The Study Of The Sedative And Hypnotic Activities And The Mechanism Of Cortex XX Extracts

Objective:××is a kind of Albizia plant. At present , there is not any report about its sedative and hypnotic activities. In this study, we systematically search the sedative and hypnotic fraction of the 95% ethanol fraction of cortex××(SJ) on the base of the screening result of SJ. We expect to find out new natural product molecule, which has sedative and hypnotic activity, and study its mechanism.In the guidance of sedative and hypnotic screening result, we search the active fraction of the 95% ethanol fraction of cortex××, and further study its sedative and hypnotic activity and the mechanism.Methods: (1) The sedative and hypnotic activities of SJ was observed by the effect on locomotor activity and incidence of falling asleep of male Kunming mice induced by sodium pentobarbital. (2) Screening different extracts derive from SJ by the effect on incidence of falling asleep induced by sodium pentobarbital. (3) Screening different extracts derive from SJ by the effect on mortality of mice in a week after administration. (4) The further sedative and hypnotic effect of the most active fraction was evaluated by the effects on locomotor activity and the sleeping time of mice induced by sodium pentobarbital. (5) To study the mechanism of the most active fraction by the effects on the convulsion of mice induced by picrotoxin or bicuculline.Results: (1) Above 0.01mg/kg (ip.), SJ restrained locomotor activity of mice significantly (p<0.05). Above 10-4mg/kg (ip.), SJ obviously increased the incidence of falling asleep of mice induced by sodium pentobarbital (p<0.05). And after intraperitoneal injection at the dose of 100mg/kg , the mice were all dead. (2) All extracts of SJ (SJ-a, b, c, d, e), separated with light petroleum, ethanol, acetic ether, n-butanol and water, significantly increased the incidence of falling asleep of mice induced by sodium pentobarbital. At 300mg/kg (ip.), the incidence of falling asleep of SJ-a, b, c, d were all 100% (p<0.001), while SJ-e was 83% (p<0.001). And the mortality of mice in a week after SJ-a, b, c, d, e administration were 50%, 67%, 33%, 100%, 83%. At 60 mg/kg (ip.), the incidence of falling asleep of SJ-a, d were 50% (p<0.05) and 100% (p<0.001), while SJ-b, c were both 17% (p>0.05). And the mortality of mice in a week after SJ-a, b, c, d, e administration were 0%, 0%, 0%, 50%. Therefore, among SJ-a, b, c, d, e, SJ-d was the most active and poisonous extract. (3) As a result of screening the activity of four extracts of SJ-d separated with different concentration of ethanol (SJ-d-0, 3, 6, 9), the effect of SJ-d-3, 6 on the incidence of falling asleep of mice induced by sodium pentobarbital were similar with SJ-d at the corresponding dosage. While the effect of SJ-d-0, 9 were weaker than SJ-d at the corresponding dosage. And the mortality of mice intraperitoneally injected SJ-d-3 at 10mg/kg, 5mg/kg were 100% and 70%. In addition, our study of the effect of different dosage of SJ-d-6 did not indicate the dose-effect relationship. And the mortality of mice intraperitoneally injected SJ-d-6 at 150mg/kg, 100mg/kg and 25mg/kg were 57%, 29% and 14%. These results suggested that SJ-d-6 was the most active extract of SJ-d, and SJ-d-3 was the most poisonous extract of SJ-d. (4) The fractions further separated from SJ-d-6 with column chromatography (SJ-d-6-7-13, 24-30, 31-41, 48-51) had no obvious effect on the incidence of falling asleep of mice induced by sodium pentobarbital (p>0.05) except SJ-d-6-7-13. And the effect of SJ-d-6-7-13 was similar with SJ-d-6 at the corresponding dosage. (5) At low dosage 0.13mg/kg, 0.2mg/kg, 0.3mg/kg (ip.), SJ-d-6-7-13 had no obvious effect on locomotor activity of mice (p>0.05). And at high dosage 15mg/kg, 30mg/kg, 60mg/kg (ip.), it significantly restrained locomotor activity of mice (p<0.05), prolonged the sleeping time induced by sodium pentobarbital (p<0.05) and increased the incidence of falling asleep induced by sodium pentobarbital of mice (p<0.05). But it had no dose-effect either. In addition, in a week after intraperitoneal injecting SJ-d-6-7-13 at 1g/kg, there was not any obvious toxic reaction on mice. (6) Among five fractions further separated from SJ-d-3 with column chromatography (SJ-d-3-8, 25, 40, 65, 85), the lethality of SJ-d-3-85 was similar with SJ-d-3 at the corresponding dosage. And the lethality of other four fractions were far lower than SJ-d-3 at the corresponding dosage. (7) Three fractions of SJ-d-6-7-13, SJ-d-6-7-13-Ⅰ,Ⅱ,Ⅲ, significantly increased the incidence of falling asleep of mice induced by sodium pentobarbital. Moreover, their effect were similar with SJ-d-6-7-13 at the corresponding dosage. And SJ-d-6-7-13-Ⅰ,Ⅱwere identified as monomer by HPLC. (8) At the range of 6.5×10^-6mg/kg～0.65mg/kg (ip.), SJ-d-6-7-13-Ⅰcould significantly increased the incidence of falling asleep of mice induced by sodium pentobarbital (p<0.05) without dose-effect relationship. But it had no the effect at 6.5mg/kg (ip.). At 6.5×10^-3mg/kg and 0.65mg/kg (ip.), it could not prolong the sleeping time of mice induced by sodium pentobarbital (p>0.05). (9) SJ-d-6-7-13 could not restrain the convulsion of mice induced by picrotoxin at 15mg/kg and 60mg/kg,and could not restrain the convulsion of mice induced by bicuculline at 15mg/kg, 30mg/kg and 60mg/kg. Diazapam could significant restrain the convulsion of mice induced by picrotoxin and bicuculline (p<0.001).Conclusion: (1) Above the dosage of 0.01mg/kg (ip.), SJ has sedative and hypnotic effect on mice. And it has strong toxicity—after intraperitoneal injection at the dose of 100mg/kg , the mice were all dead. (2) The n-butanol extract (SJ-d) was the most active and poisonous extract among different extracts of SJ separated with light petroleum, ethanol, acetic ether, n-butanol, water. (3) Among four extracts of SJ-d separated with different concentration of ethanol (SJ-d-0, 3, 6, 9), SJ-d-6 was the most active one and SJ-d-3 was the most poisonous one. (4) SJ-d-6-7-13 was proved to be the most active fraction of SJ-d-6, which was further separated into several fractions with column chromatography. It significantly restrained locomotor activity of mice, prolonged the sleeping time induced by sodium pentobarbital and increased the incidence of falling asleep induced by sodium pentobarbital. And SJ-d-3-85 was proved to be the most poisonous fraction of SJ-d-3, which was further separated into several fractions with column chromatography. (5) Three fractions of SJ-d-6-7-13, SJ-d-6-7-13-Ⅰ,Ⅱ,Ⅲ, significantly increased the incidence of falling asleep of mice induced by sodium pentobarbital. And SJ-d-6-7-13-Ⅰ,Ⅱwere monomer. But at the dosage used in our experiment, it has not obvious effect on the sleeping time of mice induced by sodium pentobarbital. Therefore, SJ-d-6-7-13-Ⅰmay be not the unique active fraction of SJ-d-6-7-13. Otherwise, the sedative and hypnotic effect of SJ-d-6-7-13-Ⅱremains to study. (6) On the opposite of diazepam, SJ-d-6-7-13 could not restrain the convulsion of mice induced by picrotoxin or bicuculline. It suggests that the mechanism of SJ-d-6-7-13 is different from diazepam. And the mechanism remains further study.