| Objective:Aspirin (acetylsalicylic acid, ASA) is a widely employed as analgesic, antiinnammatory, and anti-platelet agent. Aspirin blocks thromboxane A2 synthesis from arachidonic acid in platelets by irreversibly acetylating and thus inhibiting cyclooxygenase, so aspirin is currently used in the prophylactic treatment of transient cerebral ischemia. However, a proportion of patients experience ischaemic events despite aspirin therapy.The absorbed ASA is rapidly hydrolyzed mainly by hepatic enzymes, whereas some is also degraded by an enzyme system present in serum and intestine, documented as aspirin esterase, to salicylic acid(SA) and acetic acid in the body. The metabolite SA has no the activity like ASA on preventing cardiovascular disease. Whether aspirin can play the role of antiplatelet effect, the strength of effect and the aspirin resistance could be related to the aspirin esterase in the human body. Furthermore, the aspirin resistance may be related to aspirin esterase activity. Some study found that Danshen could decrease the incidence rate of aspirin resistance, so we suspected whether the change of aspirin esterase activity caused by Danshen could account for decrease of aspirin resistancerate, and guide the rationalization clinical administration of aspirin. The orthogonal design analysis was used to optimize the incubating conditions and the aspirin esterase kinetics was study in the Liver Microsome of rat, and to study the influence on the enzyme activity by Compound Danshen Dripping Pill and its main compounds.Methods:A new RP-HPLC method to determine the aspirin and its metabolites in the plasma or the Liver Microsome of rat:the Shim-pack VP-COD column (4.6mmx150mm,5μm) and Shim-pack C18 pre-column were used. The mobile phase was acetonitrile and 1%o phosphoric acid (78:22, v/v). The flow rate was 1mL-min-1, and the column temperature was 30℃. The detective wavelength was at 228nm. The concentrations of aspirin and it's metabolite (salicylic acid) in the culture in vitro were determined by RP-HPLC. The ratio of CSA/C(ASA+SA) was used to reflect the activity of aspirin esterase. Orthogonal experimental design was used to optimize the incubating conditions of aspirin esterase and the aspirin esterase kinetics was study in the Liver Microsome of rat. The impact of Compound Danshen Dripping Pill and its main compounds on aspirin esterase in human plasma(or in the Liver Microsome of rat) was evaluated by comparing aspirin esterase activities between control group(without other drug) and experimental group(with other drug).Results:The RP-HPLC method of aspirin and its metabolite(salicylic acid) was used. The optimal incubating conditions of asipirin esterase in the liver microsome of rat is the A1B3C1D3 using orthogonal design analysis, which were 20μL aspirin(0.8mmo1·L-1) and 100μL the liver microsome of rat were incubated in the incubating solution(pH=7.4)for 40 minutes. Enzyme kinetics studies of aspirin esterase activity in the liver microsome of rat were determined using various aspirin concentrations, the Lineweaver-Burke linear showed that the Michealis equation was y=5.996x+1.359 (r=0.9698) and Michaelis constant (Km) was 4.42mmol·L-1,Vmax was 0.736 mmol-min-1·mL-1,the optimum reaction temperature is 37℃and the optimum pH is 7.4. The enzyme is more sensitive to pH changes and is extremely unstable to the heat. Compound Danshen Dripping Pill have no significant effect on the activity of aspirin esterase in plasma (P>0.05). increases aspirin esterase activity in plasma (P<0.05).The other main constituents in Compound Danshen Dripping Pill (Tanshinol sodium,protocatechuic aldehyde, protocatechuic acid, salvianolic acid A, and salvianolic acid B, et al) have no significant effect on the activity of aspirin esterase (P>0.05). Tanshinol sodium increases the value of aspirin esterase activity in the liver microsome of rat; while cryptotanshinone decreases the value of aspirin esterase activity in the liver microsome of rat (P<0.05). The other main constituents in Compound Danshen Dripping Pill have no significant effect on the aspirin esterase in the liver microsome of rat (P>0.05).Conclusions:The method is simple, accurate and rapid, suitable for the determination of aspirin and salicylic acid in plasma. The method can be used to assay the activity of aspirin esterase in vitro, and open up a new way for carrying out in-depth studies of aspirin-metabolizing enzymes. By analyzing aspirin esterase in the liver microsome of rat showed that it was sensitive to the pH change and extremely unstable to heat. Compound Danshen Dripping Pill have no significant effect on the activity of aspirin esterase in plasma, Tanshinol sodium and Cryptotanshinone have significant effect on the activity of aspirin esterase in the liver microsome of rat. The interactions should be pay attention to and providing accurate and clinically relevant advice to patients regarding the possibility of interactions. The result could not count for that the combination can decrease the incidence rate of aspirin resistance, so further research should be carried out to find the reason. |
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