Determination of Salvianolic Acid B in Yiqi Huayu Prescription by HPLC

[Objectives]To determine the content of salvianolic acid B in Yiqi Huayu Prescription by HPLC.[Methods]The chromatographic column was ZORBAX Eclipse Plus C 18(4.6 nm×250 nm,5μm);the mobile phase was acetonitrile-0.1%phosphoric acid(21:79),the detection wavelength was 286 nm,the column temperature was 30℃,and the flow rate was 1.0 mL/min.A method for determination of salvianolic acid B in Yiqi Huayu Prescription was established.[Results]The linear relationship of salvianolic acid B was good in the range of 0.0214-0.4064 mg/mL.The regression equation was Y=5995.98984 X-0.07332,r=0.9999.The average recovery rate was 98.88%(RSD=1.6%).[Conclusions]The method is reliable,accurate and specific,and can be used for the determination of salvianolic acid B in Yiqi Huayu Prescription.

Effect of salvianolic acid B-loaded mesoporous silica nanoparticles on myocardial ischemia-reperfusion injury

Background:Currently,no drugs can specifically improve clinical cardiac ischemia-reperfusion injury or the prognosis of hemodialysis.Salvianolic acid B(SalB)is a widely used cardiac protectant;however,its clinical application is limited by its low oral bioavailability and poor intestinal absorption.The exploration of its preparation and clinical applications has become a research hotspot in recent years.Methods:To determine whether mesoporous silica nanoparticles(MSNs)efficiently delivered SalB to the heart and SalB@MSNs-RhB reduced myocardial ischemia-reperfusion injury,we constructed a myocardial ischemia-reperfusion male rat model,hypoxia/reoxygenation cardiomyocytes,and treated them with SalB@MSNs-RhB.Results:SalB@MSNs-RhB showed improved bioavailability,therapeutic effect,heightened JAK2/STAT3-dependent pro-survival signaling,and antioxidant responses,thereby protecting cardiomyocytes from ischemia-reperfusion injury-induced oxidative stress and apoptosis.Conclusion:This use of SalB-loaded nanoparticles and investigation of their mechanism of action may provide a new strategy for treating cardiomyocytes.Thus,hypoxia/reoxygenation promotes the clinical application of SalB.

Effect of salvianolic acid B on Smad3 expression in hepatic stellate cells

BACKGROUND: Salvianolic acid B (SA-B), one of water soluble compounds derived from Radix salviae miltiorrhizae, had good action against liver fibrosis of patients with chro- nic hepatitis. Hepatic stellate cells (HSCs) is the cellular re- source for liver fibrogenesis, while transforming growth factor-β1 (TGF-β1) is most potent fibrogenic factor. In this study we investigated the mechanism of SA-B action against liver fibrosis relating to the interference with TGF- β1 signaling at HSC. METHODS: Hepatic stellate cells (HSCs) were isolated, cultured, and incubated with SA-B. The TGF-β1 content in the supernatant of subcultured HSCs was assayed with ELISA. Type I collagen and Smad3 protein in TGF-β1-sti- mulated primarily cultured HSCs for 4 days were detected by Western blot. RESULTS: TGF-β1 secreted in activated HSCs was more than in primary HSCs, and SA-B significantly decreased TGF-β1 secretion in activated HSCs. TGF-β1 increased the expression of type I collagen and Smad3 protein in d4 pri- mary HSCs, while SA-B inhibited their expression. CONCLUSIONS: SA-B inhibits TGF-β1 secretion in activa- ted HSCs and counteracts the expression of TGF-β1 stimu- lated type I collagen and Smad3. These actions are associat- ed with the effect of SA-B on liver fibrosis.

Salvianolic acid B modulates the expression of drug-metabolizing enzymes in HepG2 cells

BACKGROUND: Enzymes involved in drug and xenobiotic metabolism have been considered to exist in two groups: phase I and phase II enzymes. Cytochrome P450 isoenzymes (CYPs) are the most important phase I enzymes in the metabolism of xenobiotics. The products of phase I metabolism are then acted upon by phase II enzymes, including glutathione S-transferases (GSTs). Herbs that inhibit CYPs such as CYP3A4 or that induce GSTs may have the potential to protect against chemical carcinogenesis since the mutagenic effects of carcinogens are often mediated through an excess of CYP-generated reactive intermediates. This study was designed to investigate the effects of salvianolic acid B (Sal B), a pure compound extracted from Radix Salviae Miltiorrhizae, a Chinese herb, on cell proliferation and CYP1A2 and CYP3A4 mRNA expression in the presence or absence of rifampicin, a potent inducer of CYPs and GST protein expression in HepG2 cells. METHODS: HepG2 cells were incubated with different concentrations of Sal B. Cell proliferation was determined by SYTOX-Green nucleic acid staining. CYP3A4 and CYP1A2 mRNA expression was assayed by real-time PCR. GST protein expression was analyzed by Western blotting. RESULTS: Low concentrations of Sal B (0-20 μmol/L) had no significant effects on cell proliferation, while higher concentrations (100-250 μmol/L) significantly inhibited proliferation in a concentration-dependent manner. Ten μmol/L Sal B, but not 1 μmol/L, down-regulated CYP3A4 and CYP1A2 mRNA expression after 24 hours of incubation, whereas both 1 and 10 μmol/L Sal B down-regulated CYP3A4mRNA expression after 96 hours of incubation; moreover, 1 and 10 μmol/L Sal B inhibited CYP3A4 mRNA expression induced by rifampicin. Both 1 μmol/L and 10 μmol/L Sal B increased GST expression. CONCLUSION: Sal B inhibits CYP3A4 and CYP1A2 mRNA expression and induces GST expression in HepG2 cells.

Effects of salvianolic acid B on in vitro growth inhibition and apoptosis induction of retinoblastoma cells

AIM: To observe the effects of salvianolic add B (SalB) on in vitro growth inhibition and apoptosis induction of retinoblastoma HXO-RB44 cells. METHODS: The effects of SalB on the HXO-RB44 cells proliferation in vitro were observed by MTT colorimetric method. The morphological changes of apoptosis before and after the treatment of SalB were observed by Hoechst 33258 fluorescent staining method. Apoptosis rate and cell cycle changes of HXO-RB44 cells were detected by flow cytometer at 48 hours after treated by SalB. The expression changes of Caspase-3 protein in HXO-RB44 cells were detected by Western Blot. RESULTS: SalB significantly inhibited the growth of HXO-RB44 cells, while the inhibition was in a concentration-and time-dependent manner. The results of fluorescent staining method indicated that HXO-RB44 cells showed significant phenomenon of apoptosis including karyorrhexis, fragmentation and the formation of apoptotic bodies, etc. after 24, 48 and 72 hours co-culturing of SalB and HXO-RB44 cells. The results of flow cytometer showed that the apoptosis rate and the proportion of cells in S phase were gradually increased at 48 hours and 72 hours after treated by different concentrations of SalB. Western Blot strip showed that the expression of Caspase-3 protein in HXO-RB44 cells was gradually increased with the increase of the concentration of SalB. CONCLUSION: SalB can significantly affect on HXO-RB44 cells growth inhibition and apoptosis induction which may be achieved through the up-regulation of Caspase-3 expression and the induction of cell cycle arrest.

Evaluation of pharmacokinetics and pharmacodynamics relationships for Salvianolic Acid B micro-porous osmotic pump pellets in angina pectoris rabbit

The work aims to investigate the in vitro release,pharmacokinetics(PK),pharmacodynamics(PD)and PK-PD relationships of Salvianolic Acid B micro-porous osmotic pump pellets(SalB-MPOPs)in angina pectoris New Zealand White(NZW)rabbits,compared with those of SalB immediate-release pellets(SalB-IRPs).The SalB plasma concentrations and Superoxide dismutase levels(PD index)were recorded continuously at predetermined time interval after administration,and the related parameters were calculated by using Win-Nonlin software.The release profile of MPOPs was more sustained than that of IRPs.PK results indicated that the mean C_(max) was significantly lower,the SalB plasma concentrations were steadier,both area under concentration-time curve from 0 to 24 h(AUC_(0-24 h))and from 0 to infinity(AUC_(0-∞))were presented larger,and both the peak concentration time(T_(max))and mean residence time(MRT)were prolonged for MPOPs,as compared with those of IRPs.PD results suggested that peak drug effect(E_(max))was lower and the equilibration rate constant(k_(e0))between the central compartment and the effect compartment was higher of MPOPs vs.those of IRPs.PKePD relationships demonstrated that the effectconcentration-time(ECT)course of MPOPs was clockwise hysteresis loop,and that of IRPs was counter-clockwise hysteresis loop.Collectively,those results demonstrated that MPOPs were potential formulations in treating angina pectoris induced by atherosclerosis.

Salvianolic acid B protects hepatocytes from H2O2 injury by stabilizing the lysosomal membrane

AIM To investigate the capability of salvianolic acid B(Sal B) to protect hepatocytes from hydrogen peroxide(H_2O_2)/carbon tetrachloride(CCl_4)-induced lysosomal membrane permeabilization. METHODS Cell Counting Kit-8 assay was used to measure cell viability. Apoptosis and death were assayed through flow cytometry. Brd U incorporation was used to detect cell proliferation. Serum alanine aminotransferase activity and liver malondialdehyde(MDA) content were measured. Liver histopathological changes were evaluated using hematoxylin-eosin staining. Lysosomal membrane permeability was detected with Lyso Tracker Green-labeled probes and acridine orange staining. The levels of protein carbonyl content(PCC), cathepsins(Cat)B/D, and lysosome-associated membrane protein 1(LAMP1) were evaluated through western blotting. Cytosol Cat B activity analysis was performed with chemiluminescence detection. The m RNA level ofLAMP1 was evaluated through quantitative real-time polymerase chain reaction. RESULTS Results indicated that H_2O_2 induced cell injury/death. Sal B attenuated H_2O_2-induced cell apoptosis and death, restored the inhibition of proliferation, decreased the amount of PCC, and stabilized the lysosome membrane by increasing the LAMP1 protein level and antagonizing Cat B/D leakage into the cytosol. CCl_4 also triggered hepatocyte death. Furthermore, Sal B effectively rescued hepatocytes by increasing LAMP1 expression and by reducing lysosomal enzyme translocation to the cytosol.CONCLUSION Sal B protected mouse embryonic hepatocytes from H_2O_2/CCl_4-induced injury/death by stabilizing the lysosomal membrane.

Salvianolic acid B dry powder inhaler for the treatment of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis(IPF)is a serious and fatal pulmonary inflammatory disease with an increasing incidenceworldwide.The drugs nintedanib and pirfenidone,are listed as conditionally recommended drugs in the“Evidence-Based Guidelines for the Diagnosis and Treatment of Idiopathic Pulmonary Fibrosis”.However,these two drugs have many adverse reactions in clinical application.Salvianolic acid B(Sal B),a water-soluble component of Salvia miltiorrhiza,could alleviate bleomycin-induced peroxidative stress damage,and prevent or delay the onset of IPF by regulating inflammatory factors and fibrotic cytokines during the disease’s progression.However,Sal B is poorly absorbed orally,and patient compliance is poor when administered intravenously.Therefore,there is an urgent need to find a new non-injection route of drug delivery.In this study,Sal B was used as model drug and l-leucine(LL)as excipient to prepare Sal B dry powder inhaler(Sal B-DPI)by spray drying method.Modern preparation evaluation methods were used to assess the quality of Sal B-DPI.Sal B-DPI is promising for the treatment of IPF,according to studies on pulmonary irritation evaluation,in vivo and in vitro pharmacodynamics,metabolomics,pharmacokinetics,and lung tissue distribution.

Salvianolic acid B protects the myelin sheath around injured spinal cord axons

Salvianolic acid B,an active pharmaceutical compound present in Salvia miltiorrhiza,exerts a neuroprotective effect in animal models of brain and spinal cord injury.Salvianolic acid B can promote recovery of neurological function;however,its protective effect on the myelin sheath after spinal cord injury remains poorly understood.Thus,in this study,in vitro tests showed that salvianolic acid B contributed to oligodendrocyte precursor cell differentiation,and the most effective dose was 20 μg/m L.For in vivo investigation,rats with spinal cord injury were intraperitoneally injected with 20 mg/kg salvianolic acid B for 8 weeks.The amount of myelin sheath and the number of regenerating axons increased,neurological function recovered,and caspase-3 expression was decreased in the spinal cord of salvianolic acid B-treated animals compared with untreated control rats.These results indicate that salvianolic acid B can protect axons and the myelin sheath,and can promote the recovery of neurological function.Its mechanism of action is likely to be associated with inhibiting apoptosis and promoting the differentiation and maturation of oligodendrocyte precursor cells.

Neuroprotective effects of salvianolic acid B on secondary spinal cord damage

Salvianolic acid B （Sal B）, an effective ingredient of Danshen （salvia miltiorrhiza root）, has been shown to exhibit anti-oxidative and anti-inflammatory effects. The present study investigated whether Sal B has a neuroprotective effect on secondary spinal cord injury when administrated alone. In addition, the effects of Sal B on attenuating expression of tumor necrosis factor-α （TNF-α） following acute spinal cord injury were analyzed, as well as the effects of combined treatment of Sal B and etanercept. Immunohistochemical staining demonstrated that Sal B significantly reduced matrix metalloproteinase-1 and c-Fos expression at 24 hours after spinal cord injury, and decreased tissue edema was detected using the dry-wet weight method at 3 days after injury. In addition, Sal B significantly promoted recovery of motor function in rats. These effects were most significant at a dose of 20 mg/kg Sal B. At 24 hours after spinal cord injury, reverse transcription-polymerase chain reaction and western blot assay results showed that Sal B, etanercept, or the combination significantly suppressed increased TNF-α mRNA and protein expression, although the combination resulted in more significant outcomes. These results suggested that Sal B exerted neuroprotective effects against secondary spinal cord injury by reducing expression of matrix metalloproteinase-1, c-Fos, and TNF-α. Moreover, Sal B combined with etanercept resulted in more significant anti-inflammatory effects.

Interactions of acetylcholinesterase with salvianolic acid B and rosmarinic acid from Salvia miltiorhiza water extract investigated by NMR relaxation rate

In order to understand whether the ameliorating effect on old ages memory disorder by the root of Salvia miltiorhiza is related to the acetylcholinesterase （ACHE） inhibition, two main ingredients, salvianolic acid B （1） and rosmarinic acid （2）, which were isolated from S. miltiorhiza water extract, were investigated in vitro by NMR relaxation rate in this work. The results showed that the proton selective relaxation rates and the molecular rotational correlation time of proton pairs for compounds 1 and 2 increased significantly by adding of AChE in mixing solution. The study reveals that the two compounds might bind to the enzyme and have ACHE inhibitory effect, which could contribute to the ameliorating effect at some extent on old ages memory disorder.

Neuroprotective effects of salvianolic acid B against oxygen-glucose deprivation/reperfusion damage in primary rat cortical neurons

Background Cerebral ischemia-reperfusion injury is the main reason for the loss of neurons in the ischemic cerebrovascular disease. Therefore, to deeply understand its pathogenesis and find a new target is the key issue to be solved. This research aimed to investigate the neuroprotective effects of salvianolic acid B （SalB） against oxygen-glucose deprivation/reperfusion （OGD/RP） damage in primary rat cortical neurons.Methods The primary cultures of neonatal Wister rats were randomly divided into the control group, the OGD/RP group and the SalB-treatment group （10 mg/L）. The cell model was established by depriving of oxygen and glucose for 3 hours and reperfusion for 3 hours and 24 hours, respectively. The neuron viability was determined by MTT assay. The level of cellular reactive oxygen species （ROS） was detected by fluorescent labeling method and spin trapping technique respectively. The activities of neuronal Mn-superoxide dismutase （Mn-SOD）, catalase （CAT） and glutathione peroxidase （GSH-PX） were assayed by chromatometry. The mitochondria membrane potential （△ψm） was quantitatively analyzed by flow cytometry. The release rate of cytochrome c was detected by Western blotting. The neuronal ultrastructure was observed by transmission electron microscopy. Statistical significance was evaluated by analysis of variance （ANOVA）followed by Student-Newman-Keuls test.Results OGD/RP increased the level of cellular ROS, but decreased the cell viability and the activities of Mn-SOD, CAT and GSH-PX; SalB treatment significantly reduced the level of ROS （P ＜0.05）; and enhanced the cell viability （P ＜0.05）and the activities of these antioxidases （P ＜0.05）. Additionally, OGD/RP induced the fluorescence value of △ψm to diminish and the release rate of cytochrome c to rise notably; SalB markedly elevated the level of △ψm （P ＜0.01） and depressed the release rate of cytochrome c （P ＜0.05）; it also ameliorated the neuronal morphological injury.Conclusion The neuroprotection of SalB may be attributed to the elimination of ROS and the inhibition of apoptosis.

Antioxidant Effect of Salvianolic Acid B on Hippocampal CA1Neurons in Mice with Cerebral Ischemia and Reperfusion Injury

Objetive： TO investigate the neuroprotective effects and underlying mechanisms of salvianolic acid B （Sal B） extracted from Salvia miltiorrhiza on hippocampal CA1 neurons in mice with cerebral ischemia reperfusion injury. Methods： Forty male National Institute of Health （NIH） mice were randomly divided into 4 groups with 10 animals each, including the sham group, the model group, the SalB group （SalB 22.5 mg/kg） and the nimodipine （Nim） group （Nim 1 mg/kg）. A mouse model of cerebral ischemia and reperfusion injury was established by bilateral carotid artery occlusion for 30 rain followed by 24-h reperfusion. The malondialdehyde （MDA） content, the nitric oxide synthase （NOS） activity, the superoxide dismutase （SOD） activity and total anti- oxidant capability （T-AOC） of the pallium were determined by biochemistry methods. The morphologic changes and Bcl-2 and Bax protein expression in hippocampal CA1 neurons were observed by using hematoxylin- eosin staining and immunohistochemistry staining, respectively. Results： In the SalB group, the MDA content and the NOS activity of the pallium in cerebral ischemia-reperfusion mice significantly decreased and the SOD activity and the T-AOC significantly increased, as compared with the model group （P〈0.05 or P〈0.01）. The SalB treatment also rescued neuronal loss （P〈0.01） in the hippocampal CA1 region, strongly promoted Bcl-2 protein expression （P〈0.01） and inhibited Bax protein expression （P〈0.05）. Conclusions： SalB increases the level of antioxidant substances and decreases free radicals production. Moreover, it also improves Bcl-2 expression and reduces Bax expression. SalB may exert the neuroprotective effect through mitochondria-dependent pathway on hippocampal CA1 neurons in mice with cerebral ischemia and reperfusion injury and suggested that SalB represents a promising candidate for the prevention and treatment of ischemic cerebrovascular disease.

Salvianolic Acid B Inhibits the TLR4-NFκ B-TNFα Pathway and Attenuates Neonatal Rat Cardiomyocyte Injury Induced by Lipopolysaccharide

Objective： To investigate the role of the TLR4-NF K B-TNFa inflammation pathway on lipopolysaccharide （LPS）-induced neonatal rat cardiomyocyte injury and the possible protective effects of salvianolic acid B （Sal B）. Methods： Wistar rat （1-2 days old） cardiomyocytes were isolated and cultured. Sal B 10-5mol/L, 10-6mol/L and 10-7mol/L were pro-treated for 6 h in the culture medium. LPS （1 μg/mL） was added to the culture medium and kept for 6 h to induce inflammation injury. The concentration of lactate dehydrogenase （LDH） in the supernatant was detected by spectrophotometry. The concentrations of tumor necrosis factor （TNF a） and heat shock protein 70 （HSP70） in the supernatant were detected by enzyme linked immunosorbent assay. The protein expressions of toll, such as receptor 4 （TLR4） and nuclear factor kappa B （NF K B） were detected by immunohistochemistry. The mRNA expressions of TLR4 and NF K B were detected by realtime reverse transcription polymerase chain reaction （RT-PCR）. Results： （1） The concentrations of LDH and TNF a in the LPS control group were significantly higher than those in the control group （561.41 ± 67,39 U/L and 77.94± 15.08 pg/mL, versus 292.13± 26.02 U/L and 25.39 ±16.53 pg/mL, respectively, P〈0.01, P〈0.05）. Compared with the LPS control group, the concentrations of LDH and TNF α were significantly decreased in the Sal B 10-5mol/L pro-treated group （451.76 ± 83.96 U/L and 34.00± 10.38 pg/mL, respectively, P〈0.05）. （2） The TLR4 and NF K B protein expression area in the LPS control group were significantly higher than those in the control group （1712.41 ± 410.12 μm2 and 2378.15 ± 175.29 μm2, versus 418.62 ± 24.42 μ m2 and 1721.74 ± 202.87μ m2, respectively, P〈0.01）. The TLR4 and NF K B protein expression internal optical density （IOD） values in the LPS control group were also significantly higher than those in the control group （3.06 ±0.33 and 7.20± 1.04, versus 0.91 ±0.21 and 4.24±0.48, respectively, P〈0.05 and P〈0.01）. Compared with the LPS control group, the TLR4 and NF K B protein expression areas were significantly decreased in the Sal B 10Smol/L pre-treated group （1251.54± 133.82 μ m2 and 1996.37 ± 256.67 μ m2, respectively, P〈0.05）, the TLR4 and NF K B protein expression IOD values were also significantly decreased in the Sal B 10-5mol/L pretreated group （1.92 ±0.28 and 5.17 ±0.77, respectively, P〈0.05）. （3） The TLR4 and NF K B mRNA expressions （2△△CT value） in the LPS control group were significantly higher than those in the control group （3.16 ± 0.38 and 5.03±0.43 versus 1.04±0.19 and 1.08±0.21, respectively, P〈0.01）. Compared with the LPS control group, the TLR4 and NF KB mRNA expressions （2△△CT value） were significantly decreased in the Sal B 10-5mol/L pre- treated group （1.34 ±0.22 and 1.74 ± 0.26, respectively, P〈0.05）. The concentration of HSP70 did not show any statistical differences in all groups （P〉0.05）. Conclusions： The TLR4-NF K B-TNF α pathway was quickly activated and was independent of HSP70 in the early phase of neonatal cardiomyocyte injury induced by LPS. The protective effects of Sal B may be through inhibiting the TLR4-NF K B-TNF a pathway and are dose-dependent.

Salvianolic Acid B Down-regulates Matrix Metalloproteinase-9 Activity and Expression in Tumor Necrosis Factor-α-induced Human Coronary Artery Endothelial Cells

Background：Salvianolic acid B （Sal B） is a bioactive water-soluble compound of Salviae miltiorrhizae,a traditional herbal medicine that has been used clinically tor the treatment of cardiovascular diseases.This study sought to evaluate the effect of Sal B on matrix metalloproteinase-9 （MMP-9） and on the underlying mechanisms in tumor necrosis factor-α （TNF-α）-activated human coronary artery endothelial cells （HCAECs）,a cell model of Kawasaki disease.Methods：HCAECs were pretreated with 1 l0 μmol/L of Sal B,and then stimulated by TNF-α at different time points.The protein expression and activity of MMP-9 were determined by Western blot assay and gelatin zymogram assay,respectively.Nuclear factor-κB （NF-κB） activation was detected with immunofluorescence,electrophoretic mobility shift assay,and Western blot assay.Protein expression levels of mitogen-activated protein kinase （c-Jun N-terminal kinase [JNK],extra-cellular signal-regulated kinase [ERK],and p38） were determined by Western blot assay.Results：After HCAECs were exposed to TNF-α,1-10 μtmol/L Sal B significantly inhibited TNF-α-induced MMP-9 expression and activity.Furthermore,Sal B significantly decreased IκBα phosphorylation and p65 nuclear translocation in HCAECs stimulated with TNF-α for 30 min.In addition,Sal B decreased the phosphorylation of JNK and ERK1/2 proteins in cells treated with TNF-α for 10 min.Conclusions：The data suggested that Sal B suppressed TNF-α-induced MMP-9 expression and activity by blocking the activation of NF-κB,JNK,and ERK1/2 signaling pathways.

The effects of borneol on the pharmacokinetics and brain distribution of tanshinone IIA,salvianolic acid B and ginsenoside Rg1 in Fufang Danshen preparation in rats

Fufang Danshen preparation(FDP)is consisted of Salviae Miltiorrhizar Radix et Rhizoma(Danshen),Notoginseng Radix et Rhizoma(Sanqi)and Borneolum Syntheticum(borneol).FDP is usually used to treat myocardial ischemia hypoxia,cerebral ischemia and alzheimer’s disease,etc.In the treatment of cerebrovascular diseases,borneol is usually used to promote the absorption and distribution of the bioactive components to proper organs,especially to the brain.The purpose of this study is investigating the effects of borneol on the pharmacokinetics and brain distribution of tanshinone IIA(TS IIA),salvianolic acid B(SAB)and ginsenoside Rg1 in FDP.Male healthy Sprague-Dawley(SD)rats were given Danshen extracts,Sanqi extracts(Panax notoginseng saponins)or simultaneously administered Danshen extracts,Sanqi extracts and borneol.Plasma and brain samples were collected at different points in time.The concentration of TS IIA,SAB and Rg1 was determined by UPLC-MS/MS method.The main pharmacokinetics parameters of plasma and brain tissue were calculated by using Phoenix WinNolin 6.1 software.In comparison with Danshen and Sanqi alone,there were significant differences in pharmacokinetic parameters of TS IIA,SAB and Rg1,and the brain distribution of SAB and TS IIA when Danshen,Sanqi and borneol were administrated together.Borneol statistically significant shortened tmax of TS IIA,SAB and Rg1 in plasma and brain,increased the bioavaiability of Rg1,inhibited metabolism of Rg1 and enhanced the transport of TS IIA and SAB to brain.These results indicated that borneol could affect the multiple targets components and produce synergistic effects.Through accelerating the intestinal absorption and brain distribution,borneol caused the effective ingredients of Danshen and Sanqi to play a quicker therapeutic role and improved the therapeutic effect.

Insights into salvianolic acid B biosynthesis from chromosome-scale assembly of the Salvia bowleyana genome

Salvia bowleyanais a traditional Chinese medic-inal plant that is a source of nutritional supple-ments rich in salvianolic acid B and a potentialexperimental system for the exploration ofsalvianolic acid B biosynthesis in the Labiatae.Here,we report a high‐quality chromosome‐scalegenome assembly of S.bowleyana covering 462.44 Mb,with a scaffold N50 value of 57.96 Mband 44,044 annotated protein‐coding genes.Evolutionary analysis revealed an estimateddivergence time betweenS.bowleyanaand its close relativeS.miltiorrhiza of~3.94 millionyears.We also observed evidence of a whole‐genome duplication in theS.bowleyanagenome.Transcriptome analysis showed that SbPAL1(PHENYLALANINE AMMONIA‐LYASE1)ishighlyexpressed in roots relative to stem and leaves,paralleling the location of salvianolic acid Baccumulation.The laccase gene family in S.bowley anaoutnumbered their counterparts inbothS.miltiorrhiza and Arabidopsis thaliana,suggesting that the gene family has undergoneexpansion inS.bowleyana.Several laccasegenes were also highly expressed in roots,wheretheir encoded proteins may catalyze the oxida-tive reaction from rosmarinic acid to salvianolicacid B.Thesefindings provide an invaluable ge-nomic resource for understanding salvianolicacid B biosynthesis and its regulation,and will beuseful for exploring the evolution of the Labiatae.

Effect of Salvianolic Acid B on Mitochondrial Function of Cerebral Ischemia in Mice

The effects of salvianolic acid B （SalB） on the mitochondrial membrane potential （MMP）, calcium, and apoptosis of neurons with cerebral ischemia in mice were investigated using an acute cerebral ischemia model established by ligating the bilateral common carotid arteries in mice. The MMP, the intracellular cal- cium concentration, and the apoptosis rate of cortical neurons were measured at 6 min, 12 rain, 18 rain, 24 min, and 30 min after cerebral ischemia by a flow cytometer. The experiments show that SalB increases the MMP and reduces the intracellular calcium and the apoptosis rate at different stages of the cerebral ischemia in mice. The results show that the protective mechanism of SalB on cerebral ischemia enhances the MMP and maintains intracellular calcium homeostasis.

Attenuation of Oxidative Stress-Induced Cell Apoptosis and Pyroptosis in RSC96 Cells by Salvianolic Acid B

Objective:To determine whether salvianolic acid B(Sal B)exerts protective effects on diabetic peripheral neuropathy by attenuating apoptosis and pyroptosis.Methods:RSC96 cells were primarily cultured with DMEM(5.6 mmol/L glucose),hyperglycemia(HG,125 mmol/L glucose)and Sal B(0.1,1,and 10μmol/L).Cells proliferation was measured by 3-(4,5-cimethylthiazol-2-yl)-2,5-dilphenyltetrazolium bromide assay.Reactive oxygen species(ROS)generation and apoptosis rate were detected by flow cytometry analysis.Western blot was performed to analyze the expressions of poly ADP-ribose polymerase(PARR),cleaved-caspase 3,cleavedcaspase 9,Bcl-2,Bax,NLRP3,ASC,and interleukin(IL)-1β.Results:Treatment with HG at a concentration of125 mmol/L attenuated cellular proliferation,while Sal B alleviated this injury(P<0.05).In addition,Sal B inhibited HG-induced ROS production and apoptosis rate(P<0.05).Furthermore,treatment with Sal B down-regulated HG-induced PARP,cleaved-caspase 3,cleaved-caspase 9,Bax,NLRP3,ASC,and IL-1βexpression,but mitigated HG-mediated down-regulation of Bcl-2 expression(P<0.05).Conclusion:Sal B may protect RSC96 cells against HG-induced cellular injury via the inhibition of apoptosis and pyroptosis activated by ROS.

Effect of astragaloside IV and salvianolic acid B on antioxidant stress and vascular endothelial protection in the treatment of atherosclerosis based on metabonomics

Vascular endothelial cells and oxidation reduction system play an important role in the pathogenesis of atherosclerosis(AS).If these conditions are disordered,it will inevitably lead to plaque formation and even rupture.Astragaloside IV(AsIV)and salvianolic acid B(Sal B)are the main active ingredients of Astragalus membranaceus and Salvia miltiorrhiza,respectively,and found to ameliorate vascular endothelial dysfunction and protect against oxidative stress in recent studies.However,it is still unknown if the combination of AsIV and Sal B(AsIV+Sal B)can inhibit the development of plaque through amplifying the protective effect of vascular endothelial cells and anti-oxidative stress effect.To clarify the role of AsIV+Sal B in AS,we observed the efficacy of each group(Control,Model,AsIV,Sal B,and AsIV+Sal B)by biomolecular assays,such as observing the pathological morphology of the aorta by oil red O staining,evaluating the level of oxidative stress and endothelial cells in the serum by the Elisa test,and analyzing the changes of all small molecule metabolites in liver tissue by UPLC-QTOF-MS.Results showed that AsIV,Sal B and AsIV+Sal B decreased the deposition of lipid in the arterial wall,so as to exert the effect of anti-oxidant stress and vascular endothelial protection,where the inhibitory effect of AsIV+Sal B was the most obvious.Metabonomics analysis showed that Sal B regulated the metabolic pathways of arginine and proline.AsIV regulated glycerol metabolism and saturated fatty acid biosynthesis metabolism.AsIV+Sal B is mainly related to the regulation of the citrate cycle(TCA cycle),alanine,aspartic acid,and glutamate metabolism,cysteine,and methionine metabolism.Succinic acid and methionine are synergistic metabolites that exert an enhancing effect when AsIV and Sal B were used in combination.In conclusion,we demonstrated that AsIV acompanied with Sal B can be successfully used for anti-oxidative stress and vascular endothelial protection of AS,and succinic acid and methionine are the synergistic metabolites.