Salvianolic Acid A Research Articles

Competitive Inhibition of Human Organic Anion Transporters 1 (SLC22A6), 3 (SLC22A8) and 4 (SLC22A11) by Major Components of the Medicinal Herb Salvia miltiorrhiza (Danshen)

When herbal products are used in combination therapy with drugs, alterations in pharmacokinetics, pharmacodynamics, and toxicity can result. Many active components of herbal products are organic anions, and human organic anion transporter 1 (hOAT1, SLC22A6), hOAT3 (SLC22A8), and hOAT4 (SLC22A11) have been identified as potential sites of drug-drug interactions. Therefore, we assessed the effects of lithospermic acid (LSA), rosmarinic acid (RMA), salvianolic acid A (SAA), salvianolic acid B (SAB), and tanshinol (TSL), components of the herbal medicine Danshen, on the function of these transporters. Kinetic analysis demonstrated a competitive mechanism of inhibition for all five. K(i) values (µM) were estimated as 20.8 ± 2.1 (LSA), 0.35 ± 0.06 (RMA), 5.6 ± 0.3 (SAA), 22.2 ± 1.9 (SAB), and 40.4 ± 12.9 (TSL) on hOAT1 and as 0.59 ± 0.26 (LSA), 0.55 ± 0.25 (RMA), 0.16 ± 0.03 (SAA), 19.8 ± 8.4 (SAB), and 8.6 ± 3.3 (TSL) on hOAT3. No significant inhibition of hOAT4 activity by TSL was observed. Using published human pharmacokinetic values, unbound C(max)/K(i) ratios were calculated as an indicator of in vivo drug-drug interaction potential. Analysis indicated a strong interaction potential for RMA and TSL on both hOAT1 and hOAT3 and for LSA on hOAT3. Thus, herb-drug interactions may occur in vivo in situations of co-administration of Danshen and clinical therapeutics known to be hOAT1/hOAT3 substrates.

The stability investigation of compound Danshen injection (a traditional medicine) with a new high-performance liquid chromatography method

Background:Compound Danshen injection (CDSI, a traditional medicine) is an effective drug for the treatment of cardiovascular and cerebrovascular diseases. However, the research about its stability is absent.Objective:A new high-performance liquid chromatography method was developed to assay its main effective constituents, i.e., propanoid acid (PA), protocatechuic aldehyde (PHA), salvianolic acid B (SAB), salvianolic acid A (SAA), and rosmarinic acid (RA). Through the newly found method, the stability of CDSI was to be investigated.Materials and Methods:The analysis was performed by a reverse-phase gradient elution using an aqueous mobile phase (containing 0.1% acetic acid) modified by acetonitrile, and detection was made simultaneously at 280 nm and 325 nm. The method was validated for accuracy, precision and limits of detection. The effects of some environmental storage conditions (light and temperature) on the stability of CDSI were investigated.Results:This method is precise, simple, and convenient. The result showed that illumination and temperature had an obvious effect on CDSI's stability. SAA is the most unstable one among the five components. In the condition of common light, it decomposed rapidly to almost 50% after only 4 h, and 100% after 8 h. PA, RA, and PHA might come from Danshen, was also the transformed products from other components in store process.Conclusion:The result indicated that the main active constituents in CDSI suffered from the illumination and temperature greatly. CDSI should be stored at low temperature and kept away from light.

Simultaneous determination of six hydrophilic components in rat plasma after oral administration of Jitai tablet by liquid chromatography–electrospray ionization–tandem mass spectrometry: Application to a pharmacokinetic study

A liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method was developed and validated for the simultaneous determination of amygdalin (ADL), danshensu (DSS), ferulic acid (FA), hydroxysafflor yellow A (HSYA), salvianolic acid A (SAA) and salvianolic acid B (SAB) in rat plasma. Plasma samples were pretreated by protein precipitation with acetonitrile. LC separation was performed on a Zorbax Eclipse Plus C18 column (3.0mm×100mm I.D, 1.8μm) with gradient elution using a mobile phase consisting of acetonitrile-0.1% formic acid in water at a flow rate of 0.3mL/min. ESI-MS spectra was acquired in negative ion multiple reaction monitoring mode. The mass transition ion-pair was followed as m/z 456.0→323.1, m/z 197.3→178.8, m/z 193.0→133.9, m/z 611.1→325.2, m/z 493.0→295.0, and m/z 717.0→519.0 for ADL, DSS, FA, HSYA, SAA and SAB, respectively. All analytes showed good linearity over a wide concentration range (r>0.99). The lower limit of quantification was 7ng/mL, 2ng/mL, 4ng/mL, 1ng/mL, 2ng/mL, and 4ng/mL for ADL, DSS, FA, HSYA, SAA and SAB, respectively. The mean recovery of the analytes ranged from 86.29% to 93.16%. The intra- and inter-day precisions were in the range of 1.50–9.98% and the accuracies were between 91.17% and 99.46%. The validated method was successfully applied to a pharmacokinetic study of the six hydrophilic components in rat plasma after oral administration of Jitai tablet.

Protection of salvianolic acid A on rat brain from ischemic damage via soluble epoxide hydrolase inhibition

Epoxyeicosatrienoic acids (EETs) and their regulating enzyme soluble epoxide hydrolase (sEH) have been associated with ischemic stroke. Salvianolic acid A (SAA) is proved to display potent cerebroprotection. However, little information is available about the link between them. This study aimed to investigate whether SAA exhibits its protective effects in rats subjected to middle cerebral artery occlusion (MCAO) through sEH and EETs. The results showed that SAA treatment ameliorated neurological deficits and reduced infarct volume. Notably, the beneficial effects of SAA were attenuated by co-administration of (14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE)), a putative selective EETs antagonist. Furthermore, SAA increased the 14,15-EET levels in the blood and brain of sham and MCAO rats. Assay for hydrolase activity showed that 1 and 3 mg/kg of SAA significantly diminished brain sEH activity of MCAO rats. A fluorescent assay in vitro indicated that SAA could inhibit recombinant human sEH activity in a concentration-dependent manner (IC50 = 1.62 μmol/l). Immunohistochemical analysis showed that SAA at the doses of 1 and 3 mg/kg significantly decreased sEH protein expression in hippocampus CA1 region of MCAO rats. In conclusion, cerebral protection of SAA is mediated, at least in part, via inhibiting sEH to increase EETs levels.

Application of Multivariate Curve Resolution Method in the Quantitative Monitoring Transformation of Salvianolic Acid A Using Online UV Spectroscopy and Mass Spectroscopy

This work presents an exploratory study of monitoring the transformation process of salvianolic acid A (SAA) using an online UV spectroscopic analysis system. A chemometrics approach, based on multivariate curve resolution of spectrophotometric matrix, was applied to resolve the concentration and spectra profiles of reactant species and to evaluate the kinetic profile. Kinetic runs have been developed at temperatures ranging from 25 to 90 °C and pH values ranging from 2.5 to 10 in order to investigate the effects of these two variables during the transformation process of SAA. The degradation reaction of SAA was considered as first-order and the degradation kinetic constant of SAA increases when temperature increases. It was also found that SAA transforms rapidly when pH is higher than 7, but remains stable when pH is lower than 6.5. Two transformation products were auxiliarily identified by direct mass spectrometric analysis using Mass Work software. Our results suggest that the manufacturing process of ...

Active Hydrophilic Components of the Medicinal HerbSalvia miltiorrhiza(Danshen) Potently Inhibit Organic Anion Transporters 1 (Slc22a6) and 3 (Slc22a8)

Many active components of herbal products are small organic anions, and organic anion transporters were previously demonstrated to be a potential site of drug-drug interactions. In this study, we assessed the inhibitory effects of six hydrophilic components of the herbal medicine Danshen, lithospermic acid, protocatechuic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, and tanshinol, on the function of the murine organic anion transporters, mOat1 and mOat3. All of Danshen components significantly inhibited mOat1- and mOat3-mediated substrate uptake (P < 0.001) with lithospermic acid (LSA), protocatechuic acid, rosmarinic acid (RMA), and salvianolic acid A (SAA) producing virtually complete inhibition under test conditions. Kinetic analysis demonstrated that LSA, RMA, and SAA were competitive inhibitors. As such, K i values were estimated as 14.9 ± 4.9 μM for LSA, 5.5 ± 2.2 μM for RMA, and 4.9 ± 2.2 μM for SAA on mOat1-mediated transport, and as 31.1 ± 7.0 μM for LSA, 4.3 ± 0.2 μM for RMA, and 21.3 ± 7.7 μM for SAA on mOat3-mediated transport. These data suggest that herb-drug interactions may occur in vivo on the human orthologs of these transporters in situations of polypharmacy involving Danshen and clinical therapeutics known to be organic anion transporter substrates.

Cardioprotective Effects of Salvianolic Acid A on Myocardial Ischemia-Reperfusion InjuryIn VivoandIn Vitro

Salvianolic acid A (SAA), one of the major active components of Danshen that is a traditional Chinese medicine, has been reported to possess protective effect in cardiac diseases and antioxidative activity. This study aims to investigate the cardioprotection of SAA in vivo and in vitro using the model of myocardial ischemia-reperfusion in rat and hydrogen peroxide (H2O2)-induced H9c2 rat cardiomyoblasts apoptosis. It was found that SAA significantly limited infarct size of ischemic myocardium when given immediately prior to reperfusion. SAA also significantly suppressed cellular injury and apoptotic cell death. Additionally, the results of western blot and phospho-specific antibody microarray analysis showed that SAA could up-regulate Bcl-2 expression and increase the phosphorylation of proteins such as Akt, p42/p44 extracellular signal-related kinases (Erk1/2), and their related effectors. The phosphorylation of those points was related to suppress apoptosis. In summary, SAA possesses marked protective effect on myocardial ischemia-reperfusion injury, which is related to its ability to reduce myocardial cell apoptosis and damage induced by oxidative stress. The protection is achieved via up-regulation of Bcl-2 expression and affecting protein phosphorylation. These findings indicate that SAA may be of value in cardioprotection during myocardial ischemia-reperfusion injury, which provide pharmacological evidence for clinical application.

Spectroscopic Studies on the Interaction between Salvianolic Acid a and Human Serum Albumin

The interaction between human serum albumin (HSA) and salvianolic acid A (SA-A) was investigated by means of spectroscopy. The thermodynamic parametersΔHandΔS, had been calculated to be 148.94KJ·mol-1&gt; 0, and 640.85 J·mol-1·K-1&gt; 0, respectively. The distance between the donor (HSA) and receptor (SA-A),r, was obtained to be 2.49nm according to Förster's non-radiative energy transfer theory. The experimental results revealed that SA-A could quench the fluorescence of HSA by a static quenching mechanism. Furthermore, the binding constants between HSA and SA-A were obviously affected by metal ions, decreased in the presence of Co(Ⅱ) and Mg(Ⅱ), increased with Ni(Ⅱ) and Zn(Ⅱ), and no change with Cu(Ⅱ). The interaction between SA-A and HSA was driven mainly by hydrophobic force.

Blocking effect of salvianolic acid a on calcium channels in isolated rat ventricular myocytes

To study the effect of salvianolic acid A (SAA) on L-type calcium current (I-CaL) in isolated ventricular myocytes of Sprague-Dawley rats. SAA powder was dissolved in normal Tyrode's solution to reach the concentrations of 1, 10, 100, and 1000 μmol/L. The traditional whole-cell patch-clamp recording technique was employed to evaluate the effects of SAA on I-CaL in single ventricular myocytes which were prepared by Langendorff perfusion apparatus from Sprague-Dawley rats. SAA (1, 10, 100, and 1000 μmol/L) inhibited I-CaL peak value by 16.23%±1.3% (n=6, P<0.05), 22.9%±3.6% (n=6, P<0.05), 53.4%±3.0% (n=8, P<0.01), and 62.26%±2.9% (n=6, P<0.01), respectively. SAA reversibly inhibited I-CaL in a dose-dependent manner and with a half-blocking concentration (IC(50)) of 38.3 μmol/L. SAA at 100 μmol/L elevated the I-V curve obviously, and shifted the half-active voltage (V(0.5)) from (-15.78±0.86) mV to (-11.24 ±0.77) mV (n=6, P<0.05) and the slope (K) from 5.33±0.74 to 4.35±0.74 (n=6, P>0.05). However, it did not alter the shapes of I-V curve, steady-state inactivation curve, or recovery from inactivation curve. SAA inhibited I-CaL in a dose-dependent manner. It shifted the steady-state activation curve to a more positive voltage, which indicated that the drug affected the activated state of calcium channels, and suggested that the Ca(2+) antagonistic effect of SAA be beneficial in the treatment of myocardial ischemia reperfusion injury.

Antiplatelet and antithrombotic activities of salvianolic acid A

Introduction Salvianolic acid A (SAA), the water-soluble phenolic acids in Salvia miltiorrhiza, has shown the most potent bioactivities, including protection against cerebral lesion, defense from oxidative damage and improvement of remembrance. In the present study, we studied the antiplatelet and antithrombotic effects of a newly synthesized SAA with different methods both in vitro and in vivo. Materials and Methods We tested the effect of antithrombotic activity of SAA in arterio-venous shunt model. The effects of SAA on adenosine diphosphate (ADP)–, Thrombin–, Arachidonic acid– induced rat platelets aggregation were tested both in vivo and in vitro. The activity of SAA on washed human platelet aggregation was determined by ADP stimulation. We also evaluated its property of modulation of hemorheology, assessed its bleeding side effect by measuring coagulation parameters after intravenous administration for 5 days and investigated the potential mechanisms underlying such activities. Results and Conclusions In vivo, SAA significantly reduced thrombus weight in the model of arterio-venous shunt. Meanwhile, SAA increased plasma cAMP level determined by radioimmunoassay in the same model. Intravenously administrated SAA (2.5–10 mg/kg) inhibited platelet aggregation induced by ADP in a dose-dependent manner. Notably, SAA did not affect coagulation parameters in rats after intravenous administration SAA for successive 5 days. In vitro, pretreatment with SAA on washed rat and human platelets significantly inhibited various agonists stimulated platelet aggregation and caused an increase in cAMP level in platelets activated by ADP. These findings support our hypothesis that SAA possesses antithrombotic activities. The antithrombotic effect might be related to its antiplatelet action and ability to modulate hemorheology without affecting coagulation system. The mechanisms underlying such activities may involve the induction of cAMP.

Salvianolic acid A inhibits platelet activation and arterial thrombosis via inhibition of phosphoinositide 3-kinase.

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia miltiorrhiza Bunge, a herb that is widely used for atherothrombotic disease treatment in Asian medicine. As platelets play pivotal roles in atherothrombogenesis, we studied the effect of SAA on platelet activation and its underlying mechanisms. SAA dose-dependently inhibited platelet aggregation induced by ADP, thrombin, collagen and U46619. It reduced ADP-enhanced platelet P-selectin expression and fibrinogen binding, which consequently hampered ADP-induced platelet-leukocyte aggregation. SAA also inhibited platelet spreading on fibrinogen, a process mediated by outside-in signaling. Under an arterial shear rate of 1000 s(-1), SAA decreased platelet adhesion on collagen surfaces by approximately 40%. Western blot analysis showed that SAA, like the phosphoinositide 3-kinase (PI3K) inhibitors LY294002 and TGX-221, potently inhibited PI3K, as shown by reduced Akt phosphorylation. The in vitro findings were further evaluated in the mouse model of arterial thrombosis, in which SAA prolonged the mesenteric arterial occlusion time in wild-type mice (35 + or - 2 min without SAA and 56 + or - 4 min with SAA; P < 0.01). Interestingly, SAA could even counteract the shortened arterial occlusion time in Ldlr(tm1Her) mutant mice (21 + or - 2 min without SAA and 45 + or - 4 min with SAA; P < 0.01). SAA inhibits platelet activation via the inhibition of PI3K, and attenuates arterial thrombus formation in vivo. Our data suggest that SAA may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.

Effect of salvianolic acid A on vascular reactivity of streptozotocin-induced diabetic rats

Aims The present study aims to evaluate the beneficial effect of salvianolic acid A (SAA) on the alterations in vascular reactivity of streptozotocin (STZ)-induced diabetic rats. Main methods Diabetes was induced by a single intraperitoneal injection of STZ (60 mg/kg). Following 16 weeks of SAA treatment (1 mg/kg/day), thoracic aortic rings of rats were mounted in organ baths. Contractile responses to noradrenaline (NA) and KCl and relaxant responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were assessed. Key findings Loss of weight, hyperglycemia, elevated content of malondialdehyde (MDA) and decline of total antioxidant capacity (TAC) were observed in diabetic rats. SAA could reverse these metabolic and biochemical abnormalities. Compared to the control, the maximum contraction ( E max) to NA, but not sensitivity (pD 2), increased significantly in diabetic aortas, which was prevented by SAA treatment. However, the response of rat aortas to KCl ( E max and pD 2) was not altered either in diabetic group or SAA treatment compared with that of normal control group. We also observed the significant decrease in relaxation to ACh rather than SNP in diabetic group compared with controls, and SAA treatment could revert the ACh response. Significance It is concluded that oral administration of SAA can significantly improve glucose metabolism and inhibit oxidative injury as well as protect against impaired vascular responsiveness in STZ-induced diabetic rats.

A sensitive method for determination of salvianolic acid A in rat plasma using liquid chromatography/tandem mass spectrometry

Salvianolic acid A (SAA), a major effective constituent of Salvia miltiorrhizas, is widely used in traditional Chinese medicine. A sensitive rapid analytical method was established and validated for SAA in rat plasma, which was further applied to assess the pharmacokinetics of SAA in rats receiving a single oral dose of SAA. The method used liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode with chloramphenicol as the internal standard. A simple liquid-liquid extraction based on ethyl acetate was employed. The combination of a simple sample cleanup and short chromatographic run time (3 min) increased the throughput of the method substantially. The method was validated over the range 1.4-1000 ng/mL with a correlation coefficient >0.99. The lower limit of quantification was 1.4 ng/mL for SAA in plasma. Intra- and inter-day accuracies for SAA were 95-113 and 98-107%, and the inter-day precision was less than 12%. This method is more sensitive and faster than previous methods. After a single oral dose of 100 mg/kg of SAA, the mean peak plasma concentration (Cmax) of SAA was 318 ng/mL at 0.5 h, the area under the plasma concentration-time curve (AUC0-12 h) was 698 +/- 129 ng.h/mL, and the elimination half-life (T1/2) was 3.29 h.

Effects of salvianolic acid-A on NIH/3T3 fibroblast proliferation, collagen synthesis and gene expression.

AIM:To investigate the mechanisms of salvianolic acid A (SA-A) against liver fibrosis in vitro.METHODS:NIH/3T3 fibroblasts were cultured routinely, and incubated with 10(-4) mol/L-10(-7)mol/L SA-A for 22h. The cell viability was assayed by (3)H proline incorporation, cell proliferation by (3)H TdR incorporation, cell collagen synthetic rate was measured with (3)H proline impulse and collagenase digestion method.The total RNA was prepared from the control cells and the drug treated cells respectively, and alpha(1) I pro-collagen mRNA expression was semi-quantitatively analyzed with RT-PCR.RESULTS:10(-4)mol/L SA-A decreased cell viability and exerted some cytotoxiciy,while 10(-5)mol/L -10(-7)mol/L SA-A did not affect cell viability, but inhibited cell proliferation significantly, and 10(-6)mol/L SA-A had the best effect on cell viability among these concentrations of drugs. 10 (-5)mol/L -10(-6)mol/L SA-A inhibited intracellular collagen synthetic rate, but no significant influence on extracellular collagen secretion. Both 10(-5)mol/L and 10(-6)mol/L SA-A could decrease alpha(1)I pro-collagen mRNA expression remarkably.CONCLUSION:SA-A had potent action against liver fibrosis. It inhibited NIH/3T3 fibroblast proliferation, intracellular collagen synthetic rate and type I procollagen gene expression, which may be one of the main mechanisms of the drug.

Protective actions of salvianolic acid A on hepatocyte injured by peroxidation in vitro.

Salvianolic radix, one of the most commonly used traditional Chinese herbs, was widely studied about its actions against liver injury and fibrosis, and was one of the focuses of recent research[1,2]. Salvianolic acid-A (SA-A) was an aqueous soluble component of Salvianolic radix. In our previous work[2], SA-A was found to have protective effects against liver injury and fibrosis induced by carbon tetrachloride (CCl4) in rats. In order to investigate the effect of SA-A on peroxidation in hepatocytes, we induced the injured hepatocyte model by CCl4 fumigation in vitro, treated the cell model with SA-A or aqueous soluble vitamin E (Vitamin E), the latter served as the control drug, and observed the influences of the drugs on the functions of the hepatocytes injured by peroxidation.