ORAC when compared with the acetone extract. NADES extracts had higher CCA, than the acetone extract. The phenolic composition of the NADES extracts was less complex than the conventional extracts, but the proportions of major antioxidants, such as flavonols and flavan-3-ols, were similar in all the solvents. Myricitrin was the major flavonoid in all of the samples, while gallic acid was the main phenolic acid in the conventional extracts and present in a greater amount in ChCl:fructose. Results suggest that NADES containing ChCl and sucrose/fructose can replace conventional solvents, especially acetone, in the extraction of antioxidants from sea knotgrass.

The efficacy of raw and processed products of Polygonum multiflorum (PM) varies greatly. \"Nine cycles of steaming and sunning\" (NCSS) is recognized as an effective technology in enhancing efficacy and reducing toxicity for PM. In this paper, PM was prepared differently into three groups (including group R, M, and \"9\"), which represent raw PM, PM processed using the method of Chinese Pharmacopoeia (ChP) and PM processed using traditional NCSS, respectively. The purpose is to establish an effective method to distinguish raw PM from different processed products and highlight the rationality of processing technology. The main organic compounds that could distinguish these three groups of samples were identified by in-depth mining of mass spectral information and various chemometric methods. Level of related metal cations have been quantified and used as another important distinguishing markers. The electronic tongue was utilized to determine the taste traits of aqueous extract from PM. Furthermore, the material basis that caused the difference in taste was discovered according to correlation analysis. In detail, saltiness has the most important contribution associated with the concentrations of K+ and Na+, however, bitterness and astringency were mainly associated with the contents of epicatechin gallate, catechin, procyanidin B1, procyanidin B2 and epicatechin. This study proposed a novel and effective strategy for identification of processing technology of PM. It lays the foundation for clarifying the modern scientific recommendations of processing technology to PM. On the other hand, it also provides a reference for related researches on other traditional Chinese medicine (TCM).

Polygoni Multiflori Radix Praeparata (PMRP), as the processed product of tuberous roots of Polygonum multiflorum Thunb., is one of the most famous traditional Chinese medicines, with a long history. However, in recent years, liver adverse reactions linked to PMRP have been frequently reported. Our work attempted to investigate the chemical constituents of PMRP for clinical research and safe medication. In this study, an effective and rapid method was established to separate and characterize the constituents in PMRP by combining ultra-high performance liquid chromatography with hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS). Based on the accurate mass measurements for molecular and characteristic fragment ions, a total of 103 compounds, including 24 anthraquinones, 21 stilbenes, 15 phenolic acids, 14 flavones, and 29 other compounds were identified or tentatively characterized. Forty-eight compounds were tentatively characterized from PMRP for the first time, and their fragmentation behaviors were summarized. There were 101 components in PMRP ethanol extract (PMRPE) and 91 components in PMRP water extract (PMRPW). Simultaneously, the peak areas of several potential xenobiotic components were compared in the detection, which showed that PMRPE has a higher content of anthraquinones and stilbenes. The obtained results can be used in pharmacological and toxicological research and provided useful information for further in vitro and in vivo studies.

Heshouwu, derived from root tubers of Fallopia multiflora (Thunb.) Harald., is a well-known herb used for millennia in traditional Chinese medicine. However, different forms of root tubers of Heshouwu have occurred in current Chinese herbal market and used in clinic, although it is still unknown whether their quality is consistent. In the present study, a mass spectrometry imaging and laser microdissection combined with UPLC-Q/TOF-MS were therefore used for the metabolite profiling on the whole and different parts of root tubers of F. multiflora and F. multiflora var. angulata. Our results suggested that the character of \"woody heart\" root tubers of F. multiflora was similar to that of F. multiflora var. angulata, but the latter had more phloem fibers and larger diameter vessel in the normal vascular bundle. Moreover, 140 compounds including stilbenes, anthraquinones, phenolic acids, naphthalenes, and other compounds were identified or putatively characterized from F. multiflora and F. multiflora var. angulata. Both unsupervised principal component analysis (PCA) and supervised Orthogonal Partial Least Squares Discrimination Analysis (OPLS-DA) multivariate statistics allowed discriminating F. multiflora and F. multiflora var. angulata. And a total of 32 potential markers were identified. The tissue-specific study indicated that the compounds in the phelloderm of F. multiflora and F. multiflora var. angulata were the most abundant. This is the first study on metabolite profiling and comparison of root tubers between F. multiflora and F. multiflora var. angulata, which would provide reasonable basis for further quality evaluation and safe medication of F. multiflora.

UNASSIGNED: As the skin is the largest organ of the human body, it is aging inevitably and produces cosmetic and psychological problems, and even disease. Therefore, the molecular mechanisms related to the prevention of skin aging need to be further explored.
UNASSIGNED: Aging models were constructed by D-galactose. Mice were administrated with polygoni multiflori radix preparat (PMRP), PMRP and 3-methyladenine, or PMRP and rapamycin intragastrically. The apparent and viscera index of aged rats was measured. Then, the physicochemical property, antioxidant ability, histological structure, mitochondrial membrane potential, ATP and ROS levels, and mitophagy of aged skins were determined. Finally, the expression of PINK1, Parkin, P62, and LC3II/I; apoptosis-related proteins; and the percentage of apoptotic cells were measured.
UNASSIGNED: PMRP relieved skin aging with reducing of thymus index, improvement of pathological damage and histological structure, increase of the expression area of fibrous tissue, the ratio of type I to type III collagen, and antioxidant ability of aged skins. Importantly, PMRP also improved mitochondrial dysfunction with an increase in the content of mitochondrial membrane potential and ATP and a decrease of ROS levels. Moreover, mitophagy was enhanced with the treatment of PMRP when observed using electron microscopy, and the expression of PINK1, Parkin, and LC3I/II was increased with PMRP treatment but P62 expression was decreased. Meanwhile, PMRP alleviated apoptosis with a decrease of apoptotic cell and the expression of Cleaved-cas3, Bax, Cyt-c, AIF, and Smac as well as an increase of Bcl-2 expression.
UNASSIGNED: The results demonstrated that the polygoni multiflori radix preparata may delay skin aging by inducing mitophagy.

Fallopia multiflora is an important Oriental herb belonging to the family Polygonaceae. The F. multiflora mitochondrial genome consists of two circular chromosomes that were 200,352- and 112,098-nucleotides long. The mitochondrial genome encodes 57 genes, including 34 protein-coding genes, 20 transfer RNA (tRNA) genes, and three ribosomal RNA (rRNA) genes. When accounting for 3 overlapping genes, 38 genes were found in chromosome I, and 22 in chromosome II. The phylogenetic analysis suggests that F. multiflora is closely related to Beta macrocarpa and Silene latifolia.

BACKGROUND: Polygonum multiflorum Thunb.(PM), (known as Heshouwu () in China) is one of the most important and well mentioned Chinese medicinal herbs in the literature for its use in blackening hair, nourishing liver and kidney, anti-aging, anti-hyperlipidemia, antioxidant, anti-inflammatory, anticancer, hepatoprotection, cardio-protection and improving age-related cognitive dysfunction. The purpose of this review is to give a comprehensive and recent update on PM: new compounds or isolated for the first time, potential hepatotoxic compounds and their mechanisms. Moreover, future perspectives and challenges in the future study of this plant are conversed which will make a new base for further study on PM.
METHODS: A comprehensive review of relevant published literature on PM using the scientific databases SCOPUS, PubMed, and Science Direct was done.
RESULTS: PM is broadly produced in many provinces of China and well known in other Eastern Asian Countries for its ethno-medical uses. Previous phytochemical investigation of PM had led to the isolation of more than 175 compounds including recently isolated 70 new compounds. Most of the new compounds isolated after 2015 are majorly dianthrone glycosides and stilbene glycosides. Processing has also a significant effect on chemical composition, pharmacological activities, and toxicity of PM. PM-induced liver injury is increasing after the first report in Hong Kong in 1996. Hepatotoxicity of PM was constantly reported in Japan, Korea, China, Australia, Britain, Italy, and other countries although its toxicity is related to idiosyncratic hepatotoxicity. More interestingly, although there is indispensable interest to predict idiosyncratic hepatotoxicity of PM and understand its mechanisms, the responsible hepatotoxic compounds and mechanisms of liver damage induced by PM are still not clear. There is a big controversy on the identification of the most responsible constituent. Anthraquinone and stilbene compounds in PM, mainly emodine and TSG are mentioned in the literature to be the main responsible hepatotoxic compounds. However, comparing the two compounds, which one is the more critical toxic agent for PM-induced hepatotoxicity is not well answered. Affecting different physiological and metabolic pathways such as oxidative phosphorylation and TCA cycle pathway, metabolic pathways, bile acid excretion pathway and genetic polymorphisms are among the mechanisms of hepatotoxicity of PM.
CONCLUSIONS: Deeper and effective high throughput experimental studies are still research hotspots to know the most responsible constituent and the mechanism of PM-induced hepatotoxicity.

BACKGROUND: Polygoni Multiflori Radix, the dried root of Polygonum multiflorum Thunb., and its processed products have been used as restoratives for centuries in China. However, the reports of Polygoni Multiflori Radix-induced liver injury (PMR-ILI) have received wide attention in recent years, and the components and mechanism of PMR-ILI are not completely clear yet. Our previous studies found that the PMR-ILI was related to the down-regulation of some drug metabolism enzymes (DME).
OBJECTIVE: To explore the effect of the inhibition of CYP3A4 or UGT1A1 on PMR-ILI, screen the relevant hepatotoxic components and unveil its mechanism.
METHODS: RT-qPCR was used to detect the effects of water extract of Polygoni Multiflori Radix (PMR) and its main components on the mRNA expression of CYP3A4 and UGT1A1 in human hepatic parenchyma cell line L02. High-performance liquid chromatography (HPLC) was employed to detect the content of major components in the PMR. And then, the stable CYP3A4 or UGT1A1 knockdown cells were generated using short hairpin RNAs (shRNA) in L02 and HepaRG cells. Hepatotoxic components were identified by cell viability assay when PMR and its four representative components, 2,3,5,4\'-tetrahydroxy stilbene glycoside (TSG), emodin (EM), emodin-8-O-β-D-glucoside (EG), and gallic acid (GA), acted on CYP3A4 or UGT1A1 knockdown cell lines. The PMR-ILI mechanism of oxidative stress injury and apoptosis in L02 and HepaRG cells were detected by flow cytometry. Finally, the network toxicology prediction analysis was employed to excavate the targets of its possible toxic components and the influence on the metabolic pathway.
RESULTS: PMR and EM significantly inhibited the mRNA expression of CYP3A4 and UGT1A1 in L02 cells, while TSG and GA activated the mRNA expression of CYP3A4 and UGT1A1, and EG activated CYP3A4 expression while inhibited UGT1A1 expression. The contents of TSG, EG, EM and GA were 34.93 mg/g, 1.39 mg/g, 0.43 mg/g and 0.44 mg/g, respectively. The CYP3A4 or UGT1A1 knockdown cells were successfully constructed in both L02 and HepaRG cells. Low expression of CYP3A4 or UGT1A1 increased PMR cytotoxicity remarkably. Same as PMR, the toxicity of EM and GA increased in shCYP3A4 and shUGT1A1 cells, which suggested EM and GA may be the main components of hepatotoxicity in PMR. Besides, EM not only inhibited the expression of metabolic enzymes but also reduced the cytotoxicity threshold. EM and GA affected the level of ROS, mitochondrial membrane potential, Ca2+ concentration, and dose-dependent induced hepatocyte apoptosis in L02 and HepaRG cells. The network toxicology analysis showed that PMR-ILI was related to drug metabolism-cytochrome P450, glutathione metabolism, and steroid hormone biosynthesis.
CONCLUSIONS: The inhibition of mRNA expression of CYP3A4 or UGT1A1 enhanced hepatotoxicity of PMR. EM and GA, especially EM, may be the main hepatotoxic components in PMR. The mechanism of PMR, EM and GA induced hepatotoxicity was proved to be related to elevated levels of ROS, mitochondrial membrane potential, Ca2+ concentration, and induction of apoptosis in liver cells.