Objective: To observe the protective effect of Salvia miltiorrhizae and Ligusticum chuanxiong effective constituents: danshensu, protocatechuic aldehyde, ligustrazine, and ferulic acid combination on primary cultured hippocampal neurons injured by oxygen glucose deprivation, and find out an optimized combination. Methods Primary cultured rats hippocampal neurons waschosen as research objects by adopting immunohistochemistry of the neuron-specific enolase IgG to authenticate, then the OGD model of the hippocampal neurons injured by oxygen glucose deprivation was established. The non-cytotoxic dose range of danshensu, protocatechuic aldehyde, ligustrazine, ferulic acid, and nimodipine was studied by MTT method. The compatibility of components was arranged by L9 (34) orthogonal design. Primary cultured rats hippocampal neurons was divided into 12 groups: control group, model group, Nimodipine positive control group, and orthogonal design 1-9 group. The activity of LDH was measured by colorimetry, the activity of SOD was tested by WST-1 and the levels of MDA were examined by TBA. The levels of TNF-α, IL-1β, and IL-6 in cell culture supernate were examined by ELISA, the apoptosis of hippocampal neurons was detected by Fluorochrome Hoechst33258 staining and the cell early apoptosis rate was detected with flow cytometry. The results of orthogonal test was analyzed by using range analysis method. Results The orthogonal compatibility of reagents played significant roles against the hypoxia damage of hippocampal neurons, improved the cellular morphology obviously, reduced the activity of LDH, increased the activity of SOD and decreased the content of MDA significantly, inhibited the release of TNF-α, and reduced the content of IL-1β and IL-6, reduced the apoptosis of cells apparently. The effect of active ingredients of S. miltiorrhiza and L. Chuanxiong on LDH activity was danshensu > ligustrazine > protocatechuic aldehyde > ferulic acid, and the best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on SOD activity was ferulic acid > ligustrazine > danshensu > protocatechuic aldehyde. The best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (40 μg/mL). The order of influence on MDA content was danshensu > protocatechuic aldehyde > ferulic acid > ligustrazine, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (60 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on TNF-α content were: ligustrazine > protocatechuic aldehyde > danshensu > ferulic acid, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (60 μg/mL), ligustrazine (40 μg/mL), and ferulic acid (10 μg/mL). The order of influence on the content of IL-1β was ligustrazine > ferulic acid > danshensu > protocatechuic aldehyde, and the best combination was danshensu (30 μg/mL), protocatechuic aldehyde (30 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on the content of IL-6 was protocatechuic aldehyde > ligustrazine > ferulic acid > danshensu, and the best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (10 μg/mL). The effect on the early apoptosis rate was ferulic acid > protocatechuic aldehyde > ligustrazine > danshensu, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (30 μg/mL), ligustrazine (20 μg/mL), and ferulic acid (40 μg/mL). Conclusion The protective mechanism of the effective constituents of S. miltiorrhizae and L. chuanxiong were relied on reducing the oxidative damage, reducing inflammation damage, and antagonizing cell apoptosis. According to the experimental results, we need to change the prescription ratio and guide clinical medication for different clinical courses.

Objective: Using total quantum statistical moment analysis to evaluate the influence of the main effective constituents in Danhong Injection on the total pharmacokinetic parameters after orthogonal design to research internal compatibility rule of traditional Chinese medicine (TCM) compound. Methods: Using modified middle cerebral artery occlusion (MCAO) method to establish rat disease model of focal cerebral ischemia-reperfusion (I/R) injury. According to the orthogonal design L9 (34) composed of the main effective constituents (danshensu, protocatechuic aldehyde, salvianolic acid B, and HYSA) of Danhong Injection, nine combinations with different dosage ratios were formed, which were used for iv administration in MCAO rats to study the pharmacokinetics of effective constituents after compatibility. Using non-compartmental model of DAS 3.2.6 software to fit the pharmacokinetic parameters and using total quantum statistical moment analysis method to calculate the main total quantum statistical moment parameters. The influence of different compatibilities on the total pharmacokinetics parameters was analyzed by orthogonal test. Results: There were different effects of the main effective constituents with orthogonal compatibilities on the total quantum statistical moment parameters. Danshensu had the strongest effect on the total AUC and had a significant effect (P < 0.05). A3B3C2D1 was the preferred dose combination for AUCt. HYSA had the strongest effect on the total MRT and its optimal ingredient composition was A1B3C1D1. Conclusion: The total quantum statistical moment analysis could combine the pharmacokinetics of danshensu, protocatechuic acid, vanillic acid, salvianolic acid B, and HYSA, and express the total pharmacokinetic behaviors. The total quantum statistical moment analysis method can be used to study the pharmacokinetics of multi-component in TCM compound prescription, which can provide the method for research on internal compatibility rule for TCM compound.

Objective: To research the pharmacokinetic-pharmacodynamic (PK-PD) model of ligustrazine in cerebral ischemia reperfusion (I/R) rats. Methods: To build the middle cerebral artery embolization (MCAO) model. The blood 0.5 mL was collected from orbital venous plexus at 0.083, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0, and 6.0 h time points after ig administration of effective parts in compatibility prescription of Chuanxiong Rhizoma and Astragali Radix. The concentration of ligustrazine in serum was determined by RP-HPLC, and then the concentration-time curves were drawn. Meanwhile, the activities of LDH in serum were determined with ELISA Kit. PK-PD modeling was fitted with DAS 3.2.6 software. The PK-PD model parameters were calculated. Results: The effect of ligustrazine on inducing LDH release did not relate directly with the concentration but lagged behind the concentration of ligustrazine in serum. The relationship between effect and serum concentration fits Emax model. Conclusion: This study successfully establishes the combined PK-PD model of ligustrazine after ig administration of different combinations of the active parts in Chinese materia medica (CMM) to rats. This research can be effectively applied to predict PK- PD studies on the main effective components in other compatibility of CMM.

OBJECTIVETo compare effects of catgut embedding at "Zusanli" (ST 36) and "Shenshu" (BL 23) on Morphine analgesic tolerance and locomotor sensitization induced by chronic Morphine administration and the mechanism.

METHODSThe rats were randomly divided into a model group, a non-acupoint group, a Shenshu group and a Zusanli group. The rats, except those in the model group, were pretreated with acupoint catgut-embedding 10 days before the first Morphine injection. The Morphine-tolerance model was established and the pain threshold was detected by hot-plate test every day. Locomotor activities were recorded after the first Morphine injection and Morphine-challenging 1 week after withdrawal of Morphine. The positive neurons of nitric oxide synthetase (NOS) were showed by NADPH-d histochemical method.

RESULTSCompared with the non-acupoint group, catgut embedding at "Zusanli" (ST 36) could attenuate the Morphine analgesic tolerance and the increase of locomotor activities in rats. Meanwhile, the expression of NOS positive neurons in nucleus accumbens septi and dorsal striatum decreased in the Zusanli group. There were no significant differences between the Shenshu group and the non-acupoint group in the analgesic threshold and locomotor sensitization, but the expression of NOS positive neurons in the striatum region significantly decreased.

CONCLUSIONCatgut embedding at "Zusanli" (ST 36) can attenuate Morphine analgesic tolerance and reverse formation of locomotion sensitization induced by chronic Morphine administration, which are possibly related with inhibition of the expression of NOS positive neurons in nucleus accumbens septi and dorsal striatum.

Acupuncture Analgesia ; Acupuncture Points ; Analgesics, Opioid ; pharmacology ; Animals ; Catgut ; Drug Tolerance ; Male ; Medicine, Chinese Traditional ; Morphine ; pharmacology ; Motor Activity ; Rats ; Rats, Sprague-Dawley

AIM: To investigate the early changes of retinal function in diabetic patients detected by multifocal electroretinogram (mfERG).METHODS: The first-order kernel responses of mfERG were recorded from eyes of 33 normal control subjects,63 diabetic patients without retinopathy and 43 diabetic patients with background retinopathy. The response densities and implicit times of N1 and P1 were compared among the control, diabetic patients without retinopathy and diabetic patients with retinopathy.RESULTS: The response densities of N1 and P1 in central 3 rings were reduced significantly in diabetic eyes with and without retinopathy. And the implicit times of N1 and P1 were delayed significantly only in diabetic eyes with retinopathy.CONCLUSION: mfERG can detect the early changes of retinal function quantitatively in diabetic patients. Analysis of response densities and implicit times of N1 and P1 can reflect the progress of local retinal dysfunction in diabetes.

OBJECTIVETo explore the differences of electroacupuncture (EA) on onset time and symptom improvement for treatment of different types of functional constipation.

METHODSThirty-eight cases of constipation were selected, including 9 cases of constipation-predominant irritable bowel syndrome (IBS-C), 10 cases of slow transit constipation (STC), 10 cases of pelvic floor dyssynergia (PFD) and 9 cases of inadequate defecatory propulsion (IDP). The electroacupuncture was applied at Tianshu (ST 25), Fujie (SP 14), Shenshu (BL 23), Dachangshu (BL 25) and so on in abdominal and lumbosacral area, 5 times per week, 10 times as a treatment course. The onset time, score of clinical symptoms of constipation and improvement of every symptom in each group were compared.

RESULTS(1) The onset time was (1.78 +/- 0.83) days in IBS-C type, (3.11 +/- 1.90) days in IDP type, (4.10 +/- 1.85) days in STC type and (4.30 +/- 2.00) days in PFD type, indicating statistical differences between IBS-C type and STC type, IBS-C type and PFD type (both P < 0.05). (2) Compared before the treatment, the total scores of symptoms on the onset day in each group were all improved (P < 0.05, P < 0.01), and score of IBS-C type was superior to the rest 3 types (P < 0.05, P < 0.01). (3) EA improved desire to defecate or frequency of defecation in each type (P < 0.05, P < 0.01), in which both were improved in STC type and PFD type, and the improvement of defecation frequency was more significant in STC type (P < 0.01). EA relieved unsmooth defecation or pendant-expansion feeling in each type (all P < 0.05), in which both were improved in IBS-C type (both P < 0.05). EA relieved abdominal distension and pain in IBS-C type, STC type and PFD type (all P < 0.05), while its effects were not obvious on defecation difficulty, defecation time and defecation texture (all P > 0.05).

CONCLUSIONThe electroacupuncture for treatment of 4 types of constipation is characterized by rapid onset; the improved symptoms are not identical in the electroacupuncture treatment plan for each type of defecation; the main improvement of symptoms are lied on desire to defecate and frequency of defecation, unsmooth defecation or pendant-expansion feeling and abdominal distension and pain. Meanwhile the improvements of defecation texture, defecation difficulty and defecation time were not signi-ficant.

Adult ; Aged ; Constipation ; etiology ; physiopathology ; therapy ; Defecation ; Electroacupuncture ; Female ; Humans ; Irritable Bowel Syndrome ; complications ; Male ; Middle Aged ; Pelvic Floor Disorders ; complications ; Treatment Outcome ; Young Adult

To study the protective mechanism of Danhong injection on brain microvascular endothelial cells (rBMECs) injured by hypoxic. In the experiment, primary suckling mouse's rBMECs cells were collected and identified with factor VIII to establish the 4 h injury model. Meanwhile, rBMECs were given Danhong injection (25, 50, 100 mL . L-1), and the superoxide dismutase (SOD) activity and the malonyldialdehyde (MDA) level were detected by the biochemical method. Cell MMP-9, ICAM-1 and P53 mRNA expression levels were detected by RT-PCR method. Changes in cells' microscopic structure were observed by transmission electron microscope. According to the results, primary rBMECs were notably injured by hypoxia. Compared with model group, Danhong injection (50, 100 mL . L-1) could remarkably resist the injury induced by hypoxic, increase intracellular SOD activity, decrease MDA level and significantly down-regulate ICAM-1, MMP-9 and P53 mRNA expressions. Danhong injection (100 mL . L-1) could protect the cells' normal morphology and microscopic structure, maintain the close intercellular junction, and inhibit the hypoxia-induced cell apoptosis. The results showed that Danhong injection plays a significant role in protecting rBMECs injured by hypoxia. Its mechanism may be related to the enhancement of cells' antioxidant capacity, the inhibition of inflammatory response and the cell apoptosis.
Animals ; Antioxidants ; metabolism ; Apoptosis ; drug effects ; Brain ; metabolism ; Cell Hypoxia ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; ultrastructure ; Female ; Gene Expression Regulation ; drug effects ; Humans ; Injections ; Intercellular Adhesion Molecule-1 ; metabolism ; Male ; Malondialdehyde ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; Protective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism ; Tumor Suppressor Protein p53 ; genetics

To study the protective effect of combined administration of active ingredients of Danhong on cultured primary mice's brain microvascular endothelial cells (rBMECs) injured by hypoxia. Primary mice's brain micro-vascular endothelial cells were cultured to establish the 4 h hypoxia model. Meanwhile, active ingredients (protocatechuic aldehyde, salvianolic acid B, hydroxysafflor yellow A and tanshinol) of Danhong were administered in rBMECs. The non-toxic dosage was determined by MTT. The leakage of lactate dehydrogenase(LDH), cell superoxide dismutase (SOD) activity and MDA level were detected by the colorimetric method. The expressions of ICAM-1, MMP-9, P53 mRNA were detected by RT-PCR method. Changes in rBMECs cell cycle and early apoptosis were detected by flow cytometry. Danhong's active ingredients and prescriptions 1, 2, 3, 7, 8, 9 could be combined to significantly restrain LDH in hypoxic cells supernatant. Prescriptions 1, 2, 3, 7, 8, 9 could significantly enhance SOD activity in anoxic cells; Prescriptions 1, 2, 3, 8, 9 could significantly decrease the MDA level; Prescriptions 1, 2, 6, 7, 9 could significantly inhibit the early rB-MECs apoptosis induced by hypoxia. After hypoxia, the up-regulated P53 mRNA expression could cause retardation in G, phase and promote cell apoptosis. This proved that the regulatory function of P53 gene lay in monitoring of calibration points in G, phase. Prescriptions 1, 2, 5, 6, 7, 8, 9 could significantly down-regulate the P53 mRNA expression; Prescriptions 1, 4, 7, 8, 9 could significantly down-regulate the ICAM-1 mRNA expression; Prescriptions 1, 3, 6, 9 could significantly down-regulate the MMP-9 mRNA expression. The combined administration of Danhong's active ingredients showed a significant protective effect on primary cultured rBMECs injury induced by hypoxia Its mechanism may be related to the enhancement of cellular antioxidant capacity and the inhibition of inflammatory response and cell apoptosis. This study could provide ideas for researching prescription compatibility, and guide the clinical medication.
Animals ; Apoptosis ; drug effects ; Brain ; drug effects ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Endothelial Cells ; drug effects ; Hypoxia ; drug therapy ; Microvessels ; drug effects ; Rats ; Rats, Sprague-Dawley

To detect the in vitro probe microdialysis recoveries based on an HPLC-DAD method for simultaneous quantification of nine active ingredients (ephedrine, pseudoephedrine, methylephedrine, amygdalin, liquiritin, cinnamyl alcohol, cinnamic acid, cinnamaldehyde and glycyrrhizic acid) in Mahuang decoction, which provides reference for in vivo pharmacokinetic study. The concentrations of nine active ingredients in dialysate were detected by HPLC-DAD, to investigate the effect of flow rates (incremental method and subtraction method) and intraday stability of the probe recoveries and medium concentrations on the recoveries. Nine active ingredients could be well separated in 52 min. At the perfusion rate of 1.0 μL x min(-1), the relative recoveries of ephedrine, pseudoephedrine, methylephedrine, amygdalin, liquiritin, cinnamyl alcohol, cinnamic acid, cinnamaldehyde and glycyrrhizic acid were (50.95 ± 0.82)%, (52.74 ± 1.13)%, (51.29 ± 0.51)%, (32.56 ± 0.84)%, (45.36 ± 0.83)%, (70.94 ± 0.99)%, (69.98 ± 2.30)%, (71.68 ± 0.63)%, and (22.14 ± 0.48)%, respectively. And the probe kept steady in 7 hours. At the same medium concentration, the probe recoveries decreased exponentially with the increase in flow rates. The recoveries of seven ingredients detected by these two methods were similar at certain flow rates, except for amygdalin and cinnamaldehyde. At the same flow rate, the relative recoveries of cinnamyl alcohol, cinnamic acid and cinnamaldehyde changed greatly (9.55%-16.2%) and the others six ingredients had less change (3.27%-5.71%) with the changes in medium concentrations. Microdialysis method could be used to detect the in vitro recoveries of nine ingredients in Mahuang decoction. Reverse dialysis method could be used for the in vivo probe recovery calibration of ephedrine, pseudoephedrine, methylephedrine, liquiritin, cinnamyl alcohol and cinnamic acid at the flow rate of 2.0 μL x min(-1).
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Ephedra sinica ; chemistry ; Microdialysis ; methods ; Molecular Structure

OBJECTIVETo optimize the prescription dose of Mahuang decoction in a multi-target manner, in order to provide reference for the quantitative optimization of the prescription dose of the traditional Chinese medicine compound.

METHODThe number of diaphoretic spots in rats, the tracheal antispasmodic rate in guinea pigs and the writhing times by acetic acid in mice were taken as the indexes for evaluating the diaphoretic, antispasmodic and analgesic effects. According to the experimental results of the 16 orthogonal combination prescriptions, a mathematical dose-effect model was built by support vector regression (SVR) and quadratic response surface regression (RSR) respectively. The multi-target optimization was achieved by elitist non-dominated sorting genetic algorithm (NSGA-II) and entropy weight TOPSIS method.

RESULTThe optimal dose of Mahuang decoction after being optimized by SVR modeling contained 17.71 g of Ephedrae Herba, 9.57 g of Cinnamomi Ramulus, 11.75 g of Armeniacae Semen Amarum and 4.39 g of Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle. The optimized result by RSR modeling contained 13.37 g of Ephedrae Herba, 11.61 g of Cinnamomi Ramulus, 11.98 g of Armeniacae Semen Amarum and 5.67 g of Glycyrrhizae Radix et Rhizoma Praeparate Cum Melle. SVR was superior to RSR in both of the forecast capacity and optimization results.

CONCLUSIONSVR-NSGA-II-TOPSIS method could be adopted for the multi-target optimization for the dose of Mahuang decoction and other traditional Chinese medicine compounds. It is proved to be the optimal prescription with the best efficacy, and could provide scientific quantitative basis for determining the dose of traditional Chinese medicine compound prescriptions and developing new traditional Chinese medicines.

Animals ; Cinnamomum ; chemistry ; Drug Compounding ; methods ; Drug Dosage Calculations ; Drug Prescriptions ; Ephedra ; chemistry ; Ephedra sinica ; chemistry ; Glycyrrhiza ; chemistry ; Guinea Pigs ; Mice ; Rats