| Sleep is an important physiological function,sufficient and high-quality sleep can reactivate the body function effectively.Sleep is regulated by central nervous system(CNS),and influenced by many factors from body and environment.With the increased social pressures,insomnia happened generally and frequently,and the incidence rate of insomnia is developing,affecting people's work and life seriously.The conventional treatment of insomnia is sedative-hypnotic drugs,but there are many side effects,for example,low safety,high dependence,addiction and withdrawal effect,limiting the effective application in clinical.In recent years,numerous studies found that many natural products(traditional chinese medicine prescription,herbal extracts,active ingredients of TCM and monomers)have good sedative and hypnotic effect with low side effect,and it is valuable in the advanced development and research.Therefore,this paper seek to separate sedative-hypnotic ingredients from traditional chinese medicine,and investigate on nano-delivery system of active ingredients.Chapter ? ReviewThis part reviewed the research progress of natural product used in the treatment of insomnia,and summarized the pharmacological action and mechanism of sedativehypnotic monomers isolated from natural source.Also,this part introduced the new research directions of natural sedative-hypnotic monomers.With embedded investigation on natural products,isolating and separating sedative-hypnotic monomers from natural source is becoming a significant study direction of insomnia thearpy.Chapter ? Extraction and pharmacodynamics of JD-XM1's activefractionThis part screened the sedative-hypnotic fraction of JD-XM1 by pharmacodynamic tracing method,and evaluated the sedative-hypnotic effects of JDXM1's active fraction.The optimum extraction condition was determined by single factor method,and the optimum condition as followed,ethanol concentration: 70 %,temperature: 90 oC,time: 2 h and liquid-solid ratio: 12.The concrete of JD-XM1 was extracted with petroleum ether,dichloromethane,ethyl acetate and n-butanol respectively,and the sedative-hypnotic effects of different extracts was evaluated by pentobarbital-induced sleep in mice.Results demonstrated that compared to normal control,n-butanol extracts significantly shortened the sleep latency(182.7 ± 7.6 vs 227.7 ± 29.7 s,P < 0.01)and prolonged the sleeping time(5518.7 ± 1558.3 vs 2265.8 ± 606.0 s,P < 0.01).Also,n-butanol extracts showed dose-dependent sedative-hypnotic effects on locomotion activity by decreasing the moving distances(P < 0.01)and moving time(P < 0.05).Pretreatment with p-chlorophenylalanine(PCPA)significantly decreased the duration of pentobarbital-induced sleep,whereas n-butanol extracts could reverse this effect,compared to model group,n-butanol extracts significantly shorten the sleep latency and prolonged the sleeping time(P < 0.01)produced by pentobarbital sodium.Chapter ? Sedative-hypnotic compound in JD-XM1's active fractionThis part isolated and purified sedative-hypnotic compound from n-butanol extracts of JD-XM1 by pharmacodynamic tracing method,and the structure elucidation of active compound was carried.Different fractions were obtained by silica gel column chromatography.Fr 6(dichloromethane: methanol = 2 : 1)shortened the sleep latency and prolonged the sleeping time produced by pentobarbital sodium.Compound A was isolated and purified from Fr 6 using C18 column,Compound A was identified to be syringic acid by structure elucidation(MS and NMR).Syringic acid(high dosage,200 mg/kg)significantly shortened the sleep latency(P < 0.01)and prolonged the sleeping time(P < 0.05)produced by pentobarbital sodium in a dose-dependent manner.Chapter ? Preparation and in vitro evaluation of syringic acidloaded liposomeThis part established and validated an efficient in vitro HPLC method for the analysis of syringic acid.Results demonstrated that established HPLC method had good system suitability.Under the selected chromatographic conditions the determinations were not interfered by excipients.Linearity range of the calibration curves was 0.5-100 ?g/mL.The stability(48 h),intra-day/inter-day precisions(RSD < 2 %)and recoveries met the acceptance criterion,demonstrating the established HPLC method was suitable for the determination of SA.Equilibrium solubility of SA in different medium was low,the max solubility(78.82 ± 4.68 ?g/m L)was measured in PBS(pH = 7.4),and equilibrium solubility of SA in distilled water and pH media(pH = 1.2)were 66.05 ± 2.65 ?g/m L,66.86 ± 2.19 ?g/m L respectively.Partition coefficient of SA was determined to be 1.31,illustrating the low absorption in gastrointestinal tract.Syringic acid-loaded liposome(SAL)was prepared by thin film drying method,the optimum formulation was screened by single factor method,the optimized mass weight of each regent is 100 mg : 1200 mg : 200 mg : 800 mg : 800 mg(syringic acid: soybean phospholipids: cholesterol: sodium cholate: isopropyl mysritate),and the hydrating medium was 20 mL PBS.The prepared SAL was transparent with high entrapment efficiency(82.20 ± 2.30 %).Homogeneous,spherical and smooth surface structured liposomes were observed through TEM.The size and distribution profile of SAL revealed a mean particle diameter of 154.67 ± 7.09 nm and polydispersity index(PDI)of 0.14 ± 0.04.The low PDI demonstrated a narrow range of particle size distribution.And the zeta potential gave 27.61 ± 3.65 mV.Storage stability showed that prepared SAL had an acceptable stability without layer separations and obvious EE changes.Chapter ? Pharmacokinetic and biodistribution study of SALThis part established and validated in vivo HPLC method for the determination of SA in rats plasma and mice tissues.Linearity(r2 > 0.99),intra-day/inter-day precisions(RSD < 5 %)and recoveries met the acceptance criterion,demonstrating the established in vivo HPLC method was suitable for the determination of SA in different biosamples.Pharmacokinetics of SAL demonstrated that SAL enhanced the oral bioavailability.Compared to free SA,SAL increased Cmax,and plasma-drug concentration was significantly higher than free SA(1.81 ± 0.94 vs 0.12 ± 0.03 ?g/m L)after 95 min,demonstrating liposome could enhance the absorption and delay the elimination.SAL prolonged the t1/2(116.67 ± 14.40 vs 25.36 ± 1.99 min)and MRT(286.27 ± 18.32 vs 37.98 ± 1.02 min),showing enhanced in vivo circulation time.Meanwhile,AUC0-360 min of SAL was increased obviously,with a 2.37-fold increase.The biodistribution of SAL in mice showed that drug concentration in plasma decreased dramatically,whereas SAL enhanced the drug concentration at 0.5 h and 2 h,demonstrating liposomal formulation delayed the elimination and enhanced in vivo circulation time.Drug concentration in different tissues successively were kidney,liver,lung,spleen,heart and brain,SAL and free SA tended to distribute in kidney and liver.Meanwhile,SA was detected in brain,demonstrating SA could penetrate blood brain barrier(BBB),and liposomal formulation could delay the elimination and enhance circulation time in brain,hence possessing sedative-hypnotic effect. |
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