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Tanshinone I (Tan I) is one of the main bioactive ingredients derived from Salvia miltiorrhiza Bunge, which has exhibited antitumor activities toward various human cancer cells. However, its effects and underlying mechanisms on human chronic myeloid leukemia (CML) cells still require further investigation. This study determined the effects and mechanisms of anti-proliferative and apoptosis induction activity induced by Tan I against K562 cells. The cytotoxic effect of Tan I at varying concentrations on K562 cells was evaluated via MTT assay. Cell apoptosis was further investigated through DAPI staining and flow cytometry analysis. The expression levels of apoptosis-related proteins and activities of JNK/ATF2 and ERK signaling pathways were analyzed by western blot. Quantitative PCR was performed to further determine mRNA expression levels of JNK1/2 and ERK1/2 after Tan I treatment. The results indicated that Tan I significantly inhibited K562 cell growth and induced apoptosis in a concentration- and time-dependent manner. It induced significant cellular morphological changes and increased apoptosis rates in CML cells. Tan I promoted the cleavages of caspase-related proteins, as well as increased the expression levels of PUMA. Furthermore, Tan I significantly activated JNK and inhibited ATF-2 and ERK signaling pathways. The mRNA expression levels of JNK1/2 and ERK1/2 were up-regulated by Tan I, further confirming its regulatory effects on JNK/ERK signaling pathways. Overall, our results indicated that Tan I suppressed cell viability via JNK- and ERK-mediated apoptotic pathways in K562 cells, suggesting that it might be a promising candidate as a novel anti-leukemia drug.
Subject(s)
Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Abietanes/pharmacology , Apoptosis , Cell Line, TumorABSTRACT
Objective: To establish a method for determining the contents of schisandrin, schisandrol B, cryptotanshinone, tanshinone I and tanshinone IIA, γ-schisandrin in Zaoren Anshen Capsules (ZAC), and provide scientific method for quality control of ZAC. Methods: HPLC was used by using Thermo 120Å-C18 column, with the mobile phase consisted of acetonitrile and 0.1% acetic acid solution with gradient elution for simultaneous determination of six main index components. The detection wavelengths were set at 250 nm for schisandrin, schisandrol B, γ-schisandrin and 270 nm for cryptotanshinone, tanshinone I, and tanshinone IIA, flow rate was 1.0 mL/min, and column temperature was 30℃. Results: Schisandrin, schisandrol B, γ-schisandrin, cryptotanshinone, tanshinone I, and tanshinone IIA showed good the linear ranges relationships in the range of 4.7-3 153.6 ng (r = 0.999 9), 7.864-314.500 ng (r = 0.999 9), 14.4-1 256.9 ng (r = 0.999 9), 15.1-1 103.8 ng (r = 0.999 9), 15.3-1 532.0 ng (r = 0.999 9) and 6.134-204.500 ng (r = 0.999 9), respectively. The average recoveries were 100.4%, 98.7%, 99.4%, 100.0%, 99.3% and 100.2%, RSD were 1.4%, 2.7%, 2.2%, 2.2%, 2.5% and 2.1%, respectively. The contents of each index component in 8 batches of sample were schisandrin 1.545 7-1.909 9 mg/g, schisandrol B 0.129 8-0.235 1 mg/g, cryptotanshinone 0.508 4-0.523 4 mg/g, tanshinone I 0.111 7-0.122 3 mg/g, tanshinone IIA 0.755 8-0.874 4 mg/g, γ-schisandrin 0.120 2-0.190 1 mg/g. Conclusion: The established analytical method is highly sensitive with strong specificity and it can be used efficiently in the quality control of ZAC.
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Objective: The potency of multi-source information fusion technology was explored to improve the model calibration and prediction performance of Chinese medicine extraction process. Methods: The ethanol extraction process for isolating fat-soluble components from Salvia miltiorrhiza was taken as the research carrier. S. miltiorrhiza from different sources were collected to simulate the fluctuation of materials. The changes of process parameters were simulated by design of experimental (DOE), and the process near infrared spectra (NIRS) were used as the process state variables. The contents of tanshinone IIA, cryptotanshinone, and tanshinone I were determined by HPLC. The raw material properties, process parameters and process state variables were combined as independent variables. The content of effective components in the extract was taken as the dependent variable. The partial least squares (PLS) algorithm was used to establish the quality prediction model of the extracts. Results: The modeling results respectively showed that the RMSECV was 0.172 8 mg/g, RMSEP was 0.031 7 mg/g, RPD was 6.91 (tanshinone IIA); RMSECV was 0.153 4 mg/g, RMSEP was 0.024 2 mg/g, RPD was 4.02 (cryptotanshinone); RMSECV was 0.117 1 mg/g, RMSEP was 0.043 2 mg/g, RPD was 4.76 (tanshinone I). Conclusion: The calibration and prediction performance of multi-source information fusion model are better than the conventional model, which can effectively improve the quality predictability and controllability of S. miltiorrhiza extract.
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Objective: To explore the intestinal absorption characteristics of tanshinone components self emulsifying drug delivery system (SEDDS). Methods: In situ single-pass perfusion method was used to investigate the absorption characteristics of cryptonshinone, tanshinone I, and tanshinone IIA in rats. The absorption parameters (Ka, Papp) of cryptotanshinone, tanshinone I, and tanshinone IIA were used as indicators to study their optimum absorption site among duodenum, jejunum, ileum, and colon. The effects of verapamil hydrochloride (P-glycoprotein inhibitor, P-gp inhibitor), probenecid (multi-drug resistant protein MRP2), and 2, 4-dinitrophenol (energy inhibitor) on the absorption of cryptonshinone, tanshinone I, and tanshinone IIA were also studied, as well as the effects of their different concentration. Results: Cryptonshinone, tanshinone I, and tanshinone IIA could be absorbed at all four intestinal segments, and the optimum absorption site was the upper segment of small intestine. The absorption of cryptotanshinone and tanshinone I were concentration-dependent at experimental concentration levels (1.05-4.19 mg/L and 1.22-5.56 mg/L), while tanshinone IIA was not affected obviously by its concentrations (2.43-11.12 mg/L). Verapamil hydrochloride had no significant influence on the absorption of cryptotanshinone or tanshinone I, while the absorption of tanshinone IIA was improved remarkably. Probenecid increased the absorption of cryptonshinone and tanshinone I apparently, while had no obvious effect on that of tanshinone IIA. 2,4-Dinitrophenol could decrease the absorption of cryptonshinone, tanshinone I, and tanshinone IIA apparently. Conclusion: Cryptonshinone and tanshinone I are supposed to be the substrate of MRP2 instead of P-gp. Tanshinone IIA is supposed to be the substrate of P-gp, instead of MRP2. The energy participated in the absorption of cryptonshinone, tanshinone I, and tanshinone IIA. Active absorption maybe also involved in the absorption of cryptonshinone, tanshinone I, and tanshinone IIA.
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OBJECTIVE: To investigate the mechanism of inhibition and radiosensitization effects of combining treatment with tanshinone I and(irradiation) IR on mice bearing Lewis lung carcinoma(LLC). METHODS: The models of LLC in C57BL/6 mice were established via subcutaneous injection of mouse LLC cells, and divided into model control group, radiation alone group, Tan I (40 mg · kg-1) group, 5-Fu + IR group, Tan I (10, 20, 40 mg · kg-1) + IR groups. After irradiated, the relative volume of tumor was observed, the delay time of tumor growth and enhancement factor (EF) was calculated. After stripped, the inhibition rate was calculated. Cell apoptosis index( AI) was in vestigated by TUNEL staining. The protein expressions of Bcl-2 and Bax were detected by Western blot. RESULTS The different concentrations of Tan I (low, medium and high) + IR groups inhibited the tumor growth significantly, and the inhibition rate was 27.14%, 38.46%, 59.78%, respectively, which were significant difference as compared with IR group (P < 0.01). And also had the significant radiosensitizing effect, the enhancement factor was 1.09, 1.76, 2.03, respectively. The AI was 51.3% in Tan I (H) + IR group, which was significant difference as compared with IR group(P < 0.05). The expression of Bcl-2 protein was decreased, and Bax proteins was increased by combining treatment with Tan I and IR. CONCLUSION Combining treatment with Tan I and IR inhibited the tumor growth and had radiosensitizing effects in mice bearing LLC. One of the mechanism may be that it might induce apoptosis by regulated the expression of Bcl-2/Bax protein, so cells were more sensitive to radiation.
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Objective: To research the influence of different washing methods before drying on the contents of active components in Salvia Miltiorrhizae Radix et Rhizoma, and provide the basis for standard original processing methods of Salvia miltiorrhiza. Methods: The fresh herbs of S. miltiorrhiza were processed with different methods including no washing, water flushing, immersion cleaning, and scrubbing in water. HPLC was applied to determine the contents of active components, with Diamonsil C18 column (250 mm×4.6 mm, 5 μm), acetonitrile-0.026% phosphoric acid for gradient elution, flow rate of 1 mL/min, and detection wave length of 286 and 270 nm. Results: The contents of rosmarinic acid, salvianolic B, crypotanshinone, tanshinone I, and tanshinone IIA were all significantly decreased in S. miltiorrhiza processed with different washing methods. The contents of active components were reduced with the increasing of washing intensity, of which the lowest contents were found in scrubbing group. Conclusion: Washing with water can make loss of active components, and flushing may be a more suitable method. In order to decrease the content loss of active components, contact time with water must be controlled as short as possible and violent friction among medicinal materials and artificial rubbing must be avoided.
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Objective: A novel one-step extraction and purification method by using superparamagnetic adsorbents was established for separation of tanshinones from Salvia miltiorrhiza. Methods: Firstly, superparamagnetic poly hydroxyethyl methacrylate (PHEMA) microspheres were prepared by dispersion polymerization method, and their adsorptive performances of tanshinones were investigated, then these microspheres for purification of tanshinones were coupled into extraction process of tanshinones. The feasibility and separation efficiency of this one-step method were further studied. Results: The prepared PHEMA microspheres with the average diameters of 1.2 μm had superparamagnetic property and good adsorption performance for tanshinones. In comparison with two steps method including extraction and purification, one-step method showed significantly higher extraction efficiency of three tanshinones from 0.179, 0.093, and 0.452 mg/g in 5 h to 0.279, 0.176, and 0.575 mg/g in 0.5 h, with less separation process, greatly shorter time consumption from 5.5 h to 0.5 h, and good purification results. Conclusion: One-step method proposed is demonstrated feasible and has the merits of high extraction efficiency, less separation process, good purification, and rapid separation speed.
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Tanshinones are a large class of hydrophobic natural products isolated from traditional Chinese herb Salvia miltiorrhiza Bunge and related plants. Tanshinones include tanshinone ⅡA, cryptotanshinone, tanshinone I, dihydrotanshinone, and so on. Both the tanshinones and the salvianolic acids have been identi-fied as the characteristic and main active ingredients of Salvia miltiorrhiza Bunge with cardiovascular protective activities. Ac-cumulated data in recent years have revealed that tanshinones possess remarkable anti-cancer activities both in vivo and in vitro. In this review, we summarize the latest progress of the an-ti-cancer effect and the underlying mechanisms of tanshinones, particularly with emphasis on tanshinone ⅡA, which might pro-vide reference for the further research and development of these compounds.
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Objective: To establish a UPLC-MS/MS method for determining the plasma concentration of dihydrotanshinone I, tanshinone I, cryptotanshinone, and tanshinone IIA in rats, and to study the pharmacokinetics of Salvia miltiorrhiza diterpene quinones composition (SMDQC) and its solid dispersion micro-pellets (SDMP). Methods: Sprague-Dawley rats were ig administered with SMDQC and its SDMP, respectively. Then the blood samples were obtained at different time points. Electrospray ionization (ESI) source was applied and operated in the positive ion mode. The plasma concentration of dihydrotanshinone I, tanshinone I, cryptotanshinone, and tanshinone IIA was then detected by UPLC-MS/MS, and the pharmcoknetic parameters were calculated by DAS 2.1.1 program. Results: The RSDs of intra- and inter-day precisions of all analytes were less than 14.6%, and the average recoveries of the four active constituents were more than 74.49%. The pharmacokinetic results showed that after ig administration of SDMP, Cmax and AUC0-∞ of the four active constituents increased significantly compared with those of SMDQC. Conclusion: The method has the high sensitivity and selectivity, and proves to be suitable for the pharmacokinetic study of SMDQC and its SDMP. The results show that the SDMP could enhance the solubility of SMDQC to improve its absorption. The relative bioavailability of the four representative constituents is 138%-204% of the crude drug.
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Objective: To investigate the in vivo metabolism of the combination of tanshinone IIA (Tan IIA), cryptotanshinone (Cry), and tanshinone I (Tan I) in zebrafish and discuss the possibility and rationality of using zebrafish in multi-component metabolism of Chinese materia medica (CMM). Methods: The zebrafish was exposed to the 1% DMSO solution of the combinations with Tan IIA, Cry, and Tan I. High performance liquid chromatography coupled with ion-trap mass spectrometry (HPLC-MS) method was used to calculate the relative molecular weight of the metabolites based on the excimer ion peak. The metabolites in solution and zebrafish after the combination being exposed to zebrafish for 24 h were identified through comparing with the literature data and reference substances. Results: After the combination being exposed to zebrafish, hydroxylation and dehydrogenation products of Tan IIA and Cry were obtained while the metabolites of Tan I was not found. Conclusion: The metabolic mechanism of zebrafish against the combination highly consists with those in rats or rat liver microsomes, which indicates that the metabolism of zebrafish against the combination is rational with the advantages of less amount of compound, lower cost, simpler operation, and higher efficiency. The above results provide the reference for using zebrafish in the metabolic study on complex CMM system.