目的 基于气相色谱-质谱联用（GC-MS）技术结合动物实验探究苍术Atractylodes chinensis挥发油对失眠的药效和作用机制，并制备苍术挥发油微乳。方法 采用GC-MS对苍术挥发油的化学成分进行分析。采用对氯苯丙氨酸（300 mg/kg）诱导失眠模型，设置对照组、模型组、地西泮（1.3 mg/kg）组和苍术低、中、高剂量（23.4、46.8、93.6 mg/kg）组，评价给药前后小鼠体质量、睡眠和自主活动变化；采用苏木素-伊红（hematoxylin-eosin，HE）染色观察小鼠脑组织形态；采用ELISA测定脑组织中5-羟色胺（5-hydroxytryptamine，5-HT）、谷氨酸（glutamic acid，Glu）、γ-氨基丁酸（γ-aminobutyric acid，GABA）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1（interleukin-1，IL-1）、IL-6水平；采用qRT-PCR检测脑组织中环磷酸腺苷（cyclic adenosine monophosphate，cAMP）、蛋白激酶A（protein kinase A，PKA）、环磷腺苷效应元件结合蛋白（cAMP-response element binding protein，CREB）、脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）和酪氨酸激酶受体B（tyrosine kinase B，TrkB）mRNA表达；采用Western blotting检测脑组织中cAMP、PKA、CREB、BDNF和TrkB蛋白表达。通过滴定法制备苍术挥发油微乳，单因素实验考察不同的表面活性剂、助表面活性剂、表面活性剂与助表面活性剂的质量比（K_m）对苍术挥发油微乳形成的影响，并对微乳外观形态、类型、温度稳定性、离心稳定性、物理性质进行质量评价；通过小动物活体荧光成像实验对苍术挥发油微乳在小鼠体内的分布特性进行研究。结果 GC-MS共鉴别出苍术挥发油中50种化学成分，主要包括醇类、芳香烃类、烯烃类、酯类和酮类等。苍术挥发油可显著增加失眠小鼠体质量（P＜0.05），缩短小鼠睡眠潜伏期（P＜0.01），延长小鼠睡眠时间（P＜0.05、0.01），改善脑组织炎性细胞浸润情况，减少自主活动次数（P＜0.01），显著升高脑组织5-HT和GABA水平（P＜0.01），降低脑组织Glu、IL-1、IL-6和TNF-α水平（P＜0.05、0.01），上调脑组织cAMP、PKA、CREB、BDNF和TrkB表达（P＜0.05、0.01）。微乳最佳处方工艺为聚山梨酯80为表面活性剂、无水乙醇为助表面活性剂、K_m＝2；质量评价发现苍术挥发油微乳呈黄色，外观均一、透明、澄清，具有良好的流动性，亚甲基蓝在微乳中的扩散速度明显快于苏丹红，在1 000～4 000 r/min的转速下进行离心和5～45 ℃温度下静置，微乳外观无变化，可证明其离心稳定性、温度稳定性较好，微乳的平均粒径、多分散系数（polymer dispersity index，PDI）和pH值分别为（159.00±4.53）nm、0.451±0.012、6.74±0.02。小动物活体成像实验结果表明苍术挥发油微乳可以通过血脑屏障到达脑组织，且具有很好的缓释作用。结论 北苍术挥发油有安神的药效作用，为苍术挥发油微乳改善失眠症提供理论依据。

Objective To explore the pharmacological effects and mechanism of volatile oil from Atractylodes chinensis on insomnia based on gas chromatography-mass spectrometry (GC-MS) technology combined with animal experiments, and prepare volatile oil from A. chinensis microemulsion. Methods GC-MS was used to analyze the chemical composition of volatile oil from A. chinensis. Insomnia model was induced by chlorpheniramine (300 mg/kg), control group, model group, diazepam (1.3 mg/kg) group, and volatile oil from A. chinensis low-, medium-, high-dose (23.4, 46.8, 93.6 mg/kg) groups were set up, changes in body weight, sleep and spontaneous activity of mice before and after administration were evaluated; Hematoxylin-eosin (HE) staining was used to observe the morphology of brain tissue in mice; ELISA was used to measure the levels of 5-hydroxytryptamine (5-HT), glutamic acid (Glu), γ-aminobutyric acid (GABA), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and IL-6 in brain tissue; qRT-PCR was used to detect the mRNA expressions of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), cAMP-response element binding protein (CREB), brain-derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB) in brain tissue; Western blotting was used to detect the protein expressions of cAMP, PKA, CREB, BDNF and TrkB in brain tissue. The volatile oil from A. chinensis microemulsion was prepared by titration method, single factor experiment was used to investigate the effects of different surfactants, co-surfactants, and the mass ratio of surfactants to co surfactants (K_m) on the formation of microemulsion of A. chinensis volatile oil, the quality of microemulsion appearance, type, temperature stability, centrifugal stability and physical properties were evaluated; The distribution characteristics of microemulsion of A. chinensis volatile oil in mice was studied through live animal fluorescence imaging experiments. Results GC-MS identified 50 chemical components in volatile oil from A. chinensis, mainly including alcohols, aromatic hydrocarbons, olefins, esters and ketones. The volatile oil from A. chinensis could significantly increase the body weight of insomnia mice (P < 0.05), shorten the sleep latency of mice (P < 0.01), prolong the sleep time of mice (P < 0.05, 0.01), improve the infiltration of inflammatory cells in brain tissue, reduce the number of independent activities (P < 0.01), significantly increase the levels of 5-HT and GABA in brain tissue (P < 0.01), reduce the levels of Glu, IL-1, IL-6 and TNF-α in brain tissue (P < 0.05, 0.01), and up-regulate the expressions of cAMP, PKA, CREB, BDNF and TrkB in brain tissue (P < 0.05, 0.01). The optimal formulation process for microemulsion was to use polysorbate 80 as the surfactant, anhydrous ethanol as the co-surfactant, K_m = 2; The quality evaluation found that microemulsion of A. chinensis volatile oil was yellow in color, uniform, transparent, and clear in appearance, with good fluidity. The diffusion rate of methylene blue in the microemulsion was significantly faster than that of Sudan red. After centrifugation at a speed of 1 000—4 000 r/min and standing at a temperature of 5—45 ℃, the appearance of the microemulsion remained unchanged, which proves its good centrifugal stability and temperature stability. The average particle size, polymer dispersion index (PDI), and pH value of the microemulsion were (159.00 ±4.53) nm, 0.451 ±0.012, and 6.74 ±0.02, respectively. The results of live animal imaging experiments showed that microemulsion of A. chinensis volatile oil could reach brain tissue through the blood-brain barrier and has good sustained release effect. Conclusion The volatile oil from A. chinensis has a sedative effect, providing a theoretical basis for the improvement of insomnia by microemulsion of A. chinensis volatile oil.

R285.5

吉林省科技发展计划项目（TDZJ202401086ZYTS）；吉林省教育厅科学技术研究项目（JJKH20241092KJ）；全国中药特色技术传承人才培训项目（国中医药人教函[2023]96号）