Uncovering Mechanism and Efficacy of Salvia Miltiorrhiza-Safflower in Cerebral Ischemia-Reperfusion Injury.

Cerebral ischemia (CI) is the main cause of stroke morbidity and disability. This study aims to identify the early molecular regulation responsible for the therapeutic effectiveness of the Herb pair Danshen-Honghua (DH) for CI. The major targets of DH were identified by searching the public database of traditional Chinese medicine (TCM). In addition, GeneCards, Disgenet, and GeneMap databases in OMIM were used to determine the disease targets of CI. A total of 88 common targets of DH and CI were selected, a protein-protein interaction (PPI) network was established by Cytoscape, and 19 core targets were screened. These genes were primarily enriched in biological processes including wound healing, reaction to oxidative stress, and response to peptides, lipid and atherosclerosis, Age-rage signaling pathway, and TNF signaling pathway by KEGG and GO enrichments. The effective components of DH had stable binding to these key targets by molecular docking. Finally, it was verified that the mechanism of DH on CI treatment may be related to the activation of the TNF-α/JNK signaling pathway by establishing the middle cerebral artery occlusion (MCAO) rat model.

A comprehensive review of stroke-related signaling pathways and treatment in western medicine and traditional Chinese medicine.

This review provides insight into the complex network of signaling pathways and mechanisms involved in stroke pathophysiology. It summarizes the historical progress of stroke-related signaling pathways, identifying potential interactions between them and emphasizing that stroke is a complex network disease. Of particular interest are the Hippo signaling pathway and ferroptosis signaling pathway, which remain understudied areas of research, and are therefore a focus of the review. The involvement of multiple signaling pathways, including Sonic Hedgehog (SHH), nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE), hypoxia-inducible factor-1α (HIF-1α), PI3K/AKT, JAK/STAT, and AMPK in pathophysiological mechanisms such as oxidative stress and apoptosis, highlights the complexity of stroke. The review also delves into the details of traditional Chinese medicine (TCM) therapies such as Rehmanniae and Astragalus, providing an analysis of the recent status of western medicine in the treatment of stroke and the advantages and disadvantages of TCM and western medicine in stroke treatment. The review proposes that since stroke is a network disease, TCM has the potential and advantages of a multi-target and multi-pathway mechanism of action in the treatment of stroke. Therefore, it is suggested that future research should explore more treasures of TCM and develop new therapies from the perspective of stroke as a network disease.

An integrated strategy of spectrum-effect relationship and near-infrared spectroscopy rapid evaluation based on back propagation neural network for quality control of Paeoniae Radix Alba.

The quantitative analysis of near-infrared spectroscopy in traditional Chinese medicine has still deficiencies in the selection of the measured indexes. Then Paeoniae Radix Alba is one of the famous "Eight Flavors of Zhejiang" herbs, however, it lacks the pharmacodynamic support, and cannot reflect the quality of Paeoniae Radix Alba accurately and reasonably. In this study, the spectrum-effect relationship of the anti-inflammatory activity of Paeoniae Radix Alba was established. Then based on the obtained bioactive component groups, the genetic algorithm, back propagation neural network, was combined with near-infrared spectroscopy to establish calibration models for the content of the bioactive components of Paeoniae Radix Alba. Finally, three bioactive components, paeoniflorin, 1,2,3,4,6-O-pentagalloylglucose, and benzoyl paeoniflorin, were successfully obtained. Their near-infrared spectroscopy content models were also established separately, and the validation sets results showed the coefficient of determination (R2 > 0.85), indicating that good calibration statistics were obtained for the prediction of key pharmacodynamic components. As a result, an integrated analytical method of spectrum-effect relationship combined with near-infrared spectroscopy and deep learning algorithm was first proposed to assess and control the quality of traditional Chinese medicine, which is the future development trend for the rapid inspection of traditional Chinese medicine.

Optimization of green extraction process with natural deep eutectic solvent and comparative in vivo pharmacokinetics of bioactive compounds from Astragalus-Safflower pair.

INTRODUCTION: Natural deep eutectic solvents (NaDESs) are promising not only in componential extraction, but also in drug delivery system due to their green and safe features. In this work, NaDESs were applied to extract bioactive components from Astragalus-Safflower pair, a classic traditional Chinese medicine combination. Furthermore, ready-to-use crude extracts were administrated to SD rats. METHODS: Total 9 NaDESs composed of food grade ingredients were screened for the extraction of representative 9 components (hydroxysafflor yellow A, anhydrosafflor yellow B, eleutheroside B, calycosin-7-O-glucoside, kaempferol-3-O-rutinoside, ononin, calycosin, astraganoside, and carthamin) from Astragalus-Safflower pair. Afterwards, genetic artificial neural network (GNN) was adopted for optimizing the ultrasound-assisted extraction process. After SD rats were orally administrated with the ready-to-use crude extracts extracted under the optimized conditions, the in vivo pharmacokinetic characteristics of 5 components were evaluated comprehensively from weight, gender, solvent and modeling surgery, under a well-established UPLC-MS/MS method. RESULTS: Betaine-Lactic acid (Bet-Lac) was eventually determined as the optimal extraction solvent for subsequent experiments. The optimal ultrasound assisted extraction process was as follows: 90 min of extraction time, 65 °C of temperature, 80% of Bet-Lac content and 50 mg/ml of solid-liquid ratio. Bet-Lac enhanced to varying degrees the bioavailability of analytes in normal and cerebral ischemia/reperfusion injured (CI/RI) rats in contrast with corresponding rats administrated with water extract groups (p < 0.05). Besides, the bioavailability of active components in CI/RI rat plasma was significantly lower than that in normal rats (p < 0.05), indicating pathological damage of CI/R had a significant impact on pharmacokinetic profile of compounds in rats. However, gender and body weight had no significant effects on the pharmacokinetic profile of bioactive components. CONCLUSIONS: NaDESs exhibited higher extraction efficiency than conventional solvents. And GNN is reliable to optimize the ultrasound assisted extraction process. This study supported the potential of non-toxic NaDESs as solvents for extraction process and drug delivery systems at the same time.

Optimization of smashing tissue and ultrasonic extraction of tanshinones and their neuroprotective effect on cerebral ischemia/reperfusion injury by inhibiting parthanatos.

A green smashing tissue and ultrasonic (STU) extraction method, which combines smashing tissue and ultrasonic-assisted extraction, was developed for the first time. The extraction of tanshinones from Salvia miltiorrhiza Bunge (SM) was taken as an example to discuss the practicability of this method. Taking the total yield of eight tanshinones as an evaluation index, response surface methodology (RSM) and artificial neural network (ANN) models were used to optimize the extraction parameters, and these two models were also compared by investigating the extract yield of tanshinones and the antioxidant activity of the obtained SM extract. The optimal STU conditions by ANN were as follows: an ethanol concentration of 73%, a liquid/solid ratio of 30 mL g-1, a smashing tissue time of 97 s and an ultrasonic time of 40 min. Under these optimal conditions, the yield of the eight components was 0.30% ± 0.12, which was greater than 0.28% ± 0.03 optimized by RSM. The IC50 values of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) of the obtained extract were 55.25 ± 3.72 µg mL-1 and 67.33 ± 2.62 µg mL-1, respectively, which were better than those of 75.49 ± 4.33 µg mL-1 and 112.10 ± 5.98 µg mL-1, respectively, optimized by RSM. Furthermore, the SM extract was found to exert neuroprotective effects by inhibiting parthanatos in middle cerebral artery occlusion/reperfusion (MCAO/R)-induced rats. The results supported the use of the SM extract, which was obtained by STU, as a potential product in the cosmetics, medicine, and food industries.

Simultaneous optimization of the extraction process of Yangyin Yiqi Huoxue prescription with natural deep eutectic solvents for optimal extraction yield and antioxidant activity: A comparative study of two models.

INTRODUCTION: Natural deep eutectic solvents (NaDESs) are green and effective solvents that are used to extract 3 flavonoids from Yangyin Yiqi Huoxue prescription, a traditional Chinese prescription. METHODS: A total of 6 types of NaDESs were systematically screened and evaluated for the total extraction yield of puerarin, calycosin, and formononetin by high-performance liquid chromatography. Then, a 4-factor-three-level experimental scheme designed by the Box-Benhnken Design was applied on the basis of a single experiment to determine the extraction yield and the antioxidant property. Finally, the extraction process was optimized through response surface methodology (RSM) and the genetic neural network (GNN), respectively. RESULTS: The use of betaine-lactic acid as an extractant displayed significant advantages in the screening process. The optimum extraction parameters provided by GNN were as follows: water content 25% (v/v), liquid to material ratio 190 mg/ml, extraction time 37 min, and extraction temperature 63 °C. Under this condition, the average experimental comprehensive evaluation values of the extraction yield and antioxidant properties were 3.12 mg/g and 86.27%, and the relative deviations to the predicted values were 0.30% and 1.44%, respectively. In addition, the experimental results of GNN were better than those of RSM (p < 0.01). CONCLUSIONS: We found the application of GNN to be effective and credible for bi-objective optimization of extraction yields and antioxidant activity in this study. Moreover, our results provide a reference and a theoretical basis for experimental and future industrial extraction for multi-objective situations.

The Role of PKC and HIF-1 and the Effect of Traditional Chinese Medicinal Compounds on Cerebral Ischemia-Reperfusion Injury.

Neuronal death occurs during cerebral ischemia. However, when hemoperfusion and oxygen supply are resumed to the ischemic focus of the brain tissue, the brain tissue damage is further aggravated, resulting in cerebral ischemia-reperfusion injury (CIRI) to the patients. Protein kinase C (PKC) plays an important role in CIRI. Through the IP3/DAG/Ca2+ signaling pathway, it promotes the influx of calcium ions in neurons and causes calcium overload, which aggravates the damage. At the same time, when brain cells are hypoxic, hypoxia-inducible factor-1 (HIF-1) is expressed, which regulates the expression of Bcl-2 and Bax through the PI3K/Akt signaling pathway and reduces nerve cell injury. It also fights hypoxic-ischemic injury by increasing the production of vascular endothelial growth factor (VEGF) to promote blood vessel formation. The PKC and HIF-1 signaling pathways are also linked to CIRI. HIF-1 activates the PKC and ERK pathways via the upregulation of VEGF, leading to increased Cx43 phosphorylation and dysfunction and aggravating CIRI. Existing studies have shown that certain traditional Chinese medicine (TCM) compounds regulate the PKC and HIF-1 signaling pathways and alleviate CIRI. These compounds downregulate the PKC and the activity of the PKC-related signaling pathways to alleviate CIRI. They can also promote the expression of HIF-1, increase the content of VEGF in ischemic tissues to promote the generation of blood vessels, and improve microcirculation. TCM compounds can inhibit the cascade of reactions underlying disease occurrence and development by targeting multiple components using different herbal formulations to improve the structural and material changes in the brain cells, which alleviate CIRI and protect the brain tissue. This study briefly describes the role of PKC and HIF-1, their relationship in CIRI, and the effect of TCM on them.

Quantitative analysis of effects of salvianic acid a combined with hydroxy safflower yellow a on rat endothelial cells after hypoxic injury using the combination index method

Abstract Cerebrovascular disease is the second most serious disease in the world. It has the features of high morbidity, high mortality and recurrence rate. Numerous research on the compatibility of Chinese medicine with effective ingredients of cerebral ischemia has been made during the past decades. The purpose of this study is to quantitatively analyze the combined pharmacological effect of effective ingredients in Danshen and Honghua (Dan Hong) on rat microvascular endothelial cells after gradually oxygen-glucose deprivation. The experimental concentration range for the compatibility of two effective ingredients were determined in the preliminary experiments by Cell Counting kit-8 (CCK-8) method. Drugs were added to rat brain microvascular endothelial cells at a non-toxic dose level. After that, the cells were cultured for 12 h, and placed in a hypoxic environment. Finally, the cell survival rate was used as a measure of drug effect. In order to determine synergism or antagonism, the combination index (CI)-isobologram method was performed to analyze the data from the experiments. Based on this theory, the potencies of each drug and the shapes of their does-effect curves are both taken into account. The results show that the synergism or the antagonism between two effective ingredients compatibility change with different proportion and dosage. Furthermore, it can be seen from the results of these experiments that when these drugs are used in combination, the dosage required to achieve the same therapeutic effects is greatly reduced compared with the case of single one. It is worth mentioning that our experiments also prove that the median-effect equation and the CI method can be applied in the field of traditional Chinese medicine.

Comparison of Traditional Chinese Medicine in the Long-Term Secondary Prevention for Patients with Ischemic Stroke: A Systematical Analysis.

Background: Keeping in view the high recurrence rate and risk of ischemic stroke, combinatorial therapy involving traditional Chinese medicine (TCM) with conventional Western medicine (WM) is receiving wider scientific attention. Thus, a systematical analysis was made to explore the efficacy of TCM+WM in the long-term secondary prevention for patients with ischemic stroke. Methods: Qualified inclusion and exclusion criteria were set up beforehand, and two researchers independently read the articles, extracted data, and evaluated the quality of included articles according to Cochrane Reviewer's Handbook 5.1 method. For the sake of comprehensive data acquisition, seven databases from the time of their establishment to May 5, 2021, have been searched completely. Additionally, pairwise meta-analysis was made to compare TCM+WM vs. WM, and network meta-analysis was conducted by frequentist random effects models for the comparison of different kinds of TCM+WM via indirect evidence. The primary outcomes defined were recurrent stroke and NIHSS. Secondary outcomes were fibrinogen (Fib) fasting blood glucose (FBG), triglycerides (TG), and total cholesterol (TC). Safety outcomes were outlined as all-cause mortality and adverse events (AEs). Furthermore, Stata16.0 software was used to accomplish the systematical analysis and cluster analysis. Results: In total, 47 qualified randomized controlled trials (RCTs) including 10,732 patients were taken into consideration. Seven traditional Chinese medicines included in the study are Naoxintong capsule (NXT), Tongxinluo capsule (TXL), Buyang Huanwu decoction (BYHW), Naomaitai capsule (NMT), Dengzhan Shengmai capsule (DZSM), Naoshuantong capsule (NST), and Maixuekang capsule (MXK). With respect to their primary outcomes, all kinds of TCM+WM were significantly more effective than WM (e.g., NXT in recurrent stroke (OR=0.54, P<0.01), TXL in NIHSS (WM=-1.4, P<0.01)). Additionally, the outcomes of cluster analysis indicated that MXK+WM and NST+WM had relatively good preventive effects for recurrent stroke, NIHSS, and all-cause mortality. There was no significant difference in the comparisons of AEs; however, this may arise from the lack of sufficient data. Conclusion: According to our systematical analysis, MXK+WM and NST+WM had relatively good secondary prevention effects for patients with ischemic stroke regarding recurrent stroke, NIHSS, and all-cause mortality. Nevertheless, better, high-quality, large-sample randomized clinical trials (RCTs) are required to verify our conclusions in the future. Systematic Review Registration: [https://inplasy.com/inplasy-2021-5-0036/], identifier [INPLASY202150036].

Guhong Injection Alleviates Cerebral Ischemia-Reperfusion Injury via the PKC/HIF-1α Pathway in Rats.

Guhong injection (GHI) is a drug for ischemic stroke created by combining safflower, a traditional Chinese medicine, and aceglutamide, a Western medicine. In this study, we investigated the curative effect of GHI on cerebral ischemia-reperfusion (I/R) injury via the PKC/HIF-1α pathway in rats. Adult male Sprague Dawley rats were randomly divided into seven groups: sham-operated, middle cerebral artery occlusion (MCAO), GHI, nimodipine injection (NMDP), MCAO + LY317615 (PKC inhibitor), GHI + LY317615, and NMDP + LY317615. After establishing an MCAO rat model, we performed neurological deficit testing, 2,3,5-triphenyltetrazolium chloride staining, hematoxylin and eosin (HE) staining, enzyme-linked immunosorbent assay, Western blotting, and q-PCR to detect the brain damage in rats. Compared with the MCAO group, the GHI and GHI + LY317615 group showed neurological damage amelioration as well as decreases in serum hypoxia-inducible factor-1α (HIF-1α), protein kinase C (PKC), and erythropoietin levels; brain HIF-1α and inducible nitric oxide synthase protein expression; and brain HIF-1α and NOX-4 mRNA expression. These effects were similar to those in the positive control groups NMDP and NMDP + LY317615. Thus, our results confirmed GHI can ameliorate cerebral I/R injury in MCAO rats possibly via the PKC/HIF-1α pathway.

Pharmacokinetic-Pharmacodynamic Modeling of the Antioxidant Activity of Quzhou Fructus Aurantii Decoction in a Rat Model of Hyperlipidemia.

BACKGROUND: Quzhou Fructus Aurantii (QFA) is an herb that is commonly used to alleviate inflammation in individuals dealing with obesity.To date, however, no systematic pharmacokinetic (PK) or pharmacodynamic (PD) analyses of the clinical efficacy of QFA under hyperlipemia-associated oxidative stress conditions have been conducted. The present study, was therefore designed to construct a PK-PD model for this herb, with the goal of linking QFA PK profiles to key therapeutic outlines to guide the therapeutic use of this herb in clinical settings. METHODS: Rats were fed a high-fat diet in order to establish a model of hyperlipidemia, after which they were randomized into a normal control group (NCG), a normal treatment group (NTG), a model control group (MCG), and a model treated group (MTG) (n = 6 each). QAF decoction was used to treat rats in the NTG and MTG groups (25 g/kg), while equivalent volumes of physiological saline were administered to rats in the NCG and MCG groups. Plasma samples were collected from the mandibular vein for animals at appropriate time points and analyzed via high-performance liquid chromatography (HPLC). We evaluated PK properties for three QAF components and compared these dynamics between the NTG and MTG groups, while also measuring levels of lipid peroxidation (LPO) in the plasma of rats in all four treatment groups. We then constructed a PK-PD model based upon plasma neohesperidin, luteolin, and nobiletin concentrations and LPO levels using a three-compartment PK model together with a Sigmoid Emax PD model. This model thereby enabled us to assess the antioxidative impact of neohesperidin, luteolin, and nobiletin on hyperlipidemia in rats. RESULTS: When comparing the NTG and MTG groups, we detected significant differences in the following parameters pertaining to neohesperidin, luteolin, and nobiletin:t1/2ß, V1, t1/2γ, CL1 (p < 0.01) and AUC0-t, Tmax, Cmax (p < 0.05). Relative to NTG group rats, AUC0-t, TmaxandCmaxvalues significantly higher for MTG group rats (p < 0.01), while t1/2ß, V1, and t1/2γ values were significantly lower in MTG group rats (p < 0.01) in MTG rats. QAF decoction also exhibited excellent PD efficacy in MTG rats, with significant reductions in plasma LPO levels relative to NTG rats (p < 0.01) following treatment. This therapeutic efficacy may be attributable to the activity of neohesperidin, luteolin, and nobiletin, as LPO levels and plasma concentrations of these compounds were negatively correlated in treated rats. Based upon Akaike Information Criterion (AIC) values, we determined that neohesperidin, luteolin, and nobiletin PK processes were consistent with a three-compartment model. Together, these findings indicated that three active components in QAF decoction (neohesperidin, luteolin, and nobiletin) may exhibit antioxidant activity in vivo. CONCLUSION: Our in vivo data indicated that neohesperidin, luteolin and nobiletin components of QAF decoctions exhibit distinct PK and PD properties. Together, these findings suggest that hyperlipidemia-related oxidative stress can significantly impact QFA decoction PK and PD parameters. Our data additionally offer fundamental insights that can be used to design appropriate dosing regimens for individualized clinical QAF decoction treatment.

Response surface optimization of the water immersion extraction and macroporous resin purification processes of anhydrosafflor yellow B from Carthamus tinctorius L.

In this study,based on a developed high performance liquid chromatographic quantitative method, the suitable extraction and purification conditions of anhydrosafflor yellow B (AHSYB) from safflower were determined by response surface methodology. The optimal water immersion extraction parameters were as follows: liquid to solid ratio of 22:1; extraction temperature of 75 °C; extraction time of 35 min. Under these conditions, the maximum extraction yield of AHSYB reached 0.465%. The aqueous extract was further purified by HPD-300 macroporous resin. The optimum adsorption conditions were: pH 2.8; adsorption flow rate of 1.9 mL/min; solution concentration of 0.06 g/mL. The optimum desorption conditions were: ethanol concentrations of 74%; desorption flow rate of 1.6 mL/min; elution volume of 4.4 BV. Under these conditions, the maximum adsorption ratio and desorption ratio reached 1.095 and 0.906 mg/g, respectively. The content of AHSYB reached 6.83%, which was 2.91 times higher than that before purification. PRACTICAL APPLICATION: The suitable conditions for water immersion extraction and macroporous resin purification of AHSYB are first determined, which facilitates the further utilization of AHSYB as a food and drug.

Simultaneous Optimization of the Ultrasonic Extraction Method and Determination of the Antioxidant Activities of Hydroxysafflor Yellow A and Anhydrosafflor Yellow B from Safflower Using a Response Surface Methodology.

An evaluation of the ultrasonic extraction process and the antioxidant activities of hydroxysafflor yellow A (HSYA) and anhydrosafflor yellow B (AHSYB) from safflower are presented herein. Using response surface methodology (RSM), based on a four-factor-three-level Box-Behnken design (BBD), the extraction parameters, namely, temperature, extraction time, solvent-to-material ratio, and extraction power, were optimized for maximizing the yields of HSYA and AHSYB. The maximum yield was obtained at a temperature of 66 °C with an extraction time of 36 min, solvent-to-material ratio of 16 mL/g, and the extraction power of 150 W, which was adjusted according to the actual conditions. The HSYA and AHSYB contents were determined using high performance liquid chromatography (HPLC). The yield and the comprehensive evaluation value of HSYA and AHSYB were calculated. The antioxidant activities of the extracts were determined using a ferric reducing antioxidant power (FRAP) kit and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. The results suggested that the safflower extracts possessed obvious ferric reducing and DPPH radical scavenging activities. The antioxidant activity increased with increasing concentration. The results suggested that optimizing the conditions of ultrasonic extraction using RSM can significantly increase the yields of HSYA and AHSYB from safflower. The safflower extracts showed better antioxidant activity. This study can encourage future research on cardiovascular and cerebrovascular diseases.

Optimal extraction bioactive components of tetramethylpyrazine in Chinese herbal medicine jointly using back propagation neural network and genetic algorithm in R language.

A combinational approach of back propagation neural network (BPNN) and genetic algorithm (GA) was proposed in the present study to optimize the extraction technology of tetramethylpyrazine (TMP) in Ligusticum wallichii Franchat. Based on the single factor test, the orthogonal experiment design method of four factors and three levels was adopted, and the concentration of TMP was measured by high performance liquid chromatography (HPLC). Subsequently, BPNN model was trained for a predictive computational model of the performance indices via experimental data, and GA was exploited to find the optimization con ditions for extraction technology of TMP. Meanwhile, both the model and algorithm were implemented in R language. Ethanol concentration of 80%, extraction time of 1.5h, extraction temperature of 55℃ and liquid-solid ratio of 8:1 were derived as optimal conditions with a maximum content of TMP of 2.04 mg/g, which was confirmed with the relative error 2.63% through the validation of the experiments. This mathematical model could be used to analyze and predict the extraction technology of TMP in Ligusticum wallichii Franchat and provide a new reference for screening optimization of Chinese medicine effective parts and components.

Pharmacokinetics of Active Ingredients of Salvia miltiorrhiza and Carthamus tinctorius in Compatibility in Normal and Cerebral Ischemia Rats: A Comparative Study.

BACKGROUND AND OBJECTIVE: Dan-Hong injection, which comprises extracts of Salvia miltiorrhiza and Carthamus tinctorius, promotes blood circulation and reduces blood stasis. Combination of S. miltiorrhiza and C. tinctorius is more effective in treating cerebral ischemia than S. miltiorrhiza alone. This study aimed to examine the pharmacokinetic characteristics of four active ingredients of S. miltiorrhiza and C. tinctorius, namely danshensu (DSS), hydroxysafflor yellow A (HSYA), and salvianolic acid A (SAA) and B (SAB) in normal and cerebral ischemia rats. METHODS: Normal and cerebral ischemia rats were injected via the tail vein with each active ingredient, and blood was collected through the jaw vein at different time points. The plasma concentration of the compatibility group was analyzed by high-performance liquid chromatography, and pharmacokinetic parameters were determined using Pharmacokinetic Kinetica 4.4 software. RESULTS: The pharmacokinetics of the four active ingredients in the normal and cerebral ischemia rats were consistent with a two-compartment model. The area under the concentration-time curve was higher in normal rats than in cerebral ischemia rats, with a highly significant difference for SAA (P < 0.01). Clearance rates were lower in normal rats than in cerebral ischemia rats, with DSS showing the most significant difference (P < 0.01). Furthermore, there were significant differences between normal and cerebral ischemia rats in the distribution phase-elimination half life for DSS, SAA, and HSYA, as well as in the apparent volume of distribution for the central compartment for DSS and HSYA (P < 0.01). The plasma concentrations of the four active ingredients were higher in normal rats than in cerebral ischemia rats. CONCLUSION: Cerebral ischemia rats showed higher drug clearance rates and longer retention times than normal rats, which may be due to destruction of the blood-brain barrier during cerebral ischemia-reperfusion. The four active ingredients likely integrated and interacted with each other to affect target sites in the brain to protect against cerebral ischemic injury.

Pharmacokinetic Study on Protocatechuic Aldehyde and Hydroxysafflor Yellow A of Danhong Injection in Rats with Hyperlipidemia.

BACKGROUND: Protocatechuic aldehyde (PAL) and hydroxysafflor yellow A (HSYA) are 2 effective ingredients of Danhong Injection, which is extensively used for the clinical treatment of cardio-cerebrovascular diseases. This study aims to investigate the pharmacokinetic differences between single and combined medication of PAL and HSYA and analyze the interaction of the above effective components in hyperlipidemia rats. METHODS: Thirty male SD rats were randomly divided into the control group (n = 6) and the model group (n = 24). The hyperlipidemia model was established by feeding with superfatted forage. The successful model rats were then randomly divided into the PAL group (16 mg/kg), the HSYA group (10 mg/kg), and the combination group (16 mg/kg + 10 mg/kg). Administration through tail-vein, and orbital blood was sampled at different time points. The mass concentration of PAL and HSYA was determined by high performance liquid chromatography (HPLC-DAD). Analysis of pharmacokinetic parameters was conducted by using DAS 3.2.6 software and SPSS 19.0 statistical analysis software. RESULTS: According to the parameters of statistical moment of non-compartmental model, there was a significant difference in plasma clearance (CL) between the PAL group and the drug combination group (p < 0.01), as well as in the area under the first moment of the plasma concentration-time curve and the elimination half-life (t1/2) between the HSYA group and the drug combination group (p < 0.01) but no obvious differences about the blood concentration time curve area, the average dwell time (MRT), and the peak concentration (Cmax; p > 0.05). CONCLUSION: The combined medication of PAL and HSYA could increase the plasma CL significantly and have a great influence on the absorption of HSYA in rats with hyperlipidemia.

Optimization of Bioactive Ingredient Extraction from Chinese Herbal Medicine Glycyrrhiza glabra: A Comparative Study of Three Optimization Models.

The ultraviolet spectrophotometric method is often used for determining the content of glycyrrhizic acid from Chinese herbal medicine Glycyrrhiza glabra. Based on the traditional single variable approach, four extraction parameters of ammonia concentration, ethanol concentration, circumfluence time, and liquid-solid ratio are adopted as the independent extraction variables. In the present work, central composite design of four factors and five levels is applied to design the extraction experiments. Subsequently, the prediction models of response surface methodology, artificial neural networks, and genetic algorithm-artificial neural networks are developed to analyze the obtained experimental data, while the genetic algorithm is utilized to find the optimal extraction parameters for the above well-established models. It is found that the optimization of extraction technology is presented as ammonia concentration 0.595%, ethanol concentration 58.45%, return time 2.5 h, and liquid-solid ratio 11.065 : 1. Under these conditions, the model predictive value is 381.24 mg, the experimental average value is 376.46 mg, and the expectation discrepancy is 4.78 mg. For the first time, a comparative study of these three approaches is conducted for the evaluation and optimization of the effects of the extraction independent variables. Furthermore, it is demonstrated that the combinational method of genetic algorithm and artificial neural networks provides a more reliable and more accurate strategy for design and optimization of glycyrrhizic acid extraction from Glycyrrhiza glabra.

Simultaneous Optimization for Ultrasound-Assisted Extraction and Antioxidant Activity of Flavonoids from Sophora flavescens Using Response Surface Methodology.

The ultrasonic-assisted extraction process and antioxidant activity of flavonoids from Sophora flavescens were investigated in this study. In order to optimize the extraction of flavonoids from Sophora flavescens, the influence of extraction time, methanol concentration, ultrasonic temperature, and solvent-to-material ratio was analyzed. Results showed that the extraction yields reached a maximum with the extraction time of 30 min, methanol concentration of 80%, temperature of 80 °C, and solvent-to-material ratio of 26 mL/g. The flavonoids were determined by HPLC, and the mean yields of trifolirhizin, formononetin, isoxanthohumol, maackiain, and kurarinone under the optimal conditions were 2.570, 0.213, 0.534, 0.797, and 3.091 mg/g, respectively. The evaluation of vitro antioxidant activity exhibited Sophora flavescens flavonoids had a strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical-scavenging ability with IC50 of 0.984 and 1.084 mg/g, respectively. These results indicate that ultrasonic-assisted extraction is an efficient approach for the selective extraction of flavonoids, and response surface methodology further optimized the extraction.

Protective effects of effective ingredients of Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) compatibility after rat hippocampal neurons induced by hypoxia injury.

OBJECTIVE: To investigate the effective ingredients of Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) (Danhong) on protective properties towards neonatal rat hippocampal neurons under hypoxia condition. METHODS: Primary culture of neonatal rat hippocampal neurons was used to model hypoxia damage on the hippocampus. Methyl thiazolyl tetrazolium (MTT) assay and preliminary experiments were conducted to identify the four effective ingredients of Danhong, along with the injection of positive drug onto hippocampal neurons at a non-toxic dosage level. The cultured cells were randomly divided into 12 groups: the normal group, model group, positive drug control group and nine compatibility groups of the four effective ingredients. Different test methods were applied to determine lactate dehydrogenasein (LDH), total superoxide dismutase (T-SOD), malondialdehyde (MDA), 6-keto prostaglandin Fla (6-keto-PGF1a), thromboxane B2 (TXB2), mitochondrial membrane potential (MMP), free calcium ions concentration ([Ca2+]i) and early onset cell apoptosis. RESULTS: Different compatibility groups could inhibit the content of LDH and intracellular calcium overload, increase activity in T-SOD, decrease level of MDA and TXB2, improve level of 6-keto-PGF1a and MMP, and prevent the early onset cell apoptosis. CONCLUSION: The compatibility of four effective ingredients of Danhong had protective effect toward hippocampi hypoxia. The mechanism might be related to inhibit oxidative stress damage and cell apoptosis, resist thrombosis, and reduce the intracellular calcium ion of overload.

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.