目的 基于氣相色譜-質(zhì)譜聯(lián)用（GC-MS）技術(shù)結(jié)合動(dòng)物實(shí)驗(yàn)探究蒼術(shù)Atractylodes chinensis揮發(fā)油對失眠的藥效和作用機(jī)制，并制備蒼術(shù)揮發(fā)油微乳。方法 采用GC-MS對蒼術(shù)揮發(fā)油的化學(xué)成分進(jìn)行分析。采用對氯苯丙氨酸（300 mg/kg）誘導(dǎo)失眠模型，設(shè)置對照組、模型組、地西泮（1.3 mg/kg）組和蒼術(shù)低、中、高劑量（23.4、46.8、93.6 mg/kg）組，評價(jià)給藥前后小鼠體質(zhì)量、睡眠和自主活動(dòng)變化；采用蘇木素-伊紅（hematoxylin-eosin，HE）染色觀察小鼠腦組織形態(tài)；采用ELISA測定腦組織中5-羥色胺（5-hydroxytryptamine，5-HT）、谷氨酸（glutamic acid，Glu）、γ-氨基丁酸（γ-aminobutyric acid，GABA）、腫瘤壞死因子-α（tumor necrosis factor-α，TNF-α）、白細(xì)胞介素-1（interleukin-1，IL-1）、IL-6水平；采用qRT-PCR檢測腦組織中環(huán)磷酸腺苷（cyclic adenosine monophosphate，cAMP）、蛋白激酶A（protein kinase A，PKA）、環(huán)磷腺苷效應(yīng)元件結(jié)合蛋白（cAMP-response element binding protein，CREB）、腦源性神經(jīng)營養(yǎng)因子（brain-derived neurotrophic factor，BDNF）和酪氨酸激酶受體B（tyrosine kinase B，TrkB）mRNA表達(dá)；采用Western blotting檢測腦組織中cAMP、PKA、CREB、BDNF和TrkB蛋白表達(dá)。通過滴定法制備蒼術(shù)揮發(fā)油微乳，單因素實(shí)驗(yàn)考察不同的表面活性劑、助表面活性劑、表面活性劑與助表面活性劑的質(zhì)量比（K_m）對蒼術(shù)揮發(fā)油微乳形成的影響，并對微乳外觀形態(tài)、類型、溫度穩(wěn)定性、離心穩(wěn)定性、物理性質(zhì)進(jìn)行質(zhì)量評價(jià)；通過小動(dòng)物活體熒光成像實(shí)驗(yàn)對蒼術(shù)揮發(fā)油微乳在小鼠體內(nèi)的分布特性進(jìn)行研究。結(jié)果 GC-MS共鑒別出蒼術(shù)揮發(fā)油中50種化學(xué)成分，主要包括醇類、芳香烴類、烯烴類、酯類和酮類等。蒼術(shù)揮發(fā)油可顯著增加失眠小鼠體質(zhì)量（P＜0.05），縮短小鼠睡眠潛伏期（P＜0.01），延長小鼠睡眠時(shí)間（P＜0.05、0.01），改善腦組織炎性細(xì)胞浸潤情況，減少自主活動(dòng)次數(shù)（P＜0.01），顯著升高腦組織5-HT和GABA水平（P＜0.01），降低腦組織Glu、IL-1、IL-6和TNF-α水平（P＜0.05、0.01），上調(diào)腦組織cAMP、PKA、CREB、BDNF和TrkB表達(dá)（P＜0.05、0.01）。微乳最佳處方工藝為聚山梨酯80為表面活性劑、無水乙醇為助表面活性劑、K_m＝2；質(zhì)量評價(jià)發(fā)現(xiàn)蒼術(shù)揮發(fā)油微乳呈黃色，外觀均一、透明、澄清，具有良好的流動(dòng)性，亞甲基藍(lán)在微乳中的擴(kuò)散速度明顯快于蘇丹紅，在1 000～4 000 r/min的轉(zhuǎn)速下進(jìn)行離心和5～45 ℃溫度下靜置，微乳外觀無變化，可證明其離心穩(wěn)定性、溫度穩(wěn)定性較好，微乳的平均粒徑、多分散系數(shù)（polymer dispersity index，PDI）和pH值分別為（159.00±4.53）nm、0.451±0.012、6.74±0.02。小動(dòng)物活體成像實(shí)驗(yàn)結(jié)果表明蒼術(shù)揮發(fā)油微乳可以通過血腦屏障到達(dá)腦組織，且具有很好的緩釋作用。結(jié)論 北蒼術(shù)揮發(fā)油有安神的藥效作用，為蒼術(shù)揮發(fā)油微乳改善失眠癥提供理論依據(jù)。

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

吉林省科技發(fā)展計(jì)劃項(xiàng)目（TDZJ202401086ZYTS）；吉林省教育廳科學(xué)技術(shù)研究項(xiàng)目（JJKH20241092KJ）；全國中藥特色技術(shù)傳承人才培訓(xùn)項(xiàng)目（國中醫(yī)藥人教函[2023]96號）