Multi-omics and chemical profiling approaches to understand the material foundation and pharmacological mechanism of sophorae tonkinensis radix et rhizome-induced liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sophorae tonkinensis Radix et Rhizoma (STR) is an extensively applied traditional Chinese medicine (TCM) in southwest China. However, its clinical application is relatively limited due to its hepatotoxicity effects. AIM OF THE STUDY: To understand the material foundation and liver injury mechanism of STR. MATERIALS AND METHODS: Chemical compositions in STR and its prototypes in mice were profiled by ultra-performance liquid chromatography coupled quadrupole-time of flight mass spectrometry (UPLC-Q/TOF MS). STR-induced liver injury (SILI) was comprehensively evaluated by STR-treated mice mode. The histopathologic and biochemical analyses were performed to evaluate liver injury levels. Subsequently, network pharmacology and multi-omics were used to analyze the potential mechanism of SILI in vivo. And the target genes were further verified by Western blot. RESULTS: A total of 152 compounds were identified or tentatively characterized in STR, including 29 alkaloids, 21 organic acids, 75 flavonoids, 1 quinone, and 26 other types. Among them, 19 components were presented in STR-medicated serum. The histopathologic and biochemical analysis revealed that hepatic injury occurred after 4 weeks of intragastric administration of STR. Network pharmacology analysis revealed that IL6, TNF, STAT3, etc. were the main core targets, and the bile secretion might play a key role in SILI. The metabolic pathways such as taurine and hypotaurine metabolism, purine metabolism, and vitamin B6 metabolism were identified in the STR exposed groups. Among them, taurine, hypotaurine, hypoxanthine, pyridoxal, and 4-pyridoxate were selected based on their high impact value and potential biological function in the process of liver injury post STR treatment. CONCLUSIONS: The mechanism and material foundation of SILI were revealed and profiled by a multi-omics strategy combined with network pharmacology and chemical profiling. Meanwhile, new insights were taken into understand the pathological mechanism of SILI.

Extraction, rapid preparation and neuroprotective effect of texasin, a main active constituent from Caragana jubata (Pall.) Poir.

Searching for new anti-ischemic stroke (anti-IS) drugs has always been a hot topic in the pharmaceutical industry. Natural products are an important source of discovering anti-IS drugs. The aim of the present study is to extract, rapidly prepare and explore the neuroprotective effect of texasin, a main active constituent from Caragana jubata (Pall.) Poir., which is a kind of Tibetan medicine with a clear anti-IS effect. The results showed that 95% ethanol was the optimal extraction solvent. A three-step rapid preparation method for texasin was successfully established, with a purity of 99.2%. Texasin at the concentration of 25-100 µM had no effect on the viability of normal cultured PC12 cells; 12.5 and 25 µM texasin could enhance the viability of PC12 cells damaged by oxygen and glucose deprivation/reoxygenation (OGD/R), and their effects are comparable to the positive drug edaravone at the concentration of 50 µM. Compared with the normal group, the expression of Bcl-2 protein in OGD/R-injured PC12 cells was downregulated (p < 0.01), and that of PERK, eIF2α, ATF4, CHOP, Bax and Cleaved caspase-3 proteins were upregulated (p < 0.01, p < 0.001). Compared with the OGD/R group, 25 µM texasin could upregulate the expression of Bcl-2 protein (p < 0.01), and downregulate that of PERK, eIF2α, ATF4, CHOP, Bax and Cleaved caspase-3 proteins (p < 0.01, p < 0.001). The 7-OH and 1-O of texasin formed H-bonds with residues Cys891 of the hinge ß-strand of PERK, which is crucial for kinase inhibitors. The above results suggest that the method established in the present study achieved rapid preparation of high-purity texasin. Texasin might inhibit neuronal apoptosis via the regulation of endoplasmic reticulum stress PERK/eIF2α/ATF4/CHOP signalling pathway to exert a protective effect on OGD/R-injured PC12 cells. Aiding by molecular docking, texasin was assumed to be a potential PERK inhibitor.

SCH 644343 alleviates ischemic stroke-induced neuroinflammation by promoting microglial polarization via the IL-4/SREBP-1 signaling pathway.

Ischemic stroke (IS), a kind of acute cerebrovascular disease, is one of the most common diseases, and it endangers the lives and health of elderly individuals. Inflammation is a key factor leading to stroke, making it a potential therapeutic target. Previous studies have found that neuroinflammation is closely associated with microglial polarization. Due to the various side effects of current drugs used to treat neuroinflammation, it is important to explore alternative drugs with anti-inflammatory activity for neuroinflammation treatment. In the present study, we investigated the effect of SCH 644343 (SCH), a natural compound, on neuroinflammation induced by IS and explored the mechanism. We found that SCH meliorated the phenotypes of IS in vivo, which was correlated with the increased percentage of infiltrated M2 macrophages in brain after stroke. SCH exerted a significant effect against oxygen-glucose deprivation/reoxygenation (OGD/R) in BV2 cells in vitro by inhibiting M1 microglial polarization and promoting M2 microglial polarization. Furthermore, suppression of SREBP-1 expression by pretreatment with the SREBP-1 inhibitor 25-HC attenuated the effect of SCH on IS in vitro. Taken together, SCH exerts anti-IS effect by promoting microglial polarization via the IL-4/SREBP-1 signaling pathway.

A new biphenanthrene with cytotoxic activity from Liparis nervosa (Thunb.) Lindl.

2,7,2'-Trihydroxy-3,4,4'7'-tetramethoxy-1,1'-biphenanthrene (1), a previously undescribed biphenanthrene, and five known phenanthrenes, i.e. 2,5-dihydroxy-4-methoxy-9,10-dihydroxyphenanthrene (2), 2,4-dihydroxy -7-methoxy-9,10-dihydroxyphenanthrene (3), 7-hydroxy-2-methoxy-phenanthrene-1,4-dione (4), 7-hydroxy-2-methoxy-9,10-dihydro-phenanthrene-1,4-dione (5), and 4,4',7,7'-tetrahydroxy-2,2'-dimethoxy-9,9',10,10'-tetrahydro-1,1'-biphenanthrene (6) were isolated from the whole plant (stems, leaves, roots and fruits) of Liparis nervosa (Thunb.) Lindl., which is a medicinal plant of the genus Liparis in the Orchidaceae family. The structures of isolates were identified using spectroscopic methods, including NMR and mass spectrometry. Additionally, the cytotoxic potency of all the isolates against human lung cancer A549 cell line was evaluated by an MTT assay. All the isolated compounds showed cytotoxic activities with IC50 values in the range of 10.20 ± 0.81 to 42.41 ± 2.34 µM. The obtained data highlight the importance of L. nervosa as a source of natural lead compounds for cancer therapy.

Determining the chemical profile of Caragana jubata (Pall.) Poir. by UPLC-QTOF-MS analysis and evaluating its anti-ischemic stroke effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Caragana jubata, belonging to the Leguminosae family, is a shrubby medicinal plant distributed in high-altitude areas of China. The red heartwood of C. jubata is the original source of 'zuomuxing', a Tibetan medicine that promotes blood circulation and removes blood stasis to treat different diseases associated with the blood. AIM OF THE STUDY: To date, research on the chemical constituents of C. jubata remains very limited. The anti-ischemic stroke (anti-IS) effects of this plant have not been studied. The aim of the present study was to analyze the chemical profile of C. jubata, establish various anti-IS models to comprehensively evaluate the anti-IS effects of C. jubata, and explore the mechanism of action. MATERIALS AND METHODS: Ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was chosen to analyze the chemical profile. A middle cerebral artery occlusion reperfusion (MCAO/R) model, zebrafish cerebral thrombosis model, and oxygen-glucose deprivation/reperfusion (OGD/R) model in PC12/BV2 cells were used to thoroughly evaluate the anti-IS effects of C. jubata. Additionally, western blotting and immunofluorescence staining were used to detect the mechanism of action. RESULTS: Fifty-three compounds were identified from a 95% ethanol extract of C. jubata (ECJ) by UPLC-QTOF-MS analysis. 17 and 7 compounds were identified from C. jubata and the genus Caragana for the first time. ECJ was found to attenuate infarct size and reduce brain edema and neurological scores in MCAO/R rats. ECJ notably reduced the zebrafish cerebral thrombosis incidence in a dose-dependent manner compared with that in the model group. Surprisingly, compared to the positive control drug aspirin, 50 µg/ml ECJ exhibited a better therapeutic effect than aspirin at 30 µg/ml. Additionally, ECJ significantly increased the viability of PC12/BV2 cells injured by OGD/R. Moreover, ECJ inhibited the protein expression of M1 markers (TNF-α, iNOS, and IL-1ß) and increased that of M2 markers (Arg-1 and CD206) in OGD/R-injured BV2 cells. ECJ significantly decreased the immunofluorescence intensity of CD16 and increased that of CD206. CONCLUSIONS: The results from UPLC-QTOF-MS analysis showed that ECJ was rich in flavonoids. The results from pharmacological experiments verified the anti-IS effects of C. jubata in vivo and in vitro for the first time. In addition, ECJ could regulate the polarization of microglia. The present study highlights the medicinal value of C. jubata, thus providing a theoretical basis for the further development of new drugs from C. jubata to treat IS.

Rapid screening for acetylcholinesterase inhibitors in Selaginella doederleinii Hieron by using functionalized magnetic Fe3O4 nanoparticles.

Insufficient acetylcholine (ACh) can cause cognitive and memory dysfunction, clinically known as, Alzheimer's disease (AD). Acetylcholinesterase (AChE) can hydrolyze ACh into acetic acid and inactivate choline. Therefore, inhibiting the activity of AChE would help to improve the effectiveness of AD treatment. Currently, the methods for rapid screening of AChE inhibitors are limited. This study reports the application of AChE-immobilized magnetic nanoparticles as a drug screening tool to screen AChE inhibitors for natural products. First, AChE was immobilized on a surface of amino-modified magnetic nanoparticles using covalent binding and the AChE concentration, and the pH as well as time was optimized to obtain the maximum enzyme immobilization yield (61.4 µg/mg), and the kinetic model indicated that AChE-immobilized magnetic nanoparticles and the substrate had the high affinity and specificity. Then, a ligand fishing experiment was carried out using a mixed model of tacrine (an inhibitor of AChE) and caffeic acid (a non-inhibitor of AChE) to verify the specificity of the immobilized AChE, and the conditions for ligand fishing were further optimized. Finally, the optimized immobilized AChE was combined with UPLC-MS to screen for AChE inhibitors in Selaginella doederleinii Hieron extracts. Four compounds were confirmed to be potent AChE inhibitors. Among the four compounds, amentoflavone had a stronger AChE inhibitory effect than tacrine (positive control) with an IC50 of 0.73 ± 0.009 µmol/L. The results showed that AChE-functionalized magnetic nanoparticles can be used in the discovery of target drugs from complex matrices.

Spectrum-Effect Relationship Analysis of Bioactive Compounds in Zanthoxylum nitidum (Roxb.) DC. by Ultra-High Performance Liquid Chromatography Mass Spectrometry Coupled With Comprehensive Filtering Approaches.

Zanthoxylum nitidum (Roxb.) DC. (ZN), with strong effects of anti-inflammation and antioxidant activities is treated as a core herb in traditional Chinese medicine (TCM) preparation for treating stomachache, toothache, and rheumatoid arthritis. However, the active ingredients of ZN are not fully clarified due to its chemical complexity. In the present study, a double spectrum-effect analysis strategy was developed and applied to explore the bioactive components in herbs, and ZN was used as an example. Here, the chemical components in ZN were rapidly and comprehensively profiled based on the mass defect filtering-based structure classification (MDFSC) and diagnostic fragment-ion-based extension approaches. Furthermore, the fingerprints of 20 batches of ZN samples were analyzed by high-performance liquid chromatography, and the anti-inflammatory and antioxidant activities of the 20 batches of ZN samples were studied. Finally, the partial least squares regression (PLSR), gray relational analysis models, and Spearman's rank correlation coefficient (SRCC) were applied to discover the bioactive compounds in ZN. As a result, a total of 48 compounds were identified or tentatively characterized in ZN, including 35 alkaloids, seven coumarins, three phenolic acids, two flavonoids, and one lignan. The results achieved by three prediction models indicated that peaks 4, 12, and 17 were the potential anti-inflammatory compounds in ZN, whereas peaks 3, 5, 7, 12, and 13 were involved in the antioxidant activity. Among them, peaks 4, 5, 7, and 12 were identified as nitidine, chelerythrine, hesperidin, and oxynitidine by comparison with the standards and other references. The data in the current study achieved by double spectrum-effect analysis strategy had great importance to improve the quality standardization of ZN, and the method might be an efficiency tool for the discovery of active components in a complex system, such as TCMs.

An image-based fingerprint-efficacy screening strategy for uncovering active compounds with interactive effects in Yindan Xinnaotong soft capsule.

BACKGROUND: Yindan Xinnaotong soft capsule (YDXNT) is a clinically effective herbal prescription used for the treatment of cardiovascular and cerebrovascular diseases. Since Chinese medicines (CMs) exert their effects via a "multiple-components and multiple-targets" mode, discovery of the active compounds with interactive effects may contribute to reveal their mechanisms of action. PURPOSE: This study aimed to establish an image-based fingerprint-efficacy screening strategy to identify active compounds with interaction effects from CM prescription, using YDXNT to inhibit microglia-mediated neuroinflammation as an instance. METHODS: A multi-component random content-oriented chemical library of YDXNT was constructed by uniform design, and their chemical fingerprint was profiled by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) methods. Then the neuroinflammation activities of chemical library members of YDXNT were determined by image-based dual phenotypic quantification. Subsequently, fingerprint-efficacy correlation and random forest analysis were applied to predict the potentially active compounds with interactive effects. Finally, the interactive effects among the active compounds were confirmed by quantitative polymerase chain reaction (qPCR) and apoptosis analysis, and network pharmacology was applied to explore the possible mechanisms. RESULTS: Image-based fingerprint-efficacy correlation analysis revealed that six tanshinones (TNs) and four flavonoids (FAs) were potential anti-neuroinflammatory compounds. The inter-family of TNs and FAs possessed obvious interactive effects (combination index ≤ 0.825). Moreover, the combination of scutellarein and tanshinone I (2:1, w/w) was discovered as the possible interactive combinatorial components, which, comparing with individual scutellarein or tanshinone I, shown more powerful effects on anti-inflammatory and anti-apoptotic effects in lipopolysaccharide (LPS)-induced BV2 cells. Network pharmacology showed that the active compounds might suppress microglia-mediated neuroinflammation via multiple targets in the T cell receptor, Jak-STAT, and Toll-like receptor signaling pathways. CONCLUSION: The image-based fingerprint-efficacy strategy simplifies the screening process of efficacious component combinations in CMs for complex diseases, which also offers a promising approach to explore the integrative therapeutic mechanisms of CMs.

A validated UHPLC-MS/MS method for pharmacokinetic and brain distribution studies of twenty constituents in rat after oral administration of Jia-Wei-Qi-Fu-Yin.

A rapid ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QqQ MS/MS) approach with high sensitivity and selectivity was developed for the quantification of twenty compounds, including 9 saponins, 8 flavonoids, 2 oligosaccharide esters and 1 phenolic acid, in rat plasma and brain, which was administrated intragastrically with Jia-Wei-Qi-Fu-Yin (JWQFY), Mass spectrometric detection was operated under multiple reaction monitoring (MRM) mode. All calibration curves possessed good linearity with correlation coefficients ( r2) higher than 0.9916 in their respective linear ranges. For intra- and inter-day precision, all the relative standard deviations (RSDs) at different levels were less than 14.68 %. Based on the UHPLC-QqQ MS/MS quantitative results, pharmacokinetic study and brain distribution of multiple components in JWQFY was then successfully performed. As a result, constituents with discrepancy structures showed diverse pharmacokinetic and distribution characteristics. For instance, ferulic acid (phenolic acid), 3, 6'-disinapoyl sucrose and tenuifoliside A (oligosaccharide esters) showed short Tmax (< 10 min), whereas the Tmax of ginsenosides Rb1, Rb2 and Rc (ppd-type terpenoid saponins) were much longer (> 4 h). Besides, ferulic acid, epimedin C, icariin, glycyrrhizin, ginsenoside Rb1 and ginsenoside Rg1 were considered as the potential effective ingredients of JWQFY because of their relatively high exposure to blood and brain. Our study would provide relevant information for discovery of pharmacodynamic ingredients, as well as further action mechanisms investigations of JWQFY.

Comparatively Evaluating the Role of Herb Pairs Containing Angelicae Sinensis Radix in Xin-Sheng-Hua Granule by Withdrawal Analysis.

The present study aims to investigate the roles of herb pairs containing Angelicae Sinensis Radix (Danggui) in Xin-Sheng-Hua Granule (XSHG) on hemolytic and aplastic anemia (HAA) mice. HAA model mice were induced by acetyl phenylhydrazine and cyclophosphamide; then the samples of XSHG and its decomposed recipes (DY, DC, DT, DH, DJ, and DZ) were orally administrated to these mice. Indicators of peripheral blood routine, organ index, and ATPase activities were tested. Moreover, the main effective components in these samples were also analyzed by UHPLC-TQ-MS/MS. Clear separation between the control and model groups from score plot of principal component analysis (PCA) was easily seen, indicating that HAA model was successfully conducted. Afterwards, relative distance calculation method between dose groups and control group from PCA score plot was adopted to evaluate the integrated effects of hematinic function of different samples. And the orders of hematinic effects were as follows: XHSG > DJ > DT > DZ > DH > DC > DY. Further analysis of these samples by UHPLC-TQ-MS/MS revealed that XSHG underwent complicated changes when herb pairs containing Danggui were excluded from XSHG, respectively. Compared with XSHG, the vast majority of active compounds in sample DY (formula minus herb pair Danggui-Yimucao) decreased significantly, which could partly explain why herb pair Danggui-Yimucao made great contribution to XSHG. These findings showed that withdrawal analysis method is a valuable tool to analyze the impacts of herb pairs containing Danggui on XSHG, which could lay foundation to reveal the compatibility rules of this formula.

Comprehensive chemical profiling of Jia-Wei-Qi-Fu-Yin and its network pharmacology-based analysis on Alzheimer's disease.

Jia-Wei-Qi-Fu-Yin (JWQFY) is a newly developed anti-Alzheimer's disease (AD) prescription modified from a classical traditional Chinese medicine formula, Qi-Fu-Yin (QFY). However, a systematic understanding of its chemical constituents and molecular mechanisms is still elusive. To address this problem, comprehensive chemical profiling followed by network pharmacology-based analysis of JWQFY was performed. Firstly, a total of 136 compounds were characterized by high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF MS), 17 of them were specifically identified in JWQFY comparing with QFY. Seventy compounds were further quantified via a validated HPLC coupled with triple quadrupole tandem mass spectrometry (QQQ MS) method. Then the protein targets of the seventy compounds were gathered from public databases for network construction. As a result, fifty-seven compounds were filtered, which interacted with 655 targets. Thirty-four of them were mapped into the KEGG pathway of AD, indicating JWQFY might exert anti-AD effects by anti-inflammation, neuronal apoptosis intervening, Aß production inhibition and phosphorylating tau protein moderating. Furthermore, in the compound-target-AD network, a list of hub compounds and hub targets was identified based on their topological features, including the degree, node betweenness and closeness. Four of the hub compounds were specifically originated from JWQFY, supporting the modification rationality of this formula. This study provided a scientific basis for understanding the bioactive compounds and the multi-target mechanism of JWQFY.

Rapid profiling of alkaloid analogues in Sinomenii Caulis by an integrated characterization strategy and quantitative analysis.

Alkaloids, the principal constituents in the caulis of Sinomenium acutum, have gained an increasing interest over the past decades since they are widely employed as a clinical treatment for rheumatoid arthritis. In the present study, an integrated characterization strategy by combining mass defect filtering-based structure classification (MDFSC) and diagnostic fragment-ion-based extension (DFIBE) was firstly proposed for rapid profiling of alkaloids in Sinomenii Caulis (SC) via ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). The rectangular MDFSC window could more accurately screen the target alkaloids of different types, and the DFIBE could facilitate the acquisition of characteristic fragment ions for structural elucidation of alkaloids. High-performance liquid chromatography (HPLC) fingerprints with principal component analysis (PCA) and hierarchical clustering analysis (HCA) was established for identifying the chemical markers and simultaneous determination of sinomenine, magnoflorine, menisperine, stepharanine and ehydrodiscretine. A total of 91 alkaloids, including 82 targeted ones (26 morphinans, 22 aporphines, 20 protoberberines and 14 benzylisoquinolines) were unambiguously identified or tentatively characterized in SC, and 14 of them were reported for the first time. Sinomenine and magnoflorine could be selected as chemical markers to evaluate the quality of SC from different localities. In conclusion, the proposed method provided a potential approach for chemical profiling and holistic quality control of herbal medicines.

Comprehensive chemical profiling of Yindan Xinnaotong soft capsule and its neuroprotective activity evaluation in vitro.

Discovering effective combinational components (ECCs) and quality control markers of TCMs is still facing challenges because the holistic healing system comprises hundreds of compounds. Here, taking Yindan Xinnaotong soft capsule (YDXNT), a TCMs preparation composed by 8 herbs, as a case, a strategy that integrated multiple chromatographic analysis and bioactivity assay was proposed for potential ECCs of neuroprotection discovery. Firstly, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF MS) and gas chromatography-mass spectrometry (GC-MS) were applied for comprehensive profiling of the chemical constituents in YDXNT. Given the fact that the complex matrix interference makes it more difficult to identify potentially active compounds, we proposed a structure-diagnostic ions-oriented strategy to remove interference ions from the raw UHPLC-MS data. The proposed strategy consisted of different filtering methods, including diagnostic fragment ions filtering (DFIF), mass defect filtering (MDF) and neutral loss (NL). Using this strategy, a total of 124 compounds were rapidly identified. Among them, 62 non-volatile and 5 volatile constituents in 30 batches of YDXNT were quantified by UHPLC tandem triple quadrupole mass spectrometry (QQQ-MS) and GC-MS methods, respectively. In order to facilitate the quality control of YDXNT, candidate ECCs were selected based on the threshold setting of absolute -contents, and their neuroprotective effects were examined. Finally, a combination of 16 compounds, accounts for 2.80% (w/w) of original YDXNT, was identified as its potential ECCs, which could be considered for the improvement of quality standardization of YDXNT.

Comprehensive chemical profiling of monascus-fermented rice product and screening of lipid-lowering compounds other than monacolins.

ETHNOPHARMACOLOGICAL RELEVANCE: Monascus-fermented rice product (MFRP) has been regarded as a dietary supplement and traditional medicine with circulation-promoting effects in China and other countries for centuries. AIM OF THE STUDY: This study was carried out to profile the chemical components in MFRP, and provide available information for elucidating the potential lipid-lowering compounds other than monacolins. MATERIALS AND METHODS: High-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) and gas chromatography coupled with mass spectrometry (GC-MS) methods were applied to comprehensive analysis of chemical components in MFRP. Potential small molecules were identified by comparing with reference standards, or tentatively characterized by comparing their retention time and high-resolution mass spectral data with previous literature. The lipid-lowering properties of ten major non-monacolin compounds were evaluated in cholesterol-fed zebrafish larvae. And one with optimum lipid-lowering activity was subsequently evaluated in high fat diet-fed C57BL/6 J mice, with the dyslipidemia and ectopic lipid deposition being investigated. RESULTS: A total of 99 compounds were characterized in MFRP, including 38 monacolins, 5 decalins, 6 isoflavones, 13 pigments, 8 azaphilonoids, 11 amino acids, 4 nucleosides, 9 lipid acids, 4 phytosterols and glycerol. The preliminary screening showed that ergosterol remarkably reduced cholesterol levels in zebrafish larvae. Moreover, ergosterol delayed body weight gain and decreased circulating total cholesterol, triglyceride, low density lipoprotein cholesterol levels in high fat diet-fed mice. Ectopic lipid accumulation was also ameliorated in the liver and heart of obese mice. CONCLUSION: Global analysis of chemical components and screening of lipid-lowering non-monacolin compounds in MFRP have improved our understanding of its therapeutic material basis.

Integrated Metabolomics and Network Pharmacology Approach to Explain Possible Action Mechanisms of Xin-Sheng-Hua Granule for Treating Anemia.

As a well-known traditional Chinese medicine (TCM) prescription, Xin-Sheng-Hua Granule (XSHG) has been applied in China for more than 30 years to treat postpartum diseases, especially anemia. However, underlying therapeutic mechanisms of XSHG for anemia were still unclear. In this study, plasma metabolomics profiling with UHPLC-QTOF/MS and multivariate data method was firstly analyzed to discover the potential regulation mechanisms of XSHG on anemia rats induced by bleeding from the orbit. Afterward, the compound-target-pathway network of XSHG was constructed by the use of network pharmacology, thus anemia-relevant signaling pathways were dissected. Finally, the crucial targets in the shared pathways of metabolomics and network pharmacology were experimentally validated by ELISA and Western Blot analysis. The results showed that XSHG could exert excellent effects on anemia probably through regulating coenzyme A biosynthesis, sphingolipids metabolism and HIF-1α pathways, which was reflected by the increased levels of EPOR, F2, COASY, as well as the reduced protein expression of HIF-1α, SPHK1, and S1PR1. Our work successfully explained the polypharmcological mechanisms underlying the efficiency of XSHG on treating anemia, and meanwhile, it probed into the potential treatment strategies for anemia from TCM prescription.

Simultaneous determination of kaempferol, quercetin, mangiferin, gallic acid, p-hydroxybenzoic acid and chlorpheniramine maleate in rat plasma after oral administration of Mang-Guo-Zhi-Ke tablets by UHPLC-MS/MS and its application to pharmacokinetics.

Mang-Guo-Zhi-Ke tablets (MGZKTs) is an effective Chinese patent medicine. It contains mango leaf extract as the main raw material and the antihistamine drug, chlorpheniramine maleate is included in the formulation. However, its pharmacokinetic effect is rarely reported. A highly sensitive, reliable and rapid high-throughput method using ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was used to simultaneously determine kaempferol, quercetin, mangiferin, p-hydroxybenzoic acid, gallic acid and chlorpheniramine maleate in rat plasma after oral administration of MGZKTs. The method was successfully developed and fully validated to investigate the pharmacokinetics of MGZKTs. Chloramphenicol and clarithromycin were used as internal standards (IS). A practicable protein precipitation procedure with methanol was adopted for sample preparation. The samples were separated on an Acquity UHPLC Syncronis C18 column (100 × 2.1 mm, 1.7 µm) using 0.1% formic acid-acetonitrile as the mobile phase. The flow rate was set at 0.4 mL/min. The obtained calibration curves were linear in the concentration range of ~1-1000 ng/mL for plasma (r > 0.99). Method validation results met the criteria reported in the US Food and Drug Administration guidelines. Quercetin, p-hydroxybenzoic acid and kaempferol were absorbed rapidly and reached the peak concentration between 0.16 and 0.25 h. This validated that the UHPLC-MS/MS method was successfully applied to study the pharmacokinetic parameters of the six compounds in rat plasma after oral administration of MGZKTs. This evidence will be useful for the clinical rational use of Mang-Guo-Zhi-Ke tablets.

Pharmacokinetic Comparison of Seven Major Bio-Active Components in Normal and Blood Stasis Rats after Oral Administration of Herb Pair Danggui-Honghua by UPLC-TQ/MS.

The compatibility between Danggui (Angelicae Sinensis Radix) and Honghua (Carthami Flos) is a known herb pair, which could activate blood circulation and dissipate blood stasis effects. In this paper, we quantified seven main bio-active components (hydroxysafflor yellow A, caffeic acid, p-coumaric acid, kaempferol-3-O-rutinoside, ferulic acid, 3-n-butylphthalide, and ligustilide) in plasma samples in vivo by UPLC-TQ/MS method and investigatedwhether the pharmacokinetic (PK) behaviors of the seven components could be altered in blood stasis rats after oral administration of the Gui-Hong extracts. It was found that the Cmax and AUC0-t of these components in blood stasis rats had increasing tendency compared with normal rats. Most components in model and normal rats had significant difference in some pharmacokinetic parameters, which indicated that the metabolism enzymes and transporters involved in the metabolism and disposition of these bio-active componentsmay bealtered in blood stasis rats. This study was the first report about the pharmacokinetic investigation between normal and blood stasis rats after oral administrationof Gui-Hong extracts, and these results are important and valuable for better clinical applications of Gui-Hong herb pair and relatedTCM formulae.

Comparatively evaluating the pharmacokinetic of fifteen constituents in normal and blood deficiency rats after oral administration of Xin-Sheng-Hua Granule by UPLC-MS/MS.

Xin-Sheng-Hua Granule (XSHG), a famous traditional Chinese medicine prescription, are clinically applied for the treatment of postpartum disease through nourishing blood and promoting blood circulation. In this investigation, a multi-constituents (trigonelline, stachydrine hydrochloride, hydroxysafflor yellow A, chlorogenic acid, amygdalin, leonurine, liquiritin, ferulic acid, senkyunolide I, senkyunolide H, glycyrrhizic acid, senkyunolide A, ligustilide, butylidenephthalide and glycyrrhetinic acid) pharmacokinetic study of XSHG was conducted for the first time. These fifteen constituents in both normal and blood deficiency rat plasma were monitored by using the established and validated ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry (UPLC-TQ-MS/MS) method. The samples were prepared through removing protein from plasma with three volumes of methanol. Sufficient separation of target constituents and internal standards (chloramphenicol and clarithromycin) was obtained on a Thermo Scientific Hypersil GOLD column (100mm×3mm, 1.9µm) within a 20min gradient elution (0.1% formic acid aqueous - acetonitrile). Multiple reaction monitoring (MRM) mode was applied to monitor target analytes in both positive and negative electrospray ionization. For the fifteen selected target analytes, this method was fully validated with excellent linearity (r≥0.9925), satisfactory intra- and inter-day precisions (RSD≤11.87%), as well as good accuracies (RE, between -12.84 and 11.69). And the stabilities, matrix effects and extraction recoveries of the rat plasma samples were also within acceptable limits (RSD<15%). Compared to normal group, the pharmacokinetics of major active constituents (except liquiritin and glycyrrhetinic acid) had significant differences (P<0.05) in the model rats, indicated that several metabolite enzymes activities could be altered at disease condition.

Comparative analysis of the main bioactive components of Xin-Sheng-Hua granule and its single herbs by ultrahigh performance liquid chromatography with tandem mass spectrometry.

Xin-Sheng-Hua granule, a representative formula for postpartum hemorrhage, has been used clinically to treat postpartum diseases. Its main bioactive components comprise aromatic acids, phthalides, alkaloids, flavonoids, and gingerols among others. To investigate the changes in main bioactive constituents in its seven single herbs before and after compatibility, a rapid, simple, and sensitive method was developed for comparative analysis of 27 main bioactive components by using ultrahigh-performance liquid chromatography with triple quadrupole electrospray tandem mass spectrometry for the first time. The sufficient separation of 27 target constituents was achieved on a Thermo Scientific Hypersil GOLD column (100 mm × 3 mm, 1.9 µm) within 20 min under the optimized chromatographic conditions. Compared with the theoretical content, the observed content of each analyte showed remarkable differences in Xin-Sheng-Hua granule except thymine, p-coumaric acid, senkyunolide I, senkyunolide H, and ligustilide; the total contents of 27 components increased significantly, and the content variation degrees for the different components were gingerols > flavonoids > aromatic acids > alkaloids > phthalides. The results could provide a good reference for the quality control of Xin-Sheng-Hua granule and might be helpful to interpret the drug interactions based on variation of bioactive components in formulae.

Metabolic profile of anhydrosafflor yellow B in rats by ultra-fast liquid chromatography/quadrupole time-of-flight mass spectrometry.

Anhydrosafflor yellow B (AHSYB) is one of the major active water-soluble pigments from Carthamus tinctorius, which has been found to inhibit ADP-induced platelet aggregation and possess significant antioxidant activity. However, the metabolic fate of AHSYB in vivo remains unknown. In order to explore whether AHSYB is extensively metabolized, the metabolites of AHSYB in plasma, urine, bile, and feces samples after intravenous administration to the rats were investigated by ultra-fast liquid chromatography/quadrupole time-of-flight mass spectrometry (UFLC/Qq-TOF-MS/MS) combined with Metabolitepilot™ software. In total, AHSYB and 22 metabolites including both phase I and phase II metabolism processes were found and tentatively identified from the bio-samples. The metabolic pathways were involved in oxidation, reduction, hydroxylation, methylation, dimethylation, O-acetylation, hydrolyzation, sulfation, glucuronidaton, glutathionation and combination with glucose. The results showed that the renal and biliary routes play an important role in the clearance and excretion of AHSYB as well as hepatocyte metabolism. All of these results were reported for the first time and would contribute to a further understanding of the in vivo intermediated processes and metabolic mechanism of AHSYB and its analogs.