Ethyl gallate (Synonyms: Ethyl 3,4,5-trihydroxybenzoate, Gallic Acid, NSC 402626, Phyllemblin)

Kinase experiment:

The expression of apoptosis-related proteins (caspases-8, -9, -3; AIF; Endo G; Bid; Bax; and Bcl-2) in HL-60 cells is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of lysates followed by western blotting. For this, HL-60 cells (1.5×106) are treated with 50 μM or 75 μM Ethyl gallate for 6 h, 12 h, or 24 h. Total cell lysates are obtained by resuspending cells in ice-cold radioimmunoprecipitation assay (RIPA) buffer for 30 min followed by centrifugation. Protein concentration is determined using a NanoDrop spectrophotometer. Aliquots of lysates (100 μg protein equivalents) are resolved by 12% SDS-PAGE and transferred onto nitrocellulose membranes[1].

Cell experiment:

HL-60 cells (1×106) are treated with 50 μM or 75 μM Ethyl gallate for 24 h or 48 h at 37°C. Cells are then harvested by centrifugation and fixed in 70% ethanol at 4°C for 24 h. Fixed cells are resuspended in PBS containing 40 μg/mL Propidium iodide (PI), 100 μg/mL RNase A, and 0.1% Triton X-100 and incubated in the dark for 30 min at room temperature. Cell cycle distribution is analyzed by flow cytometry on a FACSCalibur. To investigate apoptotic cells, HL-60 cells (1×106) incubated with different concentration of 50 μM, 75 μM and 100 μM Ethyl gallate for 24 h or 48 h at 37°C, and then DAPI staining is conducted. The cells are photographed using a fluorescence microscopy[1].

Animal experiment:

Forty eight female albino Wistar rats of six to eight weeks old are used and divided into eight groups based on their body weights. Group 1 rats serve as control receiving 1.0 mL of the vehicle (0.1% ethanol); Group 2 rats receive A. nilotica (L.) leaf extract (250 mg/kg body weight); Group 3 rats receive A. nilotica (L.) leaf extract (500 mg/kg body weight); Group 4 rats receive A. nilotica (L.) leaf extract (1000 mg/kg body weight); Group 5 rats receive A. nilotica (L.) leaf extract (2000 mg/kg body weight); Group 6 rats receive Ethyl gallate (5 mg/kg body weight); Group 7 rats receive Ethyl gallate (10 mg/kg body weight); Group 8 rats receive Ethyl gallate (20 mg/kg body weight). Body weights are recorded on 0th and 14th day for each group and all rats are decapitated after an overnight fast[2].

References:

[1]. Kim WH, et al. Ethyl gallate induces apoptosis of HL-60 cells by promoting the expression of caspases-8, -9, -3, apoptosis-inducing factor and endonuclease G. Int J Mol Sci. 2012;13(9):11912-22.
[2]. Mohan S, et al. In vitro protection of biological macromolecules against oxidative stress and in vivo toxicity evaluation of Acacia nilotica (L.) and ethyl gallate in rats. BMC Complement Altern Med. 2014 Jul 21;14:257.