Study On Sedative And Hypnotic Effects Of New Drug Xin Meng Granule And Its Mechanism

1.Background Xin Meng Granule(XMG)consisits of five Chinese herbal medicine,including Mori Fructus,Ziziphus Spinosae semen,Salviae Miltiorrhizae Radix et Rhizoma,Schisandrae Chinensis Fructus and so on.It has enriching Yin to invigorate Kidney and nourishing heart to tranquillize mind effects,which aims to treat insomnia that has deficiency between heart and kidney.XMG,in accordance with the sixth class of national Chinese medicine new drug,is jointed by the Chongqing Pharscin Pharmaceutical Co.,Ltd.and Southwest University.It has access to the national technology patent,and the national major new drug creation special funding.Now it has entered the clinical trial stage II.2.Objectives To determine the content of the main active consituents of XMG;To observe the sedative and hypnotic effects of XMG,including the best dosage,the best administration time,the best administration course,drug tolerance and its mechanism.They will provide a scientific basis for its clinical application.3.Methods 3.1 Determining the content of the main active consituents of XMGThe content of the main active consituents of XMG was determined by HPLC,including protocatechuic aldehyde,spinocein,ferulic acid and schisandrin.The four consituents were separated on Xterra RP18 column(250 mm × 4.6 mm × 3.5 ?m)using the mobile phase consisted of methanol-0.1% glacial acetic acid in water at the detection wavelength of 235 nm(protocatechuic aldehyde,schisandrin)and 335 nm(spinocein and ferulic acid).The mobile phase was subjected to gradient elution.3.2 Studying the sedative and hypnotic effects of XMGThe sedative and hypnotic effects of XMG were investigated by the spontaneous locomotor activity test,the subthreshold and threshold dosage of pentobarbital sodium test.Seven dosages(0.375 g/kg,0.75 g/kg,1.5 g/kg,3 g/kg,6 g/kg,12 g/kg,24 g/kg)of XMG were observed by the spontaneous locomotor activity,the sleep onset,latency and duration in normal mice.Three dosages(0.75 g/kg,3 g/kg,12 g/kg)of XMG were observed at different administration time(8:00~10:00 or 14:00~16:00)for different courses(1 d,7 d or 15 d)to study the best administration time and course.3.3 Studying the drug tolerance of XMGThree dosages(0.75 g/kg,3 g/kg,12 g/kg)of XMG were observed by the spontaneous locomotor activity,the sleep onset,latency and duration in normal mice for different courses(15 d,30 d,45 d,60 d)to study drug tolerance.3.4 Studying the sedative and hypnotic mechanism of XMGThe contents of monoamine neurotransmitters,including NE,DA,5-HT and 5-HIAA,and amino acid neurotransmitters,including GABA and Ach,were measured by ELISA after administration dosages of 0.75 g/kg,3 g/kg,12 g/kg for 7 d.The contents of substance related to sleep in the brain of mice were observed.The effects of GABAA receptor inhibitor bicuculline,flumazen and picrotoxin on mice induced by pentobarbital sodium were observed to verify whether the sedative and hypnotic effects of XMG were dependent on the GABAergic nervous system.4.Results 4.1 The contents of the main active consituents of XMGThe contents of the main active consituents of XMG were determined by HPLC,including protocatechuic aldehyde,spinocein,ferulic acid and schisandrin.The chromatographic conditions were stable,and its specificity,precision,stability,repeatability and sample recovery were good,which were suitable for the quantitative analysis of the active consituents of XMG.The contents of protocatechuic aldehyde,spinocein,ferulic acid and schisandrin were 111.67 ± 2.36 mg/kg,83.02 ± 0.44 mg/kg,27.35 ± 0.68 mg/kg,265.27 ± 2.29 mg/kg respectively.4.2 The sedative and hypnotic effects of XMG Compared with the placebol,after administration for 7 d,five dosages(1.5 g/kg,3 g/kg,6 g/kg,12 g/kg and 24 g/kg)of XMG significantly inhibited the spontaneous locomotor activity in mice.In the tests induced by the subthreshold dosage of pentobarbital sodium in mice,three dosages(6 g/kg,12 g/kg and 24 g/kg)of XMG significantly increased the sleep onset.In the tests induced by the threshold dosage of pentobarbital sodium in mice,seven dosages(0.375 g/kg,0.75 g/kg,1.5 g/kg,3 g/kg,6 g/kg,12 g/kg and 24 g/kg)of XMG significantly reduced the sleep latency,and the three dosages(1.5 g/kg,3 g/kg and 6 g/kg)of XMG significantly prolonged the sleep duration.They suggested that 6 g/kg of XMG had the four indicators of sedative and hypnotic effects in mice,which could be used as the best recommended dosage.Converted human weight by 70 kg,the best daily consumption of XMG was 48 g.Compared with the placebol,after administration for 7 d,XMG significantly inhibited the spontaneous locomotor activity in mice and increased the sleep onset induced by the subthreshold dosage of pentobarbital sodium in mice on the morning of 8:00~10:00 and the afternoon of 14:00~16:00.In the morning it more significantly reduced the sleep latency and prolonged the sleep duration induced by the threshold dosage of pentobarbital sodium in mice(P < 0.05).They suggested that the best time of XMG was from 8:00 to 10:00 in the morning,and the best time for clinical treatment was 20: 00~22: 00 before bedtime.Compared with the placebol,3 g/kg of XMG after administration for 1 d,7 d or 15 d significantly inhibited the spontaneous locomotor activity and increased the sleep onset induced by the subthreshold dosage of pentobarbital sodium in mice.It significantly reduced the sleep latency after administration for 15 d and prolonged the sleep duration after administration for 7 d or 15 d induced by the threshold dosage of pentobarbital sodium in mice.3 g/kg of XMG had a weakly sedative and hypnotic effect after administration for 1 d(2 indicators),but it had a stable effect after administration for 7 d(3 indicators),and had a significant effect after administration for 15 d(4 indicators).They suggested that the best administration course of XMG was 15 d,which could be used as the best recommended administration course in clinical.4.3 The drug tolerance of XMG on sedative and hypnotic effectsCompared with the placebol,two dosages(3 g/kg and 12 g/kg)of XMG after administration for 15 d,30 d,45 d or 60 d,significantly inhibited the spontaneous locomotor activity in mice,increased the sleep onset induced by the subthreshold dosage of pentobarbital sodium in mice,reduced the sleep latency and prolonged the sleep duration induced by the threshold dosage of pentobarbital sodium in mice.They indicated that there was no need to increase the dosage to maintain the effect,showing that the sedative and hypnotic effects of XMG had no drug tolerance.4.4 The sedative and hypnotic mechanism of XMGCompared with the placebol,3 g/kg and 12 g/kg of XMG after administration for 7 d significantly decreased the contents of neurotransmitters,including NE,DA,5-HT,and GABA,and significantly increased the contents of 5-HIAA and Ach(P < 0.05).It suggested that XMG could significantly affect neurotransmitters related to sleep in the brain of mice,which might be the mechanisms to treat insomnia.There were inhibitory effects of three GABAA receptor inhibitors,including phytotoxin 1~2 mg/kg,flumazenil 4~8 mg/kg,bicuculline 4~8 mg/kg,on hypnotic activity of XMG(3~12 g/kg)after administration for 1 d induced by pentobarbital sodium.The results suggested that the hypnotic effect of XMG could be inhibited by GABAA receptor inhibitors.5.Conclusions5.1 The contents of the main active consituents of XMG,including protocatechuic aldehyde,spinocein,ferulic acid and schisandrin,were 111.67 ± 2.36 mg/kg,83.02 ± 0.44 mg/kg,27.35 ± 0.68 mg/kg,265.27 ± 2.29 mg/kg,respectively.5.2 XMG significantly inhibited the spontaneous locomotor activity,increased the sleep onset,reduced the sleep latency and prolonged the sleep duration in mice.The best recommended dosage of XMG was 6 g/kg/d.Converted human weight by 70 kg,the best daily consumption of XMG was 48 g.The best recommended administration time was from 8:00 to 10:00 in the morning,and the best time for clinical treatment was 20: 00~22: 00 before bedtime.The best recommended administration course was 15 d,and the best course for clinical treatment was 15 d.5.3 3 g/kg and 12 g/kg of XMG had a significantly sedative and hypnotic effect after administration for 15 d,30 d,45 d or 60 d.It indicated that the sedative and hypnotic effects of XMG had no drug tolerance.5.4 The effect of XMG on insomnia was related to decreasing the contents of NE,DA,5-HT,GABA,and increasing the contents of 5-HIAA,Ach.The sedative and hypnotic effects of XMG were dependent on the GABAergic nervous system.