Synthesis and vasodilative activity of tanshinone IIA derivatives

A series of 2,2′-(substituted methylene)bis-(1,6,6-trimethyl-6,7,8,9-tetrahydrophenanthro[1,2-b]furan-10,11-dione) derivatives were synthesized by the reaction of tanshinone IIA (D₁) and aromatic aldehyde in the presence of p-TsOH. Bromination derivative of D₁ and hydrolysis product of cryptotanshinone (D₂) were also prepared in this work. Vasodilation activity in vitro of them was valuated on the contractile response of vascular thoracic aorta smooth muscle from Wistar rats for the first time. Most of them exhibited a concentration-dependent inhibition on the contractile response of norepinephrine.     

丹参四个栽培类型的脂溶性成分的聚类分析

为给丹参优质新品种选育研究提供一定的理论依据,本文采用HPLC法测定江苏栽培的四个丹参类型的丹参酮IIA、隐丹参酮的含量,采用紫外分光光度法测定其总丹参酮的含量,以DPS软件对其脂溶性成分的HPLC的8个峰的比例、隐丹参酮与丹参酮IIA的比值、丹参酮IIA、隐丹参酮和总丹参酮的含量进行聚类分析。结果表明四个类型丹参的脂溶性成分类别基本一致,其中小叶型丹参的隐丹参酮与丹参酮IIA的比值、隐丹参酮的含量与其它三个类型相差较大,聚类结果显示小叶型丹参独自成为一个类群,说明小叶型丹参为一个较为特殊的类型,也是一个优质的丹参材料。     

Induction of CYP1A by a diterpene quinone tanshinone IIA isolated from a medicinal herb Salvia miltiorrhiza in C57BL/6J but not in DBA/2J mice

Effects of tanshinone IIA, an active diterpene quinone of the herbal medicine Salvia miltiorrhiza (Danshen), on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in the arylhydrocarbon (Ah)-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice. Oral treatment of tanshinone IIA caused a dose-dependent increase of liver microsomal 7-methoxyresorufin O-demethylation (MROD) activity in B6 but not in D2 mice. In B6 mice, tanshinone IIA increased hepatic benzo(a)pyrene hydroxylation (AHH), 7-ethoxyresorufin O-deethylation, MROD, and 7-ethoxycoumarin O-deethylation activities. The levels of Cyp1A2 protein and mRNA were elevated. On the contrary, in D2 mice, tanshinone IIA decreased hepatic AHH and nifedipine oxidation activities and the CYP3A protein level without affecting other activities determined. Cyp1A2 protein and mRNA levels were not affected by tanshinone IIA in D2 mice. Tanshinone IIA had no effects on UGT and GST activities in both B6 and D2 mice. These results demonstrated that induction of CYP1A2 by tanshinone IIA depended on the Ah-responsiveness and occurred at pre-translational level.     

Tanshinone IIA Inhibits Hypoxia-Induced Pulmonary Artery Smooth Muscle Cell Proliferation via Akt/Skp2/p27-Associated Pathway

We previously showed that tanshinone IIA ameliorated the hypoxia-induced pulmonary hypertension (HPH) partially by attenuating pulmonary artery remodeling. The hypoxia-induced proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the major causes for pulmonary arterial remodeling, therefore the present study was performed to explore the effects and underlying mechanism of tanshinone IIA on the hypoxia-induced PASMCs proliferation. PASMCs were isolated from male Sprague-Dawley rats and cultured in normoxic (21%) or hypoxic (3%) condition. Cell proliferation was measured with 3 - (4, 5 - dimethylthiazal - 2 - yl) - 2, 5 - diphenyltetrazoliumbromide assay and cell counting. Cell cycle was measured with flow cytometry. The expression of of p27, Skp-2 and the phosphorylation of Akt were measured using western blot and/or RT-PCR respectively. The results showed that tanshinone IIA significantly inhibited the hypoxia-induced PASMCs proliferation in a concentration-dependent manner and arrested the cells in G1/G0-phase. Tanshinone IIA reversed the hypoxia-induced reduction of p27 protein, a cyclin-dependent kinase inhibitor, in PASMCs by slowing down its degradation. Knockdown of p27 with specific siRNA abolished the anti-proliferation of tanshinone IIA. Moreover, tanshinone IIA inhibited the hypoxia-induced increase of S-phase kinase-associated protein 2 (Skp2) and the phosphorylation of Akt, both of which are involved in the degradation of p27 protein. In vivo tanshinone IIA significantly upregulated the hypoxia-induced p27 protein reduction and downregulated the hypoxia-induced Skp2 increase in pulmonary arteries in HPH rats. Therefore, we propose that the inhibition of tanshinone IIA on hypoxia-induce PASMCs proliferation may be due to arresting the cells in G1/G0-phase by slowing down the hypoxia-induced degradation of p27 via Akt/Skp2-associated pathway. The novel information partially explained the anti-remodeling property of tanshinone IIA on pulmonary artery in HPH.     

Tanshinone production in roots of micropropagated Salvia przewalskii Maxim

Production of tanshinones (tanshinone I and IIA) was determined in roots of Salvia przewalskii micropropagated plants. It was found that the total tanshinone content (tashinone I and tashinone IIA) was dependent on the age of the analyzed plants. The roots of 2-year-old in vitro regenerated plants at flowering stage produced highest tanshinone levels (3.8 mg/g dry weight of tanshinone I and 7.6 mg/g dry weight of tanshinone IIA).     

Induction of apoptosis and inhibition of cell adhesive and invasive effects by tanshinone IIA in acute promyelocytic leukemia cells in vitro

Tanshinone IIA, a diterpene quinone extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, is used widely and successfully in clinics in China for treating inflammatory diseases. Recently tanshinone IIA has been reported to have apoptosis inducing effects on a large variety of cancer cells. In this study, the anti-proliferation and apoptosis inducing effects of tanshinone IIA as well as its influence on cell adhesion to and invasion through the extracellular matrix (ECM) on acute promyelocytic leukemia (APL) NB4 cells in vitro were studied. Cell proliferation was assessed by MTT assay, cell apoptosis was observed by Hoechst 33258 staining and flow cytometry (FCM); The variation of caspase-3 and apoptotic related genes were assayed by Western blotting, cell mitochondrial membrane potential as well as cell adhesive and invasive effects were also investigated by using standard methods. The results showed that tanshinone IIA exhibited induction of apoptosis by activation of caspase-3, downregulation of anti-apoptotic protein bcl-2 and bcl-xl and upregulation of pro-apoptotic protein bax, as well as disruption of the mitochondrial membrane potential. Furthermore, treatment by tanshinone IIA could reduce cell adhesion to and invasion through ECM in leukemia NB4 cells. These data provide a potential mechanism for tanshinone IIA-induced apoptosis and cell growth inhibition in leukemia NB4 cells, suggesting that tanshinone IIA may serve as an effective adjunctive reagent for the treatment of APL.Contributed equally to this study.     

High-Dose Tanshinone IIA Suppresses Migration and Proliferation While Promoting Apoptosis of Astrocytoma Cells Via Notch-1 Pathway

Malignant astrocytoma is the most common malignant tumor with strong invasion in the central nervous system. Tanshinone IIA is an effective compound to suppress cell proliferation and promote cell apoptosis. However, there is little research about the role of tanshinone IIA in the treatment of astrocytoma. This study aimed to investigate the effect of tanshinone IIA on migration, proliferation and apoptosis of astrocytoma cells. The efficacy of tanshinone IIA on migration, proliferation and apoptosis of astrocytoma cells were evaluated by flow cytometry and the assays of plate clone formation, CCK-8, wound healing and transwell migration. The protein molecule and signaling pathway were detected by western blot. High-dose tanshinone IIA suppressed migration and proliferation of astrocytoma cells while promoting apoptosis of astrocytoma cells. The western blot results showed that there were high Notch-1 protein expression and low c-Myc, MMP-9 and Bcl-2 activation in the high-dose tanshinone IIA group compared with the control group. High-dose tanshinone IIA suppresses astrocytoma cell proliferation, migration while promoting apoptosis through Notch-1 pathway. Tanshinone IIA may be used to develop new drugs for the treatment of astrocytoma.     

Tanshinone IIA enhances BMP-2-stimulated commitment of C2C12 cells into osteoblasts via p38 activation

In this study, we demonstrate a stimulatory effect of tanshinone IIA isolated from the root of Salvia miltiorrhiza on the commitment of bi-potential mesenchymal precursor C2C12 cells into osteoblasts in the presence of bone morphogenetic protein (BMP)-2. At low concentrations, tanshinone IIA enhanced BMP-2-stimulated induction of alkaline phosphatase (ALP), an early phase biomarker of osteoblast differentiation, and mRNA expression of BMPs. ALP induction was inhibited by the BMP antagonist noggin, suggesting that tanshinone IIA enhances the osteogenic activity of BMP signaling. Furthermore, considering the tanshinone IIA-mediated enhancement of BMP-2-stimulated Smad-Runx2 activities, tanshinone IIA could enhance the osteogenic activity of BMP-2 via acceleration of Smad-Runx2 activation. Additionally, pharmacologic inhibition studies suggest the possible involvement of p38 in the action of tanshinone IIA. The p38 inhibitor SB202190 strongly and dose-dependently inhibited tanshinone IIA-enhanced ALP induction. SB202190 also dose-dependently inhibited the tanshinone IIA-induced p38 activation and combined tanshinone IIA-BMP-2-induced Smad activation. In conclusion, tanshinone IIA enhances the commitment of C2C12 cells into osteoblasts and their differentiation through synergistic cross talk between tanshinone IIA-induced p38 activation and BMP-2-induced Smad activation. These activations could subsequently induce the activation of Runx2, which induces osteogenesis via regulation of the osteogenic factors BMP and ALP expression.     

Tanshinone IIA suppress the proliferation of HNE-1 nasopharyngeal carcinoma an in vitro study

Nasopharyngeal carcinoma (NPC) at present is considered to be one of the fatal diseases detected commonly in the people belonging to Southeast Asia and southern China. According to the WHO reports among the detected cases of NPC worldwide, 80% are from China. The present study investigates the effect of tanshinone IIA on the migration and invasion potential of HNE-1NPC cells and studied the detailed mechanism involved. Effect of the tanshinone IIA on viability of the HNE-1NPC cells was analyzed by MTS assay. Cell matrigel invasion and wound-healing motility assays, respectively were used for the analysis of invasion and migration potential of HNE-1 cells. Tanshinone IIA inhibited the viability of HNE-1cells in a dose dependent manner. Migration and invasion potential of the tanshinone IIA treated cells was reduced significantly (P < 0.05) compared to the control cells after 48 h. Analysis of the proteins involved in migration and invasion revealed a significant decrease in the expression of matrix metalloproteinase (MMP)-2 and MMP-9 on treatment with tanshinone IIA. It also inhibited the p65 and p50 expression in the nuclear fractions of HNE-1 cells after 48 h. Thus, tanshinone IIA inhibits migration and invasion potential of the HNE-1NPC cells through reduction in the expression of matrix metalloproteinases. Therefore, tanshinone IIA can be used for the treatment of NPC.     

Tanshinone IIA improves impaired nerve functions in experimental diabetic rats

Diabetic neuropathy is one of the most common complications in diabetes mellitus. Thus far, effective therapeutic agents for restoring the impaired motor and sensory nerve functions in diabetic neuropathy are still lacking. The antioxidant and neuroprotective properties of tanshinone IIA make it a promising candidate for the treatment of diabetic neuropathy. Therefore, the present study investigated the possible beneficial effect of tanshinone IIA on the impaired nerve functions displayed by a rat diabetic model. Insulin-dependent diabetes in rats was developed by a single dose of streptozotocin (STZ) at 50 mg/kg. The diabetic rats were randomly divided into four groups (n = 10 in each group), and were intraperitoneally administrated daily for 4 weeks with tanshinone IIA (20 mg/kg, 50 mg/kg and 100 mg/kg), or normal saline from the fourth day after STZ injection, respectively. At the end of tanshinone IIA administration, thermal and mechanical nociceptive threshold were determined by a hot plate test and Von Frey hairs; motor nerve conducting velocity (MNCV) was determined by an electrophysiological method; nerve blood flow (NBF) was detected using a laser Doppler flow meter; Na⁺,K⁺ATPase activity, the level of superoxide dismutase (SOD), catalase and malondialdehyde (MDA) in sciatic nerves, and the serum total antioxidant capability were also determined. We found that tanshinone IIA was capable of restoring diabetes-induced deficit in nerve functions (MNCV and NBF), and impairment in thermal and mechanical nociceptive capability. In addition, tanshinone IIA significantly increased the serum total antioxidant capability, improved the activities of Na⁺,K⁺ATPase, increased the levels of SOD and catalase, and reduced the MDA level in sciatic nerves in diabetic rats. All the findings indicate the beneficial effect of tanshinone IIA on impaired nerve functions and raise the possibility of developing tanshinone IIA as a therapeutic agent for diabetic neuropathy.     

Tanshinone IIA and tanshinone I production by Trichoderma atroviride D16, an endophytic fungus in Salvia miltiorrhiza

In this study the isolation of an endophytic fungus from the root of the medicinal herb Salvia miltiorrhiza Bunge is reported for the first time. The fungus produced tanshinone I and tanshinone IIA in rich mycological medium (potato dextrose broth) under shake flask and bench scale fermentation conditions. The fungus was identified as Trichoderma atroviride by its morphology and authenticated by ITS analysis (ITS1 and ITS2 regions and the intervening 5.8S rDNA region). Tanshinone I and tanshinone IIA were identified by HPLC and LC-HRMS/MS and confirmed through comparison with authentic standards. This endophytic fungus has significant scientific and industrial potential to meet the pharmaceutical demands for tanshinone I and tanshinone IIA in a cost-effective, easily accessible and reproducible way.     

Tanshinone IIA from Salvia miltiorrhiza induces heme oxygenase-1 expression and inhibits lipopolysaccharide-induced nitric oxide expression in RAW 264.7 cells

Tanshinone IIA exerts anti-inflammatory effects and influences electron transfer reaction in mitochondria. In the present study, we demonstrated that tanshinone IIA increased intracellular production of reactive oxygen species (ROS), which in turn induces heme oxygenase-1 (HO-1) expression in RAW 264.7 macrophages. Tanshinone IIA inhibited COX-2 and iNOS expression in lipopolysaccharide-activated RAW 264.7 macrophages. Inhibition of HO-1 or scavenging of CO significantly reversed the inhibition of LPS-stimulated nitrite accumulation by tanshinone IIA, suggesting a novel role of HO-1 in the anti-inflammatory effect of tanshinone IIA.     

Functional proteomic and structural insights into molecular targets related to the growth inhibitory effect of tanshinone IIA on HeLa cells

Certain antitumor agents have recently been extracted from the roots of Salvia miltiorrhiza Bunge. The diterpene derivative, tanshinone IIA, possesses cytotoxic activity against several human carcinoma cell lines. It also inhibits invasion and metastasis of cancer cells. In the present study, we isolated tanshinone IIA from S. miltiorrhiza, and it exhibited strong growth inhibition against human cervical cancer cells in dose‐ and time‐dependent manners with a 50% cell growth inhibition value of 2.5 μg/mL (8.49 μM). Flow cytometric analysis of cell cycle progression revealed that G₂/M arrest was initiated after a 24 h exposure to the drug. It also resulted in DNA fragmentation and degradation of poly (ADP‐ribose) polymerase indicating that tanshinone IIA may be a potential antitumor agent. Furthermore, we performed a comprehensive proteomic analysis to survey global protein changes induced by tanshinone IIA treatment on HeLa cells. Significant changes in the levels of cytoskeleton proteins as well as stress‐associated proteins were observed. Immunoblot analysis and immunofluorescence staining were used to confirm the levels of protein expression. Overexpression of the vimentin rescued these tanshinone IIA‐induced events. Computational docking methods indicated that tanshinone IIA could stably bind to the β‐subunit of the microtubule protein. An interaction network analysis of these 12 proteins using MetaCore? software suggested that tanshinone IIA treatment regulated the expressions of proteins involved in apoptotic processes, spindle assembly, and p53 activation, including vimentin, Maspin, α‐ and β‐tubulin, and GRP75. Taken together, our results suggest that tanshinone IIA strongly inhibited the growth of cervical cancer cells through interfering in the process of microtubule assembly, leading to G₂/M phase arrest and sequent apoptosis. The success of this large‐scale effort was assessed by a bioinformatics analysis of proteins through predictions of protein domains and possible functional roles. The possible contributions of these proteins to the cytotoxicity of tanshinone IIA provide potential opportunities for the development of cancer therapeutics.     

Mutation breeding of Emericella foeniculicola TR21 for improved production of tanshinone IIA

TR21, an original tanshinone IIA-producing endophytic fungus from the root of Salvia miltiorrhiza Bunge, produces low levels of tanshinone IIA. Three techniques were used to rapidly improve tanshinone IIA production: TR21 mutation by ultraviolet radiation (UV), TR21 mutation by sodium nitrite (NaNO₂) and TR21 mutation by a combination of both UV and NaNO₂. An improved mutant, labeled as NU152, was obtained from the combination of UV and NaNO₂. The content of tanshinone IIA produced by NU152 held a relative high value of 51.44 ± 0.22 μg per g, and the NU152 produced a more than 1.46-fold increase in tanshinone IIA yield, compared with the wild type TR21 (P < 0.05). The fungus NU152 showed a possible way for the sustainable production of tanshinone IIA.     

Tanshinone IIA and astragaloside IV promote the migration of mesenchymal stem cells by up-regulation of CXCR4

Mesenchymal stem cells (MSCs) have a therapeutic potential to treat cardiovascular diseases. However, a significant barrier to MSC therapy is insufficient MSC engraftment in ischemic myocardium after systemic administration. Here, we investigated the modulatory effects of tanshinone IIA and astragaloside IV on the migration of MSCs and further defined the underlying mechanisms. CXCR4 expression in MSCs was determined by using flow cytometry, real-time PCR, and western blotting. The results showed that CXCR4 expression was significantly higher in tanshinone IIA- and astragaloside IV-stimulated MSCs than that of the control. MSC migration toward stromal cell-derived factor-1α (SDF-1α) was studied using a transwell system. MSCs treated with tanshinone IIA and astragaloside IV showed stronger migration than that of the control. Moreover, this enhanced migration ability was abrogated by a CXCR4 inhibitor. In a rat acute myocardial infarction model, MSCs stimulated with tanshinone IIA and astragaloside IV were stained with Dio and injected into model rats via the tail vein. Dio-labeled cells in myocardium sections were observed by fluorescence microscopy. Tanshinone IIA- and astragaloside IV-stimulated MSCs showed enhanced capacities to home to ischemic myocardium sites. In addition, there was no significant difference in the SDF-1α expression among groups. These data suggest that tanshinone IIA and astragaloside IV regulate MSC mobilization, at least partially via modulation of the CXCR4 expression.     

Tanshinone IIA from Salvia miltiorrhiza BUNGE inhibits human aortic smooth muscle cell migration and MMP-9 activity through AKT signaling pathway

Smooth muscle cell (SMC) migration plays an important role in normal angiogenesis and is relevant to disease-related vascular remodeling in conditions such as brain arteriovenous malformations, pulmonary hypertension, arteriosclerosis, and restenosis after angioplasty. In this present study, we showed that tanshinone IIA, the major lipid-soluble pharmacological constituent of Salvia miltiorrhiza BUNGE, inhibits human aortic smooth muscle cell (HASMC) migration and MMP-9 activity. Tanshinone IIA significantly inhibited IkappaBalpha phosphorylation and p65 nuclear translocation through inhibition of AKT phosphorylation. Tanshinone IIA inhibited TNF-alpha-induced ERK and c-jun phosphorylation, but not other MAPKs such as JNK and p38. Tanshinone IIA also inhibited NF-kappaB and AP-1 DNA-binding. Moreover, tanshinone IIA inhibited the migration of TNF-alpha-induced HASMCs. Our results provide evidence that tanshinone IIA has multiple effects in the inhibition of HASMC migration and may offer a therapeutic approach to block HASMC migration.     

回流提取过程中丹参酮的热降解行为研究

用甲醇作提取溶剂,在回流条件下考察了从丹参药材中提取丹参酮类有效成分的过程中,隐丹参酮、丹参酮I及丹参酮IIA等三种丹参酮的热降解行为。结果表明,回流提取过程中所考察的三种丹参酮均发生严重的热降解,降解速率:丹参酮IIA>丹参酮I>隐丹参酮,其热降解均具有零级反应动力学特征;同时,回流提取过程中丹参酮的热降解是在丹参酮共萃物存在下发生的。     

Roles of reactive oxygen species in methyl jasmonate and nitric oxide-induced tanshinone production in Salvia miltiorrhiza hairy roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants for treatment of coronary heart disease. Tanshinones are the main biological active compounds in S. miltiorrhiza. In this study, effects of exogenous methyl jasmonate (MJ) and nitric oxide (NO) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of reactive oxygen species (ROS) in MJ and NO-induced tanshinone production were elucidated further. The results showed that contents of four tanshinone compounds were significantly increased by 100 μM MJ when compared to the control. Application of 100 μM sodium nitroprusside (SNP), a donor of NO, also resulted in a significant increase of tanshinone production. Expression of two key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) was up-regulated by MJ and SNP. Generations of O₂ ⁻ and H₂O₂ were triggered by MJ, but not by SNP. The increase of tanshinone production and up-regulation of HMGR and DXR expression induced by MJ were significantly inhibited by ROS scavengers, superoxide dismutase (SOD) and catalase (CAT). However, neither SOD nor CAT was able to suppress the SNP-induced increase of tanshinone production and expression of HMGR and DXR gene. In conclusion, tanshinone production was significantly stimulated by MJ and SNP. Of four tanshinone compounds, cryptotanshinone accumulation was most affected by MJ elicitation, while cryptotanshinone and tanshinone IIA accumulation was more affected by SNP elicitation. ROS mediated MJ-induced tanshinone production, but SNP-induced tanshinone production was ROS independent.     

Tanshinone IIA isolated from Salvia miltiorrhiza elicits the cell death of human endothelial cells

Summary Tanshinone IIA, a major component extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, is known to exhibit potent cytotoxicity against various human carcinoma cells in vitro. However, the mechanism by which tanshinone IIA produces this anti-tumor effect remains unknown. Since anti-neovascularization has generally been regarded as an effective strategy for anti-cancer therapy, we decided to investigate the mechanism underlying tanshinone IIA-mediated death of human endothelial cells. In this study, we demonstrate that tanshinone IIA elicits human endothelial cell death independent of oxidative stress. These events are partially calcium-dependent and actually dependent upon NAD(P)H: quinone oxidoreductase (NQO1) activity. Tanshinone IIA induces an increase in intracellular calcium, which triggers the release of cytochrome c, thus causing loss of the mitochondrial membrane potential (MMP), resulting in the subsequent activation of caspases. Blocking the induction of Ca²⁺ perturbation with BAPTA-AM partially rescued cells from tanshinone IIA-induced cytotoxicity. Additionally, blocking NQO1 activity with dicoumoral or inhibiting caspase activities with the general caspase inhibitor, z-VAD-fmk, prevented cell death induced by tanshinone IIA. Therefore, our results imply that tanshinone IIA-mediated cytotoxicity against human endothelial cells may occur through activation of NQO1, which induces a calcium imbalance and mitochondrial dysfunction, thus stimulating caspase activity.These authors contributed equally to this work.