Rats were randomly divided into many different experimental groups without any sizeable variation in regular entire body weights. The animals were orally administered one hundred or 400 mg/kg EGCG two h before administration of LDOPA and carbidopa . Manage rats obtained precisely the same oral administrations of automobile only. Blood samples have been collected from your tail vein with light anesthesia at 0.5, 1, 2, three, four and six h after LDOPA + carbidopa administration. About one hundred mL blood was drawn, collected in heparincoated tube, and plasma was obtained from speedy centrifugation and at once frozen at 280uC until eventually evaluation . At two and 6 h following blood assortment, rats have been euthanized with CO2 followed by decapitation. The striatal regions of your brain were dissected, weighed, and stored at 280uC till evaluation. Plasma LDOPA and its metabolites were measured utilizing a procedure described previously with small modifications.
Plasma samples from each rat have been spiked with 5 mL inner standard . To precipitate proteins, 20 mL of 1.2 M perchloric acid was additional. Tubes have been mixed, placed on ice for 10 min, and centrifuged for 4 min at 1250 g at 4uC. Aliquots of your supernatant had been added to 60 mL potassium citrate buffer to precipitate the perchlorate. pop over here Every tube was vortexed for one min, left on ice for ten min, centrifuged for 4 min at 1250 g at 4uC, and after that an aliquot in the supernatant was injected onto the HPLC for examination of composition. Concentrations of 3OMD and dopamine in striatum have been measured by HPLC with electrochemical detection in accordance to a previously described procedure . Briefly, the thawed tissues have been homogenized working with PowerGen 700 in 0.
4 M perchloric acid, plus the homogenates have been centrifuged and filtered for HPLC analysis. The HPLC process consisted of the Shimadzu pump , an electrochemical detector , in addition to a HR80 C18 reversephase column . The mobile phase consisted of 50 mM sodium phosphate, Protein Kinase C inhibitor one mM sodium dodecyl sulfate, 0.67 mL triethylamine, 13.three mM EDTA, and 8% acetonitrile in water, adjusted to pH three.0 with phosphoric acid. Before use, the mobile phase was filtered by 0.45 mm filter , and degassed below vacuum. An isocratic elution at a flow fee of one mL/min was used. LDOPA and its metabolites have been recognized by comparing their retention times with people of traditional compounds. Concentrations had been calculated from your peak height with the help of an inner standard . Linearity on the detector responses were examined for all catecholamines, as well as coefficients of correlation for all standards have been .
0.999. The relative conventional deviation to the intraday repeatability was below 5%, representing good precision on the analytical technique employed.