Haitong Wan

Synergistic protective effect of astragaloside IV–tetramethylpyrazine against cerebral ischemic-reperfusion injury induced by transient focal ischemia

ETHNOPHARMACOLOGICAL RELEVANCE Astragaloside IV and tetramethylpyrazine have been extensively used in the cardio-cerbrovascular diseases of medicine as a chief ingredient of glycoside or alkaloid formulations for the treatment of stroke and myocardial ischemia diseases. AIM OF THE STUDY To investigate the effects of astragaloside IV (ASG IV) and tetramethylpyrazine (TMPZ) on cerebral ischemia-reperfusion (IR) injury model in rat model. MATERIALS AND METHODS Rats were randomly divided into the following five groups: sham group, IR group and treatment group including ASG IV, ASG IV-TMPZ and nimodipine treatment. The therapeutic effect was evaluated by micro-positron emission tomography (Micro-PET) using (18)F-fluoro-2-deoxy-d-glucose. The neurological examination, infarct volume and the levels of oxidative stress- and cell apoptosis-related molecules were assessed. RESULTS Micro-PET imaging showed that glucose metabolism in the right hippocampus was significantly decreased in the IR group compared to the sham group (P<0.01). ASG IV and ASG IV-TMPZ treatments reversed the decreased glucose metabolism in the model group (P<0.05 and P<0.01, respectively). IR induced the increase of Caspase-3 mRNA levels, MDA content and iNOS activity, but it caused the decrease of SOD activity and Bcl-2 expression compared the sham group (P<0.01). ASG IV-TMPZ and ASG IV reversed the IR-induced changes of these parameters, i.e. the down regulation of Caspase-3 mRNA, MDA content and iNOS activity, and the up regulation of SOD activity and Bcl-2 expression (P<0.05). CONCLUSION This study showed that ASG IV-TMPZ played a pivotal synergistic protective role against focal cerebral ischemic reperfusion damage in a rat experimental model.

Evaluation of the hepatoprotective and antioxidant activities of Rubus parvifolius L.

The hepatoprotective and antioxidant activities of the n-butanol extract of Rubus parvifolius L. (RPL), a widely used medicinal plant, were evaluated. Results demonstrated that RPL extract possessed pronounced hepatoprotective effects against carbon tetrachloride (CCl4)-induced hepatic injury in mice, which was at least partially attributed to its strong antioxidant capacity. Treatment with RPL extract markedly attenuated the increases in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels caused by CCl4 intoxication. It also significantly prevented the decrease in superoxide dismutase (SOD) activity and the increase in malondialdehyde (MDA) content of liver tissue. Meanwhile, histopathological changes of hepatic damage were also remarkably ameliorated. Phytochemical analysis based on high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) revealed the presence of various phenolic compounds, including caffeic acid conjugates, ellagic acid glycosides, and flavonol glycosides, which might be responsible for the hepatoprotective and antioxidant activities of RPL.

Effects of herbal drugs in Mahuang decoction and their main components on intestinal transport characteristics of Ephedra alkaloids evaluated by a Caco-2 cell monolayer model.

ETHNOPHARMACOLOGICAL RELEVANCE Mahuang decoction, Ephedra combined with Cassia twig, Bitter apricot kernel and Prepared licorice, has been widely used as a multi-herb prescription in traditional Chinese medicine (TCM). Many modern pharmacological studies have shown that the compatibility application of these four herbs has promising therapeutic effects on respiratory infection, acute glomerulonephritis and chronic renal failure. However, the underlying principles for governing the formulation of Mahuang decoction remain unknown. In this study, we used a Caco-2 cell monolayer model to explicate the possible compatibility mechanism of Mahuang decoction from the perspective of intestinal absorption. MATERIAL AND METHODS Firstly, the apical-to-basolateral and basolarteral-to-apical transport of the main characteristic active alkaloids in Ephedra, l-ephedrine (LEP), d-pseudoephedrine (DPEP) and l-methylephedrine (LMEP), as a single compound, was investigated. Secondly, the influence of main components in Cassia twig, Bitter apricot kernel and Prepared licorice on the transport of LEP, DPEP and LMEP was investigated. Finally, the bidirectional transport of these three alkaloids in single Ephedra extract, in Mahuang decoction and in drug pair extracts, such as Ephedra-Cassia twig, Ephedra-Bitter apricot kernel, Ephedra-Prepared licorice, was assessed. RESULTS The investigated LEP, DPEP and LMEP could transport through the Caco-2 cell monolayer at a high level, with the efflux ratio (ER) of 1.41, 1.33 and 1.30, respectively, when the cells were treated with each single compound solution. In the presence of verapamil, the permeability from apical side to basolateral side (PAB) of the three alkaloids increased significantly (P<0.05), and their ERs decreased. The treatment of cells with Mahuang decoction and the drug pair extracts from Ephedra-Cassia twig, Ephedra-Bitter apricot kernel and Ephedra-Prepared licorice appreciably decreased PAB of LEP, DPEP and LMEP with increased ERs, compared to the treatment with single Ephedra extract. When concomitant administration with herbal drugs and their main ingredients (including cinnamaldehyde-cinnamyl alcohol-cinnamic acid group, volatile oil from Cassia twig, liquiritin-glycyrrhizic acid group from Prepared licorice, Cassia twig extract, Bitter apricot kernel extract and Prepared licorice extract), was adopted, PAB of LEP, DPEP and LMEP were reduced significantly and the ERs of the corresponding compounds were promoted appreciably. Only amygdalin (from Bitter apricot kernel) had little influence on the transport of Ephedra alkaloids. CONCLUSION The findings indicate that LEP, DPEP and LMEP in Ephedra extract have similar absorption as in the pure solution of each compound. The intestinal absorption of LEP, DPEP and LMEP is through passive diffusion and these compounds may be P-gp substrates. The compatibility application of Cassia twig, Bitter apricot kernel and Prepared licorice, and their main components except amygdalin can suppress the absorption of the three main Ephedra alkaloids across the Caco-2 cell monolayer. On the basis of our results, Cassia twig, Bitter apricot kernel and Prepared licorice in Mahuang decoction decrease the absorption of Ephedra alkaloids, which may alleviate the drastic diaphoretic function and toxicity of Ephedra.

Neuroprotective effect of parthenocissin A, a natural antioxidant and free radical scavenger, in focal cerebral ischemia of rats.

Neuroprotective effects of parthenocissin A (PA), a novel antioxidant and free radical scavenger, were studied in a transient middle cerebral artery (MCA) occlusion model in rats for the first time. The animals were treated intraperitoneally with PA at 2.5, 5 or 10 mg/kg, for both 30 min before MCA occlusion and 6 h after reperfusion. The MCA was occluded for 1 h in anesthetized Sprague‐Dawley rats. Compared with vehicle‐treated controls, MCA occluded animals treated with PA showed dose‐dependent reductions in brain infarction size with improved neurological and motor outcome. Biomedical assay showed that the PA treatment suppressed lipid peroxidation and restored superoxide dismutase (SOD) activity in brain tissue. In addition, the ischemia/reperfusion (I/R) induced elevation of nitric oxide (NO) production and nitric oxide synthase (NOS) activity in brain tissue was also inhibited. Thus, PA demonstrated a neuroprotective effect in the I/R model and the beneficial effects of the compound may result from the reduction of oxidative stress and the inhibition of NO production induced by I/R. The neuroprotective effects of PA have highlighted the potential use of stilbene oligomers in stroke therapy. Copyright

A neuroprotective polysaccharide from Hyriopsis cumingii.

A water-soluble polysaccharide from Hyriopsis cumingii (HCp) was isolated by hot-water extraction and ethanol precipitation. We investigated the preliminary neuroprotective activity of HCp on cerebral ischemia/reperfusion (I/R)-induced rat brain tissue injury using the middle cerebral artery occlusion (MCAO) model. The structural analysis showed that the major component of HCp (HP-I) is an α-(1→4)-d-glucan branched with a single α-d-glucose at C-6 every five residues on average and has a weight-average molecular weight of about 2.65 × 10(5). Pharmacological studies revealed that HCp improves and restores nerve function impairment, significantly increases brain tissue total superoxide dismutase activity, lowers malondialdehyde content effectively, and reduces brain levels of caspase-3 mRNA expression in a dose-effect manner, indicating that H. cumingii polysaccharide possesses significant neuroprotective effects.

Simultaneous quantification of nine major active components in traditional Chinese prescription Mahuang decoction and the influence of herbal compatibility on their contents.

Background: Mahuang decoction (MHD), a famous classic traditional Chinese formula, has been extensively applied for treating cold, influenza, asthma, acute bronchitis, and other pulmonary diseases. However, the interaction among four drugs of MHD has not been clearly deciphered from the aspect of molecular composition. Objective: To assess the quality of MHD and explore the interplay among different prescription drugs. Materials and Methods: A reversed-phase high performance liquid chromatography (RP-HPLC) coupled with diode array detector (DAD) method for the simultaneous separation and determination of nine bioactive components was developed. A somatomedin A (SMA)-phenyl column (4.6 mm × 250 mm, 5 μm) was eluted by a gradient mobile phase contained acetonitrile and 0.05% formic acid-0.05% triethylamine aqueous solution. Four detection wavelengths (210, 252, 278, and 291 nm) were utilized for the quantitative analysis due to the different ultraviolet (UV) spectra of these compounds. Results: Satisfactory separation was obtained for all the components, and the assay was fully validated in respects of linearity, precision, stability, and accuracy. It was found that the calibration curves for all analytes showed good linearity (R2≥ 0.9991) within the test ranges. The relative standard deviations (RSDs) for intra- and interday repeatability were not more than 1.70 and 2.66%, respectively. The spike recoveries of nine components varied from 97.50 ± 1.69 to 99.27 ± 1.37%. Conclusion: The established method was successfully applied to analyze nine active compounds in decoction samples of various drug compatibilities of MHD. The variations of contents were obvious for different combinations, which hinted the mutual promotion or inhibition of componential dissolution among four herbs of MHD.

Effect of ligustrazine on levels of amino acid neurotransmitters in rat striatum after cerebral ischemia-reperfusion injury

This study aimed to evaluate the effect of ligustrazine on levels of amino acid transmitters in the extracellular fluid of striatum following cerebral ischemia/reperfusion (I/R) in male Sprague-Dawley rats. A microdialysis cannula guide was implanted into the right striatum. After recovery, animals underwent a sham operation or middle cerebral artery occlusion (MCAO). Those that developed cerebral ischemia after MCAO were randomized to receive propylene glycol salt water and ligustrazine respectively. Striatal fluid samples were collected from all animals at 15-min intervals after treatment and were subjected to HPLC analysis of aspartic acid, glutamic acid, taurine, and γ-amino butyric acid. Upon the last sample collection, animals were sacrificed and brain tissue specimens were collected for triphenyltetrazolium chloride staining and NeuN staining. Compared with the sham operation, MCAO induced significant neurological deficits and increased striatal concentrations of the four neurotransmitters assessed in a time-dependent manner (P < 0.01). Ligustrazine effectively attenuated the detrimental effects of MCAO on the brain. These observations suggest that ligustrazine as a novel cerebral infarction-protective agent may have potential clinical implications for I/R-related brain damage.

Molecular cloning and characterization of a cDNA encoding cycloartenol synthase from Fritillaria thunbergii Miq.

Fritillaria thunbergii Miq., known as the bulbous plants of the genus fritillaria , produces a large amount of sterols. Homology based polymerase chain reactions (PCRs) with degenerate primers designed from the conserved sequences among the known cycloartenol synthase (CAS) resulted in cloning of a CAS from the young leaves of F. thunbergii Miq . A putative cycloartenol synthase cDNA ( FtCAS ) consists of a 2271 bp open reading frame, which encodes for 756 amino acids. The deduced amino acid sequence shows 82% homology to cycloartenol synthases from Dioscorea zingiberensis . Heterologous expression of the FtCAS in the methylotrophic yeast, Pichia pastoris , resulted in production of cycloartenol. Our results indicate that FtCA S encode cycloartenol synthase.

Effects of Danhong Injection on platelet aggregation in hyperlipidemia rats

ETHNOPHARMACOLOGICAL RELEVANCE Danhong Injection (DHI), a Chinese medical product extracted from Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese), has been reported to have effects on inflammatory, anti-fibrinolytic properties, antithrombotic and decrease blood-lipid. It is extensively used for the clinical treatment of cardiovascular disease. This study aimed to investigate the effects of DHI on blood-lipid levels and platelet aggregation rate in hyperlipidemia rats. MATERIALS AND METHODS Rats were randomly divided into 6 groups: normal control (NC), model control (MC), DHI-treated control at doses of 1.0mL/kg, 2.0mL/kg, 4.0mL/kg, respectively, and Simvastatin positive control at dose of 2.0mg/kg. All DHI treated groups were intraperitoneally injected for 7 days. The effects of DHI on serum triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were evaluated. And platelet activating factor (PAF), platelet membrane glycoprotein IIb/IIIa (GP IIb/IIIa) and 6-keto-prostaglandin F1а (6-K-PGF1а) were determined by enzyme-linked immunosorbent assay (ELISA). Moreover, the expression of prostaglandin I-2 (PGI2), prostaglandin E-2 (PGE2) and thromboxane A2 (TXA2) in liver was determined by real-time PCR. RESULTS Compared with the MC group, the rats treated with DHI had significantly reduced TC, TG, LDL-C, FIB, GP IIb/IIIa and platelet aggregation. Meanwhile, the thrombin time (TT), activated partial thrombin time (APTT), prothrombin time (PT), 6-K-PGF1а was significantly increased. Expression of PGI2 and PGE2 mRNA was significantly increased, whereas the TXA2 was significantly reduced. CONCLUSIONS This study demonstrated that the blood lipid and platelet aggregation has a regulatory effect after DHI treatment. The insights gained from this study will improve understanding of the mechanisms involved in the effect of DHI on hyperlipidemia and the pharmacological rationale for the use of DHI in diseases caused by formation of thrombosis and lipid metabolic disorders.

Protective effect of Danhong Injection combined with Naoxintong Capsule on cerebral ischemia-reperfusion injury in rats

ETHNOPHARMACOLOGICAL RELEVANCE Danhong Injection (DHI) and Naoxintong Capsule (NXT) are renowned traditional Chinese medicine in China. The drug combination of DHI and NXT is frequently applied for the treatment of cardiovascular and cerebrovascular diseases in clinic. However, there had been no pharmacological experiment studies of interaction between DHI and NXT. Due to the drug interactions, exploring their interaction profile is of great importance. MATERIAL AND METHODS In this study, focal cerebral I/R injury in adult male Sprague-Dawley rats were induced by transient middle cerebral artery occlusion (tMCAO) for 1h followed by reperfusion. Rats were divided into 5 groups: sham group, ischemia reperfusion untreated group (IRU), DHI group (DHI 10mL/kg/d), NXT group (NXT 0.5g/kg/d), DHI plus NXT group (DHI-NXT, DHI 10mL/kg/d plus NXT 0.5g/kg/d). All drug-treated groups were respectively successive administrated for 7 days after ischemia/ reperfusion (I/R) injury. The effects on rat neurological function were estimated by neurological defect scores. Brain infarct volumes were determined based on 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Pathological changes in brain tissues were observed using hematoxylin and eosin (H&E) staining and transmission electron microscope (TEM). Levels of nitric oxide (NO), granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) in serum were determined with enzyme-linked immunosorbent assay (ELISA). Immunohisto-chemistry and Western blot were used to detect the expressions of basic fibroblast growth factor (bFGF), von Willebrand factor-microvessel vascular density (vWF-MVD), vascular endothelial cell growth factor (VEGF), transforming growth factor-β1 (TGF-β1), angiogenin-1 (Ang-1), angiogenin-2 (Ang-2) and platelet derived growth factor (PDGF) at day 7 after ischemia/reperfusion (I/R) injury. RESULTS Compared with IRU group and mono-therapy group (DHI group or NXT group), Danhong Injection combined with Naoxintong Capsule (DHI-NXT) group significantly ameliorated neurological deficits scores, infarct volume and pathological change, significantly decreased the overexpression of NO and the level of Ang-1, significantly increased the expressions of VEGF, Ang-2, G-CSF, GM-CSF, bFGF, PDGF, vWF, TGF-β1. CONCLUSION The protective benefits on rat brain against I/R injury were clearly produced when DHI and NXT were used in combination, which provided rational guidance for clinical combined application of DHI and NXT, and this protection maybe associated with the up-regulation expressions of the related chemokines and growth factors of angiogenesis.