生物活性

Cryptotanshinone is a major tanshinone that exhibits multiple activities. Exhibits antitumor activity via inhibition STAT3 activity (IC50 = 4.6 μM). Displays antibacterial and anti-inflammatory activity and acts as an antidiabetes and antiobesity agent via activation of AMPK (AMP-activated protein kinase). Also inhibits AChE (acetylcholinesterase) (IC50 = 4.09 μM) and reduction in Aβ peptide generation.Antitumor antibiotic agent that inhibits DNA topoisomerase II. DNA intercalator that inhibits nucleic acid synthesis and induces apoptosis. Reduces intracellular tau levels.

Inhibition of protein tyrosine phosphatase (PTP)[2]

Cytotoxicity on Hela cells of cervical cancer[3]

Growth inhibition of breast cancer cells[4]

Thecell viability of cancer cell lines

体外研究

体内研究

dissolve in 0.1%月桂醇硫酸酯钠溶液

激酶实验

In Vitro Phosphatase Activity Assay[2]

Glutathione S-transferase (GST) fusion proteins of SHP2 purified in-house were used as the enzyme, and a phosphopeptide corresponding to the surrounding sequence of pTyr1018in the epidermal growth factor receptor (EGFR, Asp-Ala-Asp-Glu-Tyr[PO3H2]-Leu-Ile-Pro-Gln-Gln-Gly) was used as the substrate. The assay determines free phosphate generated by dephosphorylation of the substrate using the Malachite Green reagent. An amount of 0.5 μg of GST-SHP2 PTP was used in the phosphatase assay for screening of SHP2 inhibitors. Initial rate conditions (0.2 mM substrate and 10 min incubation time) (Vmaxconcentration of substrate is approximately 0.3mM) were determined following the standard enzymatic kinetic assay and used in subsequent screening. In brief, 0.5 μg of GST-SHP2 PTP was incubated in 40 μL of assay buffer (25 mM Tris-HCl, pH 7.4, 50mM NaCl, 5 mM DTT, and 2.5 mM EDTA) with test compounds at various concentrations at room temperature for 30 min. The substrate was then added to a final concentration of 0.2 mM. The reaction system was incubated at 30 °C for 30 min. Finally, 50 μL of MalachiteGreen solution was added, and OD620was measured after 10 min. The protocols for the phosphatase assays for SHP1, PTP1B, CD45, LAR, MEG2, and TC-PTP were similar, with the exception that GST-CD45 cytoplasmic domain, GST-LAR, GST-MEG2, and GST-TC-PTP enzymes were used in the respective assays.

细胞实验

Culture of cell lines[5]

Drug-sensitive CCRF-CEM and P-glycoprotein (P-gp)-overexpressing multidrug-resistant CEM/ADR5000 leukemia cells were incubated at 5 % CO2at 37°C and cultured in RPMI medium containing 10 % fetal bovine serum and 1 % penicillin and streptomycin. CEM/ADR5000 cells were treated with doxorubicin (5 μg/mL) in each passage to maintain P-gp expression.

Cell viability

Resazurin assay was used to evaluate cytotoxicity. Cells were seeded and incubated in 96-well plates with indicated concentration of cryptotanshinone (CPT) for 72 h. Resazurin solution was added to wells for 4 h. Fluorescent resorufin, the reduced form of resazurin in the viable cells, was analyzed by Infinite M2000 Proplate reader with optical density at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.

动物实验

Animals[9]

Male Wistar rats weighing between 260 and 320 g obtained and housed individually in plastic cages under standard laboratory conditions. All experiments were performed under anesthesia (2% isoflurane) to minimize the animals’ suffering. Diabetes was induced in the rats by intravenous (i.v.) injection of streptozotocin (65 mg/kg) into fasting animals. Animals were considered diabetic once they had a plasma glucose level above 350 mg/dL. All experiments were performed 9 weeks after diabetes induction.

Rats were separated into three groups (n = 6): Wistar rats (control), vehicle- treated STZ-treated rats(STZ), and cryptotanshinone-treated STZ-treated rats(cryptotanshinone). Cryptotanshinone was prepared in a 0.1% solution of sodium lauryl sulfate. Then, the prepared solution or the vehicle solution was administered orally at 10 mg/kg cryptotanshinone daily between 09:00 and 10:00 h for 28 days.

Catheterization for hemodynamic dP/dt measurement

Hemodynamic measurements were performed. Briefly, temporary pacing leads were placed in the right atrium and the RV apex of the heart. A left ventricular (LV) pacing electrode was placed in the anterior region via the great cardiac vein. After femoral artery and venous puncture, pressure transducer catheters were inserted into the heart to provide the right ventricular, aortic, mean blood and left ventricular pressures. Pressure catheters and pacing leads were connected to an external pacing computer to monitor the heart rate and to acquire the hemodynamic signals. Body temperature in the rats was maintained at 37.5°C during the experiments.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Shin DS, Kim HN, Shin KD, et al. Cryptotanshinone inhibits constitutive signal transducer and activator of transcription 3 function through blocking the dimerization in DU145 prostate cancer cells. Cancer Res. 2009;69(1):193-202.
[2] Liu W, Yu B, Xu G, et al. Identification of cryptotanshinone as an inhibitor of oncogenic protein tyrosine phosphatase SHP2 (PTPN11). J Med Chem. 2013;56(18):7212-7221.
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体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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