生物活性

Resveratrol is a natural polyphenol derived from red wine; in Asian folk-medicine it and it's glycoside, polydatin are the primary ingredients of Kojo-Kon. A phytoestrogen with antitumor, antioxidant, antiplatelet, anti-inflammatory and antifungal effects. This trihydroxystilbene is derived from various sources and has been studied with regard to numerous applications including reducing serum lipids, inhibition of platelet aggregation, and actions as a chemopreventative agent and antioxidant. Inhibits cytochrome P450 1A1 (IC50 = 23 μM) and displays mixed agonist/antagonist actions at ERα and ERβ estrogen receptors. Resveratrol has been shown to increase the longevity of yeast and to display antiproliferative effects on cancer stem cells in vitro. Resveratrol is a specific inhibitor of Cox-1 via inhibition of the hydroperoxidase activity of Cox-1. Converted into the anticancer agent piceatannol by cytochrome P450 1B1.

体外研究

体内研究

0.5% CMC Na

激酶实验

Caspase activity[1]

This assay is based on the cleavageactivity of caspase-3 (DEVDase) on the substrate (Ac-DEVD-pNA), and then thep-nitroaniline (pNA) is released from substrate. CaspACE™ Assay kit was used.The cell lysates were prepared and incubated with specific caspase-3 antibody. Immunocomplexeswere incubated with peptide substrate in assay buffer (100mM NaCl, 50mM

4-(2-hydroxyethyl)-1-pipera-zine-ethanesulphonicacid (HEPES), 10mM dithiothreitol, 1mM EDTA, 10% glycerol, 0.1%3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate (CHAPS), pH 7.4) for2 h at 37 °C. The p-nitroaniline release was monitored at 405 nm. Results arerepresented as the fold change of the activity compared to the control group.

SIRT1activity

SIRT 1 activity was determined with SIRT1Deacetylase Fluorometric Assay kit. Nuclear proteins were extracted using theNuclear and Cytoplasmic Protein Extraction kit according to the manufacturer’s instructions.The resulting fluorescence was measured at 340 nm excitation and 440 nmemission wavelengths with a fluorescent microplate reader.

细胞实验

Cell cultures and treatments[2]

The rat C6 glioma cell line was maintainedin DMEM supplemented with 10% FBS and 1% antibiotics solution. The human T98Gglioblastoma cell line was maintained in EMEM supplemented with 10% FBS, 1%antibiotics solution, 2mM glutamine, 1% non-essential amino acids and 1% sodiumpyruvate. Cells were incubated at 37 °C in an atmosphere consisting of 95% air and5% CO₂ in a humidified incubator until they reached 70% confluency. Atotal of 1× 10⁶ cells were seeded in ø 40 mm culture dishes. After24 h of preincubation in culture medium containing 5% of FBS, the cells weretreated with resveratrol, its analogs or tannic acid. The incubation wascontinued for a further 24 h to assess cell cycle distribution, apoptosis orp53 induction. Control cells were treated with a vehicle (DMSO). Cells treatedwith 50 nM of camptothecin were used as a positive control. The concentrationof DMSO in culture medium did not exceed 0.1%.

MTTassay for cell viability

The effect of tested polyphenols on cellviability was assessed with an MTT assay. Briefly, the cells were seeded in96-well plates at a density of 1× 10⁴ cells/well in 100 μL of growthmedium. They were allowed to attach overnight and resveratrol, its appropriateanalog or tannic acid was then added to the culture medium in variousconcentrations (0 to 200 μM) for 24 and 48 h at 37 °C. The cells weresubsequently incubated with MTT (0.5 mg/mL) solution for another 4 h. The waterinsoluble formazan crystals were solubilized in acidic isopropanol before themeasurement of absorbance using a microplate reader at 570 nm. All theexperiments were repeated three times, with at least three measurements perassay.

Flowcytometric cell cycle analysis

Cell cycle distribution was evaluated byflow cytometric analysis. After treatment, C6 or T98G cells were collected andfixed in 70% ethanol at 4 °C for 30 min. Since then, the cells were washedtwice in PBS and resuspended in 250 μL of PBS containing 50 μg/mL propidium iodide(PI) and 100 μg/mL RNase A. After incubation in the dark at37 °C for 30 min,the fluorescence of cells was analyzed with a FACSCanto flow cytometer (BectonDickinson, USA). Data analysis and acquisition was performed using FACS Divasoftware.

动物实验

Animals[3]

Male C57BL/6J mice were at 6 weeks of ageand housed in a 23 ± 3°C environment, with free access to food and water. Aftera 1-week acclimation period, 18 mice were randomly divided into three groups: anormal diet group served as a control (10% of calories from fat, 20% ofcalories from protein and70% of calories from carbohydrates); a high-fat diet(HFD) group (60% of calories from fat, 20% of calories from protein and 20% ofcalories from carbohydrates); and a resveratrol-treated HFD group. Resveratrolwas orally administered at an average dose of 100mg/kg/day, whereas the normaldiet and HFD group supplemented with 6.67% DMSO/physiological saline for 12weeks. Body weights were measured biweekly. Fasting and non-fasting plasmasamples were obtained from the tail vein; blood samples were collected usinganticoagulant EDTA tubes. Fasting plasma total cholesterol (TC), triglyceride(TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoproteincholesterol (HDL-C) levels were determined using kits. The plasma PAF-AH levelswere assayed using the PAF-AH kit. At the end of the experiments (12 weeks),the mice were euthanized via cervical dislocation, and their livers werecollected to detect Lp-PLA2 expression and inflammatory cells infiltration(H&E staining).

Westernblot analysis

For the western blot analysis, we washedthe treated cells and collected livers with cold phosphate-buffered saline(PBS), lysed the cells and the grinding livers in lysis buffer containing a100× phosphatase inhibitor cocktail and 1mM PMSF for 30 min on ice or at 4°C, followedby centrifugation. Protein concentrations were determined using an enhancedbicinchoninic acid (BCA) protein assay kit. Equal amounts of proteins wereseparated via 12% or 8% SDS-PAGE. Then, the separated proteins were transferredto a nitrocellulose membrane, blocked with 5% (wt/vol) nonfat milk in TBST for2 h at room temperature, probed with primary antibodies (the antibody againstLp-PLA2, β-actin, SIRT1, SIRT2 and SOD2, 1:2000 dilution) overnight at 4°C, washedwith TBST three times, incubated with the appropriate secondary antibodies(1:1000 dilution) for 1 h at room temperature, and then washed with TBST threetimes. The resulting signals werevisualized using the Pierce enhancedchemiluminescence (ECL) Plus Western Blotting Substrate scanned with the Azurec600 Western Blot Imaging System. When incubated with another primary antibody,the membranes were stripped. Each treatment group was analyzed in triplicate.

H&EStaining

The inflammatory cells infiltration inlivers was measured by H&E staining Kit, following the manufacturer’sinstructions.

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] Chao SC, Chen YJ, Huang KH, et al. Induction of sirtuin-1 signaling by resveratrol induces human chondrosarcoma cell apoptosis and exhibits antitumor activity. Sci Rep. 2017;7(1):3180.
[2] Zielinska-Przyjemska M, Kaczmarek M, Krajka-Kuzniak V, Luczak M, Baer-Dubowska W. The effect of resveratrol, its naturally occurring derivatives and tannic acid on the induction of cell cycle arrest and apoptosis in rat C6 and human T98G glioma cell lines. Toxicol In Vitro. 2017;43:69-75.
[3] Sun S, Zhang M, Yang Q, et al. Resveratrol suppresses lipoprotein-associated phospholipase A2 expression by reducing oxidative stress in macrophages and animal models. Mol Nutr Food Res. 2017.

体内溶解度
约32 mg/mL

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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