Famous Traditional Chinese Medicine Prescription Research Articles

Dingxian pill alleviates hippocampal neuronal apoptosis in epileptic mice through TNF-α/TNFR1 signaling pathway inhibition

Ethnopharmacological relevanceDingxian Pill (DXP), a famous traditional Chinese medicine prescription, and has been widely proven to have positive therapeutic effects on “Xianzheng” (the name of epilepsy in ancient China). However, the anti-epileptic molecular mechanisms of DXP are not yet fully understood and remain to be further investigated. Aim of the studyTo elucidate the molecular mechanism of DXP's improvement in epileptic neuronal loss, damage and apoptosis by regulating TNF-α/TNFR1 signaling pathway. Materials and methodsSixty Kunming mice were randomly divided in 6 groups: control group (equal volume of normal saline), model group (180 mg kg−1 pilocarpine hydrochloride – used to establish the epilepsy animal model), carbamazepine group (30 mg kg−1), and low, medium, and high-dose Dingxian Pill groups (4.08, 8.16, and 16.32 g kg−1, respectively – oral administration once daily for 2 weeks). Successful establishment of the epileptic mouse model was monitored with electroencephalography. Pathological changes in hippocampal tissue were analyzed with hematoxylin-eosin staining. Hippocampal neuronal apoptosis was analyzed with TUNEL staining. TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein expression levels in hippocampal tissue were analyzed with real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot, respectively. Cleaved caspase-8 protein levels in hippocampal tissue were measured with immunohistochemistry and Western blot. ResultsCompared to control, the model group showed an increase in continuous epileptic discharge waves on EEG, a damaged hippocampal neuron morphological structure, increased hippocampal neuronal apoptosis, and significantly increased TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein levels, and increased caspase-8 cleavage (P < 0.05). Compared to the model group, the carbamazepine group as well as the low-, medium-, and high-dose Dingxian Pill groups showed decreased epileptic discharges on EEG, an obvious hippocampal neuron morphological structure restoration, varying degrees of attenuated hippocampal neuronal apoptosis, and significantly decreased TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein levels as well as decreased caspase-8 cleavage (P < 0.05). ConclusionsDingxian Pill exerts an anti-epileptic effect through inhibition of TNF-α/TNFR1 signaling pathway-mediated apoptosis in hippocampal neurons.

Applications and Mechanisms of Taohong Siwu Decoction in the Treatment of Ischemic Stroke

Ischemic stroke is a kind of sudden vascular disease of the brain brought on by ischemic hypoxic necrosis of the brain tissue which is becoming a significant global source of death and long-term impairment. Present common treatment options for cerebral ischemia including thrombolytic therapy, mechanical embolization, and intracranial arterioplasty can only benefit a few patients due to the narrow therapeutic time windows. Traditional Chinese Medicine (TCM) prescriptions possess the qualities of multi-ingredients, multi-targets, synergistic and multi-efficacy advantages as a means of treating ischemic stroke. Taohong Siwu Decoction (THSWD) is a famous TCM prescription that promotes blood flow, removes blood clots, adds nourishment, and tonifies blood. Clinical and laboratory research results show the significant advantages of THSWD in ischemic stroke treatment. This review summarizes THSWD’s natural materials and processing methods, pharmacological effects, and related mechanisms to treat ischemic stroke. Its mechanism of action focuses on improving hemorheology, anti-inflammatory response, reducing excitotoxicity, anti-calcium overload, anti-oxidative stress, etc. Moreover, we systematically summarize THSWD and its use in combination with other treatments. Finally, the research obstacles of THSWD in the treatment of ischemic stroke are summarized, and the application prospects in the medical field are prospected.

Comprehensive characterization of the chemical constituents of Lianhua Qingwen capsule by ultra high performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry

Lianhua Qingwen capsule is a famous traditional Chinese medicine (TCM) prescription that is widely used for the treatment of respiratory diseases in China. To facilitate in-depth and global characterization of the chemical constituents of Lianhua Qingwen capsule, a profiling method based on ultra-high performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) was applied in both positive and negative ion modes for the comprehensive characterization of the chemical profiles of Lianhua Qingwen capsule. A total of 596 compounds were identified or tentatively characterized, including 137 flavonoids, 46 phenylpropanoids, 43 phenylethanoid glycosides, 145 terpenoids, 83 organic acids and their derivatives, 15 quinones, 39 alkaloids, 32 alcohol glycosides and 56 other compounds. Thus, this results widely extended and enriched the chemical constituents of Lianhua Qingwen capsule, which will provide comprehensive and valuable information for its quality control and further pharmacological study, facilitate understanding the effective substance and pharmacodynamic material basis, thereby providing a solid foundation for further development of the Lianhuaqingwen capsule.

Huang-Lian-Jie-Du decoction drug-containing serum inhibits IL-1β secretion from D-glucose and PA induced BV2 cells via autophagy/NLRP3 signaling

Ethnopharmacological relevanceHuang-Lian-Jie-Du decoction (HLJDD), a famous traditional Chinese medicine prescription with heat-clearing and detoxifying effects, has been widely used to treat diabetes, dementia, stroke, and other diseases. However, the detailed mechanisms of HLJDD against type 2 diabetes associated cognitive dysfunction (DACD) through inhibiting interleukin-1β (IL-1β) mediated neuroinflammation remain to be further elucidated. Aim of the studyThe aim of this study was to investigate the effect and potential mechanism of HLJDD on IL-1β secretion in a DACD model of BV2 cells induced by D-glucose and palmitic acid (PA). Materials and methodsUltra-performance liquid chromatography-quadrupole/electrostatic field orbital well high-resolution mass spectrometry technology was used to analyze the compounds in HLJDD drug-containing serum. The cytotoxicity was detected by cell counting kit-8. Enzyme-linked immunosorbent assay was used to measure the secretion of IL-1β in BV2 cells. Reactive oxygen species, glutathione, superoxide dismutase, and malondialdehyde kits were used to detect the intracellular oxidative stress levels. The autophagy level was determined by autophagy staining kit and transmission electron microscope. The expression levels of autophagy-related 7 (Atg7), P62, LC3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3(NLRP3), Caspase1, and IL-1β were detected by real-time PCR, immunofluorescence, and western blotting. The Atg7siRNA was transfected into BV2 cells to produce autophagy inhibitory effect. Then the effect of HLJDD drug-containing serum on IL-1β secretion in D-glucose and PA induced BV2 cells and the potential mechanism of autophagy-NLRP3 inflammasome activation were further observed. ResultsEighty-eight compounds were preliminarily identified in HLJDD drug-containing serum, among which geniposide, baicalin, palmatine, berberine, wogonoside, wogonin, and geniposidic acid were identified as the main prototype components of HLJDD into the blood. In this study, the DACD model of BV2 cells induced by high concentrations of glucose and PA was successfully constructed. HLJDD drug-containing serum significantly reduced the secretion of IL-1β and the activity of NLRP3 inflammasome with improving the oxidative stress level. Interestingly, the enhanced autophagy level was also found. After transfection of Atg7siRNA into BV2 cells, the effect of HLJDD drug-containing serum on autophagy promotion was reversed, but the inhibitory effects on IL-1β secretion, NLRP3 inflammasome activation, and oxidative stress were reduced. ConclusionsThese results indicated that the inhibition of HLJDD drug-containing serum on the IL-1β secretion in D-glucose and PA induced BV2 cells was related to autophagy promotion, the decreased NLRP3 inflammasome activation, and the improved oxidative stress. Moreover, the improvement of HLJDD drug-containing serum on IL-1β secretion, NLRP3 inflammasome activation, and oxidative stress were all closely associated with Atg7 mediated autophagy promotion. Geniposide, baicalin, palmatine, berberine, wogonoside, wogonin, and geniposidic acid may be the potential active ingredients of HLJDD drug-containing serum.

The protective effects of Zhi-Gan-Cao-Tang against diabetic myocardial infarction injury and identification of its effective constituents.

Cardiovascular complications are highly prevalent in patients with diabetes. Zhi-Gan-Cao-Tang (ZGCT), a famous traditional Chinese medicine (TCM) prescription, can be used for the treatment of diabetes with cardiovascular disease complications. ZGCT is composed of nine Chinese herbs: the radix and rhizoma of Glycyrrhiza uralensis Fisch. (Gancao in Chinese, 12g), the radix of Rehmannia glutinosa Libosch. (Dihuang in Chinese, 50g), the radix and rhizoma of Panax ginseng C. A. Mey. (Renshen in Chinese, 6g), the radix of Ophiopogon japonicus (L. f.) Ker-Gawl. (Maidong in Chinese, 10g), the fructus of Ziziphus jujuba Mill. (Dazao in Chinese, 18g), the fructus of Cannabis sativa L. (Maren in Chinese, 10g), Donkey-hide gelatine (Ejiao in Chinese, 6g), the ramulus of Cinnamomum cassia Presl (Guizhi in Chinese, 9g), and the fresh rhizoma of Zingiber officinale Rosc. (Shengjiang in Chinese, 9g). Many of these Chinese herbs are also used in other systems of medicine (Japan, India, European, etc.). However, the effects and effective constituents of ZGCT against diabetic cardiovascular disease remain unclear. This study aimed to investigate the protective effect of ZGCT against diabetic myocardial infarction (DMI) injury in vivo and in vitro and to identify the effective constituents of ZGCT. The in vivo effect on DMI injury was evaluated in a DMI mouse model. The in vitro effect and effective constituent screening experiments were conducted in an H9c2 cardiomyocyte injury model induced by high glucose and hypoxia. It was found that ZGCT significantly reduced myocardial infarction size and serum lactate dehydrogenase (LDH) levels in DMI mice. Myocardial histopathological experiments showed that ZGCT alleviated the disordered arrangement and fracture of muscle fibers and cell disappearance and reduced inflammatory cell infiltration. Cellular experiments showed that ZGCT inhibited cardiomyocyte apoptosis by decreasing the expression of the proapoptotic factor Bax. In addition, it inhibited inflammatory reactions by suppressing the activation of the IκBα/NF-κB pathway and the expression of iNOS. Eight constituents from six Chinese herbs in the recipe of ZGCT were found to enhance the viability of injured cardiomyocytes, and six effective constituents played protective roles through anti-apoptotic and/or anti-inflammatory activities. In addition, one of the effective constituents, glycyrrhizic acid, was verified in vivo to have cardioprotective effect on DMI mice. The TCM prescription ZGCT protects against DMI by inhibiting cardiomyocyte apoptosis and reducing inflammatory reactions. Eight effective constituents of ZGCT were identified. This study provides a scientific basis for the clinical application of ZGCT and is valuable for quality marker research on this prescription.

Molecular mechanisms of Huanglian Jiedu decoction in treating Alzheimer's disease by regulating microbiome via network pharmacology and molecular docking analysis.

Huanglian Jiedu decoction (HLJDD) is a famous traditional Chinese medicine prescription, which is widely used in the treatment of Alzheimer's disease (AD). However, the interaction between bioactive substances in HLJDD and AD-related targets has not been well elucidated. A network pharmacology-based approach combined with molecular docking was performed to determine the bioactives, key targets, and potential pharmacological mechanism of HLJDD against AD, through the regulation of microbial flora. Bioactives and potential targets of HLJDD, as well as AD-related targets, were retrieved from Traditional Chinese Medicine Systems Pharmacology Analysis Database (TCMSP). Key bioactive components, potential targets, and signaling pathways were obtained through bioinformatics analysis, including protein-protein interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Subsequently, molecular docking was performed to predict the binding of active compounds with core targets. 102 bioactive ingredients of HLJDD and 76 HLJDD-AD-related targets were screened. Bioinformatics analysis revealed that kaempferol, wogonin, beta-sitosterol, baicalein, acacetin, isocorypalmine, (S)-canadine, (R)-canadine may be potential candidate agents. AKT1, TNF, TP53, VEGFA, FOS, PTGS2, MMP9 and CASP3 could become potential therapeutic targets. 15 important signaling pathways including the cancer pathway, VEGF signaling pathway, and NF-κB signaling pathway might play an important role in HLJDD against AD. Moreover, molecular docking analysis suggested that kaempferol, wogonin, beta-sitosterol, baicalein, acacetin, isocorypalmine, (S)-canadine, and (R)-canadine combined well with AKT1, TNF, TP53, VEGFA, FOS, PTGS2, MMP9, CASP3, respectively. Our results comprehensively illustrated the bioactives, potential targets, and possible molecular mechanisms of HLJDD against AD. HLJDD may regulate the microbiota flora homeostasis to treat AD through multiple targets and multiple pathways. It also provided a promising strategy for the use of traditional Chinese medicine in treating human diseases.

Development of a three-step-based novel strategy integrating DMPK with network pharmacology and bioactivity evaluation for the discovery of Q-markers of traditional Chinese medicine prescriptions: Danlou tablet as an example

BackgroundQuality marker (Q-marker) serves an important role in promoting the standardization of the quality of traditional Chinese medicine (TCM) prescriptions. However, discovering comprehensive and representative Q-markers from TCM prescriptions composed of multiple components remains difficult. PurposeA three-step-based novel strategy integrating drug metabolism and pharmacokinetics (DMPK) with network pharmacology and bioactivity evaluation was proposed to discover the Q-markers and applied to a research example of Danlou tablet (DLT), a famous TCM prescription with remarkable and reliable clinical effects for coronary heart disease (CHD). MethodsFirstly, the metabolic profile in vivo of DLT was systemically characterized, and the pharmacokinetic (PK) properties of PK markers were then investigated. Secondly, an integrated network of “PK markers – CHD targets – pathways – therapeutic effects” was established to screen out the crucial PK markers of DLT against CHD. Thirdly, the crucial PK markers that could exhibit strong myocardial protection activity in the H9c2 cardiomyocyte model were selected as the candidate Q-markers of DLT. According to the proportion of their Cmax value in vivo, the candidate Q-markers were configured into a composition; the bioactivity was then evaluated to confirm their synergistic effect and justify their usage as Q-markers. ResultsFirst of all, a total of 110 DLT-related xenobiotics (35 prototypes and 75 metabolites) were detected in bio-samples, and the pharmacokinetic properties of 13 PK markers of DLT were successfully characterized, revealing the quality transitivity and traceability from prescription to in vivo. Then, 6 crucial PK markers with three topological features (degree, betweenness, and closeness) greater than the average values in the pharmacology network were screened out as the key components of DLT against CHD. Furthermore, among these 6 crucial PK markers, 5 components (puerarin, alisol A, daidzein, paeoniflorin, and tanshinone IIA) with strong myocardial protection activity were chosen as the candidate Q-markers to constitute a new composition. The composition activated the expression of the PI3K/AKT pathway and exhibited strong myocardial protection activity, and the effective concentrations (nM level) of these components in the composition were significantly lower than their individually effective concentrations (μM level), indicating that there was a certain synergistic effect between them. Hence, the 5 components with multiple properties, including testability, quality transitivity and traceability from prescription to in vivo, effectiveness, and compatibility contribution, were defined as comprehensive and representative Q-markers of DLT. ConclusionThis study not only presented a novel idea for the revelation of comprehensive and representative Q-markers in quality control research of TCM prescriptions, but also identified the reasonable Q-markers of DLT for the first time to improve the quality control level of DLT.

Effect of Huanglian Decoction on the Intestinal Microbiome in Stress Ulcer (SU) Mice.

Background Stress ulcer (SU) is a serious gastrointestinal mucosal lesion under acute stress. Huanglian decoction is a famous traditional Chinese medicine prescription, which has been used to treat digestive system diseases for thousands of years. Many clinical cases have proved that Huanglian decoction has a good effect on SU. Some studies have shown that the intestinal bacteria will be changed accordingly when the gastrointestinal mucosa is damaged. However, there are few published reports on the effect of the intestinal microbiome with SU mice that were treated by Huanglian decoction. In this study, we investigated the effect of the fecal microbiome in mice with SU by the 16S rDNA sequencing technology. Methods Male KM mice were induced by cold-restraint stress except for the normal control group and then treated by Huanglian decoction (Group HD) and Esomeprazole magnesium solution (Group ES) separately for 7 days. 16S rDNA sequencing technology analysis was applied to evaluate the changes of bacterial flora in mice feces. And, histopathological methods and molecular biological detection methods were also performed. Results Huanglian decoction could help to repair the gastric mucosal injury and regulate the relative content of TNF-α and IL-1β. Moreover, Huanglian decoction could increase the relative abundance of intestinal probiotics in the intestine of mice with SU, especially in Verrucomicrobiae and Akkermansia. Conclusions Huanglian decoction might effectively promote the repair of gastrointestinal mucosal injury and regulate the number and species of intestinal bacteria to maintain the stability of gastrointestinal microecology.

Comprehensive Analysis of Fecal Microbiome and Metabolomics in Hepatic Fibrosis Rats Reveal Hepatoprotective Effects of Yinchen Wuling Powder From the Host-Microbial Metabolic Axis.

Hepatic fibrosis (HF) is a typical consequence in the development of multiple chronic liver diseases, which is intimately related to the composition and metabolic status of gut microbiota. A myriad of evidence has indicated that traditional Chinese medicine can treat HF by regulating gut microbiota. Yinchen Wuling powder (YCWLP) is a famous traditional Chinese medicine prescription, which has been used to relieve liver diseases for thousands of years. YCWLP has demonstrated protective function on HF, but its effect on the alterations of gut microbiota is still unclear, and its explicit therapeutic mechanism also needs to be further elucidated. In this study, 16S rRNA gene sequencing and fecal metabolomics analysis were combined to investigate the influence of YCWLP on gut microbiota in HF rats and the interactions between gut microbiota and host metabolism. The results showed that YCWLP treatment significantly improved the disorder of multiple organ indices, HF-related cytokines and plasma LPS induced by HF. Masson’s trichrome stainings also showed that YCWLP treatment could significantly alleviate the severity of HF in rats. Additionally, YCWLP could reverse the significant changes in the abundance of certain genera closely related to HF phenotype, including Barnesiella [Ruminococcus] and Christensenella. Meanwhile, YCWLP significantly increased the abundance of Bifidobacterium, Coprococcus and Anaerostipes, which are closely related to butyrate production. Metabolomics and Spearman’s correlation analysis showed that YCWLP could regulate the disorder of arginine biosynthesis, sphingolipid metabolism and alanine, aspartate and glutamate metabolism in HF rats, and these regulations were intimately related to Barnesiella, [Ruminococcus], Christensenella, Coprococcus and Anaerostipes. By explaining the biological significance of the above results, we concluded that YCWLP might ameliorate HF by regulating the imbalance of gut microbiota, increasing the abundance of butyrate-producing bacteria to reduce ammonia production, promote ammonia degradation, and regulate pro-inflammatory cytokines and immune function.

The Herbal Formula Granule Prescription Mahuang Decoction Ameliorated Chronic Kidney Disease Which Was Associated with Restoration of Dysbiosis of Intestinal Microbiota in Rats.

Chronic kidney disease (CKD) has become a global health issue, and there is increasing evidence showing the beneficial roles of traditional Chinese medicine (TCM) in CKD treatment. Here, we studied the renoprotective role of Mahuang decoction, a famous TCM prescription, in a rat CKD model induced with the combination of doxorubicin and adenine. Our data showed that intragastric administration of Mahuang decoction inhibited the loss of bodyweight and attenuated proteinuria, serum creatinine, and blood urea nitrogen in CKD rats. Kidney histological analysis revealed decreased tubulointerstitial injury and fibrosis in CKD rats treated with Mahuang decoction accompanied with suppressed expression of TGF-β1 and phosphorylated NF-κB/P65 (p-P65) as indicated by immunohistochemistry. ELISA analysis demonstrated reduced serum levels of proinflammatory cytokines TNFα and IL-6. Most importantly, intestinal microbiota analysis by 16s rRNA-seq showed that Mahuang decoction restored the impaired richness and diversity of intestinal microflora and recovered the disrupted microbial community through reducing the abundance of deleterious microbes and promoting the expansion of beneficial microbes in CKD rats. Collectively, our findings demonstrated that Mahuang decoction mitigated kidney functional and structural impairment in CKD rats which were associated with the restoration of dysbiosis of intestinal microbiota, implying its potential in clinical CKD treatment.

Investigation of the therapeutic effect of Yinchen Wuling Powder on CCl4-induced hepatic fibrosis in rats by 1H NMR and MS-based metabolomics analysis

Hepatic fibrosis (HF) is a typical consequence of various chronic liver diseases, and there is still no ideal drug for its treatment. Yinchen Wuling Powder (YCWLP), a famous traditional Chinese medicine prescription, is effective for the treatment of icteric hepatitis, hepatic fibrosis, non-alcoholic fatty liver disease and other liver diseases in clinical practices, however, the underlying mechanisms of YCWLP on HF is still unclear. In this study, 1H NMR and MS-based metabolomics analysis along with body weight change, serum liver function indexes, serum liver fibrosis index and histopathological observations of liver were applied to evaluate the therapeutic effect of YCWLP on hepatic fibrosis and the mechanism associated with this. The results of the pharmacodynamics study show that YCWLP has a significant therapeutic effect on hepatic fibrosis. As for the metabolomics research, 7 metabolites in the plasma samples, 28 in the urine samples and 6 in the liver samples were significantly altered due to the protective effect of YCWLP on CCl4-induced hepatic fibrosis. These endogenous metabolites are involved in amino acid metabolism, carbohydrate metabolism, glycerophospholipid metabolism and gut bacteria metabolism. These findings suggest that YCWLP could treat hepatic fibrosis by promoting urea circulation and reducing blood ammonia accumulation, improving carbohydrate metabolism and reducing oxidative stress, improving glycerophospholipid metabolism and protecting cell membrane, and regulating intestinal flora metabolism.

Uncovering the protective mechanism of Taohong Siwu decoction against diabetic retinopathy via HIF-1 signaling pathway based on network analysis and experimental validation

BackgroundDiabetic retinopathy (DR) is a common and serious microvascular complication of diabetes. Taohong Siwu decoction (THSWD), a famous traditional Chinese medicine (TCM) prescription, has been proved to have a good clinical effect on DR, whereas its molecular mechanism remains unclear. Our study aimed to uncover the core targets and signaling pathways of THSWD against DR.MethodsFirst, the active ingredients of THSWD were searched from Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database. Second, the targets of active ingredients were identified from ChemMapper and PharmMapper databases. Third, DR associated targets were searched from DisGeNET, DrugBank and Therapeutic Target Database (TTD). Subsequently, the common targets of active ingredients and DR were found and analyzed in STRING database. DAVID database and ClueGo plug-in software were used to carry out the gene ontology (GO) and KEGG enrichment analysis. The core signaling pathway network of “herb-ingredient-target” was constructed by the Cytoscape software. Finally, the key genes of THSWD against DR were validated by quantitative real-time PCR (qRT-PCR).ResultsA total of 2340 targets of 61 active ingredients in THSWD were obtained. Simultaneously, a total of 263 DR-associated targets were also obtained. Then, 67 common targets were found by overlapping them, and 23 core targets were identified from protein-protein interaction (PPI) network. Response to hypoxia was found as the top GO term of biological process, and HIF-1 signaling pathway was found as the top KEGG pathway. Among the key genes in HIF-1 pathway, the mRNA expression levels of VEGFA, SERPINE1 and NOS2 were significantly down-regulated by THSWD (P < 0.05), and NOS3 and HMOX1 were significantly up-regulated (P < 0.05).ConclusionTHSWD had a protective effect on DR via regulating HIF-1 signaling pathway and other important pathways. This study might provide a theoretical basis for the application of THSWD and the development of new drugs for the treatment of DR.

Holistic quality evaluation of Qingwen Baidu Decoction and its anti-inflammatory effects

Ethnopharmacological relevanceQingwen Baidu Decoction (QBD), a famous traditional Chinese medicine prescription with heat-clearing and detoxifying efficacies, is widely used in the treatment of inflammatory diseases. However, due to lack of holistic quality evaluation research, the further study on the detailed molecular mechanisms of action are still insufficient. Aim of the studyThis study aimed to evaluate the overall quality of QBD and to explore the anti-inflammatory effects and associated intracellular signaling pathways. Materials and methodsa comprehensive method of chemical fingerprint analysis and simultaneous multi-component quantification was firstly developed by high performance liquid chromatography with diode array detector (HPLC-DAD). Similarity analysis, principal component analysis and hierarchical cluster analysis with heatmap were also applied to screen out the markers components in QBD samples. Moreover, its anti-inflammatory effects and mechanisms were further investigated by survival analysis, hematoxylin-eosin staining (H&E), neutrophil observation, quantitative real-time PCR analysis (qRT-PCR), Western blotting and confocal microscopy. ResultsTwenty-one characteristic peaks from 11 herbs were chemically identified in the chromatographic fingerprint. Fifteen quantitative markers from 11 herbs, such as baicalin, wogonoside, geniposidic acid, oxypaeoniflora and so on, were screened out with the aid of chemometrics to further quantitatively assess the quality of QBD. The results of survival analysis, H&E and neutrophil observation in zebrafish inflammatory models consistently showed that QBD exerts potent anti-inflammatory effects in a dose-dependent manner. Additionally, QBD inhibited the activation of NF-κB and STAT3 signal pathways in LPS-induced zebrafish and RAW 264.7 macrophage cells. ConclusionCollectively, our investigations firstly described the chemical profile of QBD and its possible mechanism of anti-inflammation, which provides a preferred strategy for monitoring the overall quality of QBD and supports its clinical application in treating inflammation-related diseases.

Simultaneous determination of nineteen compounds of Dahuang zhechong pill in rat plasma by UHPLC-MS/MS and its application in a pharmacokinetic study

Dahuang zhechong pill (DHZCP) is a famous traditional Chinese medicine prescription, which is widely used in the treatment of liver diseases. However, due to the lack of a dynamic DHZCP profile, the in vivo pharmacokinetics of active ingredients within this medicine remains unknown. In this paper, a rapid, sensitive and reliable UHPLC-MS/MS method was used to determine the content of 19 characteristic constituents of DHZCP in rat plasma, including rhein, emodin, chrysophanol, physcion, aloeemodin, p-methoxyphenylacetic acid, hypoxanthine nucleoside, wogonin, wogonoside, baicalin, norwogonin, naringenin, nutmeg acid, paeoniflorin, verbascoside, rhodiola glucoside, forsythoside A, formononetin, and glycyrrhizic acid. An Agilent Extend-C18 column (2.1 mm × 100 mm, 1.8 μm) was used to separate the 19 characteristic constituents, with a mobile phrase of (A) 0.1% formic acid and (B) acetonitrile. The constituents were detected in negative ion mode with multiple reactions monitoring (MRM). The established UHPLC-MS/MS method had good linearity, with a coefficient of determination (r2) of >0.99. The daytime and intra-day precision were less than 12%, and the accuracy ranged from −9.56% to 7.82%. The stability, extraction recovery, and matrix effect met the requirements. The method was successfully applied to the pharmacokinetic study of these nineteen characteristic constituents after oral administration of DHZCP. UHPLC-MS/MS was used for the first time to study the pharmacokinetics of the characteristic chemical constituents in DHZCP, which provided reference and theoretical guidance for further clarification of its pharmacodynamic basis.

Simultaneous Quantitative Analysis of Multiple Biotransformation Products of Xian-Ling-Gu-Bao, a Traditional Chinese Medicine Prescription, with Rat Intestinal Microflora by Ultra-Performance Liquid Chromatography Tandem Triple Quadrupole Mass Spectrometry.

Xian-Ling-Gu-Bao (XLGB), a famous traditional Chinese medicine prescription consisted of six herbal medicines, was used for prevention and treatment of osteoporosis in China. As an oral formulation, the multiple components contained in XLGB were inevitably biotransformed by the intestinal microflora before absorption via the gastrointestinal tract. However, the dynamic profiles of biotransformation products of XLGB remain unknown. In this paper, a rapid and sensitive ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry method was developed for the simultaneous quantitative analysis of multiple biotransformation products of XLGB with rat intestinal microflora. For 10 selected quantitative compounds, all calibration curves revealed good linearity (r2>0.99) within the sampling ranges considered. The whole intra- and inter-day precisions (as relative standard deviation) of all analytes were <13.5%, and the accuracies (as relative error) were in the range from -11.3 to 11.2%. The lower limits of quantification were 20, 10, 5, 20, 2, 2, 2, 5, 2 and 2ng/mL for sweroside, timosaponin BII, epimedin C, asperosaponin VI, psoralen, isobavachin, icariside II, timosaponin AIII, isobavachalcone and icaritin, respectively. The matrix effects, extraction recoveries and stabilities were all satisfactory. Meanwhile, dynamic profiles of 21 additional biotransformation products were also monitored by their area-time curves. The analytical method was successfully applied to describe dynamic profiles of 31 biotransformation products of XLGB and the recipes with removal of a definite composed herbal medicine (Anemarrhenae Rhizoma or Rehmanniae Radix).

Characterization of chemical profile and quantification of representative components of DanLou tablet, a traditional Chinese medicine prescription, by UHPLC-Q/TOF-MS combined with UHPLC-TQ-MS

DanLou tablet (DLT), a famous traditional Chinese medicine prescription (TCMP) consisting of 10 herbal medicines, is extensively used for the treatment of angina pectoris and acute coronary syndrome in China. However, active chemical constituents responsible for the therapeutic effects still remain unclear, due to the fact that the complex composition in DLT have not been holistically clarified. Therefore, this study aimed to characterize the chemical profile and simultaneously quantify the representative components in DLT. First, 157 chemical constituents including flavonoids, triterpenoids, tanshinones, lactones, phenolic acids, paeoniflorins and the other types of components were detected, among which 39 were exactly identified by comparing their retention times and MS fragmentation behaviors with those of authentic standards by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q/TOF-MS). Moreover, 33 representative components were simultaneously quantified by ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-TQ-MS), which were selected based on following three principles: qualitative and quantitative markers in the Chinese Pharmacopeia (2015 edition), bioactive components possessing cardiovascular-related in vivo or in vitro activities and those derived from 10 consisted herbs in DLT with a diversity of representative structure types. The method was validated in terms of linearity, precision, repeatability and recovery and successfully applied for the quality evaluation of 20 batches of DLT samples. Further chemometric analysis indicated that danshensu and salvianolic acid B were the most significant quantitative markers for the content fluctuation of DLT. In summary, the chemical profiles of DLT were systematically characterized and a practical quantitative method combined with chemometrics was developed to evaluate the intrinsic quality of multiple DLT samples in this study. The present work would be helpful for guaranteeing the safety, efficacy, and controllability in clinical medication of DLT.

Protective effect of Jie-Geng-Tang against Staphylococcus aureus induced acute lung injury in mice and discovery of its effective constituents

Ethnopharmacological relevanceJie-Geng-Tang (JGT), a famous traditional Chinese medicine prescription, consists of Platycodonis Radix and Glycyrrhizae Radix et Rhizoma. According to traditional medicinal theory, JGT exerts various effects, including apocenosis, detoxifying, moisturizing the lung and relieving sore throat. It is often used to treat throat inflammation and lung diseases. Aim of the studyTo determine the protective effect of JGT on Staphylococcus aureus (S. aureus)-induced acute lung injury (ALI) in mice and to identify the compounds in the prescription that may be responsible for antibacterial activity. Materials and methodsThe protective effect of JGT was assessed using S. aureus-induced ALI mice (i.g., 2.7 g/kg/day). Bacterial burden, pathological morphology, cytokine levels of TNF-α, IL-1β, KC, and MIP-2 were evaluated in the lung and bronchoalveolar lavage fluid at 24 h post-infection, respectively. Twenty three compounds in the prescription were evaluated for their minimum inhibitory concentration (MIC) in vitro by means of microbroth dilution method against S. aureus. The antibacterial effects in vitro of licochalcone A and isoliquiritigenin were also investigated by transmission electron microscopy. In vivo antibacterial activities of licochalcone A and isoliquiritigenin were evaluated by survival rates, bacterial burden, and pathological morphology of lung tissues on S. aureus-induced ALI in mice (i.p., 160 mg/kg/day). ResultsPretreatment with JGT significantly improved the pathological morphology of lung tissues on S. aureus-induced ALI in mice, accompanied with the reduced bacterial burden in the lungs and inhibited expression of inflammatory cytokine levels at 24 h post-infection. Five compounds, namely licochalcone A, licoisoflavone B, glyasperin A, isoliquiritigenin, and licochalcone B from Jie-Geng-Tang displayed good antibacterial activities against S. aureus (MIC < 128 μg/mL). Furthermore, applications of licochalcone A and isoliquiritigenin resulted in the increased survival rates, reduced bacterial burden in the lungs, and improved pathological morphology of lung tissues in S. aureus infected mice. ConclusionThe study demonstrated that Jie-Geng-Tang presented protective role of acute lung injury, which supported its traditional use for the treatment of lung diseases. Licochalcone A, isoliquiritigenin, licoisoflavone B, glyasperin A, and licochalcone B might contribute to the antibacterial activity of JGT on S. aureus-induced acute lung injury. The anti-S. aureus activity of licoisoflavone B, glyasperin A, and licochalcone B in vitro, as well as the anti-S. aureus activity of licochalcone A in vivo, were first reported in this study.

In vivo metabolic profiles of Bu-Zhong-Yi-Qi-Tang, a famous traditional Chinese medicine prescription, in rats by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry

Bu-Zhong-Yi-Qi-Tang (BZYQT), a famous traditional Chinese medicine prescription (TCMP), has been extensively used for conditioning sub-health status and diseases caused by spleen-qi deficiency in China for over 700 years. BZYQT is prevalent not only in China, but also in Japan and South Korea for the clinical treatment of chronic diseases, such as fatigue, tuberculosis and loss of appetite after surgery. However, due to a lack of research on the holistic metabolism of BZYQT, the in vivo bioactive components of BZYQT remain unclear, hindering further study of its in vivo mechanism of action and quality control. In the present study, a four-step integrated strategy based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS) was established to systematically screen the in vivo xenobiotics of BZYQT. Ultimately, a total of 162 xenobiotics (59 prototypes and 103 metabolites) were identified or tentatively characterized, including 48 in plasma, 147 in urine and 58 in feces, while the in vivo metabolic profile of atractylenolide III (a major component of BZYQT) was elucidated for the first time. The xenobiotics of BZYQT mainly included flavonoids from Astragali Radix, Glycyrrhizae Radix et Rhizoma and Citrus reticulatae Pericarpium; lactones from Angelicae Sinensis Radix and Atractylodis Macrocephalae Rhizoma; and triterpenoid saponins from Cimicifugae Rhizoma. After oral administration, BZYQT-related components underwent diverse metabolic pathways. Among them, flavonoids mainly underwent glucuronidation, sulfation and demethylation, while lactones mainly underwent hydroxylation and acetylcysteine conjugation, and deglycosylation was the major metabolic reaction of saponins. Our investigation gives a comprehensive analysis of the metabolic characteristics of BZYQT and will provide an important basis for further studying the pharmacokinetics of BZYQT to explore its in vivo disposal features and discover its in vivo bioactive components.

1H NMR-based metabolomics revealed the protective effects of Guilingji on the testicular dysfunction of aging rats

Ethnopharmacological relevanceGuilingji (GLJ), a famous and classical traditional Chinese medicine (TCM) prescription, has been used to extend the lifespan and improve the life qualities of the elderly for hundreds of years in China.Aim of the study: We aimed to explore the protective effects of GLJ on the testicular dysfunction of aging rats, as well as the regulating effects of GLJ on the metabolic disturbance and metabolite changes in natural aging rats. Materials and methodsForty 23-month-old rats were divided randomly into four groups, including the old control group and three groups of GLJ treatment at 37.5, 75, and 150 mg/kg doses, respectively. Additionally, 10 four-month rats were included as the youth control group. Testicular dysfunction was first evaluated by measuring the changes in the wet weights of the testicles, concentration of serum testosterone (T), and morphologic changes of the testis. Subsequently, an 1H NMR-based metabolomics approach coupled with multivariate analysis, including partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) was applied to monitor the metabolite changes. ResultsCompared with the old control group, the wet weights of the testicles and T concentration were significantly increased, while the morphologic abnormality of testicular tissues was improved by a 4-week treatment course with GLJ. Furthermore, compared with the old control group, the urinary levels of alanine, pantothenate, phenylalanine, β-hydroxybutyrate and pyruvate were significantly decreased after a 4-week treatment course with GLJ. Additionally, we found that amino acid metabolism and pyruvate metabolism were significantly involved in the regulatory effect of GLJ. ConclusionsThe current findings provided, for the first time, sound evidence of the protective effects of GLJ on testicular dysfunction from both biochemical and metabolomics perspectives. The mechanisms of GLJ could be related to regulating amino acid metabolism and pyruvate metabolism. The current study lays an important foundation for further research and for the broad clinical application of GLJ.

Brain metabonomics study of the antidepressant-like effect of Xiaoyaosan on the CUMS-depression rats by 1H NMR analysis

Ethnopharmacological relevanceXiaoyaosan (XYS), a famous TCM prescription with a long history of clinical use for relieving a wide variety of depression symptoms, consists of Radix Bupleuri (Bupleurum chinense DC.), Radix Angelicae Sinensis (Angelica sinensis (Oliv.) Diels), Radix PaeoniaeAlba (Paeonia lactiflora Pall.), Rhizoma Atractylodis Macrocepha lae (Atractylodes macrocephala Koidz.), Poria (Poria cocos (Schw.)Wolf), Radix Glycyrrhizae (Glycyrrhiza uralensis Fisch.), Herba Menthae Haplocalycis (Mentha haplocalyx Briq.), and Rhizoma Zin-giberis Recens (Zingiber officinale Rosc.). Aim of the studyWe aimed to characterize the diversity and variation of two kinds metabolites of brain, i.e. aqueous and lipophilic metabolites, gaining comprehensive insights into the metabolic processes of depression-like behavior, and to reveal the mechanisms of antidepressant-like effects of XYS. Materials and methodsWe first established a CUMS (Chronic Unpredictable Mild Stress)-induced depression-like behavior model. We then extracted both aqueous and lipophilic metabolites of rat brains by a two-phase extraction method, which were subsequently characterized by two differential sequences of 1H nuclear magnetic resonance (NMR). Multivariate analysis including Principal Components Analysis (PCA) and Orthogonal Partial Least Squares-Discriminate Analysis (OPLS-DA) was applied. ResultsMetabolic profiling by PCA indicated that XYS significantly reversed the metabolic perturbation caused by CUMS. OPLS-DA showed a total of 15 metabolites including 6 lipophilic and 9 aqueous metabolites was screened as potential biomarkers involved in CUMS-induced depression-like behavior. On top of this, five pathways including (1)D-glutamine and D-glutamate metabolism, (2) valine, leucine and isoleucine biosynthesis, (3) alanine, aspartate and glutamate metabolism, (4) taurine and hypotaurine metabolism and (5) arginine and proline metabolism were recognized as the most influenced pathways associated with the process of CUMS-induced depression-like behavior. Notably, XYS significantly reversed abnormality of 5 aqueous and 4 lipophilic metabolites to normal, suggesting that XYS synergistically mediated abnormalities of multiple pathways (1), (3), (4) and (5). ConclusionsIt is the first report to investigate the antidepressant-like effects and underlying mechanisms of XYS from the perspective of brain metabolites. In a broad sense, this study brings novel and valuable insights to evaluate the efficacy of traditional Chinese medicine (TCM), to interpret mechanisms, and to provide the theoretical basis for further research on therapeutic mechanisms in clinical practice.