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.

Effects of salvianolic acids on endothelial cells against damage induced by cholestane-3β-5α-6β-triol

To investigate the effects of salvianolic acids on human umbilical vein endothelial cells (HUVEC) against damage induced by cholestane-3β-5α-6β-triol (chol-triol) Methods The viability of HUVEC was measured by MTT method The apoptosis of HUVEC induced by chol-triol was detected by flow cytometry and TUNEL assay The production of malondialdehyd (MDA) in HUVEC was tested by thiobarbaturic acid (TBA) assay Results The viability of HUVEC treated with chol-triol 100 μmol/L decreased by 39 8% while salvianolic acids 100 μg/ml increased by 27 9% The apoptotic rate of HUVEC measured by PI staining increased from 6%-8% to 17%-20% after chol-triol treatment for 12 h Salvianolic acids 100 μg/ml reduced the apoptotic rate to 10%-14% after treatment HUVEC for 1 h prior to chol-triol treatment In another experiment, chol-triol increased the number of TUNEL-positive cells 5 times, but salvianolic acids 10 μg/ml and 100 μg/ml reduced the number of TUNEL-positive cells by 36 9% and 61 2%, respectively The production of MDA in HUVEC increased by 120 7% after chol-triol treatment for 12 h Salvianolic acids 10 μg/ml and 100 μg/ml also decreased the concentration of MDA by 28 7% and 39 8%, respectively Conclusion Salvianolic acids has protective effect on endothelial cells against damage induced by chol-triol

EETs mediate cardioprotection of salvianolic acids through MAPK signaling pathway

Salvianolic acids,including salvianolic acid A(SAA)and salvianolic acid B(SAB),are.the main water-soluble bioactive compounds isolated from the Chinese medicinal herb Danshen and have been shown to exert in vitro and in vivo cardiovascular protection.Recent studies suggest that epoxyeicosatrienoic acids(EETs),the primary cytochrome P4502J(CYP2J)epoxygenase metabolites of arachidonic acid,are involved in the progression of ischemic injury in diverse organs.Here,we investigated the relation between the protective effects of salvianolic acids and EETs/sEH as well as MAPK signaling pathway.In the present study,the rat acute myocardial infarction(AMI)model was established by the left anterior descending coronary artery occlusion.Our results showed that salvianolic acids significantly reduced ST-segment elevation and serum levels of CK-MB,LDH,and ALT in AMI rats,and significantly attenuated the caspase 3 expression and reduced the ratio of Bax/Bcl-2.ELISA measurement showed that salvianolic acids significantly increased the 14,15-EET levels in blood and heart,and attenuated hydrolase activity of sEH in heart of AMI rat.Western blotting analysis suggested that salvianolic acids significantly attenuated the phosphorylation of JNK and p38,and increased phosphorylation of ERK in heart.In conclusion,these results indicate that EETs/sEH and MAPK signaling pathways are important processes in cardioprotection of salvianolic acids.

Effects of LaCl_3 on photosynthesis and the accumulation of tanshinones and salvianolic acids in Salvia miltiorrhiza seedlings

The effects of LaCl3 on the growth,photosynthetic gas-exchange characteristics,chlorophyll fluorescence,and the accumulation of tanshinones and salvianolic acids in Salvia miltiorrhiza seedlings were investigated. The results showed that the increase in photosynthesis induced by LaCl3 might be attributed to the enhanced stomatal conductance of the leaves and the increased level of the photochemical efficiency of PS II. The accumulation of tanshinone IIA and cryptotanshinone was markedly increased with the application of LaCl3 at 20 and 60 mg/L,while tanshinone I was only slightly increased. The content of salvianolic acid B was,however,decreased with the treatment of LaCl3 at 200 mg/L.

Study on Optimization of Ethanol Reflux Extraction of Phenolic Acids from Salvia miltiorrhiza

The extraction technology of phenolic acid compounds from Salvia miltiorrhiza by ethanol reflux was studied. In this experiment, salvianolic acid B standard was used to make the standard curve. Single factor experiment and orthogonal experiment were used to study the extraction of different ethanol concentrations, reflux times and material-to-liquid ratios. The OD value of salvianolic acid compounds was measured with a spectrophotometer. The extraction rate of phenolic acid compounds under different extraction conditions was calculated through a regression equation, so as to obtain the optimal conditions for the ethanol reflux extraction process of Salvia miltiorrhiza. The experimental data can provide a reference for the ethanol reflux extraction process of salvianolic acids in the industry. According to the experiment, the extraction rate of phenolic acids in Salvia miltiorrhiza was the highest when the ethanol concentration was 60%, the reflux time was 1.5 hours, and the ratio of material-to-liquid was 1:10.

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.

Role of CD36 in central nervous system diseases

CD36 is a highly glycosylated integral membrane protein that belongs to the scavenger receptor class B family and regulates the pathological progress of metabolic diseases.CD36 was recently found to be widely expressed in various cell types in the nervous system,including endothelial cells,pericytes,astrocytes,and microglia.CD36 mediates a number of regulatory processes,such as endothelial dysfunction,oxidative stress,mitochondrial dysfunction,and inflammatory responses,which are involved in many central nervous system diseases,such as stroke,Alzheimer’s disease,Parkinson’s disease,and spinal cord injury.CD36 antagonists can suppress CD36 expression or prevent CD36 binding to its ligand,thereby achieving inhibition of CD36-mediated pathways or functions.Here,we reviewed the mechanisms of action of CD36 antagonists,such as Salvianolic acid B,tanshinone IIA,curcumin,sulfosuccinimidyl oleate,antioxidants,and small-molecule compounds.Moreover,we predicted the structures of binding sites between CD36 and antagonists.These sites can provide targets for more efficient and safer CD36 antagonists for the treatment of central nervous system diseases.

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.

SALVIANOLIC ACID H,A NEW DEPSIDE FROM SALVIA CAVALERIEI VAR.SIMPLICIFOLIA

A new depside named salvianolic acid H was isolated from the aqueous extract of Salvia cavaleriei vat.simplicifolia,along with salvianolic acid A,B,C,isosalvianolic acid C,lithospermic acid, rosmarinic acid,R-(+)-β-(3,4-dihydroxyphenyl)lacticacid and protocatechualdehyde.Rosmarinic acid was obtained as the major component.

SALVIANOLIC ACID G,A CAFFEIC ACID DIMER WITH A NOVEL TETRACYCLIC SKELETON

Salvianolic acid G,a caffeic acid dimer with a novel tetracyclic skeleton was isolated from the roots of Salvia miltiorrhiza.Its structure was elucidated by chemical and spectral analysis,especially by 2D NMR analysis.

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.

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.

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 A ameliorates AGEs-induced glomerular endothelial dysfunction and protects against diabetic nephropathy

OBJECTIVE Diabetic nephropathy(DN)has been one of the most common complications of diabetes and the leading cause of end-stage renal disease.Glomerular hyperfiltrationis central in earlystage of DN and leads to the progression of renal architectonic and functional abnormalities.Salvianolic acid A(SalA)has been proved to protect diabetic complications such as hepatic fibrosis and neuropathy.The present study was designed to investigate the effects of SalA on glomerular endothelial dysfunctionand diabetic nephropathy.METHODS Primary glomerular endothelial cells were subjected to assess permeability under injury of advanced glycation end-products(AGEs).AGEs-induced changes of Rho A/ROCK pathway and cytoskeleton rearrangement were assessed bywestern blotandimmunofluorescence.The beneficial effects of SalA on diabetic nephropathy were investigated in a rat model induced by high-fat and high-glucose diet combined with low dose of streptozocin(35 mg·kg^(-1),ip).Renal function and architectonic changes were evaluated by biochemical assay and PAS staining.RESULTS SalA 3μMameliorated AGEs-induced glomerular endothelial permeability(P<0.05)and suppressed rearrangement of cytoskeleton through inhibiting AGE-RAGE-Rho A/ROCK pathway.SalA1 mg·kg^(-1)markedly reduced endothelium loss(P<0.01)and glomerular hyperfiltration(P<0.05)in diabetic kidney.Subsequently,SalA 1 mg·kg^(-1) suppressed glomerular hypertrophy and mesangial matrix expansion,eventually reduced 24 h-urinary albumin and ameliorated renal function by decreasing blood urine nitrogen(BUN),serum creatinine(Scr)and serum n-acetyl-β-d-glucosaminidase(NAG).AGEs-RAGE-Nox4-induced oxidative stress was suppressed by the treatment of SalA 1 mg·kg^(-1).CONCLUSION SalA ameliorated AGEs-induced glomerular endothelial hyperpermeability,and effectively protected against early-stage diabetic nephropathy by reducing hyperfiltration and alleviating renal structural deterioration through inhibiting AGEs and its downstream pathway.Thus,SalA might be a promising therapeutic agent for the treatment of diabetic nephropathy.

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.

Preparation and Certification of a New Salvianolic Acid A Reference Material for Food and Drug Research

Salvianolic acid A(Sal A),a water-soluble ingredient in Danshen,has various biological activities.Sal A and its impurities have similar physical and chemical properties,as well as strong reducibility;therefore,they are difficult to prepare and purify.In this study,high-purity Sal A was obtained by purification of sephadex chromatography and preparative chromatography.Furthermore,HPLC-DAD tandem ECD and HPLC-DAD tandem MS methods were used for non-volatile organic impurity analysis,ICP-MS method was used for non-volatile inorganic impurities and mass balance method and quantitative nuclear magnetic resonance were employed to certify the product.The structures of Sal A and its relative impurities were validated by nuclear magnetic resonance spectroscopy and mass spectrometry,and their contents were quantified as well.Following the principles of ISO Guides 34:2009 and 35:2005,a Sal A reference material was certified,covering homogeneity studies,stability studies,characterization,and uncertainty estimations.

The Synthesis of Hexamethyl Ether of Salvianolic Acid A Benzyl Ester

A salvianolic acid A derivative, hexamethyl ether of salvianolic acid A benzyl ester, was synthesized in fourteen steps. The overall yield was 3.4 parts per thousand. The compound was identified by (1)HNMR and MS. This is the first report of building this structural skeleton.