Chemical profile and miscarriage prevention evaluation of Jiao-Ai Decoction, a classical traditional Chinese formula.

Jiao-Ai Decoction (JAD), a classical traditional Chinese formula composed of seven Chinese herbs, has been widely used in clinical practice for the treatment of abortion for a long time. However, the material basis and pharmacological mechanism remain unclear. An integrative method combining ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) analysis and therapeutic effect evaluation based on the hypothalamus-pituitary-ovarian axis (HPOA) was employed to elaborate these problems. Firstly, the chemical profile of JAD was identified by UPLC-Q-TOF-MS. Secondly, the main target ingredients from JAD were determined by UPLC-T-Q-MS. Finally, the miscarriage prevention of JAD on threatened abortion pregnant rats induced by mifepristone was investigated. Threatened abortion model in rats were replicated, uterine bleeding quantity (UBQ) and histopathological sections were measured, the contents of luteinizing hormone (LH), follicular stimulating hormone (FSH), estradiol (E2) and progesterone (P) were determined by ELISA, related genes and protein expression levels were detected by RT-PCR and western blotting. As a result, a total of 101 compounds were identified and 27 ingredients were determined to evaluate the quality of JAD. In the model rats, JAD could effectively regulate the HPOA to achieve miscarriage prevention, and the mechanism might be related to the regulation of gene and protein expression on the HPOA. This work could provide a novel and valuable approach for the quality evaluation of JAD and were expected to provide ideas and methods for the basic research on the scientific application of similar traditional Chinese medicine prescriptions.

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.

Simultaneous determination of twelve quinones from Rubiae radix et Rhizoma before and after carbonization processing by UPLC-MS/MS and their antithrombotic effect on zebrafish.

Rubiae Radix et Rhizoma (called "Qiancao", QC), the root and rhizome of Rubia cordifolia L., has been widely used in clinical practice for its excellent performance in removing blood stasis and haemostasis. However, after carbonization processing, significant changes occurred in chemical components of the charcoal of Rubiae Radix et Rhizoma (called "Qiancaotan", QCT), which enhanced the performance in haemostasis and weakened the performance in removing blood stasis in clinic. In order to study the material basis of function variation during processing, a rapid, reliable, accurate and validated UPLC-MS/MS approach was established to determine twelve quinones in QC and QCT simultaneously. Meanwhile, the antithrombotic effect of target components on zebrafish thrombus model induced by phenylhydrazine (PHZ) was investigated. Chromatographic separation was accomplished on an ACQUITY UPLC C18column with acetonitrile-water containing 0.2 % (v/v) formic acid as mobile phase, at a flow rate of 0.30 mL/min. Quantitation was performed using multiple reaction monitoring (MRM) in positive and negative ion electrospray ionization (ESI). Furthermore, the activity evaluation studies showed that the reduction of removing blood stasis effect of QCT was due to the decrease of dehydro-α-lapachone, lapachol, rubioncolin C and mollugin. This study demonstrated that the method has been successfully applied to determine the content of twelve quinones responsible for the function variation of QCT, and provided a new insight into the material basis and the effect of eliminating stasis before and after processing of QC.

Simultaneous Determination of Quercitrin, Afzelin, Amentoflavone, Hinokiflavone in Rat Plasma by UFLC-MS-MS and Its Application to the Pharmacokinetics of Platycladus orientalis Leaves Extract.

Leaves of Platycladus orientalis have been used as blood cooling and homeostatic therapy for thousands of years in traditional Chinese medicine. Emerging evidences of modern pharmacology have proved flavonoids as the key elements responsible for the efficacies. However, there has been no report on pharmacokinetic study of the flavonoids from Platycladus orientalis leaves extract. In this study, a sensitive and rapid ultra-flow liquid chromatography-tandem mass spectrometry method was established and validated for the simultaneous determination of amentoflavone, afzelin, hinokiflavone and quercitrin in rat plasma. The four flavonoids and luteolin (internal standard, IS) were recovered from rat plasma by methanol-ethyl acetate (v:v, 50:50). Chromatographic separation was performed on a C18 column with gradient elution. Our results showed that the recoveries from spiked control samples were more than 85% for all analytes and IS. The relative standard deviations of intra-day and inter-day precision were within 15% while the REs ranged from -6.6% to 8.0%. The validated method in this study was successfully applied to pharmacokinetic study in healthy rats after oral administration of P. orientalis leaves extract.

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.

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.

[Comparative study on acute toxicity of different active ingredient fractions from Kansui stir-baked with vinegar on zebrafish embryos].

The 24 h normal developing zebrafish embryos were used to evaluate the acute toxicity and the compounds of respective fractions were analyzed by UFLC-Q-TOF-MS simultaneously. Nine concentration groups with respective concentration and a blank control group were designed for each fraction to investigate their effect on survival rates of zebrafish embryos 96 h after drug administration, and calculate the median lethal concentration (LC50) of different fractions to zebrafish embryos. The results showed that all of the fractions had acute toxicity to zebrafish embryos except VEKD, and the order was as follows: VEKB, VEKC, VEKA and VEKD. According to the results of UFLC-Q-TOF-MS, the chemical ingredients contained in VEKB and VEKC were mainly composed of ingenane-type and japhane-type diterpenoids, respectively. It could be speculated that japhane-type diterpenoids might be the active compounds with lower toxicity associated with the results of toxicity study, providing some references for the further research on effective material basis of Kansui stir-baked with vinegar according to the principle of "drastic medicine, no death risks".

[Changes of chemical constituents in Rubiae Radix et Rhizoma before and after carbonized by UPLC-Q-TOF-MS method].

In order to explore the effect on chemical constituents after carbonized, the changes of chemical constituents in raw and carbonized Rubiae Radix et Rhizoma were analyzed by UPLC-Q-TOF-MS. The research also used principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) for data statistics to find out the main differences on components before and after carbonized. The accurate m/z values of Q-TOF-MS and Q-TOF-MS-MS fragments were applied to identify the structures. The results showed that 6 more discrepant constituents were existed between raw and carbonized Rubiae Radix et Rhizoma. Three constituents were selected as the main discrepant components according to the peak area (276 nm) and identified, as lucidin, xanthopurpurin and 1,3,6-trihydroxy-2-methylanthraquinone. After carbonized, contents of xanthopurpurin and 1,3,6-trihydroxy-2-methylanthraquinone were observably increasing, while lucidin was obviously decreasing. They could be used as the chemical markers for the differentiation between raw and carbonized Rubiae Radix et Rhizoma. The results of this experiment played an important role in the study of processing principle of carbonized Rubiae Radix et Rhizoma. It also provided important evidences for the interpretation of effective material based on carbonized Rubiae Radix et Rhizoma.

[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.

[Protective effect of different polar fractions of Carbonized Rubiae Radix et Rhizoma (cRRR) against oxidative injury].

The protective effect of different polar fractions of Carbonized Rubiae Radix et Rhizoma (cRRR) against ox-LDL-induced damage to human umbilical vein endothelial cells (HUVECs) was investigated by MTT assay, and the components were identified by using UPLC-Q-TOF-MS. According to the study, ethyl acetate extract and n-butanol extract could increase cell viability (P<0.01), while petroleum ether extract had no influence, and water extract could even inhibit the cell viability to some degree. Moreover, 32 compounds in four polar fractions were analyzed, including 31 quinones and their glycosides, and one rubiprasins C. Petroleum ether extract, ethyl acetate extract, n-butanol extract and water extract contained 23, 32, 26, 15 compounds, respectively. According to cell experiments in vitro, active fractions were ethyl acetate extract and n-butanol extract. The results could provide scientific references for further studies on effective material basic of cRRR, and lay a foundation for studies on the relationship between efficacies and materials.

Anti-inflammatory effect of volatile oil from Schizonepeta tenuifolia on carrageenin-induced pleurisy in rats and its application to study of appropriate harvesting time coupled with multi-attribute comprehensive index method.

ETHNOPHARMACOLOGICAL RELEVANCE: Schizonepeta tenuifolia Briq. (ST) herbs have been used as a Traditional Chinese Medicine (TCM) for treating colds for thousands of years. The volatile oil is considered as the main material basis responsible for the efficacy of ST and has attracted lots of attention on its anti-inflammatory effect recently. AIM OF STUDY: This paper investigated the anti-inflammatory effects of the distilled volatile oils from Schizonepeta tenuifolia (STVO) that were collected at different harvesting times on carrageenin-induced pleurisy in rats. Based on the anti-inflammatory effects, instead of contents of marker components, we aim to evaluate the quality and determine the appropriate harvesting time of ST. MATERIALS AND METHODS: There were eleven groups with eight male rats in each randomly. They were model group, control group, positive group (dexamethasone) and eight groups treated with eight STVOs at different harvesting times. All treatments were performed by gavage and administered once a day on four consecutive days. One hour after the last treatments, except the rats in control group, those in other groups were treated with carrageenin to induce the pleurisy. Four hours later, all the rats were sacrificed and their pleurisy exudates and lung tissues were collected for further analysis. To evaluate the comprehensive anti-inflammatory effect of the eight STVOs, multi-attribute comprehensive index method (MACIM) was used to obtain the integration of various effects. RESULTS: All the eight STVOs could decrease the seven indicators relating to pleurisy, which were exudate volume, leukocytes, protein level, myeloperoxidase (MPO), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α) and interleukine-1ß (IL-1ß). Majority of these decreases were significant (P<0.01, P<0.05). As far as each indicator was concerned, each STVO showed different effect from others. With MACIM, it was found that STVO in Group 6 was the most effective one with the highest Vs and appropriate harvesting time of ST was in late September. CONCLUSIONS: The study may provide scientific basis to further understanding of the mechanism of STVO in anti-inflammatory effect of carrageenin-induced pleurisy. Meanwhile, this study also provides a new access to determining the appropriate harvesting time of TCM and even evaluating the quality of TCM.

[Study on toxicity of vinegar-processed Kansui Radix on basis of symptom-based prescription theory].

OBJECTIVE: To study the differences in the toxicity of vinegar-processed Kansui Radix on normal and cancerous ascites model rats. METHOD: Normal and cancerous ascites model rats were taken as the research objects and orally administered with different doses of vinegar-processed Kansui Radix for 7 d. Pathological sections were prepared to observe the damages in liver, stomach, intestinal tissues in rats and detect the impacts on serum, liver, stomach and intestinal tissues and the oxidative damage index. RESULT: Compared with the blank group, all of normal administration groups and model groups showed significant damages in liver, stomach and intestinal tissues. Compared with the model groups, all of normal administration groups revealed notable alleviation in damages. Compared with the blank group, the model groups showed significant increases in AST, ALT and MDA in serum and liver (P < 0.01) and a significant decrease in GSH in serum and liver, stomach, intestinal tissues (P < 0.01). Compared with the blank group, the results showed significant decreases in ALT, AST in serum and ALT in liver in model low, medium and high dose groups and AST activity in liver tissues in the normal high dose group (P < 0.05, P < 0.01); significant decreases in GSH in serum and stomach tissues in normal low, medium and high dose groups and GSH content in liver and intestinal tissues in normal medium and high dose groups (P < 0.05, P < 0.01); notable rises in MDA in liver tissues in normal low, medium and high dose groups and MDA content in serum and stomach and intestinal tissues in normal medium and high dose groups (P < 0.05, P < 0.01). Compared with model groups, data revealed significant decreases in ALT, AST in serum in model low, medium and high dose groups, AST in liver tissues of model medium and high dose groups and ALT activity in liver in the model high dose group (P < 0.05, P < 0.01); significant increases in GSH content in serum and stomach tissues of model low, medium and high dose groups, GSH in liver tissues in model medium and high dose groups and GSH in intestinal tissues in the high dose groups (P < 0.05, P < 0.01); and notable declines in MDA content in serum in model low, medium and high dose groups, MDA in liver tissues of model medium and high dose groups and MDA in stomach and intestinal tissues the high dose group (P < 0.05, P < 0.01). CONCLUSION: According to the study, vinegar-processed Kansui Radix showed a significant lower toxicity liver, stomach, and intestines of cancerous ascites model rats, which provided a basis for clinical safe application of vinegar-processed Kansui Radix based on symptom-based prescription theory.

[Filtration of active fractions with function of expelling water retention with drastic purgative from Kansui Radix stir-baked with vinegar].

To study the function of expelling water retention with drastic purgative of different polarities of Kansui Radix stir-baked with vinegar on the cancerous ascites model rats, the furosemide was taken as positive control drug, and the cancerous ascites model rats were respectively orally administered with different polarities of Kansui Radix stir-baked with vinegar for 7 d. The amount of urine and ascites, the level of urinary sodium, potassium, chloride ion and pH, and the content of PRL1, AII, ALD in serum were investigated. Compared with model groups, ethyl acetate extract group showed a decreasing trend in ascites; the amount of urine of showed a significant increase (P < 0.05); the level of urinary sodium, potassium, chloride ion (P < 0.05, P < 0.01), pH (P < 0.05), and the content of PRL1, AII, ALD in serum all showed a significant decrease (P < 0.01). The effects of petroleum ether extract and n-butanol extract were weaker than that of ethyl acetate extract. The water exact was the weakest. The results showed that ethyl acetate extract is the active part of Kansui Radix stir-baked with vinegar on the function of expelling water retention with drastic purgative on the cancerous ascites model rats, alleviating the water-electrolyte disorder and body fluid acid-base imbalance, regulating the renin angiotensin aldosterone system.

[Filtration of active fractions with hemostasis effect from platycladi cacumen carbonisatum].

OBJECTIVE: To discuss the hemostasis of the different polarities of Platycladi Cacumen Carbonisatum (PCC) on the blood heat and hemorrhage syndrome rat model induced by dry yeast. METHOD: The SD rats were divided into seven groups. Yunnan Baiyao was taken as the positive control drug. The rats in the control group and model group were fed with CMC-Na for 7 days, and the rats in other groups were fed with corresponding drugs simultaneously. On day 7, the blood heat and hemorrhage syndrome rat model was established. Indexes including the whole blood viscosity, plasma viscosity, thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen content (FIB), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), blood platelet count (PLT), thrombocytocrit (PCT), mean platelet volume (MPV), platelet distribution width (PDW) and the rate of platelet aggregation induced by ADP were detected. Additionally, the pathological examinations of lungs among each group were compared. RESULT: Compared with the control group, the RBC, HGB and HCT of rats in the model group increased significantly, with distinct increase in high, middle and low whole blood viscosity and plasma viscosity of rats in the model group; TT and APTT were notably prolonged, while PT was notably shortened, with significant increase in FIB content; PLT, PCT, MPV and PDW remarkably increased; Additionally, the rate of platelet aggregation induced by ADP significantly decreased. After ig administration of the ethyl acetate extract of PCC, the low whole blood viscosity and plasma viscosity remarkably decreased; TT and APTT were significantly shortened, with notable reduction in PDW and in FIB content Additionally, the rate of platelet aggregation induced by ADP significantly increased. The injury of lungs was also improved in ethyl acetate extract group. The rate of platelet aggregation induced by ADP of n-butanol extract group notablly increased. Plasma viscosity of water extract group remarkably decreased, with TT being significantly shortened. But the effects of n-butanol extract or water extract were weaker than that of ethyl acetate extract. And the effect of petroleum ether extract was the weakest. CONCLUSION: Ethyl acetate extract is the active part of PCC, showing the effect of hemostasis by reducing the low whole blood and plasma viscosity, improving coagulation function mainly by acting on the endogenous coagulation, and ameliorating the function of platelet aggregation.

[HPLC specific chromatograms of drug pair of schizonepetae herba and saposhnikoviae radix].

In this experiment, the HPLC specific chromatogram was adopted, with Agilent Extend-C18 (4.6 mm x 250 mm, 5 microm) as the chromatographic column, and 0.5 per thousand trifluoroacetic acid and acetonitrile as the mobile phase for gradient elution, so as to establish specific chromatograms for drug pair of Schizonepetae Herba and Saposhnikoviae Radix from different producing area, identify 12 common characteristic peaks, and obtain the comparison specific chromatography of drug pair of Schizonepetae Herba and Saposhnikoviae Radix. The method is simple, accurate and highly reproducible, and thus can be used as the basis for the quality control of the drug pair.

[Comparative study on effects of Rubiae Radix et Rhizoma and carbonized Rubiae Radix et Rhizoma on acute blood stasis rat model].

OBJECTIVE: To observe the effects of Rubiae Radix et Rhizoma (RRR) and carbonized Rubiae Radix et Rhizoma (CRRR) on the acute blood stasis rat model, and reveal their differences in efficacy. METHOD: The acute blood stasis model was induced by subcutaneously injecting adrenaline hydrochloride and soaking in ice water. Yunnan Baiyao was used as the positive control drug, and administered for consecutively seven days. This model was adopted to observe the effect of high, middle and low dose RRR and CRRR groups on hemorheology, thrombin activity, and blood platelet system. RESULT: RRR could significantly reduce the wholeblood viscosity and plasma viscosity of blood stasis rats under different shear rates, and showed certain two-way regulating function in hemostasis. It also showed certain effect on ADP-induced platelet aggregation rate, but which was lower than CRRR. CRRR achieved the main hemostatic mechanism by stimulating intrinsic and extrinsic blood coagulation and fibrinogen, and could significantly enhance the platelet aggregation rate of rats in the acute blood stasis model (P <0. 01). CONCLUSION: RRR had the effect of removing blood stasis and hemostasis, while CRRR mainly has the hemostatic effect. This further demonstrates the traditional processing theory of "promoting blood circulation with crude herbs and stopping bleeding with processed herbs".

[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.

Platycladus orientalis leaves: a systemic review on botany, phytochemistry and pharmacology.

Platycladus orientalis leaves (Cebaiye) have been used for thousands of years as traditional Chinese medicine (TCM). According to the theory of TCM, they are categorized as a blood-cooling and hematostatic herb. In clinical practice, they were usually prescribed with heat-clearing herbs to reinforce the efficacy of hemostasis. The review provides the up-to-date information from 1980 to present that is available on the botany, processing research, phytochemistry, pharmacology and toxicology of the leaves. The information is collected from scientific journals, books, theses and reports via library and electronic search (Google Scholar, Pubmed and CNKI). Through literature reports, we can find that the leaves show a wide spectrum of pharmacological activities, such as anti-inflammatory, antioxidant, antimicrobial, disinsection, anticancer, diuretic, hair growth-promoting, neuroprotective and antifibrotic activities. Diterpene and flavonoids would be active constituents in P. orientalis leaves. Many studies have provided evidence for various traditional uses. However, there is a great need for additional studies to elucidate the mechanism of blood-cooling and hematostatic activity of the leaves. Therefore, the present review on the botany, traditional uses, phytochemistry and toxicity has provided preliminary information for further studies of this herb.

Analysis of isorhamnetin-3-O-neohesperidoside in rat plasma by liquid chromatography/electrospray ionization tandem mass spectrometry and its application to pharmacokinetic studies.

AIM: To establish an LC-MS/MS method for determination of isorhamnetin-3-O-neohesperidoside and investigate its application on pharmacokinetic study in rats. METHODS: Eight rats were given 5 mg·kg(-1) isorhamnetin-3-O-neohesperidoside after intravenous administration. A highly sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of isorhamnetin-3-O-neohesperidosidein rat plasma using rutin as internal standard. The analytes and rutin (internal standard) were extracted with methanol followed by a rapid isocratic elution with 10 mmol·L(-1) ammonium acetate buffer/methanol (20 : 80, V/V) on a C18 column (150 mm × 2.1 mm, I.D., 5 µm) and subsequent analysis by mass spectrometry in the multi-eaction-monitoring mode. RESULTS: The assays were linear over the concentration range of 0.01-10 µg·mL(-1) for isorhamnetin-3-O-neohesperidosidein rat plasma. The lower limit of quantifications for isorhamnetin-3-O-neohesperidoside was 0.01 µg·mL(-1). CONCLUSION: The validated method is successfully applied to determine the plasma concentrations of isorhamnetin-3-O-neohesperidosidein in rats.

[Study on reducing mechanism of hepatotoxicity induced by ethyl acetate fractions of kansui radix stir-baked with vinegar in mice].

OBJECTIVE: To study the mechanism of the reducing mechanism of hepatotoxicity induced by ethyl acetate fractions of Kansui Radix stir-baked with vinegar in mice. METHOD: Mice with normal ICR were orally administered with ethyl acetate fractions of Kansui Radix and Kansui Radix stir-baked with vinegar. Their blood and liver homogenate were collected to detect the level of AST, ALT, LDH, SOD, activities of Na(+) -K(+) -ATPase and Ca(2+) -Mg(2+) -ATPase, GSH and MDA. Liver tissues were collected for HE staining and morphological observation under light microscope. RESULT: According to the results of pathological sections, compared with the control group, all of Kansui groups showed a significant increase in the hepatic tissues injury (P < 0.01). Compared with Kansui groups, all of vinegar-baked groups showed a significant decrease in the hepatic tissues injury (P < 0.01). Compared with the control group, all of Kansui groups showed a significant increase in ALT, AST and LDH (P < 0.05, P < 0.001) in serum and hepatic tissues, and significantly decrease in the activity of SOD (P < 0.001) and the content of GSH. They also showed a significant increase in MDA (P < 0.001) and a significant decrease in the level of Na(+) -K(+) -ATPase and Ca(2+) -Mg(2+) -ATPase (P < 0.01) in hepatic tissues, with a certain dose-effect relationship. Compared with all of Kansui groups, all of vinegar-baked groups showed a significant decrease in ALT, AST and LDH (P < 0.05, P < 0.001), and a notable increase in SOD (P < 0.001) and GSH in serum and hepatic tissues. They also showed a remarkable decrease in MDA (P < 0.001), and a significant increase in the level of Na(+) -K(+) -ATPase and Ca(2+) - Mg(2+) -ATPase (P < 0.01) in hepatic tissues, with a certain dose-effect relationship. CONCLUSION: Being stir-baked with vinegar can significantly reduce the hepatotoxicity of Kansui Radix. Its mechanism may be related to the reduction of the effect of Kansui Radix on the permeability of hepatic tissues cell membranes and the oxidative injury.