[Effects of Euphorbiae Pekinensis Radix before and after processing with vinegar on liver and gastrointestinal toxicity of zebrafish embryos].

To study the effects of different fraction of Euphorbiae Pekinensis Radix before and after processing with vinegar on liver and gastrointestinal toxicity of zebrafish embryos,the zebrafish embryos after fertilized 12 h(12 hpf) were exposed to different concentrations of solution until 96 h(96 hpf),for observation of the toxicity response of the liver and gastrointestinal of individual zebrafish embryos. The results showed that toxicity increased in a dose-dependent manner. The liver and gastrointestinal toxicity of the zebrafish embryos in various polar fractions of Euphorbiae Pekinensis Radix before and after processing with vinegar was mainly manifested as slow liver development,smaller liver area,edema of yolk sac,delayed absorption,slowing of gastrointestinal motility,abnormal function of gastrointestinal goblet cell secretion. In addition,the toxicity of different polarity was followed by petroleum ether,dichloromethane,ethyl acetate. The above results indicated that the toxicity was reduced after processing with vinegar,and the fractions of petroleum ether and methylene chloride were the main sites responsible for liver and gastrointestinal toxicity.

[Acute toxicity of Euphorbiae Pekinensis Radix and vinegar-processing Euphorbiae Pekinensis Radix on zebrafish embryo].

The embryos of model organism zebrafish were used to evaluate the acute toxicity of the extracts of Euphorbiae Pekinensis Radix and vinegar-processing Euphorbiae Pekinensis Radix, and the total terpene content of each extract was determined by using euphol as the reference standards. Twenty-four h normally developed zebrafish embryos were chosen, and 8 concentrations were adopted for each extract. Then the growth and death of zebrafish embryos were observed at 96 h after administration, and median lethal concentrations (LC50) of the different samples on zebrafish embryos were calculated. The results showed that all of the extracts (before and after vinegar processing) had acute toxicity on zebrafish embryos. The toxicity of vinegar-processing Euphorbiae Pekinensis Radix was significantly lower than that of crude Euphorbiae Pekinensis Radix. Among different extraction methods, ethanol extract was more poisonous than water extract; in different polarity fractions, the toxicity was in the following order: petroleum ether>dichloromethane>ethyl acetate>n-butyl alcohol and remaining part. Combined with the results of the determination of terpene components, it can be concluded that the terpenoids are the main toxic components of Euphorbiae Pekinensis Radix, positively correlated with toxicity degree. It indicates that the zebrafish embryo model is appropriate for the toxicity evaluation of Euphorbiae Pekinensis Radix and provides appropriate research methods and theoretical basis for the further study of the toxic components and the mechanism of reducing toxicity.

[Application of gut microbiota in research of Chinese medicines].

This article summarizes the research progress in recent years on interactions between Chinese medicines and gut microbiota based on the physiological functions of gut microbiota, including imbalance impacts of toxic/irritating Chinese medicines on gut microbiota, prognosis effects of Chinese medicines on gut microbiota imbalance, metabolism effects of gut microbiota on Chinese medicine components, and co-metabolism effects between gut microbiota and host. We would think and prospect the specific biological effects of Chinese medicines, gut microbiota structures and the relations between endogenous metabolites from "gut microbiota and host co-metabolism". All of these aim to investigate biological mechanisms and effective components of Chinese medicines based on gut microbiota and offer a new strategy for promoting safe and effective application of Chinese medicines.

[Study on detoxication of euphorbia pekinensis radix processed with vinegar on rat small intestinal crypt epithelial cells IEC-6].

OBJECTIVE: To compare the difference of Euphorbia Pekinensis Radix before and after being processed with vinegar in the toxicity on rat small intestinal crypt epithelial cells IEC-6, and make a preliminary study on the mechanism of detoxication of Euphorbia Pekinensis Radix processed with vinegar. METHOD: With rat small intestinal crypt epithelial cells IEC-6 as the study object, the MTT method was adopted to detect the effect of Euphorbia Pekinensis Radix before and after being processed with vinegar on IEC-6 cell activity. The morphology of cells were observed by the inverted microscope. The down-regulated mitochondrial apoptosis pathway of enterocytes caused by the vinegar processing was analyzed by using the high content screening. RESULT: Compared with the negative control group, the proliferation inhibition experiment showed that Euphorbia Pekinensis Radix showed a relatively high intestinal cell toxicity (P < 0.01). The results of HCS analysis showed that Euphorbia Pekinensis Radix could significantly reduce the cell nucleus Hoechst fluorescence intensity and mitochondria membrane (P < 0.05, P < 0.01), and increase Annexin V-FITC and PI fluorescence intensity and membrane permeability (P < 0.01, P < 0.01, P < 0.01). After being processed with vinegar, compared with Euphorbia Pekinensis Radix groups with different doses, Euphorbia Pekinensis Radix processed with vinegar could significantly decrease the cell proliferation inhibition effect on enterocytes, increase the cell nuclear Hoechst fluorescence intensity and mitochondria membrane (P < 0.05, P < 0.05), and decrease Annexin V-FITC and PI fluorescence intensity and membrane permeability (P < 0.01, P < 0.01, P < 0.05), and showed a certain dose-effect relationship. CONCLUSION: The vinegar processing can further reduce the toxicity of Euphorbia Pekinensis Radix on enterocytes. Its possible mechanism can decrease the effect of Euphorbia Pekinensis Radix on the permeability of IEC-6 cell membrane, so as to provide a basis for further explanation of the detoxication mechanism of Euphorbia Pekinensis Radix processed with vinegar.

Novel pretreatment nomograms based on pan-immune-inflammation value for predicting clinical outcome in patients with head and neck squamous cell carcinoma.

Background: The prognostic value of an effective biomarker, pan-immune-inflammation value (PIV), for head and neck squamous cell carcinoma (HNSCC) patients after radical surgery or chemoradiotherapy has not been well explored. This study aimed to construct and validate nomograms based on PIV to predict survival outcomes of HNSCC patients. Methods: A total of 161 HNSCC patients who underwent radical surgery were enrolled retrospectively for development cohort. The cutoff of PIV was determined using the maximally selected rank statistics method. Multivariable Cox regression and least absolute shrinkage and selection operator (LASSO) regression analyses were performed to develop two nomograms (Model A and Model B) that predict disease-free survival (DFS). The concordance index, receiver operating characteristic curves, calibration curves, and decision curve analysis were used to evaluate the nomograms. A cohort composed of 50 patients who received radiotherapy or chemoradiotherapy (RT/CRT) alone was applied for generality testing of PIV and nomograms. Results: Patients with higher PIV (≥123.3) experienced a worse DFS (HR, 5.01; 95% CI, 3.25-7.72; p<0.0001) and overall survival (OS) (HR, 5.23; 95% CI, 3.34-8.18; p<0.0001) compared to patients with lower PIV (<123.3) in the development cohort. Predictors of Model A included age, TNM stage, neutrophil-to-lymphocyte ratio (NLR), and PIV, and that of Model B included TNM stage, lymphocyte-to-monocyte ratio (LMR), and PIV. In comparison with TNM stage alone, the two nomograms demonstrated good calibration and discrimination and showed satisfactory clinical utility in internal validation. The generality testing results showed that higher PIV was also associated with worse survival outcomes in the RT/CRT cohort and the possibility that the two nomograms may have a universal applicability for patients with different treatments. Conclusions: The nomograms based on PIV, a simple but useful indicator, can provide prognosis prediction of individual HNSCC patients after radical surgery and may be broadly applicated for patients after RT/CRT alone.

[Effect of Kansui Radix prepared by different processes on LO2 cell cycle and apoptosis].

OBJECTIVE: To discuss the effect of Kansui Radix prepared by different processes on cell cycle and apoptosis of normal human liver cell lines LO2. METHOD: With normal human liver cell lines LO2 as the study object, the MTT method was adopted to study the effect of Kansui Radix prepared by different processes, including Kansui Radix, stir-baking Kansui Radix, Kansui Radix moistening with vinegar and Kansui prepared by different processes, on LO2 cell activity. The cellular morphological changes were observed by inverted microscope. The effect of Kansui Radix stir-baked with vinegar on LO2 cell cycle and apoptosis was observed by flow cytometry. RESULT: Compared with the negative control group, Kansui could obviously inhibit the activity of human normal liver cell lines LO2 (P <0.01) , and significantly increase the percentage of LO2 cells in S phase (P <0.05) , notably decrease the percentage of LO2 cells in G2/M phase (P <0.01) , significantly increase the early apoptosis rate, late apoptosis rate and necrosis rate and total apoptosis rate of human normal liver cell lines LO2 (P <0.01). Compared with the Kansui group, all of the other processed Kansui samples could significantly decrease the cell proliferation inhibition (P <0.01) , and the trend of morphological degradation. Besides, they could significantly increase the percentage of LO2 cells in G2/M phase (P <0.05, P <0.05, P <0. 01) , significantly decrease the early apoptosis rate, late apoptosis rate and necrosis rate, and total apoptosis rate of human normal liver cell lines LO2 (P < 0.01). The order of the increase in the percentage of cells in G2/M phase and the decrease in apoptosis rate was Kansui Radix stirbaked with vinegar > Kansui Radix moistening with vinegar > stir-baking Kansui Radix. CONCLUSION: The toxicity of processed Kansui could be reduced by affecting LO2 cell cycle and apoptosis. The processes of stir-baking and moistening with vinegar can play a synergistic effect in the detoxication of human normal liver cell lines LO2, which provides a basis for unveiling the rationality of stirbaking with vinegar of Kansui in the detoxication, as well as the optimizing the process.

[Study on detoxication and mechanism of vinegar-processed Euphorbia pekinensis on normal liver cells LO2].

OBJECTIVE: To compare the toxicity of Euphorbia pekinensis before and after being processed by vinegar on normal liver cells LO2, and discuss its possible mechanism. METHOD: LO2 cells were cultured in vitro, and processed with different concentrations of crude and vinegar-processed E. pekinensis. MTT assay was used to measure the inhibitory effect of LO2 cell; Hoechst 33258 staining was used to observe the morphological changes in apoptosis cell; Annexin V-FITC flow cytometry was used to analyze the apoptotic rate of LO2 cell; PI staining flow cytometry was used to analyze its impact on cell cycle. The level or content of ALT, AST, LDH, SOD, MDA and GSH were observed as well. RESULT: Compared with the negative control group, crude E. pekinensis at all concentrations could obviously inhibit LO2 cell proliferation, induce LO2 cell apoptosis and cause cell arrest in S phase, with significant differences (P <0.05). E. pekinensis could significantly increase the levels of ALT, AST and LDH (P <0.05) in the supernatant of cell culture fluid, significantly decrease the level of SOD and the content of GSH (P <0.05) , and significantly increase the content of MDA (P <0.05). Compared with the crude E. pekinensis group, E. pekinensis after being vinegar-processed can significantly reduce cell apoptotic rate, cell cycle arrest, activities of ALT, AST, LDH in the supernatant of cell culture fluid (P <0.05) , and remarkably increase the level of SOD and the content of GSH, but reduce the content of MDA in the supernatant of cell culture fluid. CONCLUSION: Vinegar-processed E. pekinensis can release the cytotoxicity of LO2 cell. Its mechanism may be related to the decrease in the oxidative damage of LO2 cells, thereby reducing the cell cycle arrest and apoptosis.

Predictive Value of Heart-Type Fatty Acid-Binding Protein for Mortality Risk in Critically Ill Patients.

Objective: Our study assessed the predictive value of heart-type fatty acid-binding protein (H-FABP) for critically ill patients. Methods: 150 critically ill patients admitted to the emergency department of Beijing Chaoyang Hospital, Capital Medical University, were included in our study from August 2021 to April 2022. Serum H-FABP, procalcitonin (PCT), lactate (LAC), and other markers were determined within 1 h after admission. The Sequential Organ Failure Assessment (SOFA) score and the Acute Physiology and Chronic Health Evaluation II (APACHE II) were calculated. The independent predictors of 28-day mortality in critically ill patients were analyzed by logistic regression, and the receiver operating characteristic curve (ROC) was used to analyze the predictive value for 28-day mortality in critically ill patients. Results: Age, APACHE II, SOFA, GCS, LAC, H-FABP, IL-6, Scr, and D-dimer were significantly different in the nonsurvivor vs. survivor groups (P < 0.05), with H-FABP correlating with cTNI, Scr, PCT, and SOFA scores (P < 0.05). Logistic regression analysis showed that H-FABP, APACHE II, LAC, and age were independent predictors for 28-day mortality in critically ill patients (P < 0.05). The AUC of ROC curve in H-FABP was 0.709 (sensitivity 72.9%, specificity 66.1%, and cut-off 4.35), which was slightly lower than AUC of ROC curve in LAC (AUC 0.750, sensitivity 58.3%, specificity 76.1%, and cut-off 1.95) and APACHE II (AUC 0.731, sensitivity 77.1%, specificity 58.7%, and cut-off 12.5). However, statistically, there was no difference in the diagnostic value of H-FABP compared with the other two indicators (Z 1 = 0.669, P = 0.504; Z 2 = 0.383, P = 0.702). But H-FABP (72.9%) has higher sensitivity than LAC (58.3%). The combined evaluation of H-FABP+APACHE II score (AUC 0.801, sensitivity 71.7%, and specificity 78.2%; Z = 2.612, P = 0.009) had better diagnostic value than H-FABP alone and had high sensitivity (71.7%) and specificity (78.2%). Conclusion: H-FABP, LAC, APACHE II, and age can be used as independent risk factors affecting the prognosis of critically ill patients. Compared with using the above indicators alone, the H-FABP+APACHE II has a high diagnostic value, and the early and rapid evaluation is particularly important for the adjustment of treatment plans and prognosis.

The toxicity mechanism of toxic compounds from Euphorbiae pekinensis Radix on zebrafish embryos.

Euphorbiae pekinensis Radix (EP) is effective in treating various diseases, but it's toxicity is a major obstacle in use in clinical. Although EP was processed with vinegar to reduce it's toxicity, the detailed mechanism of toxicity in EP have not been clearly delineated. This study investigate the toxicity attenuation-mechanism of Euphorbiae pekinensis after being processed with vinegar (VEP) and the toxic mechanism of four compounds from EP on zebrafish embryos. The contents of four compounds decreased obviously in VEP. Correspondingly, slower development on embryos can be seen as some symptoms like reduction of heart rate, liver area and gastrointestinal peristalsis after exposed to the compounds. Some obvious pathological signals such as pericardial edema and yolk sac edema were observed. Furthermore, the compounds could increase the contents of MDA and GSH-PX and induce oxidative damage by inhibiting the activity of SOD. Also, four compounds could provoke apoptosis by up-regulating the expression level of p53, MDM2, Bax, Bcl-2 and activating the activity of caspase-3, caspase-9. In conclusion, the four compounds play an important role in the toxicity attenuation effects of VEP, which may be related to the apoptosis induction and oxidative damage. This would contribute to the clinical application and further toxicity-reduction mechanism research.

Radix Kansui Stir-Fried with Vinegar Reduces Radix Kansui-Related Hepatotoxicity in Mice via Mitochondrial Pathway.

OBJECTIVE: To investigate the mechanism of Radix Kansui (RK) stir-fried with vinegar (VRK) decreased hepatotoxicity in mice. METHODS: According to a random number table, 40 mice were randomly divided into negative control group (0.5% carboxymethylcellulose sodium, 20 mL/kg), positive control group (0.1% mixture of carbon tetrachloride in soybean oil, 20 mL/kg), RK group (the ethyl acetate extracts of RK, 250 g crude drug/kg) and VRK group (the ethyl acetate extracts of VRK, 250 g crude drug/kg) with 10 mice per group. All mice were administered orally by gavage daily for 7 continuous days. The morphology of liver tissues was examined to assess the liver injury by a transmission electron microscope. Hepatocyte apoptosis in vivo was determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nickend labeling (TUNEL) assay. Immunohistochemical technique was adopted to detect the expression of particular antiapoptotic and proapoptotic proteins in the mitochondrial pathways, including B-cell lymphoma (Bcl-2) and caspase-3, as well as the expression of inflammatory mediators, including nuclear factor kappa B (NF- κ B) and intercellular adhesion molecule-1 (ICAM-1). RESULTS: Liver injury and hepatocyte apoptosis were observed in RK mice, and the liver injury were significantly reduced in VRK-treated mice. In immunohistochemistry study, compared with the negative control group, RK inhibited dramatically the Bcl-2 protein expression and significantly increased the expression of caspase-3, NF- κ B and ICAM-1 (all P<0.01). Compared with the RK group, VRK group induced significant increase on Bcl-2 protein expression, and decreased the caspase-3, NF- κ B and ICAM-1 protein expression (P<0.05 or P<0.01). CONCLUSION: The mechanism of reduced hepatotoxicity of VRK may be associated with the reduced inflammation, regulation of antiapoptotic and proapoptotic mediators in the mitochondrial pathway.

Comparison of content-toxicity-activity of six ingenane-type diterpenoids between Euphorbia kansui before and after stir-fried with vinegar by using UFLC-MS/MS, zebrafish embryos and HT-29 cells.

The dried roots of Euphorbia kansui (EK) are especially beneficial for the treatment of edema, but the severe toxicity limits their clinical applications. Euphorbia kansui stir-fried with vinegar (VEK) is traditionally employed to reduce the toxicity of EK. However, the material basis for the toxicity reduction with effectivity conservation is still unclear. Therefore, in this study, a rapid, sensitive, and reliable ultra-fast liquid chromatography tandem mass spectrometry (UFLC-MS/MS) method was firstly established to simultaneously determine six ingenane-type diterpenoids, i.e. kansuiphorin C (1), 5-O-benzoyl-20-deoxyingenol (2), 20-deoxyingenol (3), 3-O-(2'E,4'E-decadienoyl)-20-O-acetylingenol (4), 20-O-(2'E,4'Z-decadienoyl)ingenol (5), and ingenol (6), in EK and VEK based on the processing conversion. Then, the toxicity evaluation on zebrafish embryos and modulation of the expression of aquaporin-3 (AQP3) proteins in HT-29 cells were employed to investigate the toxicity-activity of six compounds. Chromatographic separation was obtained on Waters BEH RP18 column (2.1 mm × 100 mm, 2.5 µm) with the mobile phase composed of 0.1 % formic acid in acetonitrile and water, respectively. The column temperature was 35 ℃ at a flow rate of 0.4 mL min-1. Multiple reaction monitoring was conducted in both positive and negative modes for quantitative analysis. The method was then successfully used for the determination of six compounds in EK and VEK. In addition, 1, 2, 4, and 5 had evident cardiotoxicity, intestinal irritation and nutrient absorption disorders on zebrafish larvae, while no in-vivo toxicity was seen for groups given 3 and 6 (LC50 > 200 µM). Meanwhile, 1, 2, 4, 5, and 6 significantly increased the expression of AQP3 protein (p < 0.05) to promote the excretion of water in the colon. This study demonstrated that toxic ingenane-type diterpenoids converted into the less toxic compounds with the same core structure through the breakage of multiple ester bonds in the side chain. At the same time, the laxative effect was retained, providing useful information for the optimization of the process of EK and quality evaluation of other similar toxic Chinese herbal medicines.

Abnormal apelin-ACE2 and SGLT2 signaling contribute to adverse cardiorenal injury in patients with COVID-19.

BACKGROUND: Angiotensin converting enzyme 2 (ACE2) has recently been identified as the functional receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent response for novel coronavirus disease 2019 (COVID-19). This study aimed to explore the roles of ACE2, apelin and sodium-glucose cotransporter 2 (SGLT2) in SARS-CoV-2-mediated cardiorenal damage. METHODS AND RESULTS: The published RNA-sequencing datasets of cardiomyocytes infected with SARS-CoV-2 and COVID-19 patients were used. String, UMAP plots and single cell RNA sequencing data were analyzed to show the close relationship and distinct cardiorenal distribution patterns of ACE2, apelin and SGLT2. Intriguingly, there were decreases in ACE2 and apelin expression as well as marked increases in SGLT2 and endothelin-1 levels in SARS-CoV-2-infected cardiomyocytes, animal models with diabetes, acute kidney injury, heart failure and COVID-19 patients. These changes were linked with downregulated levels of interleukin (IL)-10, superoxide dismutase 2 and catalase as well as upregulated expression of profibrotic genes and pro-inflammatory cytokines/chemokines. Genetic ACE2 deletion resulted in upregulation of pro-inflammatory cytokines containing IL-1ß, IL-6, IL-17 and tumor necrosis factor α. More importantly, dapagliflozin strikingly alleviated cardiorenal fibrosis in diabetic db/db mice by suppressing SGLT2 levels and potentiating the apelin-ACE2 signaling. CONCLUSION: Downregulation of apelin and ACE2 and upregulation of SGLT2, endothelin-1 and pro-inflammatory cytokines contribute to SARS-CoV-2-mediated cardiorenal injury, indicating that the apelin-ACE2 signaling and SGLT2 inhibitors are potential therapeutic targets for COVID-19 patients.

Kansuiphorin C and Kansuinin A ameliorate malignant ascites by modulating gut microbiota and related metabolic functions.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia kansui is a toxic Chinese herbal medicine and exhibits promising treatment to the malignant ascites (MA) in its traditional use. Ingenane-type and jastrophane-type diterpenes are demonstrated to be responsible for the toxicity and efficacy of kansui. Two representative compounds, kansuiphorin C (KPC) and kansuinin A (KA) in each type were proved to effectively reduce the ascites. The biological and toxicological effects are closely associated with the gastrointestinal tract, but the possible mechanism and related metabolic functions of KPC and KA treating MA through modulating the gut microbiota remain unclear. AIM OF THE STUDY: To investigate the possible mechanism and related metabolism of KPC and KA ameliorating malignant ascites through modulating gut microbiota. MATERIALS AND METHODS: MA rats and normal rats were divided into different groups and administrated with KPC, KA, and positive drug, respectively. 16S rDNA gene sequencing and metagenomes analysis combined with the quantification of short-chain fatty acids of feces were performed to reflect the modulation of gut microbiota. Then, the metabolites of KPC and KA in rat feces under the normal and pathological circumstances were detected by ultra-fast liquid chromatography coupled with MS/MS detector (UFLC-MS/MS) to explore the in-vivo bacterial biotransformation. RESULTS: KPC and KA were modulatory compounds for gut microbiota. The richness of Lactobacillus and the decreased abundance of Helicobacter involved in the carbohydrate metabolism and amino acid metabolism could be responsible for their prohibitory effects on malignant ascites. KPC exhibited stronger modulation of gut microbiota through making the abundance of Helicobacter about 3.5 times lower than KA. Besides, in-vivo microbial biotransformation of KPC and KA contained oxidation, hydrolysis, dehydration, and methylation to form metabolites of lower polarity. Besides, at the dosage of 10 mg kg-1, the toxicity of both compounds had weaker influences on the gut microbiota of normal rats. CONCLUSION: KPC and KA could ameliorate malignant ascites by modulating gut microbiota mainly containing the increase of Lactobacillus and the decrease of Helicobacter and related carbohydrate and amino acid metabolism, providing a basis for their promising clinical usage.

Determination of kansuiphorin C and kansuinin A in rat feces using UFLC-MS/MS and its application in the comparative excretion study on normal and malignant ascites rats.

Malignant ascites (MA) is one of the severe complications of gastrointestinal tumors, affecting the patients' survival time and quality of life. Euphorbia kansui is a commonly used toxic Chinese herbal medicine for malignant ascites. Our previous study showed that the biological and toxicological effects of kansui were closely related to the gastrointestinal tract. The ingenane-type and jastrophane-type diterpenoids are both toxic and active components of kansui. The contents of kansuiphorin C (KPC) and kansuinin A (KA) take highest accounts in each type of diterpene. Hence, in this study, the efficacy and toxicity of KPC and KA on normal rats and MA rats were firstly evaluated by serum liver enzymes (ALT and AST), oxidative damage indicators (GSH, SOD, MDA and LDH), inflammatory indexes (TNF-α, IFN-γ and IL-2) and the volume of ascites. Changes in the levels of these indices showed that although the toxicity of KPC on normal rats was stronger than KA, KPC exhibited better efficacy to the malignant ascites with no obvious side effects at the dose of 10 mg·kg-1. Then, accurate and reliable methods for the determination of KPC and KA in the rat feces by ultra-fast liquid chromatography coupled with MS/MS detector (UFLC-MS/MS) were established, detected by the multiple reaction monitoring mode. The chromatographic separation was conducted on an XBbridge C18 column (50 mm × 2.1 mm, 2.5 µm) using gradient elution composed of 0.1% formic acid in water and acetonitrile. The flow rate was 0.5 mL·min-1 and column temperature was 30 °C. The method was finally applied to the comparative study on normal and malignant ascites rats given KPC and KA, respectively. Interestingly, the results showed that KPC's accumulative fecal excretion rate (normal, 19.22%±5.36%; model, 15.96%±3.47%) were much higher than that of KA (normal, 2.928%±0.741%; model, 2.835%±0.873%) at the same dose within 48 h. This suggested KPC had higher in-vivo transformations in comparison with KA, providing guidance for the further preclinical research of KPC and KA as promising compounds treating MA.

A review on traditional uses, phytochemistry and pharmacology of Eclipta prostrata (L.) L.

ETHNOPHARMACOLOGICAL RELEVANCE: Eclipta prostrata, a traditional herbal medicine, has long been used in Asia and South America for the therapy of hemorrhagic diseases (e.g. hemoptysis, hematemesis, hematuria, epistaxis and uterine bleeding), skin diseases, respiratory disorders, coronary heart disease, hair loss, vitiligo, snake bite and those caused by the deficiency of liver and kidney. AIM OF THE REVIEW: In this review, we highlight relatively comprehensive and up-to-date information of E. prostrata on traditional uses, phytochemistry, pharmacology and toxicity, along with featuring the gaps in current knowledge, aiming to provide references for future research and possible opportunities for well applications of this medicinal plant. MATERIALS AND METHODS: Information on E. prostrata was gathered from scientific databases (Google Scholar, Web of Science, Scifinder, Baidu Scholar, PubMed and CNKI). Information was also obtained from local books, Ph.D. theses and M.Sc. dissertations and Chinese Pharmacopoeia. The plant taxonomy was validated by the database "The Plant List". RESULTS: Various phytochemical classes has been identified and isolated from the plant covering triterpenes, flavonoids, thiopenes, coumestans, steroids and others. Among these, coumestans are reported as the most common ingredients. The isolated crude extracts and individual compounds have been reported to exhibit promising pharmacological properties, such as hepatoprotective, osteoprotective, cytotoxic, hypoglycaemic, anti-inflammatory, anti-microbial, hypolipidemic, promoting hair growth, rejuvenative and neuroprotective effects. CONCLUSIONS: Until now, significant progress has been witnessed in phytochemistry and pharmacology of E. prostrata. Thus, some traditional uses has been well supported and clarified by modern pharmacological studies. Moreover, E. prostrata also showed therapeutic potential in some refractory diseases such as cancer, dementia and diabetes. But, present findings are still insufficient that cannot satisfactorily explain some mechanisms of action. More well-designed studies in vitro especially in vivo are required to establish links between the traditional uses and bioactivities, discover new skeletons and activity molecules, as well as ensure safety before clinical use.

A Novel Integrative Processing Technology for the Preparation of Rehmanniae Radix Slices.

The traditional processing method for the slices preparation of Rehmanniae roots is time- and energy-consuming and is prone to result in loss of active components during twice water-treatment (once for wash and the other for softening) and drying steps. In this study, we firstly explored an integrative processing technique for Rehmanniae Radix by 2x3 factorial experiment based on the contents of catalpol and verbascoside as measured by HPLC. The potential differences between the traditional stepwise processing technique and the integrative processing technique for catalpol and verbascoside in the prepared slices were investigated. To further confirm the effectiveness of drugs using the integrative processing technique, some pharmacological variables, such as rectal temperature, hematologic parameters (RBC, HGB, HCT, and blood viscosity), and coagulation parameters (TT, APTT, PT and FIB), were detected in a blood-heat and hemorrhage syndrome rat model. Two-way ANOVA analysis showed that drying for 18 h at 50°C was considered as the best combination of process conditions. The mean catalpol and verbascoside contents in the integrative method-processed samples (4.30% and 0.33%, respectively) were higher than those in the traditional method-processed samples (2.61% and 0.21%, respectively). Significant increases in rectal temperature, and hematologic parameters, TT, APTT, and FIB, were observed in the model group rats, compared to the blank group animals (P<0.01). Both in the integrative groups and traditional groups, the extracts caused significant decreases in rectal temperature, RBC, HGB, and HCT with increased concentration compared to the model group animals. All coagulation parameters tested were shortened in model rats received two kind prepared slices. There were no significant therapeutic differences between the integrative and the traditional method-processed slices on the hemostasis and hemorheological parameters in this blood-heat and hemorrhage syndrome rat model, indicating that our integrative method may be a feasible technique for processing Rehmanniae Radix slices.

The toxicity and efficacy evaluation of different fractions of Kansui fry-baked with vinegar on Walker-256 tumor-bearing malignant ascites effusion rats and normal rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Kansui, the root of Euphorbia kansui S.L.Liou ex S.B.Ho (E.kansui), is a classical traditional Chinese medicine (TCM) with certain toxicity. According to the theory of TCM, kansui fry-baked wtith vinegar (VEK) possesses low toxicity and mild diuretic and purgative efficacy. In clinical practice, it is commonly used for the treatmtablent of ascites and oliguria. The present study aimed to evaluate the toxicity and efficacy of different fractions of VEK and reveal the underlying material basis by employing an animal model of malignant ascites effusion (MAE) in rats. MATERIALS AND METHODSTA: The MAE rats as the model were constructed in SPF male wistar rats by intraperitoneal injection of Walker-256 tumor cells. The MAE rats were used and randomly divided into the control group (normal rats), control groups with different fractions (VEKA, VEKB, VEKC and VEKD), model group (MAE rats), positive control group (model group with furosemide), model groups with different fractions (VEKA, VEKB, VEKC and VEKD). Histopathological observation was used to confirm Walker-256 tumor-bearing organ injuries in rats. For the efficacy evaluation, the ascites and urine volumes, the urinary electrolyte concentrations (Na+, K+ and Cl-) and pH, the ascites levels of pro-inflammatory cytokines (IL-2, IL-6, TNF-α, IFN-γ and VEGF), PRA, the serum levels of Ang II, ALD and ADH, as well as AQP8 protein expression in the gastrointestinal tract were detected. Furthermore, different levels of indicators were measured in the toxicity evaluation of different fractions both on normal and model rats, including serum liver enzymes (AST and ALT), serum oxidative damage parameters (GSH, MDA, LDH and SOD), expressions of inflammatory parameters (NF-κB, ICAM-1 and E-cadherin) and apoptosis signals (caspase-3, -8, -9, Bcl-2 and Bax) in the liver and gastrointestinal tract. RESULTS: Walker-256 tumor-bearing malignant ascites effusion rats showed obvious hepatic and gastrointestinal injuries by histopathological observation. In the efficacy evaluation, model rats treated with VEKB and VEKC showed significant urine increase (VEKB, P < 0.01; VEKC, P < 0.01) and ascites reduction (VEKB, P < 0.01; VEKC, P < 0.01). These two fractions also balanced the concentrations of Na+, K+ and Cl- in urine (VEKB, all P < 0.05; VEKC, all P < 0.05), remarkably decreased urinary pH (VEKB, P < 0.01; VEKC, P < 0.01), and reduced the ascites levels of IL-2, IL-6, TNF-α, IFN-γ and VEGF (VEKB, all P < 0.01; VEKC, all P < 0.01) in the model rats. Moreover, levels of PRA, the serum Ang II, ALD and ADH of model rats were decreased after treated by VEKB and VEKC (VEKB, all P < 0.05; VEKC, all P < 0.05). Meanwhile, the expression of gastrointestinal AQP8 of the model rats was also enhanced after treated by VEKB and VEKC (VEKB, P < 0.01; VEKC, P < 0.01). In the toxicity evaluation, although VEKB and VEKC caused toxic indexes moved to the worse aspects in normal rats, nearly all of these indicators notably improved in the model rats. Additionally, VEKA showed no effect on the indicators, either in the efficacy evaluation or in the toxicity evaluation. And VEKD could significantly improve some indicators (urine volume, concentration of K+ in urine, serum MDA, AI and caspase-9) in MAE rats. CONCLUSIONS: VEKB and VEKC were demonstrated a significant efficacy in treating malignant ascites effusion, which could reduce hepatic and gastrointestinal damage on the model rats but cause the same damage to the normal. These data embody the traditional Chinese medicine application principle: You Gu Wu Yun. And these results will provide reference for the safer and better clinical utilization of kansui.

Anti-thrombotic and pro-angiogenic effects of Rubia cordifolia extract in zebrafish.

ETHNOPHARMACOLOGICAL RELEVANCE: Rubia cordifolia is a common traditional Chinese medicine that promotes blood circulation and eliminates blood stasis, and has been used to cure diseases related to blood stasis syndrome (BSS) clinically for many years. It has been previously demonstrated that anti-thrombosis and pro-angiogenesis can improve BSS. However, the anti-thrombotic and pro-angiogenic activities of Rubia cordifolia have not been well investigated. AIM OF STUDY: To determine the potential anti-thrombotic and pro-angiogenic activities of Rubia cordifolia and to elucidate the underlying mechanisms. In addition, the major chemical constituents of Rubia cordifolia extract (QC) were qualitatively analysed by UPLC-Q-TOF/MS to explore the association between pharmacological activity and chemical constituents. MATERIAL AND METHODS: The QC samples were composed of a 95% ethanol extract and an aqueous extract following extraction using 95% ethanol. UPLC-Q-TOF/MS was used to analyse the major chemical constituents of QC. For the anti-thrombotic experiment of QC, a phenylhydrazine (PHZ)-induced AB strain zebrafish thrombosis model was used. The zebrafish larvae were stained using O-dianisidine, and the heart and caudal vein of the zebrafish were observed and imaged with a fluorescence microscope. The staining intensity of erythrocytes in the heart (SI) of each group and the morphology of thrombus in the caudal vein were used to assess the anti-thrombotic effect of QC. For the pro-angiogenic assay of QC, the intersegmental blood vessel (ISV) insufficiency model of Tg(fli-1: EGFP)y1 transgenic zebrafish (Flik zebrafish), which was induced by the VEGF receptor tyrosine kinase inhibitor II (VRI), was used. The morphology of the intact ISVs and defective ISVs was observed to evaluate the pro-angiogenic activity of QC. The mechanism involved in promoting angiogenesis was studied with real-time PCR. RESULTS: A total of 12 components in QC were identified based on standard compounds and references, including nine anthraquinones and three naphthoquinones. After treatment with QC, the PHZ-induced thrombosis in AB strain zebrafish larvae decreased to a certain degree, which we believe was related to its dosages, and the therapeutic effect within the 50-200 µg/mL QC treatment groups was especially prominent (P < 0.01, P < 0.001) compared to that in the PHZ model group. Similarly, QC also recovered the loss of the ISVs, which was induced by VRI in Flik zebrafish larvae, which have a certain dose-effect relationship. The pro-angiogenic activity of QC was also conspicuous (P < 0.01, P < 0.001) compared to that of the VRI model group. The following real-time PCR assay proved that QC significantly restored the VRI-induced downregulation of vWF, VEGF-A, kdrl, and flt-1 in Flik zebrafish (P < 0.05, P < 0.01, P < 0.001). CONCLUSIONS: A total of 12 compounds from QC were analysed by UPLC-Q-TOF/MS. The data of the pharmacological experiments demonstrated that QC presented anti-thrombotic and pro-angiogenic activities in zebrafish, and the principal active components were likely anthraquinones and naphthoquinones. Thus, the current study provided a theoretical basis for the clinical use of Rubia cordifolia as a traditional Chinese medicine in promoting blood circulation and eliminating stasis.