[Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma alleviates acute lung injury in rats by regulating autophagy via PI3K/Akt/mTOR pathway].

This study aims to reveal the effects of the herb pair Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma(AR-SMRR) on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR) pathway and autophagy in the lung tissue of the rat model of acute lung injury(ALI). Fifty adult male SD rats were randomized into sham, model, autophagy inhibition(intraperitoneal injection of chloroquine at 10 mg·kg~(-1)), autophagy induction(intraperitoneal injection of rapamycin at 15 mg·kg~(-1)), and AR-SMRR(5 g·kg~(-1), gavage) groups. The rats in the sham group received intratracheal instillation of normal saline, and those in other groups received intratracheal instillation of lipopolysaccharide(LPS, 5 mg·kg~(-1)) for the modeling of ALI. Seven days before the operation, the rats in the sham and model groups were administrated with normal saline, and those in other groups with corresponding drugs. Specimens were collected 24 h after modeling. The pathological changes of the lung tissue were observed under a light microscope. The lung wet/dry weight ratio and the lactate dehydrogenase(LDH) activity and total protein concentration in the bronchoalveolar lavage fluid(BALF) were measured. Western blot was employed to measure the protein levels of microtubule-associated protein 1-light chain 3(LC3), beclin-1, p62, PI3K, Akt, and mTOR. Compared with the sham group, the model group showed increased histopathological score of the lung tissue, lung wet/dry weight ratio, and LDH activity and protein concentration in BALF. Autophagy inhibition further increased these indicators compared with the model group, while autophagy induction and AR-SMRR lowered the levels. In addition, AR-SMRR up-regulated the protein levels of LC3-Ⅱ and beclin-1, down-regulated the expression of p62, and inhibited the expression of p-PI3K, p-Akt, and p-mTOR in the lung tissue of ALI rats. The findings suggest that AR-SMRR can alleviate the lung injury and edema in the rat model of ALI induced by LPS by enhancing autophagy via down-regulating PI3K/Akt/mTOR signaling pathway.

[Experimental study on treatment of retinitis pigmentosa by inducing Müller cell reprogramming with Lycii Fructus and Salviae Miltiorrhizae Radix et Rhizoma].

This study aims to explore the effect of Lycii Fructus and Salviae Miltiorrhizae Radix et Rhizoma(LFSMR), a drug pair possesses the function of nourishing Yin, promoting blood circulation, and brightening the eyes, in treating retinitis pigmentosa(RP)by inhibiting the gliosis of Müller cells(MCs) and inducing their reprogramming and differentiation into various types of retinal nerve cells. Twelve C57 mice were used as the normal control group, and 48 transgenic RP(rd10) mice were randomly divided into the model group, positive control group, and low and high dose LFSMR groups, with 12 mice in each group. HE staining was used to detect pathological changes in the retina, and an electroretinogram was used to detect retinal function. Retinal optical coherence tomography was used to detect retinal thickness and perform fundus photography, and laser speckle perfusion imaging was used to detect local retinal blood flow. Digital PCR was used to detect gene expression related to retinal nerve cells, and immunofluorescence was used to detect protein expression related to retinal nerve cells. LFSMR could significantly improve the pathological changes, increase the amplitude of a and b waves, increase the retinal thickness, restore retinal damage, and increase retinal blood flow in mice with RP lesions. LFSMR could also significantly inhibit the m RNA expression of the glial fibrillary acidic protein( GFAP) during the pathogenesis of RP and upregulate m RNA expression of sex determining region Y box protein 2(SOX2), paired box protein 6(Pax6),rhodopsin, protein kinase C-α(PKCα), syntaxin, and thymic cell antigen 1. 1(Thy1. 1). LFSMR could significantly inhibit GFAP protein expression and enhance protein expression of SOX2, Pax6, rhodopsin, PKCα, syntaxin, and Thy1. 1. It could also reverse the pathological changes in the retina of rd10 mice, improve retinal function and fundus performance, increase retinal thickness, enhance local retinal blood flow, and exert therapeutic effects on RP. The mechanism of action of LFSMR may be related to inhibiting the gliosis of MCs and promoting their reprogramming and differentiation into various types of retinal nerve cells.

[Correlations between tanshinones and soil factors of Salviae Miltiorrhizae Radix et Rhizoma from different producing areas].

Soil nutrients and inorganic elements affect not only the growth and development of medicinal plants but also the formation and accumulation of active ingredients in traditional Chinese medicines. The content of tanshinones and 28 inorganic elements in Salviae Miltiorrhizae Radix et Rhizoma samples from 18 producing areas in 6 provinces was determined, and 35 physical and chemical properties of the corresponding soil samples were determined. The enrichment characteristics of inorganic elements in Salviae Miltiorrhizae Radix et Rhizoma were analyzed. The correlation analysis and stepwise regression analysis were performed to screen out the main soil factors affecting the content of tanshinones in Salviae Miltiorrhizae Radix et Rhizoma. The results showed that the content of tanshinones in the samples from different areas varied significantly, being the highest in the samples from Shandong, the second in the samples from Henan, and low in the samples from Shanxi and Sichuan. K, Mg, Ca, and Na were rich in Salviae Miltiorrhizae Radix et Rhizoma samples, among which Na and K had the highest enrichment coefficients. The results of correlation and regression analyses showed that soil K, Na, Ti, and total nitrogen were the main soil factors affecting the tanshinones in Salviae Miltiorrhizae Radix et Rhizoma. Specifically, the content of tanshinones was positively correlated with Ti and negatively correlated with Na, K, and total nitrogen in the soil. Therefore, during the planting of Salvia miltiorrhiza, the land should be selected with full consideration to the salinity and saline land should be avoided. Secondly, the application of nitrogen and potassium fertilizers can be appropriately reduced, and water-soluble elemental fertilizers for S. miltiorrhiza should be developed.

Soluble polyvinylpyrrolidone-based microneedles loaded with Sanguis draconis and Salvia miltiorrhiza for treatment of diabetic wound healing.

BACKGROUND: Nowadays, diabetic wound healing remains a crucial challenge due to their protracted and uncertain healing process. Traditional Chinese medicine (TCM) has demonstrated the therapeutic value of Sanguis draconis (SD)-Salvia miltiorrhiza (SMR) Herb Pair in diabetic wound healing. However, new administration modes are urgently needed for their convenient and wide-ranging applications. OBJECTIVE: We propose a soluble polyvinylpyrrolidone-based microneedle patch containing the herbal extracts of SD and SMR (MN-SD@SMR) for diabetic wound healing. METHODS: The herbal extracts of SD and SMR are purification and concentration via traditional lyophilization. SD endowed MN-SD@SMR with functions to improve high glycemic blood environment and migration of keratinocyte and fibroblast cells. RESULTS: SMR in MN-SD@SMR could improve blood flow velocity and microcirculation in the wound area. The effectiveness of transdermal release and mechanical strengths of MN-SD@SMR were verified. CONCLUSION: Integrating the advantages of these purified herbal compositions, we demonstrated that MN-SD@SMR had a positive healing effect on the wounds in vitro and vivo. These results indicate that soluble polyvinylpyrrolidone-based microneedle patch containing the herbal extracts of SD and SMR has a promising application value due to their superior capability to promote diabetic wound healing.

Advances in Pharmacological Research on Anti-knee Osteoarthritis of Active Components of Salviae Miltiorrhizae Radix et Rhizoma / 中药新药与临床药理

Knee osteoarthritis(KOA)is a degenerative joint disease with a high incidence,which has seriously affected the quality of life of middle-aged and elderly people.Salviae Miltiorrhizae Radix et Rhizoma,a commonly used traditional Chinese medicinal for activating blood and removing stasis,has been proved to have a certain preventive effect on KOA in basic research and clinical application.Studies have found that the active ingredients of Salviae Miltiorrhizae Radix et Rhizoma have a variety of anti-KOA effects,such as regulation of inflammatory factors,anti-oxidative stress,inhibition of chondrocyte apoptosis,regulation of cartilage matrix degradation,and promotion of chondrocyte autophagy.It can reduce joint pain and inflammatory edema,delay articular cartilage degeneration,and maintain cartilage matrix homeostasis.This paper reviews the effect and mechanism of the effective components from Salviae Miltiorrhizae Radix et Rhizoma on the prevention and treatment of KOA,and analyzes the shortcomings of its related research.Our aim is to provide reference for the clinical treatment of KOA.

Network Pharmacological Analysis and Experimental Verification of the Mechanism of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma Drug Pair in the Treatment of Hypertension / 中药新药与临床药理

Objective To investigate the mechanism of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair in the treatment of hypertension based on the network pharmacology method and animal experiment verification.Methods(1)TCMSP,BATMAN and TCMIP databases were used to screen the active components and targets of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair.The hypertension-related targets were obtained by searching the Drugbank,Genecard,TTD and Disgenet databases.The intersection(common target)of the active component target and the target related to hypertension disease was taken,and the obtained intersection target was the potential target of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair for the treatment of hypertension.The active ingredients and their targets of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair were imported into Cytoscape 3.9.1 software to construct a'Chinese medicines-active ingredients-targets'network and screen key active ingredients.The protein-protein interaction(PPI)network of potential targets was constructed to screen potential core targets.The Metascape platform was used to analyze the GO function and KEGG pathway enrichment of potential targets.The key active components and potential core targets were selected for molecular docking verification.(2)Thirty male spontaneously hypertensive rats(SHR)were randomly divided into model group,western medicine group(Candesartan Cilexetil,0.72 mg·kg-1)and low-,medium-and high-dose groups of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma(2.25,4.50,9.00 g·kg-1).Another male WKY rats were selected as blank group,with 6 rats in each group,once a day for 8 weeks.The systolic blood pressure of rat tail artery was detected before administration and 2,4,6 and 8 weeks after drug intervention.The pathological changes of thoracic aorta were observed by HE staining.The protein expression levels of GRP78,CHOP and Caspase-12 in aorta abdominalis were detected by Western Blot.Results(1)A total of 83 active components of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma were obtained,and 158 potential targets(intersection targets)for the treatment of hypertension were screened out.Five key active ingredients:p-hydroxybenzoic acid,4-hydroxybenzylamine,tanshinone I,tanshinone,γ-sitosterol;6 potential core targets:IL6,TNF,CASP3,JUN,PTGS2,IL1B;GO functional enrichment analysis obtained 1 826 biological process items,89 cell component items,and 199 molecular function items.KEGG pathway enrichment analysis obtained 186 pathways,mainly involving neuroactive ligand-receptor interaction,calcium signaling pathway,inflammatory response(such as TNF and MAPK signaling pathway),vascular protection(such as HIF-1 and cAMP signaling pathway),oxidative stress(such as PI3K-Akt signaling pathway)and other signaling pathways.Tanshinone I and tanshinone had strong binding force to 6 potential core targets,and γ-sitosterol had strong binding force to IL6,CASP3,JUN,PTGS2 and IL1B.(2)Compared with the blank group,the systolic blood pressure of the model group was significantly increased(P＜0.01).The thoracic aortic endothelial injury was obvious,the endothelial cell morphology was abnormal,swelling and exfoliated cells could be seen,the intima of the tissue was disordered,the intima structure was incomplete,and the intima was thickened.The protein expressions of GRP78,CHOP and Caspase-12 in abdominal aorta were significantly increased(P＜0.01).Compared with the model group,the systolic blood pressure of the rats in the administration group was significantly decreased(P＜0.01);the injury of thoracic aorta was alleviated,and the morphology,intima structure and thickness of endothelial cells were improved to varying degrees.The protein expressions of GRP78,CHOP and Caspase-12 in abdominal aorta were significantly decreased(P＜0.01).Conclusion Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair may act on core targets such as IL6,TNF,CASP3,JUN,PTGS2,and IL1B through key active components such as p-hydroxybenzoic acid,tanshinone,and γ-sitosterol,and regulate key signaling pathways such as TNF signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway,and PERK signaling pathway to improve vascular endothelial dysfunction,inhibit endoplasmic reticulum stress,and lower blood pressure.

Correlation Analysis Between Anti-cerebral Ischemia Oxidative Damage and Contents of Active Components in Characteristic Processed Products with Porcine Cardiac Blood and Other Processed Products of Salviae Miltiorrhizae Radix et Rhizoma from Menghe Medical School / 中国实验方剂学杂志

ObjectiveTo analyze the correlation between 11 small molecule active components and 1 protein component of characteristic processed products with porcine cardiac blood and other products of Salviae Miltiorrhizae Radix et Rhizoma（SMRR） from Menghe medical school and anti-cerebral ischemic oxidative damage， and to identify its key component markers of characteristic processed products with porcine cardiac blood for anti-cerebral ischemic oxidative damage. MethodHigh performance liquid chromatography（HPLC） was established to simultaneously determine the contents of 11 active ingredients in SMRR and its processed products［processed with porcine cardiac blood， porcine blood， wine and transferrin（Tf） in porcine cardiac blood］， and the content of Tf in different processed products of SMRR was detected by enzyme-linked immunosorbent assay（ELISA）. Furthermore， A zebrafish ischemic stroke model was constructed to evaluate the effects of different processed products of SMRR on the behavioral trajectory of cerebral ischemic zebrafish， the neuronal damage of transgenic zebrafish Tg（elavl3：eGFP） brain， as well as the levels of malondialdehyde（MDA） and superoxide dismutase（SOD） in the brain tissues. The hippocampal neurons oxygen-glucose deprivation/reoxygenation（OGD/R）-induced ischemia-hypoxia model was constructed to evaluate the effects of different processed products of SMRR on oxidative damage of neuronal cells by taking lactate dehydrogenase（LDH）， reactive oxygen species（ROS）， MDA and SOD as indexes. Finally， principal component analysis（PCA）， partial least squares-discriminant analysis（PLS-DA） and Spearman correlation analysis were used to analyze the 11 small molecule active components and 1 protein component with efficacy indicators， in order to screen the key components of the characteristic processed products with porcine cardiac blood for cerebral ischemic oxidative damage. ResultCompared with the raw products， the contents of water-soluble and fat-soluble components in processed products of SMRR increased to different degrees， while the content of salvianolic acid A decreased. Compared with the wine-processed products， the contents of salvianolic acid B， danshensu， rosmarinic acid and other components in the porcine cardiac blood-processed products， porcine blood-processed products， Tf-processed products were increased， while the content of salvianolic acid A was decreased. ELISA results showed that there was no significant difference in Tf content between the porcine cardiac blood-processed products， porcine blood-processed products， Tf-processed products. Pharmacological results showed that different processed products of SMRR could improve the behavioral deficits， brain neuronal injury and oxidative stress after ischemic stroke in zebrafish， and the effect of the porcine cardiac blood-processed products was most pronounced. PCA results showed that salvianolic acid B， salvianolic acid A， rosmarinic acid， lithospermic acid， danshensu， tanshinone ⅡA， caffeic acid， cryptotanshinone and tanshinone Ⅰ were the main contributing components of SMRR and its processed products. And the results of correlation analysis showed that the contents of cryptotanshinone， rosmarinic acid， caffeic acid， dihydrotanshinone Ⅰ， salvianolic acid B， tanshinone ⅡA and tanshinone Ⅰ were negatively correlated with MDA level in zebrafish brain tissue， while the contents of lithospermic acid， protocatechuic aldehyde， rosmarinic acid， dihydrotanshinone Ⅰ， salvianolic acid B and Tf were positively correlated with SOD level， and the contents of rosmarinic acid， caffeic acid， dihydrotanshinone Ⅰ， salvianolic acid B， tanshinone ⅡA， tanshinone Ⅰ， danshensu， Tf were positively correlated with neuronal fluorescence intensity in the zebrafish brain. And the contents of lithospermic acid， protocatechuic aldehyde， rosmarinic acid， dihydrotanshinone Ⅰ， salvianolic acid B， tanshinone ⅡA and Tf were negatively correlated with LDH， ROS and MDA levels and positively correlated with SOD level. ConclusionThere are differences in the anti-ischemic oxidative damage effects of SMRR and its different processed products， among which the porcine cardiac blood-processed products has the strongest effect on improving oxidative damage， which may be related to the content changes of salvianolic acid B， danshensu， rosmarinic acid and other components. This study can provide a basis for clarifying the quality markers of SMRR processed with porcine cardiac blood for cerebral ischemia and elucidating its processing mechanism.

Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma Mitigate Renal Fibrosis： A Review / 中国实验方剂学杂志

Renal fibrosis， the final pathological outcome of end-stage chronic kidney diseases， is associated with inflammation， oxidative stress， epithelial-mesenchymal transdifferentiation （EMT）， and extracellular matrix deposition. It belongs to the categories of edema， ischuria， anuria and vomiting， and consumptive disease in traditional Chinese medicine （TCM）， with the key pathogenesis of Qi deficiency and blood stasis and the primary treatment principle of replenishing Qi and activating blood. Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma mainly contains astragalosides， polysaccharides， calycosin， salvianolic acid， and tanshinone， with the effect of tonifying Qi and activating blood. Studies have shown that this herb pair and its active components can delay the progress of renal fibrosis by regulating multiple signaling pathways. With consideration to the pathogenesis of Qi deficiency and blood stasis， this article reviews the research progress in the mitigation of renal fibrosis by Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma from the aspects of protecting glomerular filtration barrier， inhibiting EMT and mesangial cell proliferation， improving renal hemodynamics， and protecting renal function. Furthermore， the mechanisms were summarized. Specifically， Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma and its effective components can improve mitochondrial function and fatty acid metabolism， alleviate endoplasmic reticulum stress and autophagy disorders， and inhibit immune inflammation and oxidative stress by regulating nuclear factor E2-related factor 2 （Nrf2）/PTEN-induced kinase 1 （Pink1）， Nrf2/antioxidant response element （ARE）， tumor necrosis factor-α （TNF-α）/nuclear transcription factor-κB （NF-κB）， miR-21/Smad7/transforming growth factor beta （TGF-β）， Wnt/β-catenin， long non-coding RNA-taurine up-regulated gene 1 （lncRNA-TUG1）/tumor necrosis factor receptor-associated factor 5 （TRAF5）， Ras-related C3 botulinum toxin substrate 1 （Rac1）/cell division cycle protein 42 （CDC42）， Ras homolog （Rho）/Rho-associated coiled-coil containing protein kinase （ROCK）， phosphatidylinositol-3-kinase （PI3K）/protein kinase B （Akt）， Janus kinase （JAK）/signal transducer and activator of transcription （STAT）， peroxisome proliferator-activated receptor α （PPARα）/peroxisome proliferator-activated receptor γ coactivator l alpha （PGC-1α）， and p38 mitogen-activated protein kinase （p38 MAPK）. This review aims to provide references for the relevant research， give play to the role of Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma， and provide guidance for the clinical treatment of renal fibrosis.

[Modernization of Chinese medicine Salviae Miltiorrhizae Radix et Rhizoma: a review].

Modernization of Chinese medicine is an important development direction of traditional Chinese medical sciences. It is of great significance to understand the mechanism of Chinese medicine with basic research, which can also accelerate the development and utilization of Chinese medicine. Salviae Miltiorrhizae Radix et Rhizoma is one of the most commonly used Chinese medicines in China for the prevention and treatment of cardiovascular and cerebrovascular diseases. It has received key and extensive attention worldwide in the following aspects: main active ingredients and their pharmacological mechanism, function and regulation of their biosynthetic pathway and application of their synthetic biology as well as the clinical preparations. The new and developing chemical analysis, network pharmacology, molecular pharmacognosy and omics make the modernization research of Salviae Miltiorrhizae Radix et Rhizoma comprehensive and in-depth. This study systematically reviewed the modernization research of Salviae Miltiorrhizae Radix et Rhizoma, which focused on its pharmacological effects, preparation research, biosynthesis and regulation mechanism of the active ingredients, and expected to exert the model role of Salviae Miltiorrhizae Radix et Rhizoma in the research of Chinese medicine.

Polysaccharide from Salviae miltiorrhizae Radix et Rhizoma Attenuates the Progress of Obesity-Induced Non-Alcoholic Fatty Liver Disease through Modulating Intestinal Microbiota-Related Gut-Liver Axis.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide, thus treatments for it have attracted lots of interest. In this study, the Salviae miltiorrhizae Radix et Rhizoma (SMRR) polysaccharide was isolated by hot water extraction and ethanol precipitation, and then purified by DEAE anion exchange chromatography and gel filtration. With a high-fat-diet-induced obesity/NAFLD mouse model, we found that consumption of the SMRR polysaccharide could remarkably reverse obesity and its related progress of NAFLD, including attenuated hepatocellular steatosis, hepatic fibrosis and inflammation. In addition, we also reveal the potential mechanism behind these is that the SMRR polysaccharide could regulate the gut-liver axis by modulating the homeostasis of gut microbiota and thereby improving intestinal function.

[Effects and correlation of ligustrazine hydrochloride-Salviae Miltiorrhizae Radix et Rhizoma compatibility on pharmacokinetics and CYP450 enzyme].

The present study investigated the effects of ligustrazine hydrochloride(LH)-Salviae Miltiorrhizae Radix et Rhizoma(SM) before and after compatibility on the pharmacokinetics of acute myocardial ischemia(AMI) rats and revealed the mechanism of pharmacokinetic changes from the perspective of metabolic enzymes. AMI rats underwent single injection of SM Glucose Injection, LH Glucose Injection, and LH-SM Glucose Injection in the caudal vein, respectively(3.78 mg·kg~(-1) salvianic acid, 0.049 mg·kg~(-1) rosmarinic acid, and 13.68 mg·kg~(-1) ligustrazine). Blood samples were collected from the orbital venous plexus at different time points, and the liver of the rats was removed after the last blood sampling. The plasma concentrations of salvianic acid, rosmarinic acid, and ligustrazine were detected by UPLC-MS/MS. Western blot was used to detect the protein expression of CYP1 A2, CYP2 C11, CYP2 C19, CYP2 D4, CYP2 E1, and CYP3 A2 in the liver of rats in each group. As revealed by the pharmacokinetic results, compared with the LH Glucose Injection group, the LH-SM Glucose Injection group showed a downward trend of T_(1/2) of ligustrazine in AMI rats and decreased AUC(P<0.05). Compared with the SM Glucose Injection, there were no significant differences in the pharmacokinetic parameters of salvianic acid and rosmarinic acid in the LH-SM Glucose Injection group. Protein expression results showed that the expression levels of CYP1 A2, CYP2 C11, CYP2 D4, CYP2 E1, and CYP3 A2 in the LH-SM Glucose Injection group increased(P<0.05) and the expression level of CYP2 C19 decreased(P<0.05) compared with those in the LH Glucose Injection group. CYP1 A2, CYP2 C11, and CYP3 A2 are isoenzymes involved in ligustrazine Ⅰ metabolism. When LH and SM were used in combination, the expression of these three enzymes increased, which changed the pharmacokinetic process in rats and accelerated the metabolism of ligustrazine.

Protective Effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix Extract on SH-SY5Y Cells Injured by Oxygen-glucose Deprivation/Reoxygenation / 中国实验方剂学杂志

ObjectiveTo explore the protective effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix （SP） extract on oxygen-glucose deprivation/reoxygenation （OGD/R）-injured SH-SY5Y cells based on oxidative stress and apoptosis. MethodThe extracts of the two medicinal materials mixed in different ratios were prepared. Human neuroblastoma SH-SY5Y cells were cultured in vitro and the injury was induced by OGD/R. Cell counting kit-8 （CCK-8） assay was used to screen the optimal ratio of the two medicinals and then the extract was used for further experiment. SH-SY5Y cells were classified into normal control group， OGD/R group， and low-， medium-， and high-dose SP （2∶1） extract groups （10， 30， 100 mg·L-1， respectively）. Cells in the groups， except the normal control group， were rapidly reoxygenated for 12 h after 4 h OGD for modeling. Then cell viability was detected by CCK-8 and cell morphology was observed under the microscope. The release rate of lactate dehydrogenase （LDH）， superoxide dismutase （SOD） activity， and content of glutathione （GSH） and malondialdehyde （MDA） were determined by spectrophotometry. The level of reactive oxygen species （ROS） was detected with 2，7-dichlorodihydrofluorescein diacetate （DCFH-DA） and mitochondrial membrane potential with JC-1 assay. The nuclear morphology was observed based on Hoechst 33342 staining， and apoptosis was examined by flow cytometry combined with Annexin V-FITC/PI staining. ResultThe viability of the cells was highest in the presence of the extract of the two medicinals mixed at the ratio of 2∶1. Compared with normal control group， OGD/R group showed damaged cell morphology， high release rate of LDH and levels of ROS and MDA （P<0.01）， low SOD activity and GSH level （P<0.01）， low mitochondrial membrane potential， and high apoptosis rate （P<0.01）. Compared with OGD/R group， SP extract improved cell viability and cell morphology and reduce cell LDH release rate in a concentration-dependent manner （P<0.01）. In addition， SP extract at 30， 100 mg·L-1 reduced the level of intracellular ROS and increased SOD activity and GSH level （P<0.05， P<0.01）， and SP extract at 100 mg·L-1 decreased the content of MDA （P <0.05）. Moreover， SP extract increased mitochondrial membrane potential， and SP extract at 30， 100 mg·L-1 lowered the apoptosis rate （P<0.01）. ConclusionThe extract of Salvia miltiorrhiza Bunge and Radix Puerariae mixed at 2∶1 shows better protective effect on OGD/R-injured SH-SY5Y cells. The mechanism is the likelihood that it alleviates oxidative damage of cells and inhibits cell apoptosis.

Protective Effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix Extract on SH-SY5Y Cells Injured by Oxygen-glucose Deprivation/Reoxygenation / 中国实验方剂学杂志

ObjectiveTo explore the protective effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix （SP） extract on oxygen-glucose deprivation/reoxygenation （OGD/R）-injured SH-SY5Y cells based on oxidative stress and apoptosis. MethodThe extracts of the two medicinal materials mixed in different ratios were prepared. Human neuroblastoma SH-SY5Y cells were cultured in vitro and the injury was induced by OGD/R. Cell counting kit-8 （CCK-8） assay was used to screen the optimal ratio of the two medicinals and then the extract was used for further experiment. SH-SY5Y cells were classified into normal control group， OGD/R group， and low-， medium-， and high-dose SP （2∶1） extract groups （10， 30， 100 mg·L-1， respectively）. Cells in the groups， except the normal control group， were rapidly reoxygenated for 12 h after 4 h OGD for modeling. Then cell viability was detected by CCK-8 and cell morphology was observed under the microscope. The release rate of lactate dehydrogenase （LDH）， superoxide dismutase （SOD） activity， and content of glutathione （GSH） and malondialdehyde （MDA） were determined by spectrophotometry. The level of reactive oxygen species （ROS） was detected with 2，7-dichlorodihydrofluorescein diacetate （DCFH-DA） and mitochondrial membrane potential with JC-1 assay. The nuclear morphology was observed based on Hoechst 33342 staining， and apoptosis was examined by flow cytometry combined with Annexin V-FITC/PI staining. ResultThe viability of the cells was highest in the presence of the extract of the two medicinals mixed at the ratio of 2∶1. Compared with normal control group， OGD/R group showed damaged cell morphology， high release rate of LDH and levels of ROS and MDA （P<0.01）， low SOD activity and GSH level （P<0.01）， low mitochondrial membrane potential， and high apoptosis rate （P<0.01）. Compared with OGD/R group， SP extract improved cell viability and cell morphology and reduce cell LDH release rate in a concentration-dependent manner （P<0.01）. In addition， SP extract at 30， 100 mg·L-1 reduced the level of intracellular ROS and increased SOD activity and GSH level （P<0.05， P<0.01）， and SP extract at 100 mg·L-1 decreased the content of MDA （P <0.05）. Moreover， SP extract increased mitochondrial membrane potential， and SP extract at 30， 100 mg·L-1 lowered the apoptosis rate （P<0.01）. ConclusionThe extract of Salvia miltiorrhiza Bunge and Radix Puerariae mixed at 2∶1 shows better protective effect on OGD/R-injured SH-SY5Y cells. The mechanism is the likelihood that it alleviates oxidative damage of cells and inhibits cell apoptosis.

Molecular Mechanism of Salviae Miltiorrhizae Radix et Rhizoma against Bladder Cancer： Based on Network Pharmacology and In Vitro Experiment / 中国实验方剂学杂志

ObjectiveTo predict the potential targets and possible related signaling pathways of Salviae Miltiorrhizae Radix et Rhizoma against bladder cancer （BC） based on network pharmacology and verify the potential molecular mechanism through in vitro cell experiment. MethodActive components of Salviae Miltiorrhizae Radix et Rhizoma were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） and BC-related targets were searched from GeneCards and Online Mendelian Inheritance in Man （OMIM）. Via Venny2.1， the potential targets of Salviae Miltiorrhizae Radix et Rhizoma against BC were screened out and the Venn diagram was plotted. Protein-protein interaction （PPI） network was constructed by STRING， followed by Gene Ontology （GO） term enrichment and Kyoto Encyclopedia of Genes and Gnomes （KEGG） pathway enrichment with DAVID. Cell Counting Kit-8 （CCK-8） assay was employed to detect the inhibitory effect of tanshinone ⅡA （Tan ⅡA）， cryptotanshinone （CPT）， and luteolin （LUT） at different concentration （0， 1， 2， 4， 8， 16， 32 μmol·L-1） on the proliferation of BC T24 and 5637 cells， propidium iodide （PI） staining to analyze the apoptosis of 5637 cells induced by Tan ⅡA， CPT， and LUT （0， 4， 8 μmol·L-1）， and Western blotting to detect the regulatory effect of Tan ⅡA （0， 4， 8， 16 μmol·L-1） on the expression of key target proteins. ResultA total of 65 active components and 39 anti-BC targets of Salviae Miltiorrhizae Radix et Rhizoma were screened out. The anti-BC targets were mainly involved in the KEGG pathways of neuron-ligand-receptor interaction， phosphatidylinositol 3-kinases （PI3K）/protein kinase B （Akt） signaling pathway， epidermal growth factor receptor （EGFR） tyrosine kinase inhibitor resistance， and hypoxia inducible factor （HIF）-1 signaling pathway. As for the CCK-8 assay， compared with the blank group， Tan ⅡA， CPT， and LUT significantly inhibited the proliferation of T24 and 5637 cells， particularly the 5637 cells. The half maximal inhibitory concentration （IC50） of Tan ⅡA on 5637 cells was significantly lower than that of CPT and LUT. Moreover， compared with the blank group， Tan ⅡA， CPT， and LUT all induced the apoptosis of 5637 cells， and the effect followed the order of Tan ⅡA>CPT>LUT （P<0.05）. Western blot showed that Tan ⅡA significantly reduced the expression of EGFR， p-PI3K， and p-Akt in 5637 cells in a concentration-dependent manner compared with the blank group （P<0.05）. ConclusionSalviae Miltiorrhizae Radix et Rhizoma exerts therapeutic effect on BC through multiple components， multiple targets， and multiple pathways. The mechanism is the likelihood that it down-regulates the expression of EGFR， p-PI3K， and p-Akt proteins， thus further inhibits cell proliferation， and induces apoptosis.

[Review of chemical composition, pharmacological effects, and clinical application of Salviae Miltiorrhizae Radix et Rhizoma and prediction of its Q-markers].

Salviae Miltiorrhizae Radix et Rhizoma is a Chinese herbal medicine that promotes blood circulation to remove blood stasis, nourishes blood to tranquilize the mind, and cools blood to disperse carbuncles. Salviae Miltiorrhizae Radix et Rhizoma has microcirculation-improving, blood vessel-dilating, atherosclerosis-preventing, anti-inflammatory, anti-tumor, and blood pressure-and blood lipid-lowering activities. As research progresses, the chemical composition, pharmacological effect, and clinical application of Salviae Miltiorrhizae Radix et Rhizoma have attracted much attention. We reviewed the research progress in this field. Based on the concept of quality marker(Q-marker) in traditional Chinese medicine, the Q-markers of Salviae Miltiorrhizae Radix et Rhizoma were predicted and analyzed from the aspects of quality transfer, traceability, ingredient specificity, association between ingredients and pharmacological effects, ingredient predictability, and compounding environment. This review provides a scientific basis for the quality control of Salviae Miltiorrhizae Radix et Rhizoma and its preparations.

[Historical evolution of Salviae Miltiorrhizae Radix et Rhizoma processing methods].

China has a long history of Salviae Miltiorrhizae Radix et Rhizoma processing with multiple methods available. The pre-sent study collated and summarized the Salviae Miltiorrhizae Radix et Rhizoma processing methods recorded in 23 related herbal medicine books, all editions of Chinese Pharmacopoeia, the 1988 edition of National Regulations for Processing of Chinese Medicine, and 20 current local processing specifications and standards. The results demonstrated various processing methods of Salviae Miltiorrhizae Radix et Rhizoma, such as removing residual part of stem, plantlet, or soil, smashing, filing, cutting, decocting, washing with wine, soaking in wine, and stir-frying with wine or blood from pig heart, while raw and wine-processed products are mainly used in modern times. Due to the lack of unified standards, the phenomena of multiple methods adopted in one place and different methods in different places have led to uneven quality of Salviae Miltiorrhizae Radix et Rhizoma pieces, even affecting the safety and effectiveness of its clinical medication. This study is expected to provide a reference for the development of Salviae Miltiorrhizae Radix et Rhizoma processing and its rational medication.

[Study on extraction kinetics of Salviae Miltiorrhizae Radix et Rhizoma: model establishment and investigation of critical process parameters].

In this study, Fick's first law and partition equilibrium were used to represent the internal and external mass transfer processes of Salviae Miltiorrhizae Radix et Rhizoma at the macroscopic level, and a mass transfer model was established. The specific surface area was integrated into the mass transfer resistance, which effectively avoided the irregular shape of medicinal materials and expanded the application scope of the model. Meanwhile, the mass transfer model was further combined with the kinetic model of salvia-nolic acid degradation to establish the extraction kinetic models of salvianolic acid B, lithospermic acid and Danshensu. The model was applied to study the extraction process of Salviae Miltiorrhizae Radix et Rhizoma. According to the sensitivity analysis results, the relative error of the model prediction was within 5% near the maximum extraction rate(320 min), and the prediction performance of the model was good. According to the investigation results of different process parameters, stirring could significantly accelerate the mass transfer rate of salvianolic acid B, while the mass transfer resistance and degradation rate constant were not affected by solvent-to-solid ratio. The linear relationship between the reciprocal of temperature and the logarithm of mass transfer resistance was good(R~2=0.996), indicating that the temperature and mass transfer resistance conformed to Arrhenius formula. In addition, we also found that the concentration changes of lithospermic acid and Danshensu were weakly affected by mass transferwhen the extraction temperature was higher than 358 K. This study has provided the basis for the process optimization and quality control of traditional Chinese medicine extraction.

[Effect of Salviae Miltiorrhizae Radix et Rhizoma on diversity of intestinal flora in diabetic nephropathy rats].

This study aimed to explore the effect of Salviae Miltiorrhizae Radix et Rhizoma, its stems and leaves on the diversity of intestinal microflora in rats with diabetic kidney injury. Diabetic rats model was established by feeding high glucose and high fat diet and 5% glucose solution with intraperitoneal injection of 30 mg·kg~(-1) streptozocin(STZ). The rats were randomly divided into normal group, model group, irbesartan control group, Huangkui Capsules control group, as well as low, middle and high dose groups of Sal-viae Miltiorrhizae Radix et Rhizoma, its stems and leaves. After administration for 2 weeks, 16 S rRNA technique was used to analyze the diversity of intestinal microflora in the feces of each group. The results showed rats in the model group developed renal tubular epithelial vacuole degeneration and a large amount of inflammatory cell infiltration in the renal interstitium. A small amount of inflammatory cell infiltration was seen in each administration group. The kidney structure of rats in irbesartan group, Huangkui Capsules group, high-dose group of Salviae Miltiorrhizae Radix et Rhizoma and its stem water extract, as well as high dose group of Salviae Miltiorrhizae Radix et Rhizoma and its stem ethnol extract group was close to the normal group. The diversity and structure of intestinal flora in the model group were significantly different from those in the normal group. Each administration group improved the fecal flora diversity in rats with diabetic kidney injury to a certain extent, especially the high dose of Salviae Miltiorrhizae Radix et Rhizoma and its stems water extract. Different flora were found in feces of diabetic nephropathy model rats on class, order, family and genus levels. On families and genera levels, the relative abundance of Bifidobacterium, Turicibacter, Peptostreptococcaceae, Desulfovibrio, and SMB53 showed an upward trend in model group, but that of Lactobacillus, Clostridium, Rikenella, Rumen fungi showed a downward trend. The administration groups can improve the relative abundance of the above intestinal flora in the model rats to a normal-like level. The results of this study provide a reference for resource utilization and further development of Salviae Miltiorrhizae Radix et Rhizoma.

Challenges and strategies in progress of drug delivery system for traditional Chinese medicine Salviae Miltiorrhizae Radix et Rhizoma (Danshen).

Traditional Chinese medicines (TCMs), with a history of thousands of years, are widely used clinically with effective treatment. However, the drug delivery systems (DDSs) for TCMs remains major challenges due to the characteristics of multi-components including alkaloids, flavones, anthraquinones, glycosides, proteins, volatile oils and other types. Therefore, the novel preparations and technology of modern pharmaceutics is introduced to improve TCM therapeutic effects due to instability and low bioavailability of active ingredients. Salviae Miltiorrhizae Radix et Rhizoma, the radix and rhizomes of Salvia miltiorrhiza Bunge (Danshen in Chinese), is a well known Chinese herbal medicine for protecting the cardiovascular system, with active ingredients mainly including lipophilic tanshinones and hydrophilic salvianolic acids. In this review, this drug is taken as an example to present challenges and strategies in progress of DDSs for TCMs. This review would also summary the characteristics of active ingredients in it including physicochemical properties and pharmacological effects. The purpose of this review is to provide inspirations and ideas for the DDSs designed from TCMs by summarizing the advances on DDSs for both single- and multi-component from Danshen.

[Pharmacokinetics of eight active ingredients of Salviae Miltiorrhizae Radix et Rhizoma-Puerariae Lobatae Radix combination in rats].

To reveal the rationality of compatibility of Salviae Miltiorrhizae Radix et Rhizoma(SMRR) and Puerariae Lobatae Radix(PLR) from the perspective of pharmacokinetics, this study established a UPLC-MS/MS method for quantitative determination of PLR flavonoids(3'-hydroxy puerarin, puerarin, puerarin 6″-O-xyloside, 3'-methoxy puerarin, puerarin apioside) and salvianolic acids and tanshinones(salvianolic acid B, cryptotanshinone, and tanshinone Ⅱ_A) in plasma of rats. Rats were given SMRR extract, PLR extract, and SMRR-PLR extract by gavage and then plasma was collected at different time. UPLC separation was performed under the following conditions: Eclipse C_(18) column(2.1 mm×50 mm, 1.8 µm), 0.1% formic acid in water(A)-0.1% formic acid in acetonitrile(B) as mobile phase for gradient elution. Conditions for MS are as below: multiple reaction monitoring(MRM), ESI~(+/-). Comprehensive validation of the UPLC-MS/MS method(specifically, from the aspects of calibration curve, precision, accuracy, repeatability, stability, matrix effect, extract recovery) was performed and the result demonstrated that it complied with quantitative analysis requirements for biological samples. Compared with SMRR extract alone or PLR extract alone, SMRR-PLR extract significantly increased the AUC and C_(max) of PLR flavonoids and tanshinones in rat plasma, suggesting that the combination of SMRR and PLR promoted the absorption of the above components. The underlying mechanism needs to be further studied.