Characterization and repair effect of supramolecular conducting hydrogel carrying ligustrazine on spinal cord injury in rats / 中国组织工程研究

BACKGROUND:Based on the concept of the combination of medicine and industry and the advantages of traditional Chinese medicine treatment,the construction of a new composite material loaded with the effective active ingredient of traditional Chinese medicine is a hot research spot in the repair of spinal cord injury,and is expected to become an effective means to solve this problem. OBJECTIVE:To observe the effect of supramolecular conducting hydrogel carrying ligustrazine in repairing spinal cord injury in rats. METHODS:The supramolecular conducting hydrogel carrying ligustrazine was prepared and its microstructure,conductivity,rheology,swelling rate and in vitro release performance were characterized.45 SD rats were divided into 3 groups by random number table method,with 15 rats in each group:no spinal cord injury in the sham operation group;spinal cord injury model was established in the model group;and supramolecular conducting hydrogel carrying ligustrazine was injected into the spinal cord injury area after model establishment in hydrogel group.BBB score was used to evaluate the recovery of hind limb motor function of each group before and 1,7,14,21 and 28 days after modeling,respectively.28 days after the model establishment,the spinal cord tissues were collected and analyzed by hematoxylin-eosin staining,immunohistochemical staining and western blot assay. RESULTS AND CONCLUSION:(1)Under scanning electron microscopy,the supramolecular conducting hydrogel with ligustrazine displayed a three-dimensional micrometer-scale porous network structure with high porosity and a pore size of approximately 100 μm.The conductivity of the hydrogel was 7.66 S/m;the swelling rate was 3 764.42%,and it had certain mechanical stability and injection property.In vitro sustained release experiments demonstrated that the supramolecular conducting hydrogel with ligustrazine sustainably released ligustrazine for more than 800 hours.With the extension of time,the cumulative release of ligustrazine exhibited an increasing trend.(2)With the extension of modeling time,the hind limb motor function gradually recovered in the model group and the hydrogel group,and the hind limb motor function of the hydrogel group was better than that of the model group on 14,21,and 28 days after modeling(P<0.05).Hematoxylin-eosin staining demonstrated that the spinal cord tissue of the model group had cavities and a large number of inflammatory cells could be seen at the stump.In the hydrogel group,some inflammatory cells were infiltrated in the injured area of the spinal cord;the void area of the injured area was reduced;neuron cells appeared in the junction area,and the tissue arrangement was relatively neat.Immunohistochemical staining and western blot assay exhibited that the expression of tumor necrosis factor α and interleukin-6 protein in the rat spinal cord of the hydrogel group was lower than that in the model group(P<0.05),and the expression of neuronal nuclear antigen protein was higher than that in the model group(P<0.05).(3)These findings confirm that the supramolecular conducting hydrogel carrying ligustrazine can promote the repair of spinal cord injury.

Effect of ligustrazine on hypoxic-ischemic encephalopathy in neonatal rats by regulating autophagy through the PINK1/Parkin pathway / 中国当代儿科杂志

OBJECTIVES@#To study the effect of ligustrazine injection on mitophagy in neonatal rats with hypoxic-ischemic encephalopathy (HIE) and its molecular mechanism.@*METHODS@#Neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group with 8 rats, a model group with 12 rats, and a ligustrazine group with 12 rats. The rats in the model group and the ligustrazine group were used to establish a neonatal rat model of HIE by ligation of the left common carotid artery followed by hypoxia treatment, and blood vessels were exposed without any other treatment for the rats in the sham-operation group. The rats in the ligustrazine group were intraperitoneally injected with ligustrazine (20 mg/kg) daily after hypoxia-ischemia, and those in the sham-operation group and the model group were intraperitoneally injected with an equal volume of normal saline daily. Samples were collected after 7 days of treatment. Hematoxylin and eosin staining and Nissl staining were used to observe the pathological changes of neurons in brain tissue; immunohistochemical staining was used to observe the positive expression of PINK1 and Parkin in the hippocampus and cortex; TUNEL staining was used to measure neuronal apoptosis; Western blotting was used to measure the expression levels of the mitophagy pathway proteins PINK1 and Parkin and the autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and ubiquitin-binding protein (P62).@*RESULTS@#Compared with the sham-operation group, the model group had a significant reduction in the number of neurons, an increase in intercellular space, loose arrangement, lipid vacuolization, and a reduction in Nissl bodies. The increased positive expression of PINK1 and Parkin, apoptosis rate of neurons, and protein expression levels of PINK1, Parkin, Beclin1 and LC3 (P<0.05) and the decreased protein expression level of P62 in the hippocampus were also observed in the model group (P<0.05). Compared with the model group, the ligustrazine group had a significant increase in the number of neurons with ordered arrangement and an increase in Nissl bodies, significant reductions in the positive expression of PINK1 and Parkin, the apoptosis rate of neurons, and the protein expression levels of PINK1, Parkin, Beclin1, and LC3 (P<0.05), and a significant increase in the protein expression level of P62 (P<0.05).@*CONCLUSIONS@#Ligustrazine can alleviate hypoxic-ischemic brain damage and inhibit neuronal apoptosis in neonatal rats to a certain extent, possibly by inhibiting PINK1/Parkin-mediated autophagy.

Protective effects of twin drug ST-11 against PC12 cell injury induced by oxygen-glucose deprivation/reperfusion and its mechanism / 中国药房

OBJECTIVE To study the protective effects of ligustrazine-scutellarein twin drug ST-11 on rat adrenal medullary pheochromocytoma PC12 cell injury induced by oxygen-glucose deprivation/reperfusion （OGD/R） and its mechanism. METHODS PC12 cells were divided into blank group， model group， nimodipine group （positive control， 5 μmol/L） and different concentration groups of ST-11 （5， 10， 20 μmol/L）. After 24 hours of pre-administration intervention， all the other groups except the blank group were cultured in glucose-free DMEM culture medium containing 10 mmol/L Na2S2O4 for 4 hours with glucose deficiency and hypoxia. After 4 hours of glucose and oxygen re-introduction， the survival rate of cells in each group， the contents of lactate dehydrogenase （LDH）， catalase （CAT）， glutathione （GSH）， malondialdehyde （MDA） and superoxide dismutase （SOD） in cell supernatant， apoptosis rate， the levels of reactive oxygen species （ROS） and mitochondrial membrane potential （MMP）， the protein expressions of B-cell lymphoma 2 related X protein （Bax）， B-cell lymphoma 2 （Bcl-2） and caspase-3 were all detected in each group. RESULTS Compared with blank group， the cell survival rate， the contents of CAT， GSH and SOD in cell supernatant， MMP level， relative expression of Bcl-2 and Bcl-2/Bax ratio in model group decreased significantly （P＜0.05）， while the contents of LDH and MDA， ROS level， apoptosis rate， relative expressions of Bax and caspase-3 were significantly increased （P＜0.05）. Compared with model group， above indexes of ST-11 groups （except for the protein expression of caspase-3 in 5 μmol/L ST-11 group） were reversed signifi-cantly （P＜0.05）. CONCLUSIONS ST-11 has a certain protec-tive effect on OGD/R-injured PC12 cells， and its effects may be related to reduction of oxidative stress and inhibition of cell apoptosis.

Clinical effect observation of Yangxue-Qingnao granule combined with ligustrazine injection in the treatment of wind-phlegm entering collaterals type of cerebral infarction / 国际中医中药杂志

Objective:To evaluate the clinical efficacy of Yangxue-Qingnao granule combined with ligustrazine injection in the treatment of wind-phlegm entering collaterals syndrome of cerebral infarction. Methods:A total of 96 patients with cerebral infarction and wind-phlegm entering collaterals syndrome to Suixi County Hospital of Traditional Chinese Medicine from January 2019 to December 2019 were selected and randomly divided into two groups by random number table method, with 48 in each group. The control group was given intravenous ligustrazine injection, and the observation group was given Yangxue-Qingnao granule on the basis of the treatment of the control group. Both groups were treated continuously for 2 weeks. TCM syndrome scores were performed before and after treatment, the National Institutes of Health Stroke Scale (NIHSS) was used to assess the degree of neurological impairment, and the Activity of Daily Living Scale (ADL) was evaluated. To evaluate the patient's quality of life, to detect the high-shear viscosity, low-shear viscosity and plasma viscosity of whole blood with an automatic hemorheology instrument. Results:The total effective rate of the observation group was 95.8% (46/48) and that of the control group was 70.8% (34/48). The difference between the two groups was statistically significant ( χ2=9.08, P<0.01). After treatment, the TCM syndrome score of the observation group was significantly lower than that of the control group ( t=3.51, P<0.01), the NIHSS score was significantly lower than that of the control group ( t=34.41, P<0.001), and the ADL score was significantly higher than that of the control group ( t=57.88, P<0.01). After treatment, the observation group's whole blood high shear viscosity [(5.04 ± 0.93)mPa?s vs. (5.64 ± 1.13)mPa?s, t=2.84], whole blood low shear viscosity [(11.32 ± 1.74)mPa?s vs. (13.39 ± 2.23)mPa?s, t=5.07] and plasma viscosity [(1.51 ± 0.33)mPa?s vs. (1.73 ± 0.47)mPa?s, t=2.65] of the observation group were significantly lower than those in the control group ( P<0.05 or P<0.01). Conclusion:Yangxue-Qingnao granule combined with ligustrazine injection can improve the neurological status and quality of life of patients with cerebral infarction syndrome of wind-phlegm entering the collaterals, and improve the clinical efficacy.

Anti-tumor Effect of Ligustrazine Alone and in Combination with Western Medicine： A Review / 中国实验方剂学杂志

With serious environmental problems and the aging of population， the incidence of cancers worldwide has increased dramatically. For modern medicine， cancer is an incurable malignant disease with a high metastasis rate and strong cell proliferation and invasion ability. At present， clinical anti-cancer therapies mainly include chemotherapy and radiotherapy， but most chemotherapy drugs can cause serious side effects. Therefore， Chinese medicine with high safety has been widely used in anti-cancer treatment， which can inhibit the proliferation of cancer cells， improve the quality of life， and prolong life. Ligustrazine is a pyrazine alkaloid isolated and purified from Chuanxiong Rhizoma， which has pharmacological effects， such as anti-fibrosis， anti-oxidation， and anti-tumor. In recent years， ligustrazine， as an index active component of Chuanxiong Rhizoma alkaloids， has gradually become one of the hot spots in clinical medical research due to its pharmacological actions， and its anti-tumor effect has attracted more attention from researchers. The present study mainly investigated the anti-cancer effect of ligustrazine from the induction of tumor cell apoptosis and autophagy， inhibition of cell proliferation and migration， reversal of multidrug resistance of cancer cells， and inhibition of angiogenesis， and the anti-tumor efficacy was observed in various malignancies such as gastric cancer， lung cancer， rectal cancer， bladder cancer， breast cancer， and ovarian cancer. In addition， ligustrazine can be combined with cisplatin， paclitaxel， podophyllotoxin， chalcone， curcumin， and other drugs to exert an anti-tumor effect. This study conducted a literature review based on the anti-tumor mechanism of ligustrazine to provide a reference for the treatment of clinical malignancies.

Effect of ligustrazine on Gas/cAMP/PKA signal activity and airway inflammation in asthmatic rats / 中国药理学通报

Aim To investigate the effect of ligus- trazine on G-protein coupled receptor signal activity in ovalbumin ( OVA)-sensitized and challenged asthmatic rats.Methods This study replicated an OVA-in¬duced juvenile asthmatic rat model, and investigated the effect of ligustrazine on airway hyperresponsive- ness, airway inflammation, lung histopathology, IL-4, IL-5 , 1L-13 , TNF-cx levels in serum, cAMP concentra¬tion in plasma, CREB, PKA, Gas and GRK2 expres¬sion in lung tissues.Results It was showed that the resistance of lung (RL) in asthmatic rats increased, while the pulmonary dynamic compliance ( Cdvn) sig¬nificantly decreased ( P < 0.05 ) compared with the control rats, and the lung tissue injury was serious; the levels of IL-4, IL-5, IL-13, and TNF-cx in serum were significantly increased ( P <0.01 ).The cAMP level in plasma was reduced and the CREB and PKA expres¬ sions were down-regulated significantly ( P < 0.05 ).The Gas expression in lung in asthmatic rats was down- regulated while the GRK2 expression was up-regulated ( P <0.01 ).Ligustrazine coulrl improve the lung inju¬ry and lung function of asthmatic rats, decrease RL and increase Cdvn ( P < 0.05 ) , decrease the levels of 1L-4, 1L-5, 1L-13, and TNF-a in serum (P <0.01) , increase cAMP in plasma and up-regulate the PKA and CREB expression, down-regulate the GRK2 expression and up-regulate the Gas expression significantly ( P < 0.01).Conclusions The above study shows that li¬gustrazine can reduce the AHR, inhibit the inflamma¬tion in asthma, and the up-regulating of Gas/cAMP/ PKA signal activity may be its mechanism.

Inhibitory Effects of Ligustrazine Plus Emodin on Expression of VEGF-related Factors in Ascites Carcinoma Walker-256 Cells / 中国实验方剂学杂志

Objective:To investigate the effects of ligustrazine combined with emodin on angiogenesis of ascites carcinoma Walker-256 cells by observing their inhibition against nuclear factor-<italic>κ</italic>B （NF-<italic>κ</italic>B）， hypoxia-inducible factor-1<italic>α</italic> （HIF-1<italic>α</italic>）， and vascular endothelial growth factor-C （VEGF-C） in HIF signaling pathway. Method:Fifty SD rats were randomly divided into sham operation group， model group， ligustrazine group， emodin group and ligustrazine combined with emodin group. Following the in situ injection of rat ascites carcinoma Walker-256 cells into the liver of normal rats， they were grouped and administered with ligustrazine （10 mg·kg^-1）， emodin （10 mg·kg^-1）， and ligustrazine （10 mg·kg^-1） plus emodin （10 mg·kg^-1） for seven days. Afterwards， the tumor-inoculated liver tissue was sampled from the experimental group and prepared into pathological sections for investigating tumor cell survival and VEGF expression. The <italic>in vitro</italic> hypoxia and hypoglycemia model （oxygen-glucose deprivation model）， hypoxia model， and hypoglycemia model of Walker-256 cells were constructed respectively. In the ligustrazine group， emodin group， and ligustrazine combined with emodin group， three consecutive concentrations that did not affect the proliferation of Walker-256 cells were selected for investigation. The drugs were administered before modeling， and the model treatment lasted for 4 h. The levels of HIF-1<italic>α</italic>， VEGF-C， and NF-<italic>κ</italic>B in the cell culture supernatant of each group were tested. Result:After the rat liver was inoculated with Walker-256 cells， the total liver mass was significantly increased（<italic>P</italic><0.05）， higher than that in the ligustrazine group， the emodin group， or the ligustrazine combined with emodin group（<italic>P</italic><0.05）. Histopathological examination showed that the response of VEGF expression in the liver tissue of each administration group was lower than that of the model group. At the cellular level， the levels of HIF-1<italic>α</italic>， VEGF-C， and NF-<italic>κ</italic>B in oxygen-glucose deprivation model of the ligustrazine group and the ligustrazine combined with emodin group were significantly reduced（<italic>P</italic><0.05）， exhibiting a certain dose-dependent response， followed by the reduction in the hypoxia model. The levels of HIF-1<italic>α</italic> and NF-<italic>κ</italic>B in the oxygen-glucose deprivation model and the hypoglycemia model of the emodin（1×10^-2，1×10^-3 mol·L^-1） group and the ligustrazine combined with emodin（1×10^-2，1×10^-3 mol·L^-1） group were significantly reduced， but there was no significant change in VEGF-C level of the hypoxia model of all the administration groups. Conclusion:Ligustrazine or emodin alone or their combination inhibits the abnormal increase in the weight of rat liver after inoculation with Walker-256 cells and the expression of VEGF in the liver tissue. Ligustrazine and emodin inhibit the protein expression of NF-<italic>κ</italic>B and HIF-1<italic>α</italic>， thereby reducing the gene and protein expression of metastasis-related target VEGF-A activated by HIF-1<italic>α</italic> transcription， restricting tumor cell neovascularization， and inhibiting the invasion and spread of ascites carcinoma cells. Among them， ligustrazine has the most significant effect against hypoxia. Glucose interferes with the effect of ligustrazine. The combination of ligustrazine with emodin is conducive to diminishing the intervention of glucose and stabilizing the inhibition against tumor cells.

Preparation and in vivo and in vitro Characteristic Evaluation of Ligustrazine Ophthalmic Liposome Thermosen- sitive Gel / 中国药房

OBJECTIVE：To prepare Liguatrazine opthalmic liposome therm osensitive gel ，and to investigate its in vivo and in vitro characteristics. METHODS ：The ammonium sulfate gradient method was used to prepare Liguatrazine liposomes. The preparation technology was optimized by using orthogonal test. Using poloxamer P 407 as gel matrix ，Liguatrazine liposomes were prepared into thermosensitive gel. A membraneless model was used to study the dissolution and in vitro drug release of the gel. The modified Franz diffusion cell was used to investigate corneal permeability and further determine corneal hydration value. The effects of the gel on the proliferation of human corneal epithelial cell HCE-T. HE staining and Draize test were used to investigate the stimulatory effects of the gel on corneal cells of the rabbit ，and the histological changes of the eyes were observed. RESULTS ：The optimal preparation technology of Liguatrazine liposome was drug-lipid ratio of 1 ∶ 10（m/m），the ammonium sulfate concentration of 0.2 mol/L，phospholipid-cholesterol ratio of 4∶1（m/m），incubation temperature of 45 ℃. Then ligustrazine opthalmic liposome thermosensitive gel was prepared with 23％ poloxamer P 407 as gel matrix. The gel had good gelatinization temperature. The in vitro drug release and dissolution showed zero-order kinetic characteristics ，and in vitro drug release of the gel was mainly related to dissolution （R2＝0.993 4）. The cumulative transcorneal permeability of the gel was 43.3％ within 6 hours and corneal hydration value was 72.98％. Low and medium concentrations （1，5 mg/L）of Ligustrazine opthalmic liposome thermosensitive gel had no obvious proliferation toxicity to HCE-T cells ，but it showed cytotoxicity at high concentration （10 mg/L）. The mean Draize eyeirritation score of the gel on rabbit cornea was within non-stimulation，and there was no abnormal change in rabbit （No.2018001） corneal histology. CONCLUSIONS ： Prepared Ligustrazine opthalmic liposome thermosensitive gel has a suitable phase transition temperature ，good corneal permeability ，and low corneal irrit ation.

Study on the Effects of Ligustrazine on Gene Expression of Acute Spinal Cord Injury Model Rats Based on Transcriptome Sequencing / 中国药房

OBJECTIVE：To investigate the effects of ligustrazine on ge ne expression of acute spinal cord injury （SCI）model rats. METHODS ：Male SD rats were randomly divided into sham operation group （group A ，6 rats），model group （group B ，6 rats at each time point ，12 rats in total ）and ligustrazine intervention group （group C ，6 rats at each time point ，12 rats in total ）. Acute SCI model was established by modified Allen ’s method in group B and C. After modeling ，group C was given ligustrazine 100 mg/kg intraperitoneally ，while group A and B were given constant volume of normal saline intraperitoneally ，once a day ，for consecutive 7 d or 14 d（i.e. group B 7d and B 14d，group C 7d and C 14d）. BBB scoring was conducted in each group before modeling ， 7 and 14 days after modeling. HE and Nissl staining observation were also carried out for spinal cord specimen. The differentially expressed genes （DEGs）between group A and group B ，group B and group C were analyzed by transcriptome sequencing. The enrichment of Gene Ontology （GO）and KEGG signaling pathway was analyzed by GO database and KEGG database. RESULTS ： Compared with group A ，BBB scores of group B were decreased significantly 7 d and 14 d after modeling （P＜0.05），and the number of nerve cells and Nissl body in spinal cord tissue were decreased. Compared with group B ，BBB scores of group C were increased significantly at above time points （P＜0.05），and the number of nerve cells and Nissl body in spinal cord tissue were increased. The numbers of DEGs of group A and group B 7 d， group A and group B 14d，group B 7d and group C 7d，group B 14d FAA380076） and group C 14 d were 886，1 404，70，66，respectively. The genes with opposite expression trend included Ncmap，Prx， Gabrq， Gabrg2， etc. The enrichment cell component ， molecular function ，biological process of DEGs were different 630179114@qq.com in each group ，mainly involving lyocytosis ，lysosome，plasmamembrane，homotype protein binding ，immune response ，ion channel activity ，immune response （group A and B ）；basolateral plasma membrane ，exodeoxyribonuclease activity ，response to INF-γ （group B 7 d and C 7 d）；extracellular domain ，receptor regulatory activity ，phenolic compound metabolism process （group B 14 d and C 14 d）. DEGs enriched in cytokine-cytokine receptor interaction（group A and B ）；CAMs，complement and coagulation cascades and Hedgehog signaling pathway （group B 7d and C 7d）； retrograde endocannabinoid signaling ，neuroactive ligand-receptor interaction ，PPAR signaling pathway ，GABA ergic synapse （group B 14 d and C 14 d），etc. CONCLUSIONS ：Protective effect of ligustrazine on acute SCI model rats may be associated with inflammatory response ，immune response/regulation ，neuron ion channel ，cytokine-cytokine receptor interaction ，neuroactive ligand-receptor interaction and regulation of GABA ergic synapse activity .

Microdialysis sampling and HPLC-MS/MS quantification of sinomenine, ligustrazine, gabapentin, paracetamol, pregabalin and amitriptyline in rat blood and brain extracellular fluid / 药学学报

The treatment plan for chronic pain often proceeds from a single drug to drug combination therapy. Sinomenine and ligustrazine, natural alkaline substances derived from traditional Chinese medicines, are expected to provide a new choice for combination analgesic therapy strategies. Here we establish a microdialysis sampling and HPLC-MS/MS quantification method for sinomenine, ligustrazine, gabapentin, paracetamol, pregabalin and amitriptyline in rat blood and brain extracellular fluid. Blood and brain microdialysis probes were implanted in the jugular vein toward the right atrium and left corpus striatum zone (AP +0.2 mm, ML 3.0 mm, DV 3.5 mm) in rats. The blood and brain microdialysis probes were perfused with citric acid buffer solution and Ringer's solution, respectively. Blood and brain extracellular fluid microdialysate were collected at intervals of 20 min at a perfusion rate of 1.5 μL·min-1, and continuously collected for 24 h after administration. The liquid chromatographic separation used a C18-reversed phase chromatographic column (HSS T3 2.5 μm, 2.1 mm×50 mm), the mobile phase was methanol/water (containing 0.05‰ formic acid), and gradient elution was carried out at a flow rate of 0.3 mL·min-1. Mass spectrometric detection used an electrospray ion source, positive ion mode and multi-reaction monitoring method. The selected quantitative ions for sinomenine, ligustrazine, gabapentin, paracetamol, pregabalin, amitriptyline and internal standard naloxone were 330/181, 137/80, 172/154, 152/110, 160/142, 278/233 and 328/310 respectively. The specificity, linear range, matrix effect, accuracy, precision, stability and probe recovery were investigated and confirmed to be suitable for the determination of the above drugs in rat blood and brain extracellular fluid microdialysate. The calculated in vivo recovery of microdialysis probes ranged from 19.38% to 25.88%. After intravenous administration of sinomenine (50 mg·kg-1), ligustrazine (50 mg·kg-1), gabapentin (50 mg·kg-1), paracetamol (50 mg·kg-1), pregabalin (50 mg·kg-1) and amitriptyline (40 mg·kg-1) to rats, the peak concentration in the blood microdialysate was in the range of 0.2-10 μg·mL-1. Drug concentrations could also be detected in brain extracellular fluid microdialysate, however with lower levels (peak concentration: 0.1-6 μg·mL-1) than those of blood microdialysates at each time point. In conclusion, this method can be applied to microdialysis sampling and quantification of sinomenine, ligustrazine, gabapentin, paracetamol, pregabalin and amitriptyline in rats. The method will promote research in identifying herb-drug pharmacokinetic interactions, as well as safety concerns in combination-therapy strategies.

Preparation and in vitro release study of ligustrazine pamoate sustained-release nanosuspension / 中草药

Objective: To prepare ligustrazine pamoate (Lig-PAM) sustained-release nanosuspension (Lig-PAM-NSps) and determine its in vitro release characteristics. Methods: Lig-PAM was prepared by hydrophobic salt formation method and its physicochemical properties were characterized. Then, Lig-PAM-NSps was prepared by miniaturized medium grinding method. The prescription and preparation process of Lig-PAM-NSps were optimized by the single factor and orthogonal experiment with average particle size, polydispersity index (PDI) and stability coefficient (SI) as indicators. Lig-PAM-NSps was characterized, and its stability and in vitro release was also investigated. Results: The compound ratio of Lig-PAM prepared by Lig and PAM in the amount of 1:1 was (97.48 ± 0.04)%. Compared with Lig, the solubilities of Lig-PAM in water and simulated body fluids were decreased by 95.50% and 77.39%, respectively. Fourier transform infrared spectroscopy (FT-IR) and X-ray powder diffraction (XRD) showed that the Lig and PAM formed Lig-PAM. The optimum prescription size of Lig-PAM-NSps was (585 ± 5) nm, PDI was (0.328 ± 0.015) and SI was (0.928 ± 0.012). The scanning electron microscopy (SEM) showed that Lig-PAM-NSps was spherical with uniform size distribution, and the particle size was about 600 nm and its physical stability was good within 60 d. The results of in vitro release showed that Lig-PAM-NSps had obvious sustained-release effect compared with Lig solution within 48 h, and showed the first-order release characteristics [ln(1-Q) = 0.153 67 t + 80.458 14, r = 0.998 26]. Conclusion: The preparation progress of Lig-PAM-NSps is stable and can release Lig slowly in vitro.

Orthogonal compatibility study on main effective parts of Yangyin Tongnao Granules based on pharmacokinetics and pharmacodynamics / 中草药

Objective: To investigate the pharmacokinetic and pharmacodynamic changes of Yangyin Tongnao Granules (YTG) in cerebral ischemia reperfusion rats after the compatibility of main effective parts (total alkaloids, total flavonoids, total saponins and total phenolic acids). Methods: By using the orthogonal design to research the main effective parts of YTG, nine different dosage ratios combinations were formed, which were used for oral administration in cerebral ischemia reperfusion rats. High performance liquid chromatography-diode array detector (HPLC-DAD) was used to determine the concentration of puerarin, ferulic acid, and ligustrazine in rat plasma at different time points. The non-compartmental model was used to fit the pharmacokinetic parameters by Drug and Statistics (DAS) 3.2.6 software. The total quantum statistical moment analysis method and comprehensive evaluation method were used to evaluate the total pharmacokinetic characteristics. Meanwhile, the content of superoxide dismutase (SOD) and catalase (CAT) were determined by enzyme-linked immunosorbent assay (ELISA) kits. Finally, the PK-PD model and the quantitative equations between drug concentration and efficacy were obtained. Results: The pharmacokinetic characteristics of puerarin, ferulic acid and ligustrazine in cerebral ischemia-reperfusion rats were different. Total statistical moment and comprehensive score study showed that different combinations had different effects on ACUT, mean retention time (MRT), and comprehensive evaluation. The effective parts inhibited the reduction of oxidation indexes such as SOD and CAT. Sigmoid-Emax models were adopted in all PK-PD models, and the fitting results had a good correlation with the measured data. The R values were more than 0.85. Conclusion: Compatibility of YTG activity parts had a certain effect on their pharmacokinetic behaviors and antioxidation in model rats. The total quantum statistical moment analysis and comprehensive evaluation method can be used to study the pharmacokinetics of multi-component traditional Chinese medicine prescriptions. PK-PD model could be used to predict and evaluate the correlation between pharmacokinetics and pharmacodynamics of traditional Chinese medicine prescriptions.

Preparation and Study on in vitro Transdermal Characteristics of Tetramethylpyrazine Pellicle / 中国药房

OBJECTIVE：To preliminarily optimize the preparation technology of Ligustrazine pellicle ，and to study its in vitro percutaneous permeation characteristics. METHODS ：With the amounts of PVA- 124，ethyl alcohol ，glycerin，tween-80 and azone as factors ，single factor experiment was used to optimize the Ligustrazine pellicle matrix formulation ；modified scoring standard was used to evaluate the film formation time ，film formation ability ，ductility，uniformity and the presence of bubble. On the basis of the optimal matrix formulation ，the pellicle with different loading amount of ligustrazine （300，250，200，150，100，50 mg/mL） was prepared and its maximum loading amount was investigated. HPLC method was adopted to determine the content of ligustrazine，and methodology investigation was conducted. Isolated back skin of rats were collected ，the percutaneous permeation test was conducted for high ，medium and low loading amount （100，75，50 mg/mL）of Ligustrazine pellicle. At 15，30，45，60， 75，90，120，150，180 min，the sample was taken and the permeation rate of ligustrazine was calculated. RESULTS ：When the amounts of PVA- 124，ethyl alcohol，glycerin，tween-80 and azone were 2.5 g，7.0 mL，1.97 mL，0.07 mL，0.28 mL（in terms of 50 mL formulation amount ），the optimal matrix formulation of Ligustrazine pellicle was obtained. The maximum drug loading amount of ligustrazine was 100 mg/mL. The linear ranges of ligustrazine was 3.125-100 μg/mL. The specificity，precision， reproducibility，recovery and stability investigation of content determination method of ligustrazine were all in line with the requirements（RSD＜2％）. The permeation rate of high ，medium and low loading amount of Ligustrazine pellicle were 608.42， 384.19，158.20 μg（/ cm2·h）. CONCLUSIONS ：According to the optimized formulation ，the prepared Ligustrazine pellicle had a short film forming time ，stable and re liable quality ； the drug-loading amount was up to 100 mg/mL. The pellicle with drug-loading amount of 75 mg/mL had reached the penetration rate range of effective plasma concentration of ligustrazine treatment.

Design, synthesis, and biological evaluation of ligustrazine/resveratrol hybrids as potential anti-ischemic stroke agents / 中国天然药物

To search for potent anti-ischemic stroke agents, a series of tetramethylpyrazine (TMP)/resveratrol (RES) hybrids 6a-t were designed and synthesized. These hybrids inhibited adenosine diphosphate (ADP)- or arachidonic acid (AA)-induced platelet aggregation, among them, 6d, 6g-i, 6o and 6q were more active than TMP. The most active compound 6h exhibited more potent anti-platelet aggregation activity than TMP, RES, as well as positive control ticlopidine (Ticlid) and aspirin (ASP). Furthermore, 6h exerted strong antioxidative activity in a dose-dependent manner in rat pheochromocytoma PC12 cells which were treated with hydrogen peroxide (HO) or hydroxyl radical (·OH). Importantly, 6h significantly protected primary neuronal cells suffered from oxygen-glucose deprivation/reoxygenation (OGD/R) injury, comparable to an anti-ischemic drug edaravone (Eda). Together, our findings suggest that 6h may be a promising candidate warranting further investigation for the intervention of ischemic stroke.

Preparation of ligustrazine microemulsion and comparative study on in vitro release of different size microemulsions / 中草药

Objective: To study the preparation process of ligustrazine microemulsion delivery system and evaluate its physical pharmacy properties; Microemulsions of different particle sizes were prepared in different oil phases, and the effect of particle size factors on the release behavior of the preparation was investigated. Methods: Taking the solubility of ligustrazine as index, the oil phase, emulsifier, and co-emulsifier were screened. The microemulsion formulation was optimized by pseudo-ternary phase diagram method. The encapsulation efficiency and drug loading was studied by ultrafiltration centrifugation. The particle size and potential were detected by the particle size analyzer. The release behavior of microemulsions with different particle sizes was compared by dialysis bag method. Results: The tetramethylpyrazine microemulsion was successfully prepared, and the appearance was clear and transparent. The average pH value was about 5.46. The detection method of microemulsion encapsulation rate was successfully established. When the drug loading of ligustrazine was 1.2 mg/mL, the encapsulation efficiency was (87.43 ± 0.20)%. The microemulsions of different particle sizes were prepared by changing the oil phase (ethyl oleate, oleic acid, and IPM). When the drug loading was 1.2 mg/mL, the three particle sizes were (16.80 ± 0.91), (129.50 ± 1.21), and (18.51 ± 0.24) nm, respectively. The release test showed that the release rate of all three could reach more than 90% within 4 h, and there was no significant difference. Conclusion: The uniform and stable tetramethylpyrazine microemulsion is successfully prepared; The release behavior of different tetramethylpyrazine microemulsions is not affected by the particle size factor.

Clinical analysis of Salvia ligustrazine injection on non-arteriti anterior ischemic optic neuropathy / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To study the effect and mechanism of salvia ligustrazine on non-arteriti anterior ischemic optic neuropathy.<p>METHODS: A total of 60 cases of non-arteriti anterior ischemic optic neuropathy were randomly divided into two groups. The control group(<i>n</i>=30 cases)was treated with oral prednisone acetate tablets and injection of compound anisodine. The experimental group(<i>n</i>=30 cases)was the same to the control group and added intravenous salvia ligustrazine, the period of treatment was 14d. The levels of IL-1β and TNF-α were detected by ELISA, and the protein expression levels of Bcl-2 and Caspase-3 were detected by Western blot. <p>RESULTS: The best corrected visual acuity was improved in both groups. The plasma levels of IL-1β and TNF-α were significantly lowed in the experimental group than the control group, and the difference between the two groups was statistically significant(<i>P</i><0.05). The protein expression of Bcl-2 and Caspase-3 in the two groups were different from those before treatment(<i>P</i><0.05), and the difference between the two groups was statistically significant(<i>P</i><0.05). <p>CONCLUSION: Salvia ligustrazine promotes the recovery of visual function by reducing the level of inflammation and inhibiting cell apoptosis in body.

Content Determination of 10 Kinds of Chemical Components in Ligusticum chuanxiong under Different Storage Conditions by HPLC / 中国药房

OBJECTIVE： To establish the method for the content determination of chlorogenic acid， ligustrazine， ferulic acid， senkyunolide I， senkyunolide H， coniferly ferulate， senkyunolide A， n-butylphtalide， Z-ligustilide and n-butylidenephthalide in Ligusticum chuanxiong under different storage conditions. METHODS： HPLC method was adopted. The determination was performed on Boston C18 column with mobile phase consisted of acetonitrile-0.5％ acetic acid solution （gradient elution） at the flow rate of 1.0 mL/min. The detection wavelength was set at 285 nm， and the column temperature was 30 ℃. The sample size was 5 μL. RESULTS： The linear range of chlorogenic acid， ligustrazine， ferulic acid， senkyunolide I， senkyunolide H， coniferly ferulate， senkyunolide A， n-butylphtalide， Z-ligustilide n-butylidenephthalide were 0.030 53-0.519 01 μg （r＝0.999 5）， 0.001 02-0.017 34 μg （r＝0.999 9）， 0.012 83-0.218 11 μg （r＝0.999 5）， 0.007 63- 0.129 71 μg （r＝0.999 7）， 0.001 76-0.029 92 μg （r＝0.999 5）， 0.054 74-0.930 58 μg （r＝0.999 9）， 0.215 80-3.668 60 μg （r＝0.999 9）， 0.018 02-0.306 34 μg （r＝0.999 7）， 0.232 50- 3.952 50 μg （r＝0.999 9）， 0.002 40-0.040 80 μg （r＝0.999 5）.The limits of quantitation were 2.073 7， 0.556 6， 0.753 8， 0.231 5， 0.306 9， 0.925 2， 2.295 3， 4.624 0， 3.215 3， 0.910 5 ng， respectively. The detection limits were 0.622 1， 0.167 0， 0.226 1， 0.069 4， 0.092 1， 0.277 6， 0.688 6， 1.387 2， 0.964 6， 0.273 1 ng， respectively. RSD of precision， stability and repeatability tests were all less than 5％ （n＝6）. The recovery rates were 95.90％-103.28％ （RSD＝2.99％， n＝6）， 88.24％-107.84％ （RSD＝4.89％， n＝6）， 95.06％-102.08％ （RSD＝3.97％， n＝6）， 93.67％-101.05％ （RSD＝1.02％， n＝6）， 94.81％-104.33％ （RSD＝2.34％，n＝6）， 94.41％-105.59％ （RSD＝4.32％， n＝6）， 92.76％-104.83％ （RSD＝1.95％， n＝6）， 87.22％-102.56％ （RSD＝2.89％， n＝6）， 94.04％-99.52％ （RSD＝0.92％， n＝6）， 88.51％-103.83％ （RSD＝4.89％， n＝6）， respectively. At 5 ℃ and 15 ℃， no obvious deterioration was observed in medicinal materials. At room temperature， some samples were moth-eaten and mildewed. The content ranges of 6 batches of samples were 0.047 7％-0.160 8％， 0.006 1％- 0.022 7％， 0.048 2％-0.172 2％， 0.023 3％-0.145 2％， 0.004 6％-0.030 7％， 0.085 2％-0.835 4％， 0.182 6％-2.112 7％， 0.009 9％- 0.098 3％， 0.614 9％-3.176 2％ and 0.005 7％-0.036 9％， showing decreasing trend； the decrease rate was in descending order 5 ℃＜15 ℃＜room temperature； at the same storage temperature， the decrease rate of polyethylene plastic bag was lower than that of polypropylene woven bag. CONCLUSIONS： This method is accurate and feasible， and can be used for simultaneous determination of 10 kinds of components in L. chuanxiong. It is suggested that L. chuanxiong medicinal materials should be sealed and packed in dry and cool places and should not be stored for a long time.

Effects of Ligustrazine on miR- 20b/VEGF and BMP 2/Smad1 Pathways in Subchondral Bone of Knee Osteoarthritis Model Rats / 中国药房

OBJECTIVE： To study the effects of ligustrazine on miR-20b/VEGF and BMP2/Smad1 pathways in subchondral bone of knee osteoarthritis （KOA） model rats， and to investigate the mechanism of ligustrazine for KOA prevention and treatment. METHODS： Totally 18 healthy male SD rats were randomly divided into normal control group， model group and ligustrazine group， with 6 rats in each group. The rats in the latter two groups were used to establish KOA model by intra-articular injection of 4％ papain solution. From the 2nd day after the last injection， ligustrazine group was given intragastrical administration of Ligustrazine suspension （100 mg/kg） 2 mL； normal control group and model group were given intragastrical administration of isometrical normal saline， once a day， for consecutive 6 weeks. After the last after medication， the situation of bilateral knee articular cartilage of rats were observed after exposure. The knee joints of rats were sectioned and stained with HE. The pathological change of articular cartilage were observed by microscope and scored by modified Mankin’s score. mRNA expression of VEGF， BMP2 and Smad1， and the expression of miR-20b were detected by RT-PCR； the protein expression of VEGF， BMP2 and Smad1 were detected by Western blot assay. RESULTS： Model group and ligustrazine group suffered from cartilage injury of knee joint at varying degrees. Compared with normal control group， Mankin’s scores of knee joint and cartilage tissue were increased significantly in model group （P＜0.01）； mRNA and protein expression of BMP and Smad1， the expression of miR-20b in subchondral bone of model group were decreased significantly， while mRNA and protein expression of VEGF were increased significantly （P＜0.01）. Compared with model group， Mankin’s score of cartilage tissue were decreased significantly in ligustrazine group （P＜0.01）； mRNA and protein expression of BMP and Smad1， the expression of miR-20b in subchondral bone were increased significantly， while mRNA and protein expression of VEGF were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS： Ligustrazine can repair damaged articular cartilage in KOA model rats， the mechanism of which may be associated with inhibiting the protein expression of VEGF and activating BMP-2/Smad1 signaling pathway via up-regulating the expression of miR-20b， and promoting the degradation of VEGF mRNA in subchondral bone.

Effects of Salviae Miltiorrhizae and Ligustrazine Hydrochloride Injection on diabetic peripheral neuropathy and oxidative stress response / 中草药

Objective: To study the therapeutic effect of point injection of Salvia Miltiorrhiza and Ligustrazine Hydrochloride Injection (SMLHI) on diabetic peripheral neuropathy (DPN) patients and its effect on oxidative stress response. Methods: A total of 100 DPN patients were selected from the Neurology Department of the Fifth Affiliated Hospital of Zhengzhou University from March 2016 to February 2018. According to the random number table method, the patients were divided into observation group and control group with 50 cases in each group. Each group was treated with routine hypoglycemic drugs, diet control and exercise control, while the control group was treated with Mecobalamin Tablets. The observation group was treated with Mecobalamin Tablets combined with SMLHI at acupoints for 4 weeks as a course of treatment for two consecutive courses of treatment. The clinical symptom score, serum superoxide dismutase (SOD), gamma-glutamyltransferase (GGT), ferritin (SF), total anti-oxidant capacity (T-AOC), and malondialdehyde (MDA) levels, median nerve and peroneal nerve conduction velocity (MNCV), median nerve, peroneal nerve conduction velocity (SNCV), and curative effect were observed and compared before and after treatment. Results: After treatment, the clinical symptom score of the observation group was significantly lower, and the total effective rate of the observation group was 84.00%, which was higher than that of the control group 64.00% (P < 0.05). The SOD level in the observation group was higher, while the GGT, SF, T-AOC, and MDA levels were lower (P < 0.05). The MNCV and SNCV in the observation group were higher (P < 0.05). Conclusion: Acupoint injection of SMLHI for DPN patients can significantly reduce oxidative stress reaction in vivo, promote the recovery of nerve function, improve clinical symptoms of patients with high safety, which is worthy of clinical promotion.

Protective effects of compatibility of effective constituents in Salvia miltiorrhiza and Ligusticum chuanxiong on hippocampal neurons injured by oxygen glucose deprivation / 中草药

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.