Potential pharmacodynamic substances and mechanism of Zhizi Houpu decoction in treating insomnia based on UHPLC-QTOF-MS and network pharmacology
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摘要:
目的 建立一种超高效液相色谱-四极杆飞行时间质谱联用（UHPLC-QTOF-MS）法鉴定栀子厚朴汤活性成分，结合网络药理学预测栀子厚朴汤抗失眠作用的物质基础及作用机制。 方法 根据UHPLC-QTOF-MS离子裂解规律，结合化合物库及文献检索，对栀子厚朴汤化学成分进行鉴定。选取口服生物利用度（OB）≥30%和类药性（DL）≥0.18的成分构建栀子厚朴汤-活性成分-靶点作用网络，利用STRING及可视化和集成发现（DAVID）数据库筛选出关键作用靶蛋白，并对生物过程、分子功能、细胞组成、信号通路进行富集分析。 结果 共鉴定出化学成分107种，筛选出与失眠相关的活性化合物35种、抗失眠靶点11个，包括AKT丝氨酸/苏氨酸激酶1（AKT1）、肿瘤蛋白53（TP53）、前列腺素内过氧化物酶合酶2（PTGS2）等。基因本体功能富集分析显示，栀子厚朴汤可能通过影响突触前膜、树突、质膜等细胞组成，参与化学突触传递、G蛋白信号通路、神经元凋亡等生物过程，以及G蛋白偶联血清素受体活性等分子功能发挥作用。京都基因与基因组百科全书通路富集分析表明，其抗失眠作用主要富集在神经活性配体-受体相互作用、钙信号、血清素能突触、cAMP信号通路等。 结论 栀子厚朴汤的抗失眠作用是多成分、多靶点、多通路协同作用的结果，其物质基础和作用机制主要与调节神经递质代谢、突触功能、神经元凋亡及炎症反应等生物学过程相关。 -
关键词:
- 栀子厚朴汤 /
- 失眠 /
- 超高效液相色谱-四极杆飞行时间质谱联用技术 /
- 网络药理学
Abstract:Objective To establish an ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) method for identifying the active ingredients of Zhizi Houpu decoction, and to predict its material basis and mechanism of anti-insomnia effects using network pharmacology. Methods The chemical components of Zhizi Houpu decoction were identified according to UHPLC-QTOF-MS ion fragmentation rule combined with compound library and literature review. Components with oral bioavailability (OB)≥30% and drug-like properties (DL)≥0.18 were selected to construct the Zhizi Houpu decoction-active ingredients-target network. The STRING database and the Database for Annotation, Visualization, and Integrated Discovery (DAVID) were used to screen key target proteins. Subsequently, enrichment analysis was performed for biological processes, molecular functions, cellular components, and signaling pathways. Results A total of 107 chemical components were identified, including 35 compounds related to insomnia and 11 anti-insomnia targets such as AKT serine/threonine kinase 1, tumor protein 53, and prostaglandin-endoperoxide synthase 2. Gene Ontology functional enrichment analysis indicated that Zhizi Houpo decoction may exert its effects through cellular components such as the presynaptic membrane, dendrite, and plasma membrane, by participating in biological processes like chemical synaptic transmission, G protein signaling pathway, and neuron apoptosis, and by regulating molecular functions such as G protein-coupled serotonin receptor activity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that its anti-insomnia effects were mainly concentrated in pathways such as neuroactive ligand-receptor interaction, calcium signaling pathway, serotonergic synapse, and cyclic adenosine monophosphate signaling pathway. Conclusion The anti-insomnia effect of Zhizi Houpo decoction is the result of a synergistic action involving multiple components, multiple targets, and multiple pathways. Its material basis and mechanism of action are primarily related to regulating biological processes such as neurotransmitter metabolism, synaptic function, neuronal apoptosis, and inflammatory response. -
睡眠是生活质量的重要组成部分，对人类健康和社会生产力有着巨大影响。近年来，睡眠障碍发生率逐步上升，约30%的人存在至少一种睡眠障碍[1]。睡眠障碍可以单独发生，也会伴随双相情感障碍、抑郁症等躯体及情绪障碍发生，被认为是帕金森病、阿尔茨海默病等疾病的标志[2]。睡眠障碍发生的原因存在多种解释，睡眠连续性降低和快速动眼期时间减少是失眠发生的标志之一[2-4]。失眠患者下丘脑-垂体-肾上腺轴过度亢奋，激素释放异常[5]，处于过度觉醒状态[6-7]。松果体分泌的褪黑素[8]，神经元分泌的γ-氨基丁酸、多巴胺、血清素等神经递质[9-10]以及食欲素受体的阻断[11]都直接或间接地影响睡眠-觉醒过程。此外，压力、情绪、饮食等外在因素也会影响睡眠[12]。目前睡眠障碍缺乏有效的治疗方案，苯二氮䓬类药物存在耐受性和依赖性等安全风险[13]，失眠认知行为疗法由于治疗成本高等问题尚未广泛使用[14]，约40%的慢性失眠患者经治疗仍无法达到持续缓解[15]，睡眠障碍已成为一个被低估的公共卫生问题。
中药复方具有多成分、多靶点、多途径的特点，在复杂疾病治疗上具有优势，较低的不良反应及药物依赖性使其成为治疗失眠的优选方案。栀子厚朴汤由栀子、厚朴、枳实组成，最早记载于《伤寒论》，用于治疗“伤寒下后，心烦腹满，卧起不安”，是治疗失眠的常用经方[16]。现代药理学研究表明，栀子厚朴汤对焦虑症、失眠、抑郁症等多种情绪障碍具有良好的治疗效果，可以改善模型小鼠的行为学和神经递质水平[17]。然而，栀子厚朴汤发挥抗失眠作用的物质基础和作用机制并不明确。本研究利用超高效液相色谱-四极杆飞行时间质谱联用（ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry，UHPLC-QTOF-MS）对栀子厚朴汤的主要化学成分进行分析，结合网络药理学对发挥抗失眠作用的核心靶点、作用通路进行预测。
1 材料和方法
1.1 药材与试剂
栀子、厚朴、枳实饮片均购自上海白鹿堂中药店，由海军军医大学药学系韩婷教授分别鉴定为茜草科植物栀子（Gardenia jasminoides Ellis）的干燥成熟果实，木兰科植物厚朴（Magnolia officinalis Rehd. et Wils.）或凹叶厚朴（Magnolia officinalis Rehd. et Wils. var. biloba Rehd. et Wils.）的干燥干皮、根皮及枝皮，芸香科植物酸橙（Citrus aurantium L.）及其栽培变种或甜橙（Citrus sinensis Osbeck）的干燥幼果。乙醇（分析级）、甲酸（高效液相色谱级）、乙腈（液相色谱-质谱联用级），均购自国药集团化学试剂有限公司。
1.2 主要仪器
Agilent 1290超高效液相色谱仪、Agilent 6538四极杆飞行时间质谱仪（美国安捷伦公司）；ACQUITY UHPLC XBridge BEH C18色谱柱（2.1 mm×100 mm，2.5 µm，美国沃特世公司）；十万分之一分析天平（德国赛多利斯科学仪器有限公司）；X1R-230 V台式离心机（美国赛默飞世尔科技公司）；Milli-QA10超纯水仪（美国密理博公司）。
1.3 样品制备
将各干燥药材饮片粉碎后，过60目筛，精密称取1.2 g栀子、1.2 g厚朴、1.0 g枳实粉末，加入25 mL 70%甲醇，超声提取30 min，3 000×g离心5 min，取上清液，过0.22 μm滤膜，即供试品溶液[18-19]。
1.4 色谱-质谱条件
ACQUITY UHPLC XBridge BEH C18色谱柱，进样量3 μL，流动相为0.1%甲酸水（A）-0.1%甲酸乙腈（B），梯度洗脱程序：0~1 min，5% B；1~2 min，5%~20% B；2~8 min，20%~45% B；8~11 min，45%~50% B；11~13 min，50%~95% B；13~15 min，95% B。分析时间为15 min，流速为0.4 μL/min，电喷雾离子源，质谱扫描范围m/z 50~1 500，毛细管电压为4 000 V。
1.5 数据采集及分析
通过中药系统药理学数据库与分析平台（Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform，TCMSP；http://lsp.nwu.edu.cn/tcmsp.php）、本草组鉴（HERB，http://herb.ac.cn）数据库，结合中国知网、PubMed等数据库的相关文献，建立栀子厚朴汤成分数据库。四级杆飞行时间质谱仪采集结果导入质谱分析软件Agilent MassHunter定性分析软件B. 06. 00进行处理，利用精确分子量匹配，结合特征碎片离子进行定性分析。
1.6 成分靶点预测
在TCMSP数据库搜索“栀子”“厚朴”“枳实”，以口服生物利用度（oral bioavailability，OB）≥30%和类药性（drug likeness，DL）≥0.18为筛选条件[20]，结合UHPLC-QTOF-MS的定性结果，获取栀子厚朴汤中的活性成分。通过TCMSP数据库、PubChem（https://pubchem.ncbi.nlm.nih.gov/）和中国知网相关文献获取栀子厚朴汤中活性成分的化学结构和简化分子线性输入规范（simplified molecular input line entry system，SMILE）结构，上传至Swiss Target Prediction数据库和Super-PRED数据库中，物种选择“Homo sapiens”、以概率值（probability）＞0为筛选条件对成分靶点进行预测，采用UniProt数据库（https://www.Uniprot.org/）对靶点名称进行规范化，去除重复靶点，得到栀子厚朴汤成分潜在靶点。
1.7 失眠靶点
以“insomnia”为关键词，检索GeneCards数据库（https://www.genecards.org/）中与失眠相关的靶点，并选择相关性评分（score）＞0.9的靶点进行分析。以“insomnia”为关键词，获取在线人类孟德尔遗传（Online Mendelian Inheritance in Man，OMIM）数据库（https://www.omim.org/）、药物银行数据库（the Comprehensive Drug Database，DRUGBANK；https://go.drugbank.com/）中失眠相关靶点，使用UniProt数据库中UniProtKB搜索功能对所得到的靶点蛋白进行校正，去重后得到失眠相关靶点。
1.8 栀子厚朴汤-活性成分-靶点网络构建
将栀子厚朴汤成分潜在靶点与失眠相关靶点上传至Venny 2.1.0中绘制维恩图，获取成分基因与失眠基因交集靶点。利用Cytoscape 3.10.0软件构建可视化栀子厚朴汤-活性成分-靶点网络。
1.9 蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络构建
将141个交集基因导入STRING数据库，物种选择“Homo sapiens”，以中等置信度（medium confidence）＞0.4为筛选条件获得PPI网络。将PPI网络数据导入Cytoscape 3.10.0软件中，去除度值（degree）≤10的靶点蛋白，按照度值大小对其余核心靶点进行可视化分析，构建栀子厚朴汤治疗失眠的PPI网络。
1.10 基因本体（Gene Ontology，GO）分析和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）分析
将交集基因导入可视化和集成发现数据库（Database for Annotation，Visualization，and Integrated Discovery；DAVID）（https://david.ncifcrf.gon/）[21]，物种选择“Homo sapiens”，分别对生物过程、分子功能、细胞成分、信号通路进行富集分析。运用微生信平台[22]将GO富集和KEGG富集的结果分别绘制成条形图和气泡图，获得栀子厚朴汤预防和治疗失眠的主要作用通路。
2 结果
2.1 栀子厚朴汤化学成分分析
采用UHPLC-QTOF-MS方法在正/负离子2种扫描模式下共鉴定出107个成分（图 1），其中栀子共检测出38个成分，厚朴共检测出39个成分，枳实共检测出37个成分，2-正戊基呋喃（pentylfuran）和芹子烯（beta-selinene）是栀子与厚朴的共有成分，(+)-莰烯（d-camphene）、月桂烯（myrcene）和对伞花烃（cymol）是厚朴与枳实的共有成分，亚油酸（linoleic acid）是3种中药的共有成分。图 1中每种成分的归属及鉴定信息见表 1。
图 1 栀子厚朴汤在正（A）、负（B）离子下的总离子流图Fig. 1 Total ion chromatogram of Zhizi Houpu decoction under positive (A) and negative (B) ions
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表 1 栀子厚朴汤提取液化学成分鉴定结果Table 1 Results of chemical composition identification of extract of Zhizi Houpu decoctionNo. RT Compound Formula Adduct m/z MW Error/(×10－6) Herbal source 1 0.613 Mannitol C6H14O6 [M－H]－ 181.071 182.079 0.08 ZZ 2 0.646 Oxalic ether C6H10O4 [M+HCOO]－ 191.055 146.057 －1.73 HP 3 0.723 (+)-synephrine C9H13NO2 [M+H]+ 168.100 167.094 －11.01 ZS 4 0.731 N-methyltyramine C9H13NO [M+H]+ 152.107 151.099 1.47 ZS 5 1.954 Shanzhiside C16H24O11 [M－H]－ 391.123 392.131 －1.50 ZZ 6 2.179 Protocatechuic acid C7H6O4 [M－H]－ 153.019 154.026 －4.01 ZZ 7 2.404 Genipin 1-gentiobioside C16H22O11 [M－H]－ 389.109 390.116 3.46 ZZ 8 2.437 Scandoside_qt C11H14O6 [M－H]－ 241.071 242.079 －4.41 ZZ 9 2.444 Deacetyl asperulosidic acid methyl ester C17H24O11 [M+Na]+ 427.121 404.131 1.09 ZZ 10 2.454 Gardenoside C17H24O11 [M+Cl]－ 439.101 404.131 －0.54 ZZ 11 2.685 Caprylic acid C8H16O2 [M+Na]+ 167.105 144.115 9.79 HP 12 2.702 O-xylene C8H10 [M+H]+ 107.084 106.078 －7.49 HP 13 2.745 Heriguard C16H18O9 [M－H]－ 353.088 354.095 2.14 ZZ 14 2.760
2.779Genipingentiobioside C23H34O15 [M+Na]+
[M+Cl]－573.174
585.160550.189 －7.84
1.21ZZ 15 2.845 2-pentylfuran C9H14O [M+HCOO]－ 183.102 138.104 －2.52 ZZ 16 2.953 Caffeic acid C9H8O4 [M－H]－ 179.035 180.042 1.33 ZZ 17 2.960 Scolymoside C27H30O15 [M+H]+ 595.164 594.158 －2.21 ZS 18 2.985 Coniferol C10H12O3 [M+H]+ 181.086 180.078 5.16 ZS 19 2.995 Safrole C10H10O2 [M+HCOO]－ 207.065 162.068 －2.90 HP 20 3.037 Geniposide C17H24O10 [M+Cl]－ 423.105 388.136 －1.94 ZZ 21 3.153 Sudan Ⅲ C22H16N4O [M－H]－ 351.128 352.132 5.59 ZZ 22 3.193 Luteolin C15H10O6 [M+H]+ 287.058 286.047 8.59 ZS 23 3.195 4-ethyl-2-methoxyphenol C9H12O2 [M+HCOO]－ 197.081 152.083 －3.60 HP 24 3.334 Quercetin C15H10O7 [M+H]+ 303.049 302.042 －0.53 ZZ 25 3.351
3.395Rutin C27H30O16 [M+H]+
[M－H]－611.162
609.145610.153 2.98
－0.69ZZ 26 3.453 Prangenin C16H14O5 [M－H]－ 285.077 286.084 1.63 ZS 27 3.495 Izoforon C9H14O [M+HCOO]－ 183.102 138.104 －0.51 ZZ 28 3.595 2-nonenal C9H16O [M+HCOO]－ 185.117 140.120 －7.44 HP 29 3.612 Hyacinthin C8H8O [M+HCOO]－ 165.055 120.057 －4.43 HP 30 3.617 Isoponcimarin C19H22O5 [M+H]+ 331.157 330.146 －1.25 ZS 31 3.728 Isochlorogenic acid b C25H24O12 [M－H]－ 515.117 516.126 －1.95 ZZ 32 3.767 Rhoifolin C27H30O14 [M+H]+ 579.173 578.163 4.14 ZS 33 3.778 Naringin C27H32O14 [M－H]－ 579.172 580.179 0.51 ZS 34 3.853 Isochlorogenic acid c C25H24O12 [M－H]－ 515.118 516.126 1.19 ZZ 35 3.886 Narirutin C27H32O14 [M+Cl]－ 615.148 580.179 0.43 ZS 36 4.008 Camphor (synthetic) C10H16O [M+H]+ 153.126 152.120 －6.02 HP 37 4.011 Apigenin trimethyl ether C18H16O5 [M－H]－ 311.092 312.099 －1.16 ZS 38 4.020
4.025Crocin C44H64O24 [M+Cl]－
[M+Na]+1011.345
999.368976.378 －3.80
－0.27ZZ 39 4.033 Furfural C5H4O2 [M+H]+ 97.027 96.021 －7.34 HP 40 4.061 Hesperidin C28H34O15 [M－H]－ 609.183 610.189 0.92 ZS 41 4.074 Neohesperidin C16H14O6 [M+H]+ 303.086 302.079 0.72 HP 42 4.078 Isochlorogenic acid a C25H24O12 [M－H]－ 515.118 516.126 －2.07 ZZ 43 4.161
4.183Neohesperdin C28H34O15 [M－H]－
[M+Na]+609.183
633.180610.189 0.27
6.53ZS 44 4.208 Coumarin C9H6O2 [M+H]+ 147.043 146.036 －1.16 HP 45 4.536 Hexanal C6H12O [M+HCOO]－ 145.086 100.088 －3.97 ZZ 46 4.989 Cis-sinapyl alcohol C11H14O4 [M+Na]+ 233.079 210.089 －1.76 ZS 47 5.152 (+)-dehydrodiconiferyl alcohol C20H22O6 [M－H]－ 357.134 358.141 －2.52 ZS 48 5.294 Citrusin b C27H36O13 [M+HCOO]－ 613.213 568.215 －2.94 ZS 49 5.336 Poncirin C28H34O14 [M－H]－ 593.187 594.194 －1.48 ZS 50 5.372 Didymin C16H14O5 [M+H]+ 287.091 286.084 0.64 ZS 51 5.377 Isokaempferide C16H12O6 [M+HCOO]－ 345.060 300.063 －6.16 ZZ 52 5.497 Isosakuranetin-7-rutinoside C28H34O14 [M+H]+ 595.204 594.194 3.64 ZS 53 5.638 Germacron C15H22O [M+H]+ 219.174 218.167 －0.95 ZZ 54 6.046 Pentylfuran C9H14O [M+H]+ 139.111 138.104 －0.93 ZZ/HP 55 6.119 Apigenin C15H10O5 [M－H]－ 269.044 270.052 －2.33 ZS 56 6.144 Naringenin C15H12O5 [M－H]－ 271.060 272.068 －4.16 ZS 57 6.310 Chrysin C15H10O4 [M+HCOO]－ 299.055 254.057 －4.23 ZZ 58 6.470 2, 6, 10-trimethyl-dodecane C6H8O4 [M+H]+ 145.049 144.042 －1.98 HP 59 6.494
6.520(R)-hesperetin C16H14O6 [M－H]－
[M+H]+301.071
303.086302.079 －2.26
－1.07ZS 60 6.578 Crocetin C20H24O4 [M+H]+ 329.174 328.167 －0.09 ZZ 61 6.678 6-methoxy aurapten C20H24O4 [M+H]+ 329.174 328.167 －1.60 ZS 62 7.011 Curdione C15H24O2 [M+H]+ 237.184 236.177 －2.89 ZZ 63 7.052 Tangeretin C20H20O7 [M+H]+ 373.129 372.120 2.03 ZS 64 7.093 Auraptene C19H22O3 [M+HCOO]－ 343.154 298.156 －1.80 ZS 65 7.293 Carvacrol C10H14O [M+H]+ 151.111 150.104 －5.82 HP 66 7.351 Pentadecylic acid C15H30O2 [M+HCOO]－ 287.221 242.224 －7.13 HP 67 7.551
7.735Deacetylnomilin C26H32O8 [M+H]+
[M+HCOO]－473.216
517.205472.209 －1.00
－6.66ZS 68 7.826 (3S, 6E)-nerolidol C15H26O [M+H]+ 223.206 222.198 2.52 ZZ 69 7.834 Beta-chamigrene C15H24 [M+H]+ 205.195 204.187 2.35 HP 70 7.876 D-camphene C10H16 [M+H]+ 137.131 136.125 －4.37 HP/ZS 71 7.918
7.967Limonin C26H30O8 [M+HCOO]－
[M+Na]+515.191
493.183470.194 －2.81
2.04ZS 72 8.034 Beta-selinene C15H24 [M+H]+ 205.195 204.187 2.97 ZZ/HP 73 8.433 Borneol C10H18O [M+Na]+ 177.124 154.135 －3.22 HP 74 8.499 Alpha-cubebene C15H24 [M+H]+ 205.194 204.187 －1.07 HP 75 8.516 Tetramethoxyluteolin C19H18O6 [M+H]+ 343.120 342.110 6.26 ZS 76 8.699 Cinnamyl acetate C11H12O2 [M+H]+ 177.090 176.083 －3.43 HP 77 8.724 6-demethoxytangeretin C19H18O6 [M+H]+ 343.120 342.110 6.27 ZS 78 8.757 Nomilin C28H34O9 [M+H]+ 515.228 514.220 －0.80 ZS 79 8.876
8.932Isoimperatorin C16H14O4 [M－H]－
[M+H]+269.081
271.096270.089 －1.63
－0.94ZZ 80 9.032 Isocaryophyllene oxide C15H24O [M+H]+ 221.190 220.182 0.12 HP 81 9.348 Sinensetin C20H20O7 [M+Na]+ 395.109 372.120 6.29 ZS 82 9.365 Corymbosin C19H18O7 [M+H]+ 359.112 358.105 －2.24 ZZ 83 9.600
9.697Obacunone C26H30O7 [M+HCOO]－
[M+H]+499.195
455.206454.199 －3.20
－1.75ZS 84 11.058 [(E, 8S)-8-methyltetradec-9-enyl] acetate C17H32O2 [M+HCOO]－ 313.236 268.240 －5.94 HP 85 11.161 Gypsogenic acid C30H46O5 [M+H]+ 487.340 486.334 －6.44 ZZ 86 11.444 Hederagenol C30H48O4 [M+H]+ 473.359 472.355 －6.81 ZZ 87 11.525
11.577Schembl120923 C18H18O2 [M－H]－
[M+H]+265.123
267.137266.130 －0.28
－0.47HP 88 11.602 Syringaresinol C22H26O8 [M+H]+ 419.172 418.162 6.65 ZZ 89 11.808
11.876Eucalyptol C18H18O2 [M－H]－
[M+H]+265.123
267.137266.130 0.31
－0.59HP 90 12.383 Methyl isopalmitate C17H34O2 [M+HCOO]－ 315.252 270.255 －7.09 HP 91 12.566 Magnolol C18H18O2 [M－H]－ 265.123 266.130 －1.43 HP 92 12.591 Obovatol C18H18O3 [M－H]－ 281.118 282.125 0.43 HP 93 12.666 Honokiol C18H18O2 [M－H]－ 265.124 266.130 2.90 HP 94 12.692 Prangenin hydrate C16H16O6 [M+H]+ 305.101 304.094 －0.37 ZS 95 13.199 Alpha-cedrene C15H24 [M+H]+ 205.195 204.187 2.03 HP 96 13.307 16-heptadecenal C17H32O [M+HCOO]－ 297.242 252.245 －2.69 HP 97 13.315 Dibutyl phthalate C16H22O4 [M+H]+ 279.159 278.151 0.13 HP 98 13.407 Dehydro-p-cymene C10H12 [M+H]+ 133.100 132.093 －6.49 HP 99 13.523 Myrcene C10H16 [M+H]+ 137.131 136.125 －3.32 HP/ZS 100 13.624 Siaresinol C30H48O4 [M+Cl]－ 507.321 472.355 －6.42 ZZ 101 13.715 2-pentadecyn-1-ol C15H28O [M+HCOO]－ 269.211 224.214 －4.98 HP 102 13.939 Stigmasterol C29H48O [M+H]+ 413.377 412.370 －1.84 ZZ 103 14.190 Linoleic acid C18H32O2 [M－H]－ 279.232 280.240 －2.25 ZZ/HP/ZS 104 14.413 Elemene C15H24 [M+H]+ 205.194 204.187 －1.22 HP 105 14.681 Oleic acid C18H34O2 [M－H]－ 281.247 282.255 －5.12 ZZ 106 14.713 Cymol C10H14 [M+H]+ 135.116 134.109 －7.47 HP/ZS 107 14.738 Alpha-humulene C15H24 [M+H]+ 205.195 204.187 －0.60 HP RT: Retention time; MW: Molecular weight; ZS: Zhishi; ZZ: Zhizi; HP: Houpu. 2.2 成分靶点和失眠靶点的预测
从GeneCards数据库、OMIM数据库及DRUGBANK共获得1 280个失眠相关靶点。如图 2所示，得到703个栀子厚朴汤潜在靶点，与失眠相关的靶点有141个，其中35个靶点与栀子、厚朴、枳实均有关联；栀子12种活性成分447个潜在靶点中的91个靶点与失眠相关；厚朴4种活性成分221个潜在靶点中的50个靶点与失眠相关；枳实20种活性成分512个潜在靶点中的112个靶点与失眠相关。
图 2 栀子厚朴汤与失眠靶点的维恩图Fig. 2 Venn diagram of Zhizi Houpu decoction and insomnia targetsHP: Houpu; ZS: Zhishi; ZZ: Zhizi.下载:
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2.3 栀子厚朴汤-活性成分-靶点网络构建
栀子厚朴汤活性成分靶点与失眠相关疾病靶点的交集基因靶点共141个。将35种活性成分与141个交集靶点导入Cytoscape 3.10.0中，所构建的栀子厚朴汤-活性成分-靶点网络见图 3。
图 3 栀子厚朴汤-活性成分-失眠靶点网络图Fig. 3 Zhizi Houpu decoction-active ingredients-insomnia target network diagramComponent a1 is shared by both ZZ and ZS. HP: Houpu; ZS: Zhishi; ZZ: Zhizi.下载:
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2.4 PPI网络图构建
如图 4所示，AKT丝氨酸/苏氨酸激酶1（AKT serine/threonine kinase 1，AKT1）、肿瘤蛋白53（tumor protein 53，TP53）、前列腺素内过氧化物酶合酶2（prostaglandin-endoperoxidesynthase 2，PTGS2）、雌激素受体1（estrogen receptor 1，ESR1）、热休克蛋白90 α家族A类成员1（heat shock protein 90 alpha family class A member 1，HSP90AA1）、caspase-3是栀子厚朴汤核心靶点，AKT1、ESR1、糖原合成酶激酶3β（glycogen synthase kinase 3β，GSK3B）、缺氧诱导因子1α（hypoxia-inducible factor 1α，HIF-1A）、单胺氧化酶A（monoamine oxidase A，MAOA）、单胺氧化酶B（monoamine oxidase B，MAOB）、溶质载体家族6成员4（solute carrier family 6 member 4，SLC6A4）是3味药的共同靶点，这些可能是栀子厚朴汤发挥抗失眠作用的重要靶点。
图 4 栀子厚朴汤PPI网络图(A)及栀子(B)、厚朴(C)、枳实(D)核心靶点PPI网络图Fig. 4 PPI network diagram of Zhizi Houpu decoction (A) and core targets of Zhizi (B), Houpu (C) and Zhishi (D)PPI: Protein-protein interaction.下载:
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2.5 GO和KEGG富集分析
GO富集分析显示（图 5A），栀子厚朴汤治疗失眠时参与的生物事件和活动主要涉及化学突触传递、G蛋白信号通路、神经元凋亡等一系列分子机制及生物过程，生物学对象的位置主要在突触前膜、树突、质膜等细胞成分上；分子功能主要涉及G蛋白偶联血清素受体活性、酶结合、核受体活性等活动。KEGG富集分析显示（图 5B），栀子厚朴汤治疗失眠作用可能涉及多条信号通路，包括神经活性配体-受体相互作用、钙信号、血清素能突触、cAMP信号通路等多条信号通路。
图 5 栀子厚朴汤治疗睡眠障碍的潜在靶点GO功能富集分析图（A）及KEGG通路富集分析图（B）Fig. 5 GO functional enrichment map (A) and KEGG pathway enrichment map (B) of potential targets of Zhizi Houpu decoction in treatment of sleep disordersGO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; BP: Biological process; CC: Cellular component; MF: Molecular function; GPCR: G protein-coupled receptor; MAPK: Mitogen-activated protein kinase; HFI-1: Hypoxia-inducible factor 1; cGMP: Cyclic guanosine monophosphate; PKG: Protein kinase G; cAMP: Cyclic adenosine monophosphate.下载:
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3 讨论
栀子厚朴汤是通过多种成分之间的相互协作发挥抗失眠协同效应。本研究基于UHPLC-QTOF-MS结果和TCMSP数据库，共鉴定出107种活性成分。为进一步探究这些活性成分可能的作用机制，对其中一些关键成分的药理活性进行了文献梳理。文献报道，西红花苷、辛弗林可以通过调节5-羟色胺（又称血清素）、多巴胺等神经递质产生抗焦虑作用[23-24]；厚朴酚[25]、栀子苷[26]具有抗炎、抗氧化及神经保护等作用；木犀草素四甲醚与抗过敏和免疫调节相关[27]；芹菜素三甲醚可以通过K+通道直接或间接地影响细胞信号转导[28]；香风草苷重编程巨噬细胞极化从而治疗炎症相关疾病[29]，通过凋亡相关斑点样蛋白/caspase-1/消皮素D通路抑制小胶质细胞的凋亡及炎症[30]；甜橙黄酮在抗炎、抗氧化、脂质代谢、调节肠道菌群等方面有很好的效果[31]。这些都可能是栀子厚朴汤发挥抗失眠作用的物质基础。
为了进一步探究栀子厚朴汤治疗失眠的作用机制，把筛选出的35种主要活性成分，利用STRING数据库建立PPI网络，分析得到栀子厚朴汤抗失眠的核心靶点11个，包括AKT1、TP53、PTGS2、ESR1、HSP90AA1、caspase-3、GSK3B、HIF-1A、MAOA、MAOB、SLC6A4。这些靶点主要参与调节血清素、多巴胺，一氧化氮等神经递质的合成、代谢、分泌等生物过程[32]。此外，AKT1在多种生命活动中发挥重要作用，与cAMP信号通路[33]、环磷酸鸟苷-蛋白激酶A信号通路[34]、低氧诱导因子1信号通路[35]、多巴胺能突触[36]等多种失眠相关信号通路存在关联。腺苷A2a受体和N-甲基-D-天冬氨酸受体2B亚基（glutamate ionotropic receptor NMDA type subunit 2B，GRIN2B）参与钙信号传递[37]，GRIN2B受体与突触可塑性有关[38]；过氧化物酶体增殖物激活受体γ、PTGS2可能通过影响炎症反应来发挥抗失眠作用[39-40]；儿茶酚-O-甲基转移酶、溶质载体家族6成员3、μ型阿片受体等可能影响疼痛或情绪来调控睡眠-觉醒周期[41-42]；HIF-1A可能通过调节缺氧反应及影响昼夜调节蛋白的表达间接影响睡眠[43]。GO功能富集和KEGG通路富集分析结果发现，栀子厚朴汤可能通过突触前膜、树突、质膜等细胞成分参与化学突触传递、G蛋白信号通路、神经元凋亡等一系列生物过程以及G蛋白偶联血清素受体活性、酶结合、核受体活性等分子活动发挥抗失眠作用，可能涉及神经活性配体-受体相互作用、钙信号、血清素能突触、cAMP信号通路等多条信号通路。
本研究利用UHPLC-QTOF-MS对栀子厚朴汤中的化学成分进行分析，共鉴定出107种化合物，为进一步研究药物体内代谢、吸收提供了依据。通过网络药理学对核心活性成分进行抗失眠机制分析，预测出可能的靶点与通道，为使用新方法进一步筛选复杂样品中成分及作用靶点提供了研究基础。
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图 1 栀子厚朴汤在正（A）、负（B）离子下的总离子流图
Fig. 1 Total ion chromatogram of Zhizi Houpu decoction under positive (A) and negative (B) ions
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图 2 栀子厚朴汤与失眠靶点的维恩图
Fig. 2 Venn diagram of Zhizi Houpu decoction and insomnia targets
HP: Houpu; ZS: Zhishi; ZZ: Zhizi.
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图 3 栀子厚朴汤-活性成分-失眠靶点网络图
Fig. 3 Zhizi Houpu decoction-active ingredients-insomnia target network diagram
Component a1 is shared by both ZZ and ZS. HP: Houpu; ZS: Zhishi; ZZ: Zhizi.
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图 4 栀子厚朴汤PPI网络图(A)及栀子(B)、厚朴(C)、枳实(D)核心靶点PPI网络图
Fig. 4 PPI network diagram of Zhizi Houpu decoction (A) and core targets of Zhizi (B), Houpu (C) and Zhishi (D)
PPI: Protein-protein interaction.
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图 5 栀子厚朴汤治疗睡眠障碍的潜在靶点GO功能富集分析图（A）及KEGG通路富集分析图（B）
Fig. 5 GO functional enrichment map (A) and KEGG pathway enrichment map (B) of potential targets of Zhizi Houpu decoction in treatment of sleep disorders
GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; BP: Biological process; CC: Cellular component; MF: Molecular function; GPCR: G protein-coupled receptor; MAPK: Mitogen-activated protein kinase; HFI-1: Hypoxia-inducible factor 1; cGMP: Cyclic guanosine monophosphate; PKG: Protein kinase G; cAMP: Cyclic adenosine monophosphate.
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表 1 栀子厚朴汤提取液化学成分鉴定结果
Table 1 Results of chemical composition identification of extract of Zhizi Houpu decoction
No. RT Compound Formula Adduct m/z MW Error/(×10－6) Herbal source 1 0.613 Mannitol C6H14O6 [M－H]－ 181.071 182.079 0.08 ZZ 2 0.646 Oxalic ether C6H10O4 [M+HCOO]－ 191.055 146.057 －1.73 HP 3 0.723 (+)-synephrine C9H13NO2 [M+H]+ 168.100 167.094 －11.01 ZS 4 0.731 N-methyltyramine C9H13NO [M+H]+ 152.107 151.099 1.47 ZS 5 1.954 Shanzhiside C16H24O11 [M－H]－ 391.123 392.131 －1.50 ZZ 6 2.179 Protocatechuic acid C7H6O4 [M－H]－ 153.019 154.026 －4.01 ZZ 7 2.404 Genipin 1-gentiobioside C16H22O11 [M－H]－ 389.109 390.116 3.46 ZZ 8 2.437 Scandoside_qt C11H14O6 [M－H]－ 241.071 242.079 －4.41 ZZ 9 2.444 Deacetyl asperulosidic acid methyl ester C17H24O11 [M+Na]+ 427.121 404.131 1.09 ZZ 10 2.454 Gardenoside C17H24O11 [M+Cl]－ 439.101 404.131 －0.54 ZZ 11 2.685 Caprylic acid C8H16O2 [M+Na]+ 167.105 144.115 9.79 HP 12 2.702 O-xylene C8H10 [M+H]+ 107.084 106.078 －7.49 HP 13 2.745 Heriguard C16H18O9 [M－H]－ 353.088 354.095 2.14 ZZ 14 2.760
2.779Genipingentiobioside C23H34O15 [M+Na]+
[M+Cl]－573.174
585.160550.189 －7.84
1.21ZZ 15 2.845 2-pentylfuran C9H14O [M+HCOO]－ 183.102 138.104 －2.52 ZZ 16 2.953 Caffeic acid C9H8O4 [M－H]－ 179.035 180.042 1.33 ZZ 17 2.960 Scolymoside C27H30O15 [M+H]+ 595.164 594.158 －2.21 ZS 18 2.985 Coniferol C10H12O3 [M+H]+ 181.086 180.078 5.16 ZS 19 2.995 Safrole C10H10O2 [M+HCOO]－ 207.065 162.068 －2.90 HP 20 3.037 Geniposide C17H24O10 [M+Cl]－ 423.105 388.136 －1.94 ZZ 21 3.153 Sudan Ⅲ C22H16N4O [M－H]－ 351.128 352.132 5.59 ZZ 22 3.193 Luteolin C15H10O6 [M+H]+ 287.058 286.047 8.59 ZS 23 3.195 4-ethyl-2-methoxyphenol C9H12O2 [M+HCOO]－ 197.081 152.083 －3.60 HP 24 3.334 Quercetin C15H10O7 [M+H]+ 303.049 302.042 －0.53 ZZ 25 3.351
3.395Rutin C27H30O16 [M+H]+
[M－H]－611.162
609.145610.153 2.98
－0.69ZZ 26 3.453 Prangenin C16H14O5 [M－H]－ 285.077 286.084 1.63 ZS 27 3.495 Izoforon C9H14O [M+HCOO]－ 183.102 138.104 －0.51 ZZ 28 3.595 2-nonenal C9H16O [M+HCOO]－ 185.117 140.120 －7.44 HP 29 3.612 Hyacinthin C8H8O [M+HCOO]－ 165.055 120.057 －4.43 HP 30 3.617 Isoponcimarin C19H22O5 [M+H]+ 331.157 330.146 －1.25 ZS 31 3.728 Isochlorogenic acid b C25H24O12 [M－H]－ 515.117 516.126 －1.95 ZZ 32 3.767 Rhoifolin C27H30O14 [M+H]+ 579.173 578.163 4.14 ZS 33 3.778 Naringin C27H32O14 [M－H]－ 579.172 580.179 0.51 ZS 34 3.853 Isochlorogenic acid c C25H24O12 [M－H]－ 515.118 516.126 1.19 ZZ 35 3.886 Narirutin C27H32O14 [M+Cl]－ 615.148 580.179 0.43 ZS 36 4.008 Camphor (synthetic) C10H16O [M+H]+ 153.126 152.120 －6.02 HP 37 4.011 Apigenin trimethyl ether C18H16O5 [M－H]－ 311.092 312.099 －1.16 ZS 38 4.020
4.025Crocin C44H64O24 [M+Cl]－
[M+Na]+1011.345
999.368976.378 －3.80
－0.27ZZ 39 4.033 Furfural C5H4O2 [M+H]+ 97.027 96.021 －7.34 HP 40 4.061 Hesperidin C28H34O15 [M－H]－ 609.183 610.189 0.92 ZS 41 4.074 Neohesperidin C16H14O6 [M+H]+ 303.086 302.079 0.72 HP 42 4.078 Isochlorogenic acid a C25H24O12 [M－H]－ 515.118 516.126 －2.07 ZZ 43 4.161
4.183Neohesperdin C28H34O15 [M－H]－
[M+Na]+609.183
633.180610.189 0.27
6.53ZS 44 4.208 Coumarin C9H6O2 [M+H]+ 147.043 146.036 －1.16 HP 45 4.536 Hexanal C6H12O [M+HCOO]－ 145.086 100.088 －3.97 ZZ 46 4.989 Cis-sinapyl alcohol C11H14O4 [M+Na]+ 233.079 210.089 －1.76 ZS 47 5.152 (+)-dehydrodiconiferyl alcohol C20H22O6 [M－H]－ 357.134 358.141 －2.52 ZS 48 5.294 Citrusin b C27H36O13 [M+HCOO]－ 613.213 568.215 －2.94 ZS 49 5.336 Poncirin C28H34O14 [M－H]－ 593.187 594.194 －1.48 ZS 50 5.372 Didymin C16H14O5 [M+H]+ 287.091 286.084 0.64 ZS 51 5.377 Isokaempferide C16H12O6 [M+HCOO]－ 345.060 300.063 －6.16 ZZ 52 5.497 Isosakuranetin-7-rutinoside C28H34O14 [M+H]+ 595.204 594.194 3.64 ZS 53 5.638 Germacron C15H22O [M+H]+ 219.174 218.167 －0.95 ZZ 54 6.046 Pentylfuran C9H14O [M+H]+ 139.111 138.104 －0.93 ZZ/HP 55 6.119 Apigenin C15H10O5 [M－H]－ 269.044 270.052 －2.33 ZS 56 6.144 Naringenin C15H12O5 [M－H]－ 271.060 272.068 －4.16 ZS 57 6.310 Chrysin C15H10O4 [M+HCOO]－ 299.055 254.057 －4.23 ZZ 58 6.470 2, 6, 10-trimethyl-dodecane C6H8O4 [M+H]+ 145.049 144.042 －1.98 HP 59 6.494
6.520(R)-hesperetin C16H14O6 [M－H]－
[M+H]+301.071
303.086302.079 －2.26
－1.07ZS 60 6.578 Crocetin C20H24O4 [M+H]+ 329.174 328.167 －0.09 ZZ 61 6.678 6-methoxy aurapten C20H24O4 [M+H]+ 329.174 328.167 －1.60 ZS 62 7.011 Curdione C15H24O2 [M+H]+ 237.184 236.177 －2.89 ZZ 63 7.052 Tangeretin C20H20O7 [M+H]+ 373.129 372.120 2.03 ZS 64 7.093 Auraptene C19H22O3 [M+HCOO]－ 343.154 298.156 －1.80 ZS 65 7.293 Carvacrol C10H14O [M+H]+ 151.111 150.104 －5.82 HP 66 7.351 Pentadecylic acid C15H30O2 [M+HCOO]－ 287.221 242.224 －7.13 HP 67 7.551
7.735Deacetylnomilin C26H32O8 [M+H]+
[M+HCOO]－473.216
517.205472.209 －1.00
－6.66ZS 68 7.826 (3S, 6E)-nerolidol C15H26O [M+H]+ 223.206 222.198 2.52 ZZ 69 7.834 Beta-chamigrene C15H24 [M+H]+ 205.195 204.187 2.35 HP 70 7.876 D-camphene C10H16 [M+H]+ 137.131 136.125 －4.37 HP/ZS 71 7.918
7.967Limonin C26H30O8 [M+HCOO]－
[M+Na]+515.191
493.183470.194 －2.81
2.04ZS 72 8.034 Beta-selinene C15H24 [M+H]+ 205.195 204.187 2.97 ZZ/HP 73 8.433 Borneol C10H18O [M+Na]+ 177.124 154.135 －3.22 HP 74 8.499 Alpha-cubebene C15H24 [M+H]+ 205.194 204.187 －1.07 HP 75 8.516 Tetramethoxyluteolin C19H18O6 [M+H]+ 343.120 342.110 6.26 ZS 76 8.699 Cinnamyl acetate C11H12O2 [M+H]+ 177.090 176.083 －3.43 HP 77 8.724 6-demethoxytangeretin C19H18O6 [M+H]+ 343.120 342.110 6.27 ZS 78 8.757 Nomilin C28H34O9 [M+H]+ 515.228 514.220 －0.80 ZS 79 8.876
8.932Isoimperatorin C16H14O4 [M－H]－
[M+H]+269.081
271.096270.089 －1.63
－0.94ZZ 80 9.032 Isocaryophyllene oxide C15H24O [M+H]+ 221.190 220.182 0.12 HP 81 9.348 Sinensetin C20H20O7 [M+Na]+ 395.109 372.120 6.29 ZS 82 9.365 Corymbosin C19H18O7 [M+H]+ 359.112 358.105 －2.24 ZZ 83 9.600
9.697Obacunone C26H30O7 [M+HCOO]－
[M+H]+499.195
455.206454.199 －3.20
－1.75ZS 84 11.058 [(E, 8S)-8-methyltetradec-9-enyl] acetate C17H32O2 [M+HCOO]－ 313.236 268.240 －5.94 HP 85 11.161 Gypsogenic acid C30H46O5 [M+H]+ 487.340 486.334 －6.44 ZZ 86 11.444 Hederagenol C30H48O4 [M+H]+ 473.359 472.355 －6.81 ZZ 87 11.525
11.577Schembl120923 C18H18O2 [M－H]－
[M+H]+265.123
267.137266.130 －0.28
－0.47HP 88 11.602 Syringaresinol C22H26O8 [M+H]+ 419.172 418.162 6.65 ZZ 89 11.808
11.876Eucalyptol C18H18O2 [M－H]－
[M+H]+265.123
267.137266.130 0.31
－0.59HP 90 12.383 Methyl isopalmitate C17H34O2 [M+HCOO]－ 315.252 270.255 －7.09 HP 91 12.566 Magnolol C18H18O2 [M－H]－ 265.123 266.130 －1.43 HP 92 12.591 Obovatol C18H18O3 [M－H]－ 281.118 282.125 0.43 HP 93 12.666 Honokiol C18H18O2 [M－H]－ 265.124 266.130 2.90 HP 94 12.692 Prangenin hydrate C16H16O6 [M+H]+ 305.101 304.094 －0.37 ZS 95 13.199 Alpha-cedrene C15H24 [M+H]+ 205.195 204.187 2.03 HP 96 13.307 16-heptadecenal C17H32O [M+HCOO]－ 297.242 252.245 －2.69 HP 97 13.315 Dibutyl phthalate C16H22O4 [M+H]+ 279.159 278.151 0.13 HP 98 13.407 Dehydro-p-cymene C10H12 [M+H]+ 133.100 132.093 －6.49 HP 99 13.523 Myrcene C10H16 [M+H]+ 137.131 136.125 －3.32 HP/ZS 100 13.624 Siaresinol C30H48O4 [M+Cl]－ 507.321 472.355 －6.42 ZZ 101 13.715 2-pentadecyn-1-ol C15H28O [M+HCOO]－ 269.211 224.214 －4.98 HP 102 13.939 Stigmasterol C29H48O [M+H]+ 413.377 412.370 －1.84 ZZ 103 14.190 Linoleic acid C18H32O2 [M－H]－ 279.232 280.240 －2.25 ZZ/HP/ZS 104 14.413 Elemene C15H24 [M+H]+ 205.194 204.187 －1.22 HP 105 14.681 Oleic acid C18H34O2 [M－H]－ 281.247 282.255 －5.12 ZZ 106 14.713 Cymol C10H14 [M+H]+ 135.116 134.109 －7.47 HP/ZS 107 14.738 Alpha-humulene C15H24 [M+H]+ 205.195 204.187 －0.60 HP RT: Retention time; MW: Molecular weight; ZS: Zhishi; ZZ: Zhizi; HP: Houpu. -
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