A comprehensive investigation on the chemical changes of traditional Chinese medicine with classic processing technology: Polygonum multiflorum under nine cycles of steaming and sunning as a case study.

The processing of traditional Chinese medicine (TCM) plays an important role in the clinical application, which usually has the function of "increasing efficiency and reducing toxicity". Polygonum multiflorum (PM) has been reported to induce hepatotoxicity, while it is believed that the toxicity is reduced after processing. Studies have shown that the hepatotoxicity of PM is closely related to the changes in chemical components before and after processing. However, there is no comprehensive investigation on the chemical changes of PM during the processing progress. In this research, we established a comprehensive method to profile both small molecule compounds and polysaccharides from raw and different processed PM samples. In detail, an online two-dimensional liquid chromatography coupled with quadrupole-orbitrap mass spectrometry (2D-LC/Q-Orbitrap MS) was utilized to investigate the small molecules, and a total of 150 compounds were characterized successfully. After multivariate statistical analysis, 49 differential compounds between raw and processed products were screened out. Furthermore, an accurate and comprehensive method for quantification of differential compounds in PM samples was established based on ultra-high performance liquid chromatography/Q-Orbitrap-MS (UHPLC/Q-Orbitrap-MS) within 16 min. In addition, the changes of polysaccharides in different PM samples were analyzed, and it was found that the addition of black beans and steaming times would affect the content and composition of polysaccharides in PM significantly. Our work provided a reference basis for revealing the scientific connotation of the processing technology and increasing the quality control and safety of PM.

Potential Involvement of Organic Anion Transporters in Drug Interactions with Shuganning Injection, a Traditional Chinese Patent Medicine.

Traditional Chinese medicine injections have been widely used in China for the treatment of various diseases. Transporter-mediated drug-drug interactions are a major contributor to adverse drug reactions. However, the research on transporter-mediated Traditional Chinese medicine injection-drug interactions is limited. Shuganning injection is a widely used Traditional Chinese medicine injection for treating various liver diseases. In this study, we investigated the inhibitory effect of Shuganning injection and its four main ingredients (baicalin, geniposide, chlorogenic acid, and oroxylin A) on 9 drug transporters. Shuganning injection strongly inhibited organic anion transporter 1 and organic anion transporter 3 with IC50 values < 0.1% (v/v), and moderately inhibited organic anion transporter 2, organic anion transporting-polypeptide 1B1, and organic anion transporting-polypeptide 1B3 with IC50 values < 1.0%. Baicalin, the most abundant bioactive ingredient in the Shuganning injection, was identified as both an inhibitor and substrate of organic anion transporter 1, organic anion transporter 3, and organic anion transporting-polypeptide 1B3. Oroxylin A had the potential to act as both an inhibitor and substrate of organic anion transporting-polypeptide 1B1 and organic anion transporting-polypeptide 1B3. In contrast, geniposide and chlorogenic acid had no significant inhibitory effect on drug transporters. Notably, Shuganning injection markedly altered the pharmacokinetics of furosemide and atorvastatin in rats. Using Shuganning injection as an example, our findings support the implementation of transporter-mediated Traditional Chinese medicine injection-drug interactions in the development of Traditional Chinese medicine injection standards.

Cytochrome P450-mediated herb-drug interaction (HDI) of Polygonum multiflorum Thunb. based on pharmacokinetic studies and in vitro inhibition assays.

BACKGROUND: Polygonum multiflorum Thunb. (PM) is well known both in China and other countries of the world for its tonic properties, however, it has lost its former glory due to liver toxicity incidents in recent years. PURPOSE: The purpose of this study is to determine whether the occurrence of herb-drug interaction (HDI) caused by PM is associated with cytochrome P450 (CYP450) based on pharmacokinetic studies and in vitro inhibition assays. The objective was to provide a reference for the rational and safe use of drugs in clinical practice. METHODS: In this study, raw PM (R), together with its two processed products which included PM by Chinese Pharmacopoeia (M) and PM by "nine cycles of steaming and sunning (NCSS)" ("9"), were prepared as the main research objects. A method based on fluorescence technology was used to evaluate the inhibition levels of raw and processed PMs, as well as corresponding characteristic compounds on seven recombinant human cytochrome P450s (rhCYP450s). The pharmacokinetics of sulindac (a representative of commonly used nonsteroidal anti-inflammatory drugs) and psoralen (a major compound of Psoralea in combination with PM) in rat plasma were studied when combined with raw and different processed products of PM. RESULTS: The inhibitory level order of the three extracts on major different subtypes of CYP450 (CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, and CYP3A4) was: R > M > "9". However, the inhibition level of R and "9" is higher than that of M on CYP2C9. Further studies showed that trans-THSG and emodin could selectively inhibit CYP3A4 and CYP1A2, respectively. Epicatechin gallate mainly inhibited CYP3A4 and CYP1A2, followed by CYP2C8 and CYP2C9. Genistein mainly inhibited CYP3A4, followed by CYP2C9 and CYP2C8. CYP3A4 and CYP2C9 were also inhibited by daidzein. The inhibitory effects of all the PM extracts were associated with their characteristic compounds. The results of HDI showed that R increased sulindac exposure to rat blood, and R and M increased psoralen exposure to rat blood, which were consistent with corresponding metabolic enzymes. Overall, the in vitro and in vivo results indicated that PM, especially R, would be at high risk to cause toxicity and drug interactions via CYP450 inhibition. CONCLUSION: This study not only elucidates the scientific connotation of "efficiency enhancement and toxicity reduction" of PM by NCSS from the perspective of metabolic inhibition but also contributes to HDI prediction and appropriate clinical medication of PM.

A strategy based on gene sequencing and molecular docking for analysis and prediction of bioactive peptides in Shuxuetong injection.

Peptides are a class of protein fragments with relatively high biological activity and intense specificity, which play crucial role in the treatment of Shuxuetong injection (SXT). However, the extraordinary complexity of Chinese medicinal formulates and the lack of systematic identification methods are primary challenges for study of pharmacodynamic peptides. In addition, infinitesimal peptides contents further hinder the identification and structural characterization of polypeptide by traditional means. In this paper, we described a strategy that LC-MS combined with molecular docking to systematically illustrate the peptide components of SXT. The key to this research was used of gene sequencing to establish a SXT protein database to further achieve the separation and enrichment of chemical methods. Moreover, the ADRA2A, PAR4 and DRD3 were precisely docked with the identified peptides. The result indicated that 12 compounds had stable binding ability and were speculated to be the latent bioactive monomers for the treatment of stroke. Additionally, 12 peptides were verified by cell-based experiment. The results showed that only YLKTT could indeed protect astrocytes from oxygen glucose deprivation/reoxygenation (OGD/R). The YLKTT showed higher activity than the others in vitro. It might be a completely new compound that has never been reported before, providing the basis for further research and a new paradigm for stroke.

Biomarkers and Mechanism Analysis for Polygoni Multiflori Radix Preparata-Induced Liver Injury by UHPLC-Q-TOF-MS-Based Metabolomics.

BACKGROUND: Polygonum Multiflorum Radix Preparata (PMP), prepared from Polygonum multiflorum Thunb. (PM), is traditionally valued for its liver and kidney-tonifying effects. However, the previous studies showed that PMP was hepatotoxic, which limited its clinical use. Unfortunately, the potential hepatotoxic ingredients and the molecular mechanism are still uncertain. OBJECTIVE: The aim of this study was to find out potential biomarkers of hepatotoxicity using metabolomics profile. MATERIALS AND METHODS: 60% ethanol extract of PMP (PMPE) was prepared. Subsequently, an untargeted metabolomics technology in combination with ROC curve analysis method was applied to investigate the alteration of plasma metabolites in rats after oral administration of PMPE (40 g/kg/d) for 28 days. RESULTS: Compared to the control group, the significant difference in metabolic profiling was observed in the PMPE-induced liver injury group, and sixteen highly specific biomarkers were identified. These metabolites were mainly enriched into bile acids, lipids, and energy metabolisms, indicating that PMPE-induced liver injury could be related to cholestasis and dysregulated lipid metabolism. CONCLUSIONS: This study is contributed to understand the potential pathogenesis of PMP-induced liver injury. The metabonomic method may be a valuable tool for the clinical diagnosis of PMP-induced liver injury.

An effective strategy for distinguishing the processing degree of Polygonum multiflorum based on the analysis of substance and taste by LC-MS, ICP-OES and electronic tongue.

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).

Multiple component-pharmacokinetic studies on 10 bioactive constituents of Peiyuan Tongnao capsule using parallel reaction monitoring mode.

Peiyuan Tongnao capsule (PTC) plays an important role in clinical application due to its excellent curative efficacy in the treatment of ischemic stroke and chronic cerebral circulation insufficiency. To standardize and rationalize the clinical application of PTC, a rapid and sensitive method based on ultra-high performance liquid chromatography/quadrupole-Orbitrap mass spectrometry with parallel reaction monitoring (PRM) mode was developed and validated for the pharmacokinetic (PK) study. Ten bioactive compounds (aucubin, salidroside, echinacoside, paeoniflorin, verbascoside, liquiritin, 2,3,5,4'-tetrahydroxy stilbene-2-O-ß-d-glucoside, coumarin, glycyrrhizic acid, and emodin) were simultaneously determined in rat plasma. All calibration curves exhibited good linearity (r2 > 0.99). The lower limits of quantification were 0.082-13.291 ng mL-1 . The intra- and inter-day precision was 0.54-12.36%, whereas the intra- and inter-day accuracy ranged from 100.45 to 114.00%. The mean extraction recoveries were 81.77-117.66%, and the average matrix effects (MEs) were 86.23-109.96%. The high extraction recoveries and acceptable MEs indicated that the pretreatment method was feasible. And the stability was acceptable under various storage conditions and processing procedures. The validated method was successfully applied to the multiple components-PK studies, which lay the foundation for further pharmacological and clinical research of PTC and may provide a reference for other traditional Chinese medicines.

The composition differences between small black beans and big black beans from different habitats and its effects on the processing of Polygonum multiflorum.

INTRODUCTION: The roots of Polygonum multiflorum (PM) serve as a classical traditional Chinese medicine (TCM), which has multiple biological activities. However, many cases of hepatotoxicity in PM have been reported in recent years. Processing PM with black beans decoction is one of the typical processing methods to reduce the hepatotoxicity of PM since ancient times. OBJECTIVES: To find potential effective constituents, as well as the optimal variety and origin of black beans for the processing of PM. METHODS: Based on ultrahigh-performance liquid chromatography Q-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) analysis, we measured the contents of the two potential toxic compounds (emodin-8-O-glucoside and torachrysone-O-hexose) in raw PM (R-PM), PM processed with big black beans (B-PM) and PM processed with small black beans (S-PM). The flow cytometry method analysed the effects of different processed products of PM on apoptosis of L02 cells in different drug concentration. Proton nuclear magnetic resonance (1 H-NMR) and UHPLC-Q-Orbitrap-MS together with multivariate statistical analysis were used to systematically analyse the different components between small black beans (Small-BB) and big black beans (Big-BB) from 30 different habitats. RESULTS: The toxicity was ranked from small to large: S-PM < B-PM < R-PM. Processing PM with black beans could significantly decrease the apoptosis rate of L02 cells, especially when the drug concentration is 80 µg/mL. Besides, we find five differential compounds (α-arabinose, α-galactose, proline, isomer of daidzein and isomer of genistein) may be potential active ingredients. In terms of the black beans collected from 30 producing areas, we find that Small-BB from Weifang in Shandong province was optimum to processing PM, followed by Shangqiu in Henan province, Jilin and Liaoning province. CONCLUSION: The ingredients that affect the processing of PM may be attributed to α-arabinose, α-galactose, proline, isomer of daidzein and isomer of genistein in black beans. When the drug concentration is higher, the effect of reducing the hepatotoxicity of PM is better. Besides, Small-BB was more effective than Big-BB for reducing the toxicity of PM, especially Small-BB from Weifang in Shandong, Shangqiu in Henan province and northeast China.

Rapid identification of chemical composition and metabolites of Pingxiao Capsule in vivo using molecular networking and untargeted data-dependent tandem mass spectrometry.

Pingxiao capsule (PXC) is a herbal medicine used for adjuvant therapy in breast cancer. However, the constituents and absorbed components of the formula and their related metabolites have not been elucidated to date. PXC is a typical traditional Chinese medicine formula consisting of Strychnos nux-vomica L., Curcuma wenyujin Y. H., Agrimonia pilosa Ledeb., Toxicodendron vernicifluum, Trogopterus dung, alumen, potassium nitrate (saltpeter) and Citrus aurantium L. In this study, a ultra-high performance liquid chromatography system equipped with high resolution Q-Orbitrap mass spectrometry (MS) and comparative Global Natural Product Social molecular networking together with the Compound Discoverer software were used to identify metabolites of PXC in vitro and in vivo. Based on untargeted data-dependent MS2 and data-mining techniques, 89 peaks of alkaloids, flavonoids, organic acid and phenolic compounds were identified in a PXC 70% methanol extract. Furthermore, 15 absorbed prototype compounds and their metabolites were rapidly confirmed in rat blood. Glucuronidation, oxidation, methylation and hydroxylation were the main metabolic pathways. We fully clarified the chemical constituents of PXC and provided a scientific and efficient strategy for rapid discovery and identification of prototypes and their metabolites in rat plasma using high-resolution MS aided by Global Natural Product Social and Compound Discoverer software.

Pharmacokinetic studies unveiled the drug-drug interaction between trans-2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucopyranoside and emodin that may contribute to the idiosyncratic hepatotoxicity of Polygoni Multiflori Radix.

Polygoni Multiflori Radix (PMR) has been a reputable tonifying traditional Chinese medicine for a long history. However, clinical side effects regarding its idiosyncratic hepatotoxicity are occasionally reported. The containing anthraquinones, particularly emodin, could cause liver injury in both in vitro and in vivo experiments. It is well-known that some compounds could influence other compounds' pharmacokinetic parameters significantly. In this work, the influence of trans-2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucopyranoside (TSG) on the pharmacokinetic behavior of emodin in rats was evaluated by an ultra-high performance liquid chromatography/triple quadrupole mass spectrometry (UHPLC/MS-MS) approach. Pharmacokinetic parameters of emodin, PMR extract, and TSG-free PMR extract (prepared by a component "knock-out" strategy with TSG eliminated), in rats after one-day and seven-day administration were determined and compared. We found that, after seven-day administration of the whole PMR extract (rather than TSG-free extract), emodin in rats was accumulated. And accordingly, the exposure of emodin in rats pre-treated with single TSG for seven days could be significantly enhanced. The results indicate that TSG was able to accelerate the exposure and metabolism of emodin. The effect of TSG on the metabolic activities of cytochrome P450 enzymes was further assessed by an LC-MS cocktail method. The accelerated exposure and metabolism of emodin could result from the up-regulation activity of CYP450s, in particular CYP1A2 isozyme. The findings obtained in this work firstly unveiled DDI between TSG and emodin in the administration of PMR, thus may provide a basis for unveiling the underlying mechanism of PMR-induced liver injury.