Preventive effects of a purified extract isolated from Salvia miltiorrhiza enriched with tanshinone I,tanshinone IIA and cryptotanshinone on hepatocyte injury in vitro and in vivo

Salvia miltiorrhiza is traditionally used to treat liver disease in Asia. In this study, we tested the ability of a purified extract of S. miltiorrhiza (PF2401-SF) and its constituents, tanshinone I, tanshinone IIA, and cryptotanshinone, to protect against acute and subacute liver damage induced by carbon tetrachloride by measuring serum transaminase levels, the reduced form of glutathione (GSH), antioxidant enzyme activities, and lipid peroxidation levels in the liver. We also evaluated their ability to protect primary cultured rat hepatocytes from tertiary-butylhydroperoxide (tBH) or d-galactosamine (GalN). PF2401-SF was protective at 50–200 mg/kg per day in acute liver injury and 25–100 mg/kg per day in subacute liver injury. Tanshinone I, tanshinone IIA, and cryptotanshinon (40 μM), inhibited lactate dehydrogenase leakage, GSH depletion, lipid peroxidation and free radical generation in vitro. PF2401-SF and its major constituents, tanshinone I, tanshinone IIA and cryptotanshinone, can protect against liver toxicity in vivo and in vitro due to its antioxidant effects.     

Enhancement of solubility and dissolution rate of cryptotanshinone, tanshinone I and tanshinone IIA extracted from Salvia miltiorrhiza

Cryptotanshinone, tanshinone I and tanshinone IIA are three major components in the extract of Salvia miltiorrhiza with pharmacological significance. However, their effective utilization is limited due to poor water solubility and bioavailability. Solid dispersion (SD) of the extract of Salvia miltiorrhiza was prepared to enhance solubility and dissolution of the three major components. Various carriers were screened for SD preparation by conventional solvent method. Dissolution of the components from selected SD systems was compared with commercial tablets of the extract from Salvia miltiorrhiza. The solubility of three components viz., cryptotanshinone, tanshinone I and tanshinone IIA, after forming SD with either of povidone K-30 (PVP K-30) or poloxamer 407, exhibited enhanced solubility in pH 6.8 buffer. Dissolution test revealed that the amount of three components released was higher from SD tablets as compared to the commercial tablets. Pharmacokinetic profile was evaluated using cryptotanshinone as a representative compound. AUC of cryptotanshinone was significantly increased when administered as a solid dispersion.     

Diterpene quinone tanshinone IIA selectively inhibits mouse and human cytochrome P4501A2

1. Tanshinone IIA is the main active diterpene quinone in the herbal medicine Salvia miltiorrhiza. In untreated mouse liver microsomes, tanshinone IIA selectively inhibited 7-ethoxyresorufin O -deethylation (EROD) and 7-methoxyresorufin O -demethylation (MROD) activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, N -nitrosodimethylamine and nifedipine. Tanshinone IIA was a competitive inhibitor of MROD activity with a K i of 7.2 ± 0.7 nM. 2. In 3-methylcholanthrene-treated mouse liver microsomes, tanshinone IIA and two minor tanshinones, tanshinone I and cryptotanshinone, inhibited liver microsomal MROD activity without affecting EROD and benzo(a)pyrene hydroxylation activities at the concentrations up to 1 µ M. Tanshinone IIA induced a type I binding spectrum with a spectral dissociation constant K?s of 2.3 ± 0.8 µ M without cooperativity. 3. In human liver microsomes, tanshinone IIA decreased EROD and MROD activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, chlorzoxazone and nifedipine. 4. In Escherichia coli membranes expressing bicistronic human CYP1A enzymes, tanshinone IIA inhibited EROD activity of CYP1A1 with an IC 50 48 times higher than that for CYP1A2. Tanshinone I and cryptotanshinone had the same IC 50 ratio (1A1/1A2) of 4. 5. The results indicate that tanshinone represents a new group of CYP1A inhibitors, and tanshinone IIA had the highest selectivity in inhibition of CYP1A2.     

Diterpene quinone tanshinone IIA selectively inhibits mouse and human cytochrome p4501A2

1. Tanshinone IIA is the main active diterpene quinone in the herbal medicine Salvia miltiorrhiza. In untreated mouse liver microsomes, tanshinone IIA selectively inhibited 7-ethoxyresorufin O-deethylation (EROD) and 7-methoxyresorufin O-demethylation (MROD) activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, N-nitrosodimethylamine and nifedipine. Tanshinone IIA was a competitive inhibitor of MROD activity with a K(i) of 7.2 +/- 0.7 nM. 2. In 3-methylcholanthrene-treated mouse liver microsomes, tanshinone IIA and two minor tanshinones, tanshinone I and cryptotanshinone, inhibited liver microsomal MROD activity without affecting EROD and benzo(a)pyrene hydroxylation activities at the concentrations up to 1 microM. Tanshinone IIA induced a type I binding spectrum with a spectral dissociation constant K(s) of 2.3 +/-0.8 microM without cooperativity. 3. In human liver microsomes, tanshinone IIA decreased EROD and MROD activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, chlorzoxazone and nifedipine. 4. In Escherichia coli membranes expressing bicistronic human CYP1A enzymes, tanshinone IIA inhibited EROD activity of CYP1A1 with an IC(50) 48 times higher than that for CYP1A2. Tanshinone I and cryptotanshinone had the same IC(50) ratio (1A1/1A2) of 4. 5. The results indicate that tanshinone represents a new group of CYP1A inhibitors, and tanshinone IIA had the highest selectivity in inhibition of CYP1A2.     

Simultaneous determination of cryptotanshinone, tanshinone I and tanshinone IIA in traditional Chinese medicinal preparations containing Radix salvia miltiorrhiza by HPLC

A reversed-phase high performance liquid chromatographic method was established for the simultaneous determination of tanshinones in five kinds of traditional Chinese medicinal preparations (TCMPs) containing Radix salvia miltiorrhiza (Chinese herbal name: Danshen). Tanshinones including cryptotanshinone, tanshinone I and tanshinone IIA were successfully separated on a Diamonsil C18 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was a mixture of methanol, tetrahydrofuran, water and glacial acetic acid (20:35:44:1, v/v/v/v), employing isocratic elution at a flow rate of 1.0 mL/min. Detection was accomplished at 254 nm. The compounds were identified by comparing their retention times and UV spectra in the 200-400 nm range with authentic standards. Regression equations revealed good linear relationship between the peak areas of the constituents and their concentrations (correlation coefficients: 0.9998 for cryptotanshinone, 0.9999 for tanshinone I and 1.0000 for tanshinone IIA). The relative standard deviations (n=6) of retention time and peak area were less than 0.25% and 1.00%, respectively. The recoveries were between 96.2% and 102.5%. The proposed method has been successfully applied to the simultaneous determination of the tanshinones in five kinds of Chinese herbal preparations containing Danshen within 20 min.     

Cytotoxicity of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on HepG2 cells in relation to glutathione perturbation.

Tanshinones are abietane type-diterpene quinones isolated from the roots of Radix Salvia miltiorrhiza (Danshen), a well-known traditional Chinese medicine in the treatment of cardiovascular diseases. Among the major diterpenes isolated, including cryptotanshinone, tanshinone I, tanshinone IIA and dihydrotanshinone, tanshinone IIA had been shown to posses various pharmacological activities including antioxidant, protection/prevention from angina pectoris and myocardial infarction, and anticancer properties. Tanshinone IIA, usually the most abundant tanshinone present in the herb, has been the focus of studies in its clinical potential, among which its ability to inhibit the proliferation of cancer cell lines. The aim of this study was to study the cytotoxicity of the tanshinones on human HepG2 cells in vitro in relation to intracellular glutathione perturbation (reduced glutathione, GSH and oxidized glutathione, GSSG). Studies using MTT assay showed that all tanshinones decreased cell viability of HepG2 cells in a concentration-dependent manner, with the cell viability decreased to 60% and 35% after 24 h and 48 h treatment, respectively. Assessment of apoptotic cells with fragmented DNA by flow cytometry indicated that only tanshinone IIA (12.5 and 25 microM) induced apoptosis in the cancer cells. Tanshinone IIA and cryptotanshinone caused significant decreases in G(1) cells by 23% and 13%, respectively, after 24 h treatment. The declines in G(1) cells were compensated by increases in G(2)/M (15% for tanshinone IIA) and S cells (8% and 13% for tanshinone IIA and cryptotanshinone, respectively). All the tanshinones studied, except tanshinone IIA, elevated GSH/GSSG ratio at low concentrations (1.56 and 3.13 microM), but the ratio decreased, indicating oxidative stress at high concentrations (6.25-25 microM). Taken together, tanshinone IIA caused HepG2 cytotoxicity through apoptosis without influencing oxidative stress, while the other tanshinones showed lower efficacy in inducing apoptosis in the HepG2 cells.     

Sodium tanshinone IIA sulfonate protects cardiomyocytes against oxidative stress-mediated apoptosis through inhibiting JNK activation

Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, a well-known Chinese medicine for treating cardiovascular disorders. Cardiomyocyte apoptosis plays a major role in the development of cardiovascular diseases. The present study was designed to investigate the effects of STS on cardiomyocyte apoptosis induced by in vivo acute myocardial infarction (MI) in adult rats and by in vitro H2O2-treated neonatal rat ventricular myocytes. In MI rats, STS significantly reduced the infarct sizes, the blood lactate dehydrogenase (LDH) level, and the number of apoptotic cardiomyocytes in the infarcted hearts. In the in vitro study, STS reversed the decreased effect of cell viability induced by H2O2. In addition, STS also markedly inhibited H2O2-induced cardiomyocyte apoptosis. C-Jun N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs) and p38 MAPK are classic oxidative stress-activated protein kinases. Our further mechanistic study revealed that increased JNK phosphorylation stimulated by H2O2 was abolished by STS treatment. In conclusion, inhibition of JNK activation plays a significant role in cardioprotective effects of STS.     

Cryptotanshinone inhibits chemotactic migration in macrophages through negative regulation of the PI3K signaling pathway

BACKGROUND AND PURPOSE: Cryptotanshinone, the major tanshinone isolated from Salvia miltiorrhiza Bunge, exhibits anti-inflammatory activity. However, there is no report on the effect of cryptotanshinone on recruitment of leukocytes to inflammatory sites. We therefore assessed the effects of cryptotanshinone on macrophage chemotaxis. EXPERIMENTAL APPROACH: Macrophage migration induced by complement 5a (C5a) or macrophage inflammatory protein-1alpha (MIP-1alpha) was measured in vitro. Intracellular kinase translocation and phosphorylation was assessed by Western blotting. KEY RESULTS: RAW264.7 cell migration towards C5a (1 microg ml(-1)) was significantly inhibited by cryptotanshinone (1, 3, 10 and 30 microM) in a concentration-dependent manner. Primary human macrophages stimulated by C5a were similarly inhibited. C5a-evoked migration in RAW264.7 cells was significantly suppressed by wortmannin (phosphatidylinositol 3-kinase (PI3K) inhibitor), PD98059 (MEK1/2 inhibitor) and SB203580 (p38 mitogen-activated protein kinase (MAPK) inhibitor), but not by SP600125 (c-Jun N-terminal kinase (JNK) inhibitor), suggesting that activation of PI3K, ERK1/2 and p38 MAPK signal pathways was involved in responses to C5a. Western blotting revealed that cryptotanshinone significantly inhibited PI3K-p110gamma membrane translocation and phosphorylation of Akt (PI3K downstream effector protein) and ERK1/2 induced by C5a. However, neither p38 MAPK nor JNK phosphorylation was affected by cryptotanshinone. Wortmannin significantly attenuated C5a-induced PI3K-p110gamma translocation, Akt and ERK1/2 phosphorylation. PD98059 suppressed ERK1/2 phosphorylation but failed to modify PI3K-p110gamma translocation by C5a stimulation. Furthermore, MIP-1alpha-induced cell migration and PI3K-p110gamma translocation were also inhibited by cryptotanshinone in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: Inhibition of macrophage migration by cryptotanshinone involved inhibition of PI3K activation with consequent reduction of phosphorylation of Akt and ERK1/2.     

Inhibition of osteoclast differentiation and bone resorption by tanshinone IIA isolated from Salvia miltiorrhiza Bunge

Osteoclasts, multinuclear cells specialized for bone resorption, differentiate from the monocyte/macrophage lineage of hematopoietic cells. Intervention in osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone diseases involving osteoclasts. In this study, we found that tanshinone IIA, originating from Salvia miltiorrhiza Bunge, inhibited the differentiation of osteoclasts. Addition of tanshinone IIA to the osteoclast precursor culture caused a significant decrease in the level of calcitonin receptor, c-Src, and integrin beta3 mRNA, which are normally upregulated during the osteoclast differentiation dependent on RANKL (receptor activator of nuclear factor kappa B ligand). RANKL activated the ERK, Akt, and NF-kappaB signal transduction pathways in osteoclast precursor cells, and tanshinone IIA suppressed this activation. Tanshinone IIA also inhibited the bone resorptive activity of differentiated osteoclasts, which was accompanied with the disruption of the actin ring. Thus, tanshinone IIA has the potential to ameliorate bone-resorption diseases in vivo by reducing both the number and activity of osteoclasts.     

Comparative pharmacokinetics and tissue distribution of cryptotanshinone,tanshinone IIA,dihydrotanshinone I,and tanshinone I after oral administration of pure tanshinones and liposoluble extract of Salvia miltiorrhiza to rats

Salvia miltiorrhiza is one of the most commonly used traditional Chinese medicines in the treatment of cardiovascular and cerebrovascular diseases. Cryptotanshinone (CTS), tanshinone IIA (Tan IIA), dihydrotanshinone I (diTan I), and tanshinone I (Tan I) are the main active compounds in the liposoluble extract of Salvia miltiorrhiza. The differences in the pharmacokinetic and tissue distribution behaviors of the four tanshinones after oral administration of the liposoluble extract of Salvia miltiorrhiza and pure compounds are not clear. This study aims to compare the pharmacokinetics and tissue distribution of the four tanshinones after oral administration of pure tanshinone monomers and the liposoluble extract of Salvia miltiorrhiza. An ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) analysis method was developed for the determination of the four tanshinones. The results showed that the AUC and C_max of tanshinones in rats receiving the extract of Salvia miltiorrhiza were significantly increased compared with those receiving the pure tanshinones. In the tissue distribution experiments, the AUC of the four tanshinones in the extract was much greater than the AUC of the monomers in the lung, heart, kidney, liver, and brain, and the coexisting constituents particularly promoted the distribution of tanshinones into tissues that the drug cannot sufficiently penetrate. These findings suggested that the coexisting constituents in the liposoluble extract of Salvia miltiorrhiza play an important role in the alteration of plasma concentration and tissue distribution of the four tanshinones. Understanding these differences could be of significance for the development and application of Salvia miltiorrhiza extract and tanshinone components.     

Tanshinone congeners improve memory impairments induced by scopolamine on passive avoidance tasks in mice

Tanshinones are a group of diterpenoids found in the roots of Salvia miltiorrhiza Bunge which has been used to treat cardiac disease. In the present study, we investigated the effect of the tanshinone congeners, tanshinone I, tanshinone IIA, cryptotanshinone, and 15, 16-dihydrotanshinone I, on learning and memory impairments induced by scopolamine (1 mg/kg, i.p.), a muscarinic antagonist, using passive avoidance tasks in mice. Tacrine was used as a positive control. Tanshinone I (2 or 4 mg/kg, p.o.), tanshinone IIA (10 or 20 mg/kg, p.o.), cryptotanshinone (10 mg/kg, p.o.), and 15, 16-dihydrotanshinone I (2 or 4 mg/kg, p.o.) significantly reversed scopolamine-induced cognitive impairments (P<0.05). Tanshinone I (2 mg/kg, p.o.) and tanshinone IIA (10 or 20 mg/kg, p.o.) were also reversed diazepam-induced cognitive dysfunctions (P<0.05). In addition, cryptotanshinone and 15, 16-dihydrotanshinone I were found to have an inhibitory effect on acetylcholinesterase in vitro with IC(50) values 82 and 25 microM, respectively. Furthermore, cryptotanshinone inhibited acetylcholinesterase activity for 3 h and 15, 16-dihydrotanshinone I for 6 h in an ex-vivo study. These results suggest that tanshinone congeners may be useful for the treatment of cognitive impairment and that their beneficial effects are mediated, in part, by cholinergic signaling enhancement.     

Pharmacokinetics, absorption and tissue distribution of tanshinone IIA solid dispersion

This study was designed to elucidate the pharmacokinetics, absorption, tissue distribution and plasma protein binding properties of tanshinone IIA, a highly lipophilic compound isolated from Salvia miltiorrhiza. Tanshinone IIA was isolated using a previously well developed LC-MS/MS method. Its pharmacokinetic characteristics, absolute bioavailability, tissue distribution and plasma protein binding properties were determined. The membrane permeability was evaluated using Caco-2 cells in monolayer. The pharmacokinetic plasma profile of tanshinone IIA after a single intravenous dosing exhibited a triexponential pattern consisting of rapid distribution (t1/2 alpha, 0.024 h), slow redistribution (t1/2 beta, 0.34 h) and terminal elimination phase (t1/2 gamma, 7.5 h). Tanshinone IIA preferentially distributed into the reticuloendothelial system, especially into liver and lung, after either intravenous or oral doses. Tanshinone IIA (99.2 %) bound highly to plasma proteins, among which lipoprotein played an important role (77.5 %). Tanshinone IIA absorption was extremely poor with an absolute bioavailability below 3.5 %. Absorptive saturation was deduced from the fact that the AUC and Cmax increased less proportionally to dose and Tmax was significantly prolonged. The poor absorption of tanshinone IIA may be caused by its low aqueous solubility and limited membrane permeability. There were no significant differences of the apparent permeability coefficient for all tested concentrations and for the apical to basolateral and reverse direction transport, suggesting a passive transport mode and no involvement of an efflux protein. In conclusion, tanshinone IIA has a suitable pharmacokinetic behavior except for its poor absorption. A pharmaceutical strategy for promoting its absorption should be designed to develop tanshinone IIA as a new drug candidate.     

Effects of tanshinone IIA on the hepatotoxicity and gene expression involved in alcoholic liver disease

Tanshinone IIA is one of the most abundant constituents of the root of Salvia miltiorrhiza BUNGE which exerts antioxidant and anti-inflammatory actions in many experimental disease models. In the present study, we demonstrated that the standardized fraction of S. miltiorrhiza (Sm-SF) was able to protect RAW 264.7 cells from ethanol-and lipopolysaccharide (LPS)-induced production of superoxide radical, activation of NADPH oxidase and subsequently death of the cells. Among four main components of Sm-SF, tanshinone IIA was the most potent in protecting cells from LPS-and ethanol-induced cytotoxicity. LPS or ethanol induced the expression of CD14, iNOS, and SCD1 and decreased RXR-alpha, which was completely reversed by tanshinone IIA. In H4IIEC3 cells, 10 microM tanshinone IIA effectively blocked ethanol-induced fat accumulation as evidenced by Nile Red binding assay. These results indicate that tanshinone IIA may have potential to inhibit alcoholic liver disease by reducing LPS-and ethanol-induced Kupffer cell sensitization, inhibiting synthesis of reactive oxygen/nitrogen species, inhibiting fatty acid synthesis and stimulating fatty acid oxidation.     

Activation of CYP3A-mediated testosterone 6β-hydroxylation by tanshinone IIA and midazolam 1-hydroxylation by cryptotanshinone in human liver microsomes

This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone. The abilities of tanshinones to activate CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation in human liver microsomes (HLMs) were tested. Substrate- and effector-dependent activation of CYP3A by tanshinones were both observed. Cryptotanshinone was shown to activate CYP3A-mediated midazolam 1-hydroxylation in a concentration-dependent manner. In contrast, tanshinone IIA and tanshinone I did not activate this hydroxylation reaction. In addition, tanshinone IIA activated CYP3A-mediated testosterone 6β-hydroxylation, whereas cryptotanshinone and tanshinone I did not. The results from our study enhance the understanding of CYP3A activation by tanshinone IIA and cryptotanshinone in HLMs. Additionally, these data allow for an accurate prediction of the magnitude and likelihood of Danshen-drug interactions.     

Improved quality control method for Danshen products--consideration of both hydrophilic and lipophilic active components

The current study intends to provide an improved quality control analysis for Danshen product-a representative herbal product with known active components that are both hydrophilic and lipophilic in nature. A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of three major lipophilic components (cryptotanshinone, tanshinone I and tanshinone IIA) and three major hydrophilic components (danshensu, protocatechuic aldehyde and salvianolic acid B) of Danshen (Salvia miltiorrhiza). These six components were successfully separated using Radial-pak C18 cartridge with the elution gradient consisting of 0.5% acetic acid in water and 0.5% acetic acid in acetonitrile at a flow rate of 1 ml/min. The intra-day and inter-day precisions of the analysis were within 2.32 and 2.0%, respectively. The detection limits were 0.02, 0.01, 0.01, 0.05, 0.005 and 0.02 microg/ml for cryptotanshinone, tanshinone I, tanshinone IIA, danshensu, protocatechuic aldehyde and salvianolic acid B, respectively. The developed method has been applied to the simultaneous determination of above six major components in Fufang Danshen Tablet and Dripping Pill products by extraction with methanol and water. It has been demonstrated that salvianolic acid B and danshensu are the major components among the eight commercial Fufang Danshen products studied. The current developed method with methanol as extraction solvent provides a simple and efficient method for simultaneous detection of both lipophilic and hydrophilic major components in Danshen products.     

Growth-inhibitory and apoptosis-inducing effects of tanshinones on hematological malignancy cells and their structure-activity relationship

This study has investigated the growth-inhibitory and apoptosis-inducing effects of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone on hematological malignancy cell lines, aiming to explore their structure-activity relationship. The growth-inhibitory effects of the tanshinones on K562 and Raji cells were assessed using a modified MTT assay; the apoptosis-inducing effects were assessed by fluorescence microscopy and flow cytometry analysis. The changes in cellular morphology were observed using an inverted phase-contrast microscope. MTT results revealed that these tanshinones inhibited cell proliferation in a concentration-dependent and time-dependent manner. The IC50 values of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone for K562 cells were 3.50, 13.52, 19.32, and 47.52 μmol/l at 24 h; 1.36, 4.70, 5.67, and 22.72 μmol/l at 48 h; and 1.15, 1.59, 2.82, and 19.53 μmol/l at 72 h, and the values for Raji cells were 3.30, 4.37, 12.92, and 52.36 μmol/l at 24 h; 1.55, 1.71, 6.54, and 25.45 μmol/l at 48 h; and 1.07, 1.38, 1.89, and 18.47 μmol/l at 72 h. The flow cytometry analysis demonstrated that these tanshinones induced apoptosis of K562 cells in a concentration-dependent manner, and dihydrotanshinone as well as tanshinone I were more potent than tanshinone IIA and cryptotanshinone. Some noticeable apoptotic morphologies could be observed by fluorescence microscopy on tanshinones-treated Raji cells. Collectively, these tanshinones caused growth inhibition and apoptosis in hematological malignancy cell lines, with dihydrotanshinone being the most potent, followed by tanshinone I, tanshinone IIA, and cryptotanshinone. These results suggested that the structure of aromatic ring A enhanced the cytotoxicity and the structure of ring C may have contributed to the cytotoxicity, but the mechanisms need to be further investigated.     

Addition of tanshinone IIA to UW solution decreases skeletal muscle ischemia-reperfusion injury

AIM: To investigate whether tanshinone IIA could improve the effect of UW solution for skeletal muscle preservation and to determine the dose range of tanshinone IIA providing optimal protection during ischemia and reperfusion. METHODS: Ischemic rat limbs were perfused with UW solution or UW plus tanshinone IIA (UW+T, 0.05, 0.1, or 0.2 mg/mL) for 0.5 h before reperfusion; controls (I/R) received no perfusion. Serum creatine phosphokinase (CPK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were measured pre-ischemia and after reperfusion (2-h, 4-h, and 6-h). Muscle water content, superoxide dismutase (SOD), malondialdehyde (MDA), adenosine triphosphatase (ATPase) were assessed pre-reperfusion and after 6-h reperfusion. Intercellular adhesion molecule-1 (ICAM-1) and apoptosis were detected after 6-h reperfusion. Reperfusion blood flow was monitored during reperfusion period. RESULTS: UW and UW+T prevented luxury perfusion during reperfusion and inhibited ICAM-1 expression and apoptosis after 6-h reperfusion. Serum CPK, AST, and LDH levels in UW rats were significantly less than those in controls after 2-h reperfusion (no difference, 4-h or 6-h reperfusion). After 4-h ischemia, there were significant differences in water content, MDA, SOD, and ATPase between UW and controls, but no difference after 6-h reperfusion. All tests with UW+T rats were significantly different from control results at corresponding durations. Higher tanshinone doses improved results. CONCLUSION: UW plus tanshinone IIA increased protection against I/R injury, suggesting that tanshinone IIA has clinical value.     

Simultaneous determination of danshensu, ferulic acid, cryptotanshinone and tanshinone IIA in rabbit plasma by HPLC and their pharmacokinetic application in danxiongfang

A selective and sensitive reversed-phase high performance liquid chromatography method was developed and validated for the simultaneous determination of danshensu, ferulic acid, cryptotanshinone, and tanshinone IIA in rabbit plasma using p-hydroxybenzoic acid as internal standard. Liquid–liquid extraction was used for sample preparation. Chromatographic separation was successfully achieved on an Agilent HC-C₁₈ column using a mobile phase composed of methanol–water (from 20:80 to 80:20, v/v) containing 0.5% (v/v) glacial acetic acid. The mobile phase was employing gradient elution at a flow rate of 1.0 ml/min. The method showed good linearity and no endogenous material interfered with the marked compounds and I.S. peaks. The limit of quantification of danshensu, ferulic acid, cryptotanshinone, and tanshinone IIA were 0.1, 0.03, 0.05, and 0.05 μg/ml, respectively. The average extract recoveries of the four compounds from rabbit plasma were all over 60%. The precisions determined from 5 days were all within 10%. The established method has been successfully applied in the pharmacokinetic study and drug interaction of danshensu, ferulic acid, cryptotanshinone, and tanshinone IIA in rabbits after intravenous administration of danxiongfang, a useful compound preparation of traditional Chinese medicine.     

Anti-allergic effects of salvianolic acid A and tanshinone IIA from Salvia miltiorrhiza determined using in vivo and in vitro experiments

Salvia miltiorrhiza root has been used in Asian traditional medicine for the treatment of cardiovascular diseases, asthma, and other conditions. Salvianolic acid B from S. miltiorrhiza extracts has been shown to improve airway hyperresponsiveness. We investigated the effects of salvianolic acid A, tanshinone I, and tanshinone IIA from S. miltiorrhiza in allergic asthma by using rat RBL-2H3 mast cells and female Balb/c mice. Antigen-induced degranulation was assessed by measuring β-hexosaminidase activity in vitro. In addition, a murine ovalbumin-induced allergic asthma model was used to test the in vivo efficacy of salvianolic acid A and tanshinone IIA. Tanshinone I and tanshinone IIA inhibited antigen-induced degranulation of mast cells, but salvianolic acid A did not. Administration of salvianolic acid A and tanshinone IIA decreased the number of immune cells, particularly eosinophils in allergic asthma-induced mice. Histological studies showed that salvianolic acid A and tanshinone IIA reduced mucin production and inflammation in the lungs. Administration of salvianolic acid A and tanshinone IIA reduced the expression and secretion of Th2 cytokines (IL-4 and IL-13) in the bronchoalveolar lavage fluid and lung tissues of mice with ovalbumin-induced allergic asthma. These findings provide evidence that salvianolic acid A and tanshinone IIA may be potential anti-allergic therapeutics.