[Quality control of Tianwang Buxin Pills based on UPLC fingerprint and multi-component quantification].

Tianwang Buxin Pills have demonstrated therapeutic effects in clinical practice, whereas there is a serious lack of comprehensive quality control to ensure the safety and effectiveness of clinical medication. In this study, ultra-performance liquid chromatography(UPLC) was employed to establish the fingerprint and the method for simultaneously determining the content of seven components of Tianwang Buxin Pills. Furthermore, chemometrics was employed to identify the key factors for the stable quality, which provided a reference for the comprehensive quality control and evaluation of this preparation. There were 25 common peaks in the UPLC fingerprints of 15 batches of Tianwang Buxin Pills, from which thirteen compounds were identified. A quantitation method was established for seven pharmacological components(α-linolenic acid, salvianolic acid B, glycyrrhetinic acid, schisandrin A, ß-asarone, 3,6'-disinapoylsucrose, and ligustilide). The principal component analysis(PCA) and partial least square discriminate analysis(PLS-DA) were performed to determine the key pharmacological components for controlling the quality stability of Tianwang Buxin Pills, which included 3,6'-disinapoylsucrose, α-linolenic acid, and ß-asarone. The established fingerprint and multi-component content determination method have strong specificity, stability, and reliability. In addition, 3,6'-disinapoylsucrose, α-linolenic acid, and ß-asarone are the key pharmacological components that ensure the quality stability between batches and can be used to comprehensively control the quality of Tianwang Buxin Pills. The findings provide a scientific basis for the quality evaluation and standard establishment of Tianwang Buxin Pills.

Neuroprotective effects of the salidroside derivative SHPL-49 via the BDNF/TrkB/Gap43 pathway in rats with cerebral ischemia.

Ischemic stroke is a common intravascular disease and one of the leading causes of death and disability. The salidroside derivative SHPL-49, which we previously synthesized, significantly attenuates cerebral ischemic injury in a rat model of permanent middle cerebral artery occlusion. To explore the neuroprotective mechanism of SHPL-49, the effects of SHPL-49 on the expression levels of neurotrophic factors in neurons and microglia and the polarization of microglia were investigated in the present study. SHPL-49 activated the brain-derived neurotrophic factor (BDNF) pathway, decreased the number of degenerated neurons, and accelerated neurogenesis in rats with cerebral ischemia. In addition, SHPL-49 promoted the polarization of microglia toward the M2 phenotype to alleviate neuroinflammation. In BV2 cells, SHPL-49 upregulated CD206 mRNA and protein levels and inhibited CD86 mRNA and protein levels. SHPL-49 also increased neurotrophic factor secretion in BV2 cells, which indirectly promoted the survival of primary neurons after oxygen-glucose deprivation (OGD). Proteomics analysis revealed that SHPL-49 promoted growth-associated protein 43 (Gap43) expression. SHPL-49 enhanced synaptic plasticity and increased Gap43 protein levels via activation of the BDNF pathway in the OGD primary neuron model. These results indicate that SHPL-49 prevents cerebral ischemic injury by activating neurotrophic factor pathways and altering microglial polarization. Thus, SHPL-49 is a potential neuroprotective agent.

Multi-pathway neuroprotective effects of a novel salidroside derivative SHPL-49 against acute cerebral ischemic injury.

SHPL-49 ((2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-(4-(4-methoxyphenyl) butoxy) tetrahydro-2H-pyran-3,4,5-triol) is a novel glycoside derivative obtained from structural modification of salidroside, which is isolated from the medicinal plant Rhodiola rosea L. SHPL-49 was administered to rats with permanent middle cerebral artery occlusion (pMCAO) for 5 days, and it was found that SHPL-49 could alleviate the cerebral infarct volume and reduce the neurological deficit score. Moreover, the effective time window of SHPL-49 in the pMCAO model was from 0.5 to 8 h after embolization. In addition, the result of immunohistochemistry showed that SHPL-49 could increase the number of neurons in the brain tissue and reduce the occurrence of apoptosis. Morris water maze and Rota-rod experiments showed that SHPL-49 could improve neurological deficits, repair neurocognitive and motor dysfunction, and enhance learning and memory ability in the pMCAO model after 14 days of SHPL-49 treatment. Further in vitro experiments showed that SHPL-49 significantly reduced the calcium overload of PC-12 cells and the production of reactive oxygen species (ROS) induced by oxygen and glucose deprivation (OGD), and increased the levels of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), decreased the production of malondialdehyde (MDA). Furthermore, SHPL-49 could reduce cell apoptosis by increasing protein expression ratio of anti-apoptotic factor Bcl-2 to pro-apoptotic factor Bax in vitro. SHPL-49 also regulated the expression of Bcl-2 and Bax in ischemic brain tissue, and even inhibited the caspase cascade of pro-apoptotic proteins Cleaved-caspase 9 and Cleaved-caspase 3. Taken together, SHPL-49 exhibited neuroprotective effects against cerebral ischemic injury through multiple pathways, such as alleviating calcium overload, reducing oxidative stress damage, and inhibiting apoptosis.

[Effect of origin species, body weight, and gender on quality of Bufonis Venenum].

Based on standard sampling for Bufonis Venenum, this study analyzed the effect of the origin, and body weight and gender of Bufo bufo gargarizans on the quality of Bufonis Venenum. To be specific, mass spectrometry(MS) and the content determination methods in Chinese Pharmacopoeia(2020) were adopted. First, MS was performed on 76 Bufonis Venenum samples collected from 40 cities/counties in 17 provinces/autonomous regions which were derived from B. bufo gargarizans and B. melanostictus. Based on content determination, the body weight and gender of B. bufo gargarizans, which influenced the quality of Bufonis Venenum, were evaluated. Multivariate statistical analysis suggested huge difference in the material basis of the medicinal material derived from B. bufo gargarizans and B. melanostictus, and 9 differential compounds were identified. The content of components specified in Chinese Pharmacopoeia was higher in the medicinal material derived from B. bufo gargarizans than in the medicinal material derived from B. melanostictus. The content of the components specified in Chinese Pharmacopoeia was low in Bufonis Venenum derived from heavy B. bufo gargarizans, and higher in the Bufonis Venenum produced by male B. bufo gargarizans than in that produced by female B. bufo gargarizans irrespective of time and geographic location. In summary, this study provide new ideas and reference for the quality control of Bufonis Venenum, collection and processing of Bufonis Venenum, artificial breeding of B. bufo gargarizans, and biosynthesis mechanism of Bufonis Venenum.

A complementary chromatographic strategy for integrated components characterization of Imperatae Rhizoma based on convergence and liquid chromatography combined with mass spectrometry and molecular network.

The complexity of natural ingredients and the diversity of preparations are the major obstacles to the quality evaluation of traditional Chinese medicines (TCMs). A more comprehensive characterization of herbal compounds using different types of chromatographic separation techniques and covering a diverse polarity range can help evaluate the quality of TCMs. In this study, we first proposed a comprehensive method for characterizing compounds derived from Imperatae Rhizoma by combining the complementary strengths of UPCC-QTOF-MS (ultra-performance convergence chromatography coupled with quadrupole-time of flight mass spectrometry) with UPLC-QTOF-MS (ultra-performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry). The method based on the UNIFI scientific platform significantly shortened the analysis time and enabled a more comprehensive characterization of known and unreported compounds. Meanwhile, a feature-based molecular network (FBMN) was established on the Global Natural Product Social (GNPS) to infer potential compounds by rapidly classifying and visualizing these components. A total of 62 compounds in Imperatae Rhizoma were jointly characterizedand classified into six types. In comparison, the UPCC-QTOF-MS technology individually characterized 17 components, including lactones, phenols, aldehydes, phenylpropanoids, and small polar organic acids. The UPLC-QTOF-MS technology characterized 16 compounds mainly phenylpropionic acids, flavonoid glycosides, and chromone glycosides. Furthermore, three types of characteristic compounds could be well aggregated into an FBMN approach. Five possible potential new compounds were detected through the supplementary identification of GNPS and the correlation analysis of vicinal known compounds. The strategy was first applied to Imperatae Rhizoma and facilitated the characterization of a large quantity of data to provide comprehensive chemical composition results. This approach can be easily extended to the study of the material basis of other herbs or preparations in order to improve the accuracy of herb quality evaluation.

[Distribution characteristics of Bufo bufo gargarizans resources in China based on quality evaluation of Bufonis Venenum].

On the premise of strictly controlling the harvesting conditions of Bufonis Venenum, we studied the relationship between the quality and resource distribution of Bufonis Venenum in China, aiming to provide the data for comprehensively understanding the geographical distribution and characteristics of Bufonis Venenum in China. In this study, 105 samples of Bufonis Venenum were collected from 42 counties and cities in 19 provinces in China, and the material basis and index components were determined by mass spectrometry and high performance liquid chromatography. The obtained data formed the quality database of Bufonis Venenum from different producing areas in China. The analysis of the material basis showed that Bufonis Venenum was mainly produced in two characteristic regions(north area and south area) divided by Qinling Mountains, northern edge of Huaiyang hills and the connecting area of Huang-Huai Plain, Huangshan Mountains, and Tianmu Mountains. Eight differential components were identified in the Bufonis Venenum samples from the south area and the north area. All the Bufonis Venenum samples from the north area showed the content of index components above the requirements of Chinese Pharmacopoeia(2020 edition), while those from the south area had the content of index components lower than the standards of Chinese Pharmacopoeia(2020 edition). The quality evaluation showed uneven distribution of Bufonis Venenum quality, which was high in the north and low in the south. The results provided a research basis for the breeding base selection of Bufo bufo gargarizans.

Shexiang Baoxin Pill, a Proprietary Multi-Constituent Chinese Medicine, Prevents Locomotor and Cognitive Impairment Caused by Brain Ischemia and Reperfusion Injury in Rats: A Potential Therapy for Neuropsychiatric Sequelae of Stroke.

Ischemic stroke is a common type of cerebrovascular event and also the leading cause of disability. Post-stroke cognitive impairment occurs frequently in stroke survivors. Shexiang Baoxin Pill (SBP) is a proprietary Chinese medicine, initially used to treat cardiovascular diseases. Herein, we aim to explore the effects of SBP on oxygen glucose deprivation and reoxygenation (OGD/R) in neuronal cells (CATH.a) and cerebral ischemia/reperfusion injury induced post-stroke cognitive impairment in middle cerebral artery occlusion (MCAO) rat model. MCAO rats received two doses of oral SBP treatment (28 or 56 mg/kg) after 1 h of operation and once daily for 2 weeks continuously. Behavioral tests, immunoblotting, and immunofluorescence were examined after 14 days. Current data suggest that SBP enhanced cell viability and downregulated apoptosis via activating the PI3K/Akt signaling pathway in CATH. a cells. Furthermore, 14 days of SBP treatment promoted the recovery of learning and locomotor function in the MCAO rats. SBP up-regulated the expression of p-Akt, p-GSK3ß, as well as the expression of NMDAR1, PSD-95, and AMPAR. Also, SBP down-regulated the expression of p-CaMKII. These results indicated that long-term SBP treatment might be a potential option for cognitive impairment induced by the ischemic stroke.

Shexiang Baoxin Pill, a Traditional Chinese Herbal Formula, Rescues the Cognitive Impairments in APP/PS1 Transgenic Mice.

BACKGROUND: Shexiang Baoxin Pill (SBP), a formulated traditional Chinese medicine (TCM), has been widely used to treat cardiovascular diseases for years. This herbal mixture has been shown to promote differentiation of cultured neuronal cells. Here, we aimed to investigate the effects of SBP in attenuating cognitive impairment in APP/PS1 transgenic mice. METHODS: Ethanol and water extracts of SBP, denoted as SBPEtOH and SBPwater, were standardized and applied onto cultured rat pheochromocytoma PC12 cells. The potential effect of SBPEtOH extract in attenuating the cognitive impairments in APP/PS1 transgenic mice was shown by following lines of evidence: (i) inhibition of Aß fibril formation, (ii) suppression of secretions of cytokines, and (iii) improvement of behavioral tests by Morris water maze. RESULTS: SBPwater and SBPEtOH inhibited the formation of ß-amyloid fibrils and protected the Aß-induced cytotoxicity in cultured PC12 cells. In APP/PS1 transgenic mice, the treatment of SBPEtOH inhibited expressions of NO, NOS, AChE, as well as aggregation of Aß. Besides, the levels of pro-inflammatory cytokines were suppressed by SBP treatment in the transgenic mice. Importantly, the behavioral tests by Morris Water maze indicated that SBP attenuated cognitive impairments in APP/PS1 transgenic mice. CONCLUSION: The current result has supported the notion that SPB might ameliorate the cognitive impairment through multiple targets, suggesting that SBP could be considered as a promising anti-AD agent.

Mechanism of angiogenesis promotion with Shexiang Baoxin Pills by regulating function and signaling pathway of endothelial cells through macrophages.

BACKGROUND AND AIMS: "Shexiang Baoxin Pill" (SBP), a commonly used traditional Chinese medicine, has been used to treat angina, myocardial infarction and coronary heart disease in China for thirty years. SBP has been proven to promote angiogenesis in a rat model of myocardial infarction (MI). The aim of the present study was to determine the pro-angiogenic effects and mechanism of SBP during inflammation or ischemic pathological conditions and elucidate its regulatory effects on endothelial cell function and signaling pathways mediated by macrophages. METHODS: We used a polyvinyl alcohol (PVA) sponge implantation mouse model as an inflammatory angiogenesis model and utilized a mouse femoral artery ligation model as a hind limb ischemia model. We also performed cell proliferation, cell migration and tubule formation in vitro experiments to assess the effects of SBP on endothelial cell function and signaling pathways by stimulating macrophage activity. RESULTS: The in vitro experiment results showed that SBP could significantly increase the expression of mRNAs and proteins associated with angiogenesis in endothelial cells by activating macrophages to release pro-angiogenic factors such as Vegf-a. Activation of macrophages by SBP eventually led to endothelial cell proliferation, migration and tubule formation and increased the expression of p-Akt and p-Erk1/2 proteins in the downstream PI3K/Akt and MAPK/Erk1/2 signaling pathways related to angiogenesis, respectively. The in vivo experiment results indicated that SBP had angiogenesis effects in both inflammatory and ischemic angiogenesis models with dose- and time-dependent effects. CONCLUSION: Shexiang Baoxin Pills can promote angiogenesis by activating macrophages to regulate endothelial cell function and signal transduction pathways.

Shexiang Baoxin Pill, a Formulated Chinese Herbal Mixture, Induces Neuronal Differentiation of PC12 Cells: A Signaling Triggered by Activation of Protein Kinase A.

Background: Shexiang Baoxin Pill (SBP) is a well-known composite formula of traditional Chinese medicine (TCM), which is commonly used today in treating cardiovascular diseases. SBP consists of seven materials thereof, including Moschus, extract of Ginseng Radix et Rhizoma, Bovis Calculus Artifactus, Cinnamomi Cortex, Styrax, Bufonis Venenum, and Borneolum Syntheticum. Here, we are investigating the potential roles of SBP in inducing neuron differentiation, i.e., seeking possible application in neurodegenerative diseases. Methods: Water and ethanol extracts of SBP, denoted as SBPwater and SBPEtOH, respectively, as well as its individual herbal materials, were standardized and applied onto cultured rat pheochromocytoma PC12 cells. The potential effect of SBP extracts in neuronal differentiation was suggested by following parameters: (i) induction of neurite outgrowth of PC12 cells, (ii) increase of neurofilament expression, and (iii) activation of transcription of neurofilament. Results: The treatments of SBPwater and SBPEtOH, or extracts from individual herbal materials, with or without low concentration of nerve growth factor (NGF), could potentiate the differentiation of cultured PC12 cells. The differentiation was indicated by increase of neurite outgrowth, as well as expression of neurofilaments. In addition, application of H89, a protein kinase A (PKA) inhibitor, suppressed the SBP-induced neurofilament expressions, as well as the phosphorylation of cAMP-responsive element binding protein (CREB) in cultures. Conclusion: SBP is proposed to possess trophic activity in modulating neuronal differentiation of PC12 cells, and this induction is shown to be mediated partly by a cAMP-PKA signaling pathway. These results indicate the neurite-promoting SBP could be useful in developing potential drug in treating or preventing neurodegenerative diseases.

Simultaneous quantification of 11 active constituents in Shexiang Baoxin Pill by ultraperformance convergence chromatography combined with tandem mass spectrometry.

On account of the complexity of chemical constituents of Shexiang Baoxin Pill (SBP), a famous traditional Chinese medicine (TCM) formula, a novel and effective UPC2-MS/MS method was developed to simultaneously determine the content of 11 active compounds of SBP with outstanding separation ability. Eleven components in SBP, including 2 ginsenosides, 2 bile acids, 3 bufadienolides and 4 volatiles were detected by electrospray ionization tandem mass spectrometry in positive and negative ion modes with multiple reaction monitor (MRM). The analysis was performed at 30°C using an Acquity UPC2 Diol (3.0×50mm, 1.7µm) column with linear gradient elution (eluent A, CO2; eluent B, methanol containing 20mM ammonium acetate), back pressure of 2000 psi, flow rate of 1.2mL/min and the injection volume of 1.0µL. The method was extensively validated regarding the linearity (r≥0.9974), precision (≤3.11%), recovery (93.34-104.50%), repeatability (≤2.00%) and stability (≤4.20%). Using this method, 11 active compounds of SBP with different polarity were simultaneously quantified in one chromatography analysis within 8min. Statistical analysis of the effects of 11 compounds on the quality of SBP revealed that the content of cinnamaldehyde varied widely in different batches. This work presents an exemplary study for quality control of complex samples, especially for TCMs.

Characterization of the Principal Constituents of Danning Tablets, a Chinese Formula Consisting of Seven Herbs, by an UPLC-DAD-MS/MS Approach.

Danning Tablets are a traditional Chinese formula showing broad clinical applications in hepatobiliary diseases and containing a diversity of bioactive chemicals. However, the chemical profiling of the formula, which serves as the material foundation of its efficacy, is really a big challenge as Danning Tablets consist of seven herbs from different origins. An ultra-performance liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry (UPLC-DAD-ESI-MS/MS) approach was developed to characterize the principal polyphenol constituents in the formula. As a result, a total of 32 constituents, including 14 anthraquinones and their glucosides, four anthrones, two naphthalene glycosides, two stilbenes and 10 flavonoids were identified based on their retention time, UV absorption and MS/MS fragmentation patterns. The sources of these compounds were also illustrated. Most of the bioactive anthraquinone derivatives were found in Rhei Radix et Rhizoma or Polygoni Cuspidati Rhizoma et Radix, which are the Emperor drugs in the formula for its clinic usage. These findings indicate the merit of using this integrated UPLC-DAD-ESI-MS/MS approach to rapidly illustrate the chemical foundation of complex formulas. The present study will facilitate the quality control of Danning Tablet formulas as well as the individual herbs.

Danning tablets attenuates α-naphthylisothiocyanate-induced cholestasis by modulating the expression of transporters and metabolic enzymes.

BACKGROUND: The Danning tablets (DNts) is commonly prescribed in China as a cholagogic formula. Our previous studies showed that DNts exerted the protective effect on α-naphthylisothiocyanate (ANIT)-induced liver injury with cholestasis in a dose-dependent mannar. However, the detailed molecular mechanisms of DNts against ANIT-induced cholestasis are still not fully explored. METHODS: Danning tablet (3 g/kg body weight/day) was administered orally to experimental rats for seven days before they were treated with ANIT (60 mg/kg daily via gastrogavage) which caused cholestasis. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (T-Bil), direct bilirubin (D-Bil) and total bile acid (TBA) were measured to evaluate the protective effect of Danning tablet at 12, 24 and 48h after ANIT treatment. Meanwhile, total bilirubin or total bile acid in the bile, urine and liver were also measured at 48h after ANIT treatment. Furthermore, the hepatic or renal mRNA and protein levels of metabolic enzymes and transports were investigated to elucidate the protective mechanisms of Danning tablet against ANIT-induced cholestasis. RESULTS: In this study, we found that DNts significantly attenuated translocation of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane into an intracellular and up-regulated the hepatic mRNA and protein expressions of metabolic enzymes including cytochrome P450 2b1(Cyp2b1) and uridine diphosphate-5¢- glucuronosyltransferase (Ugt1a1)) and transporters including bile salt export pump (Bsep) and multidrug resistance protein 2 (Mdr2)) as well as renal organic solute transporter beta (Ostß), accompanied by further increase in urinary and biliary excretion of bile acid and bilirubin. CONCLUSIONS: DNts might promote bile acid and bilirubin elimination by regulating the expressions of hepatic and renal transporters as well as hepatic metabolic enzymes.

The effectiveness of borneol on pharmacokinetics changes of four ginsenosides in Shexiang Baoxin Pill in vivo.

Shexiang Baoxin Pill (SBP) is a traditional Chinese medicine, widely used for cardiovascular diseases in the clinic. Ginsenosides are important effective components in SBP, but their pharmacokinetic characteristics are still not known. In this paper, we studied the pharmacokinetics of ginsenoside Rb1, Rc, Re and Rg1 in SBP and investigated the effect of borneol on the pharmacokinetic characteristic of ginsenosides based on an Agilent G6410A triple quadrupole LC/MS system. Results showed that the pharmacokinetic parameters of ginsenoside Rb1, Rc, Re and Rg1 in rat plasma after oral administration of SBP are significantly different with oral administration of SBP without Borneolum Syntheticum. Plasma pharmacokinetic profiles after oral administration of ginsenoside Rb1, Rc, Re, Rg1 and co-administration with borneol at three different ratios (10:1, 1:1 and 1:10 ginsenoside vs borneol, w/w) were also determined. It was demonstrated that borneol can elevate the plasma concentration of ginsenosides after co-admininstration.

The serum metabolomic study of intervention effects of the traditional Chinese medicine Shexiang Baoxin Pill and a multi-component medicine polypill in the treatment of myocardial infarction in rats.

A metabolomic approach based on liquid chromatography coupled with quadrupole time-of-flight detector (LC-Q-TOF/MS) was developed to investigate the therapeutic mechanism of a traditional Chinese medicine (TCM) formula Shexiang Baoxin Pill (SBP) and a multi-component medicine polypill (consisting of simvastatin (Sim), atenolol (Ate), ramipril (Ram), hydrochlorthiazide (Hyd) and aspirin (Asp), named as SARHA). Twenty-seven biomarkers were identified in the serum of MI rats. Thirteen related pathways and 4 main pathological processes including oxidative injury, energy metabolism dysfunction, amino acid metabolism dysfunction and inflammation are involved in MI development. Our study revealed that SBP showed better therapeutic effectiveness than the polypill on MI through regulation of the energy metabolism dysfunction, oxidative injury and inflammation. The combination agent polypill had only certain therapeutic effects on inhibiting oxidative injury and inflammation induced by MI. The reverse effect of the polypill on biomarkers related to MI was much better than mono-therapy groups.

Protective effect of Danning tablet on acute livery injury with cholestasis induced by α-naphthylisothiocyanate in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Danning tablet, as a composite prescription of traditional Chinese medicine, has been used clinically to relieve liver and gallbladder diseases in China. However, the mechanisms involved are still unclear. AIM OF THE STUDY: The present investigation was designed to assess the effects and possible mechanisms of Danning tablet on α-naphthylisothiocyanate (ANIT)-induced liver injury with cholestasis. MATERIALS AND METHODS: Danning tablet (3, 1.5 or 0.75g/kg body weight/day) was intragastrically (i.g.) given to experimental rats for seven days before they were treated with ANIT (60mg/kg daily via i.g.) which caused liver injury. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltranspeptidase (γ-GTP), total bilirubin (T-Bil), direct bilirubin (D-Bil), total bile acid (TBA) and bile flow were measured to evaluate the protective effect of Danning tablet at 48h after ANIT treatment. Furthermore, protective mechanisms of Danning tablet against ANIT-induced liver injury were elucidated by assays of liver enzyme activities and component contents including myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione peroxidase (Gpx), catalase (CAT) and glutathione S-transferase (GST), as well as liver lipid peroxide (LPO) and glutathione (GSH). The biochemical observations were supplemented by histopathological examination. Phytochemical analysis of Danning tablet was performed by UPLC-MASS. RESULTS: Obtained results demonstrated that high dose (3g/kg) of Danning tablet significantly prevented ANIT-induced changes in bile flow (P<0.01), and serum levels of ALT, AST, ALP, γ-GTP, T-Bil, D-Bil (P<0.01) and TBA (P<0.05). In addition, ANIT-induced increases in hepatic MPO, GST activities and GSH, LPO contents were significantly (P<0.01) reduced, while SOD, Gpx, CAT activities in the liver tissue which were suppressed by ANIT were significantly (P<0.01) elevated in the groups pretreated with Danning tablet at the dose of 3g/kg B.W. Histopathology of the liver tissue showed that pathological injuries were relieved after Danning tablet (3g/kg) pretreatment. The results also showed that medium dose (1.5g/kg) of Danning tablet exhibited partially protective effect on ANIT-induced liver injury with cholestasis by reversing part of biochemical parameters and histopathological changes. Low dose (0.75g/kg) of Danning tablet did not show any protective effect on ANIT-induced liver injury with cholestasis. Phytochemical analyses revealed the presence of anthraquinones, flavonoids and stilbene in the Danning tablet. CONCLUSION: These findings indicate that Danning tablet exerts a dose-dependently protective effect on ANIT-induced liver injury with cholestasis in rats, and the possible mechanism of this activity is likely due to its attenuation of oxidative stress in the liver tissue and neutrophil infiltration.