Tritoqualine (Inhibostamin)

Kinase experiment:

Tritoqualine (TRQ) is suspended in 0.2% Tween 80 solution[2].Male Wistar rats weighing about 150 g are used. Tritoqualine (TRQ) is given to the rats (200 mg/kg, p.o.) daily for 7 days. Hepatocytes are isolated from a perfused liver by a modified Seglen's procedure. Briefly, perfusion of the liver is started with 150 mL of a 10 mM HEPES-buffered Ca2+ free Hanks' solution (pH 7.4) containing 0.5 mM EGTA. After 5 min, the liver is excised, and the perfusate is changed to 100 mL of a recirculating HEPES-buffered Hanks' solution (pH 7.4) containing 5 mM CaCl2, 0.05% collagenase and 0.005% soybean trypsin inhibitor instead of EGTA. The process continues for 15-20 min at 37°C with aeration of carbogen (95% O2, 5% CO2), until the liver began to disintegrate visibly. Cells are dispersed in a HEPES-buffered Hanks' solution (pH 7.4), filtered through a nylon stocking, and sedimented by triple centrifugation for 1 min at 50×g. Finally, the hepatocytes are suspended at 5×105 cells/mL in the same solution supplemented with 2% BSA. Two mL of the cell suspension is placed on 35 mm diameter culture dishes and incubated in an incubator at 37 °C. CCl4 is diluted with ethanol (10%, v/v) and added to the dishes at a concentration of 2.6 or 5.2 mM (0.25 or 0.5%, v/v, respectively). As a marker of cell membrane damage, lactate dehydrogenase (LDH) leaked out from the cells is determined using the supernatant of centrifuged samples. Total LDH activity is measured after lysis of the cells by sonication. The results are expressed as a ratio of LDH release induced by CCl4 to total LDH in the cells[2].

Animal experiment:

Rats[2] Tritoqualine (TRQ) is given p.o. to rats weighing about 200 g daily for 14 days. The same amount of 0.2% Tween 80 solution is given to the other rats. CCl4 diluted with olive oil (37.5%, v/v) is given to the rats (0.75 mL/kg, i.p.) 4 hr after the last administration of these compounds. The animals are killed 20 hr after the injection of CCl4. Serum is obtained from arterial blood. The liver is washed with 1.15% KCl solution through the portal vein, excised and homogenized in ten volumes of the same solution. Lipid peroxidation in the liver homogenates is quantified by the 1 % phosphoric acid method. All manipulations are done rapidly on ice to avoid further peroxidation. Glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in the serum are measured using commercial test kits.

References:

[1]. Ishii K, et al. Therapeutic effect of histidine decarboxylase inhibitor on chronic active hepatitis. Gastroenterol Jpn. 1978;13(2):105-10.
[2]. Yuasa S, et al. Suppressive effect of tritoqualine on lipid peroxidation and enzyme leakage induced by carbon tetrachloride in rat hepatocytes. Jpn J Pharmacol. 1986 Jun;41(2):205-10.