Simultaneous determination of danshensu,rosmarinic acid,cryptotanshinone, tanshinone IIA,tanshinone I and dihydrotanshinone I by liquid chromatographic–mass spectrometry and the application to pharmacokinetics in rats

A sensitive and specific liquid chromatography–tandem mass spectrometry method (LC–MS) was developed and validated for the separation and simultaneous determination of danshensu, rosmarinic acid and tanshinone compounds including cryptotanshinone, tanshinone I, dihydrotanshinone I and tanshinone IIA in rat plasma. Chromatographic separation of the analytes was successfully achieved on a C₁₈ column using a mobile phase composed of acetonitrile–water containing 0.5% glacial acetic acid. This method demonstrated good linearity and did not have endogenous material interfering with the active compounds and I.S. peaks. The limit of quantification of danshensu, rosmarinic acid, cryptotanshinone, dihydrotanshinone I, tanshinone I and tanshinone IIA were 5, 0.75, 0.1, 0.1, 1 and 0.5 ng/mL. The average extraction recoveries of these analytes from rat plasma were all over 60%. The precisions determined from five days were all within 10%. This method has been successfully applied in the simultaneous quantification and the pharmacokinetic studies of these six compounds in animals which were orally administered with danshen preparations.     

Simultaneous quantification of cryptotanshinone and its active metabolite tanshinone IIA in plasma by liquid chromatography/tandem mass spectrometry (LC-MS/MS)

A rapid and sensitive method for the simultaneous determination of cryptotanshinone and its active metabolite tanshinone IIA in rat plasma was developed and well validated, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. This method entailed a single step of liquid-liquid extraction with ethyl acetate from a small volume of plasmas. The analytes and internal standard diazepam were baseline separated on a Shim-pack VP-ODS analytical column. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source operated under selected reaction monitoring (SRM) mode. The method was linear in the concentration range of 1-100 ng/ml for both tanshinone IIA and cryptotanshinone. The intra- and inter-day precisions (R.S.D.%) were within 10.2% for both analytes. Deviation of the assay accuracies was within +/-12.0% for both analytes. Both analytes were proved to be stable during all sample storing, preparation and analytic procedures. The method was successfully applied to a pharmacokinetic study after an oral administration of cryptotanshinone to rats with a dose of 20 mg/kg. With the lower limits of quantification at 1.0 ng/ml for tanshinone IIA and 0.2 ng/ml for cryptotanshinone, this method was proved to be sensitive enough and reproducible for the pharmacokinetics study of both tanshinones.     

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.     

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.     

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.     

Enhanced production of tanshinone IIA in endophytic fungi Emericella foeniculicola by genome shuffling

Context: Tanshinone IIA, commercially produced from Salvia miltiorrhiza Bunge (C.Y.Wu) (Labiatae), has various biological benefits. Currently, this compound is mainly extracted from plants. However, because of the long growth cycle and the unstable quality of plants, the market demands can barely be satisfied.

Objective: The genomic shuffling technology is applied to screen the high-yield tanshinone IIA strain, which could be used to replace the plant S. miltiorrhiza for the production of tanshinone IIA. The change in the production of tanshinone IIA is clarified by comparing it with the original strain.

Materials and methods: Tanshinone IIA was extracted from Strains cells, which was prepared through 0.5?mL protoplast samples by using hypertonic solution I from two different strains. Then, it was analyzed by high-performance liquid chromatography at 30?°C and UV 270?nm. Total DNA from the strains was extracted for RAPD amplification and electrophoresis to isolate the product.

Results: In this study, a high-yield tanshinone IIA strain F-3.4 was screened and the yield of tanshinone IIA was increased by 387.56?±?0.02?mg/g, 11.07 times higher than that of the original strain TR21.

Discussion: This study shows that the genetic basis of high-yield strains is achieved through genome shuffling, which proves that genome shuffling can shorten the breeding cycle and improve the mutagenesis efficiency in obtaining the strains with good traits and it is a useful method for the molecular breeding of industrial strains.     

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.     

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.     

PF2401-SF, standardized fraction of Salvia miltiorrhiza and its constituents, tanshinone I, tanshinone IIA, and cryptotanshinone, protect primary cultured rat hepatocytes from bile acid-induced apoptosis by inhibiting JNK phosphorylation.

Bile acid-induced hepatocyte apoptosis plays an important role in cholestatic liver disease, and the role of apoptosis may be of therapeutic interest in preventing liver disease. The dried root of Salvia miltiorrhiza Bunge (Labiatae) has been used traditionally to treat liver diseases. We investigated the antiapoptotic effects of a standardized fraction of S. miltiorrhiza (PF2401-SF) and its components, tanshinone I, tanshinone IIA, and cryptotanshinone, in primary cultured rat hepatocytes. PF2401-SF was enriched with tanshinone I (11.5%), tanshinone IIA (41.0%), and cryptotanshinone (19.1%). Glycochenodeoxycholic acid (GCDC)-induced apoptosis, as shown by DNA fragmentation, poly(ADP-ribose) polymerase cleavage, and activation of caspases-8, -9, and -3. PF2401-SF and its components, tanshinone I, tanshinone IIA, and cryptotanshinone showed antiapoptotic activity. Treatment with PF2401-SF or with its components significantly inhibited the generation of intracellular reactive oxygen species. Hydrophobic bile acids activate c-Jun-NH(2)-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPK), and extracellular signal-regulated kinase 1/2, and PF2401-SF inhibited the phosphorylation of JNK and p38. All three components of PF2401-SF inhibited JNK phosphorylation. Addition of inhibitors of MAPK showed that inhibition of JNK decreased apoptosis. These data indicate that PF2401-SF and its components protect hepatocytes from GCDC-induced apoptosis in vitro by inhibiting JNK.     

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.     

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.     

精制冠心片超高效液相色谱法特征图谱的研究

目的：建立精制冠心片的特征图谱。方法：采用UHPLC法。采用Dikma Endevaorsil C₁₈色谱柱（2.1 mm×100 mm,1.8 μm）,柱温30 ℃,流速0.2 mL·min^-1,流动相为乙腈-0.05%磷酸溶液,梯度洗脱,检测波长为270 nm。结果：建立了以丹酚酸B,丹参酮IIA为参照峰的精制冠心片的特征图谱,并指认了芍药苷、迷迭香酸、丹酚酸B、藁本内酯、隐丹参酮和丹参酮IIA 6个特征峰。结论：精制冠心片特征图谱的建立,可以更好地控制精制冠心片的质量。     

Glycine increases mRNA adiponectin and diminishes pro-inflammatory adipokines expression in 3T3-L1 cells

Tanshinone IIA, one of the main active components from Chinese herb Danshen, is widely used to treat cardiovascular diseases including arrhythmia in Asian countries especially in China. However, the mechanisms underlying its anti-arrythmia effects are not clear. In this study we investigate the effects of tanshinone IIA on human KCNQ1/KCNE1 potassium channels (I(Ks)), human ether-a-go-go-related gene potassium channels (hERG), Kv1.5 potassium channels, inward rectifier potassium channels (I(K1)) expressed in HEK 293 cells using patch clamp technique. Tanshinone IIA potently and reversibly enhanced the amplitude of I(Ks) in a concentration dependent manner with an EC(50) of 64.5 microM, accelerated the activation rate of I(Ks) channels, decelerated their deactivation and shifted the voltage dependence of I(Ks) activation to negative direction. Isoproteronol, a stimulator of beta-adrenergic receptor, at 1 microM and sodium nitroprusside (SNP), a NO donor, at 1 mM, had no significant effects on the enhancement of I(Ks) by 30 microM tanshinone IIA. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), a selective protein kinase A inhibitor, at 0.1 microM and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), a selective nitric oxide-sensitive guanylyl cyclase inhibitor, at 10 microM, also had no significant effects on the enhancement of I(Ks) by 30 microM tanshinone IIA. Tanshinone IIA did not affect expressed hERG channels, Kv1.5 channels and I(K1) channels. These results indicate that tanshinone IIA directly and specifically activate human cardiac KCNQ1/KCNE1 potassium channels (I(Ks)) in HEK 293 cell through affecting the channels' kinetics.     

The effect of tanshinone IIA on renal and liver functions in ovine fetuses in utero

This was the first study in determination of the effects of the herbal medicine, Danshen, on fetal hepatic and renal functions in utero. Tanshinone IIA, an active ingredient of Danshen, was tested in the experimental fetal model. Three doses (20, 40, or 80?mg) of tanshinone IIA and 0.9% NaCl (as the control) were intravenously (i.v.) administrated into pregnant ewes. Both maternal and fetal blood samples were collected and analyzed for renal and liver functions by examining the enzymes and renal excretion. The results showed that tanshinone IIA did not alter fetal urine volume, urine electrolytes, and osmolality. Enzyme activities related to the hepatic and renal functions were not changed. In addition, maternal application of tanshinone IIA had no effect of maternal and fetal lipid profile. The results demonstrated that tanshinone IIA used during the last third of gestation did not cause the biochemical changes related to renal and liver functions in both the mother and fetus. This provides new information to guide the use of herbal medicine during pregnancy.     

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

1. This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone.

2. 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.

3. 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.

4. 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.     

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.     

Tanshinone IIA suppresses inflammatory bone loss by inhibiting the synthesis of prostaglandin E2 in osteoblasts

Tanshinone IIA isolated from Danshen is widely used in Oriental medicine. However, the action of tanshinone IIA in inflammatory bone-resorptive diseases remains unknown. Here we examined the effect of tanshinone IIA in inflammation-mediated osteoclastic bone resorption. Tanshinone IIA inhibited osteoclast differentiation in cocultures of bone marrow cells and calvarial osteoblasts. Tanshinone IIA regulated the expression of receptor activator of NF-kappaB ligand and osteoprotegerin in osteoblasts treated with lipopolysaccharide (LPS). Also, tanshinone IIA inhibited prostaglandin E(2) (PGE(2)) synthesis by inhibiting Cyclooxygenase-2 (COX-2) expression induced by LPS. Furthermore, tanshinone IIA greatly suppressed bone loss in the mouse models of bone loss. Our findings suggest that tanshinone IIA inhibits osteoclast formation by inhibiting COX-2/PGE(2) signaling and by suppressing bone erosion in vivo. These results suggest that tanshinone IIA may be of therapeutic value as an anti-bone-resorptive drug in the treatment of bone-related disease.     

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.