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中国精品科技期刊2020

基于UPLC-Q-TOF-MS技术以及网络药理学分析山西陈醋的小分子肽类成分及潜在作用机制

聂丽媛, 范三红, 曹林旭, 秦雪梅, 李震宇

聂丽媛,范三红,曹林旭,等. 基于UPLC-Q-TOF-MS技术以及网络药理学分析山西陈醋的小分子肽类成分及潜在作用机制[J]. 食品工业科技,2024,45(18):31−41. doi: 10.13386/j.issn1002-0306.2023100274.
引用本文: 聂丽媛,范三红,曹林旭,等. 基于UPLC-Q-TOF-MS技术以及网络药理学分析山西陈醋的小分子肽类成分及潜在作用机制[J]. 食品工业科技,2024,45(18):31−41. doi: 10.13386/j.issn1002-0306.2023100274.
NIE Liyuan, FAN Sanhong, CAO Linxu, et al. Small Molecule Peptide and the Potential Mechanism of Shanxi Vinegar Based on UPLC-Q-TOF-MS Technology and Network Pharmacology[J]. Science and Technology of Food Industry, 2024, 45(18): 31−41. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023100274.
Citation: NIE Liyuan, FAN Sanhong, CAO Linxu, et al. Small Molecule Peptide and the Potential Mechanism of Shanxi Vinegar Based on UPLC-Q-TOF-MS Technology and Network Pharmacology[J]. Science and Technology of Food Industry, 2024, 45(18): 31−41. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023100274.

基于UPLC-Q-TOF-MS技术以及网络药理学分析山西陈醋的小分子肽类成分及潜在作用机制

基金项目: 企业横向委托项目(01130123070083)。
详细信息
    作者简介:

    聂丽媛(1998−),女,硕士研究生,研究方向:中药活性成分研究,E-mail:nieliyuan0310@163.com

    通讯作者:

    李震宇(1980−),男,博士,教授,研究方向:中药质量控制及活性成分研究,E-mail:lizhenyu@sxu.edu.cn

  • 中图分类号: TS264.2+2

Small Molecule Peptide and the Potential Mechanism of Shanxi Vinegar Based on UPLC-Q-TOF-MS Technology and Network Pharmacology

  • 摘要: 目的:明确山西陈醋中的小分子肽类化学成分,并进一步探讨其针对心血管等疾病的潜在作用机制。方法:采用超高效液相色谱-四极杆-飞行时间串联质谱(UPLC-Q-TOF-MS)技术结合GNPS分子网络技术分析山西陈醋中的小分子肽类成分。运用Genecards、Drugbank 5.0、DAVID等数据库对小分子肽类化合物靶点及通路进行分析,并通过Cytoscape软件构建“成分-靶点-信号通路-疾病”网络。使用Autodock Dock 1.5.6和PyMol 2.5.5进行山西陈醋核心靶点与关键成分的分子对接。结果:共鉴定出41个小分子肽类化合物,其中包括27个环二肽、10个直链二肽和4个三肽类化合物。其发挥心血管和肥胖等保健作用的“成分-靶点-信号通路-疾病”网络包含14个活性成分和109个药物靶点。通路富集分析得到59条信号通路。分子对接结果表明Cyclo(Leu-Pro)、Val-Val、Pro-Phe、Cyclo(His-Pro)等成分可能与核心靶点F2、MAPK1,MMP9、VCAM1等具有较好的结合能力,初步验证了网络药理学预测结果的准确性。结论:山西陈醋可能通过多成分、多靶点、多通路协同发挥预防调节心血管疾病和肥胖等疾病的保健作用。
    Abstract: Objective: To elucidate the small molecule peptides in Shanxi vinegar and further explore their potential mechanism of action against cardiovascular and other related diseases. Methods: The small molecule peptides of Shanxi vinegar were analyzed using ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS/MS). The targets and pathways of small molecule peptides were predicted via Genecards, Drugbank 5.0, DAVID, and other databases. The "component-target-signaling pathway-disease" network was constructed using Cytoscape software. Autodock Dock 1.5.6 and Pymol 2.2.0 were used to perform molecular docking between the core targets of Shanxi vinegar and the components. Results: A total of 41 small molecule peptide compounds were identified by UPLC-Q-TOF-MS/MS, which including 27 cyclic dipeptides, 10 linear dipeptides, and 4 tripeptides. The "component-target-signaling pathway-disease" network, contributing to the health-promoting effects on cardiovascular and obesity, encompasses 14 active components and 109 drug targets. A total of 59 pathways were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Molecular docking results demonstrated favorable binding interactions between the core targets (F2, MAPK1, MMP9, VCAM1) and the corresponding active components Cyclo (Leu-Pro), Val-Val, Pro-Phe, Cyclo (His-Pro) in Shanxi vinegar, which preliminarily confirming the accuracy of the network pharmacology prediction. Conclusion: Shanxi vinegar might prevent and regulate cardiovascular diseases and obesity through multiple components, targets and pathways.
  • 我国是最早生产谷物酿造食醋的国家,酿造原料以谷物为主,在我国已有一千多年的历史,由于其酿造原料多样,酿造过程又有丰富的微生物参与[1],故酿造食醋中富含多种功能性成分[2]。研究表明,酿造食醋中富含多种生物活性成分,主要包括黄酮、氨基酸、川芎嗪、有机酸等化合物[3],具有缓解结肠炎、抗菌[45]、降低血脂[6]等作用。

    自然界中有许多结构简单的小分子肽类化合物,如微生物的发酵液中发现由常见氨基酸残基或类似物组成的二肽,包括环二肽(Cyclodipeptides,Cyclo)和直链二肽,是机体吸收蛋白质的主要形式之一[78]。小分子肽类化合物结构多样,具有调节机体生理功能,为机体提供营养的双重功效,可以提高吸收功效、减轻胃肠道消化负担、预防疾病、控制体重、调节血压以及延缓衰老等作用,逐渐成为功能性食品和药物开发的重点领域。小分子肽除参与调节机体多种生理功能外,还具有抗菌[9]、抗炎[10]、降压[11]、抗抑郁[12]等药理作用。研究表明,Cyclo(Gly-Pro)对福尔马林及醋酸诱导的小鼠有明显的抗炎作用[10],含组氨酸的Cyclo(His-Phe)和Cyclo(His-Tyr)具有抗心律失常和抗高血压活性[1314],Cyclo(His-Pro)联合锌可以改善遗传性糖尿病肥胖小鼠的糖代谢[15]。直链二肽Ala-His有益于预防和治疗肥胖、心血管功能障碍和衰老相关疾病,促进人体健康[16]。Asn-Trp显著降低高脂饮食诱导小鼠的血液总胆固醇和低密度脂蛋白含量[17]。对于酿造食醋中的二肽类成分,有文献对镇江醋粉进行分离得到两个Cyclo(Phe-Pro)和Cyclo(Pro-Leu),并对二者进行了定量方法的研究[1819]。目前对于酿造食醋中的小分子肽类成分缺乏系统的研究,这也进一步制约了小分子肽类成分与酿造食醋保健功能的相关性研究。

    UPLC-Q-TOF-MS/MS质谱技术具有高分辨率、高灵敏度的特点,能够对化学成分进行定性分析。通过对化合物相对分子质量和二级碎片信息的分析,可快速识别各类化学成分,用于食品功能成分的快速解析。全球天然产物社会分子网络(GNPS)可根据相关化合物的MS/MS二级质谱碎片的相似性,将同一类化合物的分子聚类到分子网络中并进行定性分析[20]。该技术已广泛应用于天然产物、代谢组学和药物发现等领域,如Lü等[21]基于GNPS分子网络对荔枝果肉提取物中9种原花青素和11种黄酮苷进行了表征。LC-MS和GNPS的结合可以互补性地研究复杂体系的化学组成。随着生物信息学的发展,疾病和复合靶点数据库的出现为寻找药物的潜在活性成分和靶点提供了新的策略,全面系统地揭示药物与疾病之间的关系。Genecards(https://www.genecards.org/)数据库自动整合来自约150个以基因为中心的数据库的资源,包括基因组学、转录组学、蛋白质组学、遗传学、临床和功能等多方面的信息[22]。Drugbank5.0(https://drugbank.com)数据库包含FDA批准的药物,临床研究的药物及与实验相关的药物,是一个涵盖生物和化学信息的数据库,为活性成分作用靶点的确定提供了有力的支持[23]

    山西陈醋是中国传统四大名醋之一,选用优质高粱等为主要原料,以豌豆所制大曲为发酵剂,经蒸、酵、熏、淋、晒等过程酿造而成[24]。有研究表明山西陈醋中蛋白质含量约为7.625%左右[2],但目前尚未见文献对山西陈醋中游离的小分子肽类成分进行系统研究。本研究拟采用UPLC-Q-TOF-MS/MS和GNPS对山西陈醋中的肽类化合物成分进行鉴定。利用Genecards和Drugbank 5.0等数据库研究山西陈醋中的活性成分及作用靶点,为解析山西陈醋中肽类成分与其保健作用机制的相关性提供科学依据。

    山西陈醋 阳泉市裕盛源农产品开发有限公司;甲酸、乙腈 质谱级,美国Thermo公司;Cyclo(Leu-Pro) 纯度不低于98%,上海毕得医药科技股份有限公司(批号M21AZ823A)。

    QTOF 5600+质谱仪 美国AB Sciex公司;安捷伦1290超高效液相色谱仪 美国Agilent Technologies公司;Milli-QIntegral Water Purification System纯水制备仪 美国Millipore公司。

    取山西陈醋1 mL,用0.22 μm微孔滤膜滤过,取续滤液,备用。

    取Cyclo(Leu-Pro)对照品2 mg,加甲醇制成0.1 mg·mL−1的储备液,取对照品储备液200 μL,加甲醇稀释成1 μg·mL−1的对照品溶液,进样前过0.22 μm微孔滤膜。

    色谱柱:Waters Acquity UHPLC HSS T3 column(2.1 mm×150 mm,1.8 μm);流动相为0.1 %甲酸水(A)和乙腈(B);梯度洗脱:0~5 min,2% B;5~20 min,2%~20% B;20~25 min,20%~35 %B;25~30 min,35%~55% B;30~32 min,55%~95% B;运行时间为32 min,流速为0.30 mL·min−1,进样量为5 μL,柱温为20 ℃。

    离子化模式:电喷雾离子源(ESI),扫描模式:TOF MS-IDA-Production scan;正负离子扫描;离子源温度为450 ℃;正离子模式离子源电压为5500 V、负离子模式离子源电压为−4500 V;去簇电压(DP)为60 V;碰撞能量(CE)为30 eV;碰撞能量扩展(CES)为15 eV;气帘气为30 psi;氮气为雾化气体,喷雾气(Gas1)55 psi,辅助加热气(Gas2)55 psi;一级质谱母离子扫描范围为m/z 50~1500,二级子离子扫描范围为m/z 50~1250[25]

    对文献已报道的小分子肽进行整理,将整理的小分子肽化学名称及分子式导入Analytics,设置匹配参数,提取潜在化合物色谱峰、一级质谱图、二级质谱图,并通过与HMDB(https://hmdb.ca)以及PubChem(https://pubchem.ncbi.nlm.nih.gov)数据库比较,确定目标化合物的分子结构,通过与数据库的准分子离子峰、二级质谱图对比鉴定陈醋中的小分子肽类成分。

    正离子模式下陈醋样品的UPLC-Q-TOF MS/MS质谱数据文件通过MS Convert软件转换为mzXML文件格式,登录WinSCP软件并将mzXML格式文件上传至GNPS,建立GNPS网络;设置Precursor Ion Mass Tolerance和Fragment Ion Mass Tolerance的质量误差为0.02 Da,余弦分数阈值为0.5,最小匹配碎片离子为7,其余参数均选择默认值,本研究继续采用基于MS/MS关联的分子网络分析陈醋中的小分子肽类成分。

    利用UHPLC-MS技术分析鉴定小分子肽类成分构建陈醋化学信息数据库,用于网络药理学研究。在Genecards(https://www.genecards.org/)和Drugbank5.0(https://www.drugbank.)数据库中获得化合物靶点,对蛋白进行过滤,并限定物种为“human”,将得到的化合物靶点去重复后构建与山西陈醋相关的作用靶点数据库。

    将获得的靶点上传至在线STRING 11.5数据库,选择类型为“Homo Sapiens”,设置参数评分值>0.9,其他参数为默认设置,同时去掉PPI网络中的单一节点,导出PPI分析结果,并利用Cytoscape软件提取其网络中的核心靶点蛋白。

    将目标基因输入David 6.8数据库(https://david.ncifcrf.gov/)进行基因本体(Gene Ontology,GO)功能分析和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析,选择物种为“Homo Sapiens”。R语言ggplot 2软件包用于绘制气泡图。点的颜色代表不同的P值,点的大小反映了通路中表达的靶基因的数量。

    将KEGG通路富集分析得到的前30条信号通路输入到CTD数据库(http://ctdbase.org/)进行疾病预测。将所预测到的疾病与靶点进一步构建“成分-靶点-信号通路-疾病”网络图并将疾病关联到的化合物和靶点输入到微生信(https://www.bioinformatics.com.cn/)绘制疾病的化合物和靶点共交集韦恩图。

    利用PubChem数据库(https://pubmed.ncbi.nlm.nih.gov)获取活性成分的SDF结构文件,并通过PyMol 2.5.5软件转化为PDB格式,利用RCSB PDB数据库(https://www.rcsb.org)检索并下载关键靶点的3D结构。采用AutoDock Vina 1.5.6软件平台将核心靶点分别与活性成进行分子对接验证,并通过PyMol 2.5.5软件将结果可视化。

    采用UPLC-Q-TOF MS高分辨质谱分别在正、负离子模式下采集山西陈醋样本的质谱数据(图1),通过与标准品、文献报道[2627]以及标准数据库的MS2数据的比较,共鉴定出30个小分子肽类化合物,其中25个环二肽、3个直链二肽和2个三肽类化合物(表1)。

    图  1  山西陈醋UPLC-Q-TOF-MS/MS色谱图
    注:A:正离子模式;B:负离子模式。
    Figure  1.  UPLC-Q-TOF-MS chromatograms of Shanxi vinegar
    表  1  山西陈醋肽类成分UPLC-Q-TOF-MS/MS分析结果
    Table  1.  Peptide compounds of shanxi vinegar UPLC-Q-TOF-MS/MS analysis results
    序号 化合物名称 分子式 保留时间(min) 加和方式 分子质量 质量偏差
    (ppm)
    MS/MS(m/z)
    1 Cyclo(Ala-Val) C8H14N2O2 10.63 [M+H]+ 171.1126 −1.1 100.0747,86.0954,72.0815,72.0496,55.0609
    2 Cyclo(Asp-Phe) C14H18N2O3 16.02 [M+H]+ 263.139 −0.1 190.1216,148.0810,136.0751,116.0696,107.0496
    3 Cyclo(Glu-Phe) C15H20N2O3 18.04 [M+H]+ 277.1544 −1 148.0725,136.0750,119.0481,86.0991
    4 Cyclo(Leu-Leu) C12H22N2O2 23.99 [M+H]+ 227.1754 −0.2 182.1549,154.1586,114.0900,86.0963
    5 Cyclo(Leu-Phe) C15H20N2O2 24.66 [M+H]+ 261.1597 −0.1 187.1430,170.0784,148.0827,120.0816,114.0971,70.0705
    6 Cyclo(Leu-Pro) C11H18N2O2 16.87 [M+H]+ 211.1439 −0.8 154.0726,138.1270,114.0926,98.0609,70.0670
    7 Cyclo(Leu-Ser) C9H16N2O3 7.61 [M+H]+ 201.123 −1.8 114.0906,86.0983,60.0480
    8 Cyclo(Pro-Tyr) C14H16N2O3 13.04 [M+H]+ 261.1232 −0.5 164.0681,154.0731,136.0754,119.0481,98.0597,91.0543,70.0670
    9 Cyclo(Phe-Tyr) C18H18N2O3 20.74 [M+H]+ 311.139 −0.1 164.0751,148.0735,136.0766,120.0805,107.0505
    10 Cyclo(Phe-Ala) C12H14N2O2 15.75 [M+H]+ 219.1127 −0.5 203.1460,173.0955,148.0740,120.0809,91.0539,72.0485
    11 Cyclo(Phe-Phe) C18H18N2O2 25.63 [M+H]+ 295.1442 0.3 222.1266,148.0767,120.0816,103.0511
    12 Cyclo(Phe-Pro) C14H16N2O2 19.80 [M+H]+ 245.1283 −0.5 172.1120,154.0731,148.0752,120.0810,98.0606,70.0674
    13 Cyclo(Pro-Pro) C10H14N2O2 10.64 [M+H]+ 195.1126 −1 70.0672,98.0599
    14 Cyclo(Pro-Ser) C9H16N2O2 14.87 [M+H]+ 185.1283 −0.7 156.0775,140.1046,98.0725,86.0985,69.0707
    15 Cyclo(Tyr-Gly) C11H12N2O3 6.53 [M+H]+ 221.0919 −0.7 164.0742,148.0812,136.0756,91.0555,73.0355
    16 Cyclo(Val-Leu) C11H20N2O2 21.21 [M+H]+ 213.1596 −0.5 140.1425,114.0977,100.0772,86.0962,72.0823,55.0581
    17 Cyclo(Val-Phe) C14H18N2O2 22.83 [M+H]+ 247.1442 0.3 219.1485,148.0749,120.0808,100.0782,72.0828,55.0579
    18 Cyclo(Val-Pro) C10H16N2O2 13.32 [M+H]+ 197.1283 −0.9 169.1340,100.0772,98.0607,72.0828,70.0675,68.0533
    19 Cyclo(Val-Val) C10H18N2O2 17.27 [M+H]+ 199.1439 −0.9 126.1265,100.0787,96.0807,72.0814,55.0573
    20 Cyclo(Pro-Ala) C8H12N2O2 7.00 [M+H]+ 169.0972 0.5 169.0958,141.1026,98.0609,72.0456,70.0673
    21 Cyclo(Pro-Thr) C9H14N2O3 5.82 [M+H]+ 199.1075 −1 181.0982,153.1010,125.0706,98.0608,70.0673,56.0532
    22 Cyclo(Ala-Tyr) C12H14N2O3 11.15 [M+H]+ 235.1075 −1.1 235.1204,207.1121,162.0886,136.0871
    23 Cyclo(Pro-Met) C10H16N2O2S 14.95 [M+H]+ 229.1003 −0.8 181.0959,153.0990,125.0688,84.0456,70.0669
    24 Cyclo(Tyr-Tyr) C18H18N2O4 13.75 [M+H]+ 327.1339 −0.2 164.0774,146.0596,136.0765,107.0501,98.9848
    25 Cyclo(His-Gly) C8H10N4O2 1.01 [M+H]+ 195.0871 −2.9 138.0675,122.0701,110.0714,82.0530,58.0320
    26 Cyclo(His-Phe) C15H16N4O2 11.13 [M+H]+ 285.1345 −0.3 138.0707,110.0709,82.0534
    27 Cyclo(His-Pro) C11H14N4O2 2.19 [M+H]+ 235.119 0 162.1044,110.0729,95.0596,82.0553
    28 Ala-Ile C9H18N2O3 7.52 [M+H]+ 203.1388 −0.9 203.1215,157.1411,140.0710,132.1009,130.0604
    29 Ala-Phe C12H16N2O3 10.87 [M+H]+ 237.1233 −0.2 237.1096,191.1215,166.0910,120.0809
    30 Val-Leu C11H22N2O3 11.98 [M+H]+ 231.1701 −0.8 132.1017,86.0973,72.0824
    31 Thr-Ile C10H20N2O4 8.04 [M+H]+ 233.1494 −0.6 132.0985,86.1021,69.0575,57.0711
    32 Leu-Tyr C15H22N2O4 11.84 [M+H]+ 295.1651 −0.6 182.0837,136.0806,86.0960,69.0750
    33 Val-Val C10H20N2O3 4.73 [M+H]+ 217.1547 0.2 118.0892,72.0817,55.0573
    34 Glu-Val C10H18N2O5 8.99 [M+H]+ 247.1287 −0.4 130.0641,103.0540,86.0977,74.0325
    35 Phe-Pro C14H18N2O3 12.48 [M+H]+ 263.139 0.1 217.1452,120.0809,116.0893,70.0675
    36 PyroGlu-Pro C10H14N2O4 8.60 [M+H]+ 227.1026 −0.1 209.0925,181.0964,116.0704,84.0459,70.0670
    37 PyroGlu-Val C10H16N2O4 10.96 [M+H]+ 229.1182 −0.5 183.1111,155.1209,138.0894,118.0879,84.0448,70.0829
    38 Gly-Ala-Phe C14H19N3O4 13.55 [M+H]+ 294.1449 0.3 276.1220,147.0905,114.0596,84.0453,68.0517
    39 Gly-Pro-Ala C10H17N3O4 1.00 [M+H]+ 244.129 −0.6 185.0894,147.0751,130.0488,112.0498,84.0444,70.0667
    40 Leu-Gly-Leu C14H27N3O4 18.41 [M+H]+ 302.2075 0.3 198.0965,189.1354,171.1067,143.1177,132.1012,86.0973
    41 Ile-Pro-Ile C17H31N3O4 19.20 [M+H]+ 342.2387 −0.1 229.1573,211.1425,183.1487,70.0667
    下载: 导出CSV 
    | 显示表格

    环二肽,也称为2,5-二酮哌嗪(DKPs),是自然界中最简单的肽衍生物,由两个氨基酸通过“首尾相连”的肽键组成。环二肽的裂解方式主要是羰基的断裂和氨基酸残基的丢失。化合物6在正离子模式下的准分子离子峰为m/z 211.1439 [M+H]+,主要的碎片离子包括m/z 114.0926 [M+H-Pro]+、m/z 98.0609 [M+H-Leu]+和m/z 70.0670 [M+H-Leu-CO]+,通过与标准品比较,确定该化合物为Cyclo(Leu-Pro);化合物12在正离子模式下的准分子离子峰为m/z 245.1283 [M+H]+,主要碎片离子包括m/z 148.0752 [M+H-Pro]+、m/z 120.0810 [M+H-Pro-CO]+和m/z 98.0606 [M+H-Phe]+。通过与文献报道的碎片进行比较,确定该化合物为Cyclo(Phe-Pro)。

    直链二肽在质谱中通常会丢失一分子的氨基酸残基产生[M+H-氨基酸残基]+,并进一步丢失H2O和羰基产生[M+H-氨基酸残基-H2O-CO]+的碎片离子。如化合物29,准分子离子为m/z 237.1233 [M+H]+,主要碎片离子包括m/z 166.0910 [M+H−Ala]+和m/z 120.0809 [M+H−Ala−H2O−CO]+,通过与PubChem数据库对比确认该化合物为Ala-Phe。化合物39在正离子模式下的准分子离子峰为m/z 217.1547[M+H]+,主要碎片离子包括m/z 118.0892 [M+H-Val]+、m/z 72.0817 [M+H-Val−H2O−CO]+,通过与PubChem数据库比较,确定该化合物为Val-Val。

    进一步采用基于MS/MS关联的GNPS分子网络解析醋中的肽类成分。根据分子网络中已知的肽类化合物,以及已知化合物和未知化合物在网络中的相关性和准分子离子质荷比差值,进一步推测肽类化合物11个,包括环二肽类化合物2个,直链肽类化合物7个,以及其他类型的肽类化合物2个。如化合物8在分子网络中的准分子离子为m/z 261.1232,与已鉴定的化合物12(Cyclo(Phe-Pro))存在m/z 15.99的质量差,对应于一个氧原子,结合其精确分子质量m/z 261.1232(质量误差−0.5 ppm),推测化合物 8 的分子式为C14H16N2O3。两者共有的质谱碎片包括m/z 154.07、m/z 98.06和m/z 70.06,两者之间相差m/z 15.99的质谱碎片包括m/z 136.07和m/z120.08,m/z 164.06和m/z 148.07。根据环肽化合物的裂解规律,m/z 164.0681和m/z 136.0754分别对应碎片离子[M+H−Pro]+和[M+H−Pro−CO]+,因此,推测化合物8为Cyclo(Pro-Tyr)。化合物37在分子网络中与已知化合物36(PyroGlu-Pro)相关,两者的分子质量差为m/z 2.013,差值对应于两个氢原子,结合其精确分子质量m/z 229.1182(质量误差为−0.5 ppm),推测化合物37的分子式C10H16N2O4。两者共有的质谱碎片包括m/z 84.04、m/z 70.06,两者之间相差m/z 2.013的质谱碎片包括m/z 183.11和m/z 181.09,m/z 118.08和m/z 116.07,因此推测化合物37为PyroGlu-Val。

    综上,课题组采用UPLC Q-TOF MS高分辨质谱结合GNPS分子网络,共鉴定出41个小分子肽类化合物,包括27个环二肽、10个直链二肽和4个三肽类化合物(表1)。

    分别用Drugbank和Genecards数据库对陈醋中小分子肽类化合物进行靶点检索,在Drugbank数据库中获得60个基因靶点,Genecards数据库中共获得235个基因靶点。收集基因靶点并进行去重复处理,共筛选到109个肽类化合物作用靶点,为了更清楚地显示生物活性成分和靶点之间的关系,采用Cytoscape软件构建“成分-靶点”网络(图2),包括123个节点(14个活性成分和109个靶点)和246条边,网络中的度值(Degree)表示连接该节点的条数。Degree值越大,表示该节点与其他节点相互作用越紧密,在网络中起到中心枢纽的作用[28]

    图  2  成分-靶点-疾病网络
    注:蓝色箭头代表靶点,紫色圆形代表成分。
    Figure  2.  Compound-target-disease network

    由网络图可知,同一成分可以作用于不同的靶点,如Cyclo(His-Pro)可以作用于NTS、CCK、NPY等6个靶点;Val-Val可作用于SOD2、COMT、VCAM1等45个靶点。同一靶点也可对应不同化学成分,如ANPEP对应Ala-Phe、Ile-Pro-Ile、Cyclo(Asp-Phe)3种化合物;GHRH作用于Cyclo(His-Pro)、Ala-Ile等3种化合物。提示山西陈醋可能是多成分作用于多靶点协同发挥作用。

    PPI网络是用于探索各种蛋白质之间的相互作用。将肽类活性成分的109个潜在作用靶点导入STRING数据库,在隐藏断开的节点后,该网络包含52个节点和74条边。如PPI网络(图3)所示,图中各节点大小和颜色深浅代表该靶点在陈醋中发挥生物学效应中的重要程度;靶点颜色越深、连接节点越大、靶点与靶点之间连线数目越多,代表其在网络中的协同作用越强,越有可能是陈醋发挥效果的核心靶点。由图3可知,等级值排名较前的包括F2、MAPK1、MMP9、A2M等靶点,提示这些靶点可能在网络中起着较为关键的作用,可能是山西陈醋发挥作用的关键靶点。

    图  3  蛋白-蛋白相互作用(PPI)网络
    Figure  3.  Protein-protein interaction (PPI) network

    对获得的相关基因靶点上传到DAVID数据库中,进行GO富集分析和KEGG信号通路分析。GO富集分析涉及生物学的三个方面:生物过程(BP)、细胞成分(CC)和分子功能(MF)。GO分析结果显示,312条潜在靶基因通路被富集,包括99个BPs、27个CCs和38个MFs(P≤0.05,FDR≤0.05),涉及基因数量最多的前10条通路如图4所示。

    图  4  GO功能富集分析
    Figure  4.  GO enrichment analysis of targets.

    KEGG信号通路分析显示,共获得59条信号通路。以基因数目降序排列,其中排名前30的信号通路见图5,圆圈越大,通路中包含的靶点数目越多。在30条信号通路中,包括与心血管疾病相关的信号通路糖尿病性心肌病(Diabetic cardiomyopathy)、脂质和动脉粥样硬化(Lipid and atherosclerosis),流体剪切力与动脉粥样硬化(Fluid shear stress and atherosclerosis)、补体和凝血级联反应(Complement and coagulation cascades),与内分泌与代谢性疾病相关的通路胰岛素抵抗(Insulin resistance),非酒精性脂肪肝病(Non-alcoholic fatty liver disease),II型糖尿病(Type II diabetes mellitus),肾素-血管紧张素系统(Renin-angiotensin system),松弛素信号通路(Relaxin signaling pathway),雌激素信号通路(Estrogen signaling pathway),卵巢类固醇生成(Ovarian steroidogenesis),脂肪细胞因子信号通路(Adipocytokine signaling pathway),与新陈代谢相关通路色氨酸代谢(Tryptophan metabolism)和其他信号通路15条。如图5所示,血脂与动脉粥样硬化(Lipid and atherosclerosis)、补体和凝血级联反应(Complement and coagulation cascades)、糖尿病心肌病(Diabetic cardiomyopathy)表现出较高的富集因子和较大的圆点,提示这些通路可能发挥了重要作用。

    图  5  KEGG信号通路富集分析
    Figure  5.  Enrichment analysis of KEGG signaling pathway

    将KEGG富集分析得到的前30条信号通路通过CTD数据库对山西陈醋的潜在保健功能进行预测,结果表明山西陈醋对Ⅱ型糖尿病、高血脂、肥胖、动脉粥样硬化、高血压等疾病可能具有潜在的保健作用。进一步构建“成分-靶点-信号通路-疾病”网络图,结果如图6所示。对于高血脂症,肽类化合物Val-Leu作用于F2、MAPK1、MMP9基因靶点,调控血脂与动脉粥样硬化(Lipid and atherosclerosis)信号通路[29];Pro-Phe、Val-Val作用于A2M、MAPK1、KLK8,通过调控补体和凝血级联反应(Complement and coagulation cascades)信号通路发挥作用[30]。对于高血压,Cyclo(His-Pro)、Ala-Phe共同作用于CCK、NTS、GRP、ANPEP等基因靶点,通过调控神经活性配体-受体相互作用(Neuroactive ligand-receptor interaction)、脂肪细胞因子信号通路(Adipocytokine signaling pathway)发挥治疗高血压的作用[3132]。化合物Cyclo(His-Pro)、Ala-Ile共同作用于GHRH、SOD2基因靶点,主要通过调控血脂与动脉粥样硬化(Lipid and atherosclerosis)信号通路发挥治疗Ⅱ型糖尿病的作用[3334];Pro-Phe和Val-Val共同作用于A2M、VCAM1、KLK8等靶点[35],调控补体和凝血级联反应(Complement and coagulation cascades)、血脂与动脉粥样硬化(Lipid and atherosclerosis)、流体剪切力与动脉粥样硬化(Fluid shear stress and atherosclerosis)信号通路治疗Ⅱ型糖尿病[36]。对于动脉硬化疾病,化合物Cyclo(Leu-Pro)、Pro-Phe共同作用于CYP2C19、CYP1A2靶点,调控类固醇激素生物合成(Steroid hormone biosynthesis)、色氨酸代谢(Tryptophan metabolism)发挥治疗作用。Val-Leu、Gly-Pro-Ala、Pro-Phe、Val-Val多个化合物共同作用于TGFB1靶点[3738],调控糖尿病心肌病(Diabetic cardiomyopathy)、糖尿病并发症中的AGE-RAGE信号通路(AGE-RAGE signaling pathway in diabetic complications)达到减肥的作用;化合物Cyclo(His-Pro)作用于NTS、NPY、GRP、GHRH等靶点,通过调控神经活性配体-受体相互作用(Neuroactive ligand-receptor interaction)对肥胖进行干预。课题组预测到的Ⅱ型糖尿病、高血脂、肥胖、动脉硬化、高血压5种疾病关联到的化合物和靶点取交集,结果见图7,得到10个共交集化合物和20个共交集靶点。其中共交集化合物包括Cyclo(Leu-Pro)、Val-Val、Pro-Phe、Cyclo(His-Pro)等,共交集靶点包括F2、MAPK1、MMP9、VCAM1等,提示这些均为陈醋发挥保健作用的关键成分及关键靶点。

    图  6  成分-靶点-信号通路-疾病网络
    注:橙色菱形代表成分,蓝色圆形代表靶点,绿色箭头代表通路,黄色三角形代表疾病。
    Figure  6.  Network of component-target-pathway-disease
    图  7  不同疾病成分和靶点之间交集韦恩图
    注:A. 五种疾病肽类化合物交集;B. 五种疾病交集靶点。
    Figure  7.  Venn diagram of chemical constituents and gene targets between different diseases

    为验证山西陈醋的关键成分及靶点作用机制,将上述关键成分Cyclo(Leu-Pro)、Val-Val、Pro-Phe、Cyclo(His-Pro)以及核心靶点F2、SOD2、MAPK1、MMP9进行分子对接验证。核心靶点在RCSB PDB数据库(https://www.rcsb.org/)中的编号分别对应为2bvr、1n0n、2y9q、4xct。当配体和受体相互作用时,一般认为结合能小于-5 kcal/mol 时表明有较好地结合活性,结果见图8A所示,关键成分与靶点间的对接结合能小于 -5 kcal/mol,说明陈醋中的小分子肽类成分与关键靶点间亲和力较强。进一步利用PyMol软件将对接结果进行可视化处理。如图8B所示,Cyclo(Leu-Pro)、Cyclo(His-Pro)等化合物主要是通过与2bvr、1n0n、2y9q、4xct的不同位点产生氢键相互作用。说明这些小分子肽类核心成分均能通过氢键与关键疾病靶点的不同氨基酸残基结合,从而发挥治疗心血管、肥胖、糖尿病等疾病的作用。

    图  8  核心成分与对关键靶点对接
    注:A. 对接结合能热图;B. 对接模式图(部分)。
    Figure  8.  Binding energy heat map of core active components and key targets

    本研究共鉴定出41个小分子肽类化合物,其中包括27个环二肽、10个直链二肽和4个三肽类化合物。对于食醋中的小肽类成分,目前文献中仅报道了镇江香醋醋粉中的2个环肽类化合物。与已有研究相比,本研究总结了小分子肽类化合物的质谱裂解规律,并从山西陈醋中鉴定到更多的游离小分子肽类化合物,进一步丰富了山西陈醋化学成分的多样性,为进一步深入研究其营养保健功能提供了依据。

    小分子肽具有多种活性,能够参与生理调节和人体代谢活动,具有促进吸收、调节免疫,降血脂等作用[1314]。本研究提示山西陈醋中的小分子肽类化合物Val-Val、Cyclo(His-Pro)等通过调节血脂与动脉粥样硬化(Lipid and atherosclerosis)等信号通路作用于MMP9和MAPK1等靶点。有研究表明,MAPK1在许多生理过程中发挥关键作用,如miR-378a-3p通过调节MAPK1的表达而调节脂肪生成[39],儿茶酚胺通过提高MAPK1来控制脂肪分解[40]。此外,高脂饮食诱导的肥胖小鼠显著改变了MMP9基因表达水平[41]。本研究通过分子对接表明,山西陈醋中关键成分及关键靶点间具有较好的结合,进一步说明山西陈醋中的小分子肽类成分可能对心血管及肥胖有关的靶点具有调控作用。

    综上,本研究采用液相色谱-质谱(LC-MS/MS)特征和GNPS分子网络,对山西陈醋中的小分子肽类化合物进行了系统研究,通过网络药理学筛选出山西陈醋发挥保健作用的关键活性成分、作用靶点及通路,并通过分子对接确认了上述成分与关键靶点的结合能力。然而,本研究对于小分子肽类成分的鉴定仅限于数据库比较和质谱规律分析,后续还需要进一步通过更多的标准品进行结构验证。此外,网络药理学预测的靶点通路等也需要进一步通过实验进行验证。

  • 图  1   山西陈醋UPLC-Q-TOF-MS/MS色谱图

    注:A:正离子模式;B:负离子模式。

    Figure  1.   UPLC-Q-TOF-MS chromatograms of Shanxi vinegar

    图  2   成分-靶点-疾病网络

    注:蓝色箭头代表靶点,紫色圆形代表成分。

    Figure  2.   Compound-target-disease network

    图  3   蛋白-蛋白相互作用(PPI)网络

    Figure  3.   Protein-protein interaction (PPI) network

    图  4   GO功能富集分析

    Figure  4.   GO enrichment analysis of targets.

    图  5   KEGG信号通路富集分析

    Figure  5.   Enrichment analysis of KEGG signaling pathway

    图  6   成分-靶点-信号通路-疾病网络

    注:橙色菱形代表成分,蓝色圆形代表靶点,绿色箭头代表通路,黄色三角形代表疾病。

    Figure  6.   Network of component-target-pathway-disease

    图  7   不同疾病成分和靶点之间交集韦恩图

    注:A. 五种疾病肽类化合物交集;B. 五种疾病交集靶点。

    Figure  7.   Venn diagram of chemical constituents and gene targets between different diseases

    图  8   核心成分与对关键靶点对接

    注:A. 对接结合能热图;B. 对接模式图(部分)。

    Figure  8.   Binding energy heat map of core active components and key targets

    表  1   山西陈醋肽类成分UPLC-Q-TOF-MS/MS分析结果

    Table  1   Peptide compounds of shanxi vinegar UPLC-Q-TOF-MS/MS analysis results

    序号 化合物名称 分子式 保留时间(min) 加和方式 分子质量 质量偏差
    (ppm)
    MS/MS(m/z)
    1 Cyclo(Ala-Val) C8H14N2O2 10.63 [M+H]+ 171.1126 −1.1 100.0747,86.0954,72.0815,72.0496,55.0609
    2 Cyclo(Asp-Phe) C14H18N2O3 16.02 [M+H]+ 263.139 −0.1 190.1216,148.0810,136.0751,116.0696,107.0496
    3 Cyclo(Glu-Phe) C15H20N2O3 18.04 [M+H]+ 277.1544 −1 148.0725,136.0750,119.0481,86.0991
    4 Cyclo(Leu-Leu) C12H22N2O2 23.99 [M+H]+ 227.1754 −0.2 182.1549,154.1586,114.0900,86.0963
    5 Cyclo(Leu-Phe) C15H20N2O2 24.66 [M+H]+ 261.1597 −0.1 187.1430,170.0784,148.0827,120.0816,114.0971,70.0705
    6 Cyclo(Leu-Pro) C11H18N2O2 16.87 [M+H]+ 211.1439 −0.8 154.0726,138.1270,114.0926,98.0609,70.0670
    7 Cyclo(Leu-Ser) C9H16N2O3 7.61 [M+H]+ 201.123 −1.8 114.0906,86.0983,60.0480
    8 Cyclo(Pro-Tyr) C14H16N2O3 13.04 [M+H]+ 261.1232 −0.5 164.0681,154.0731,136.0754,119.0481,98.0597,91.0543,70.0670
    9 Cyclo(Phe-Tyr) C18H18N2O3 20.74 [M+H]+ 311.139 −0.1 164.0751,148.0735,136.0766,120.0805,107.0505
    10 Cyclo(Phe-Ala) C12H14N2O2 15.75 [M+H]+ 219.1127 −0.5 203.1460,173.0955,148.0740,120.0809,91.0539,72.0485
    11 Cyclo(Phe-Phe) C18H18N2O2 25.63 [M+H]+ 295.1442 0.3 222.1266,148.0767,120.0816,103.0511
    12 Cyclo(Phe-Pro) C14H16N2O2 19.80 [M+H]+ 245.1283 −0.5 172.1120,154.0731,148.0752,120.0810,98.0606,70.0674
    13 Cyclo(Pro-Pro) C10H14N2O2 10.64 [M+H]+ 195.1126 −1 70.0672,98.0599
    14 Cyclo(Pro-Ser) C9H16N2O2 14.87 [M+H]+ 185.1283 −0.7 156.0775,140.1046,98.0725,86.0985,69.0707
    15 Cyclo(Tyr-Gly) C11H12N2O3 6.53 [M+H]+ 221.0919 −0.7 164.0742,148.0812,136.0756,91.0555,73.0355
    16 Cyclo(Val-Leu) C11H20N2O2 21.21 [M+H]+ 213.1596 −0.5 140.1425,114.0977,100.0772,86.0962,72.0823,55.0581
    17 Cyclo(Val-Phe) C14H18N2O2 22.83 [M+H]+ 247.1442 0.3 219.1485,148.0749,120.0808,100.0782,72.0828,55.0579
    18 Cyclo(Val-Pro) C10H16N2O2 13.32 [M+H]+ 197.1283 −0.9 169.1340,100.0772,98.0607,72.0828,70.0675,68.0533
    19 Cyclo(Val-Val) C10H18N2O2 17.27 [M+H]+ 199.1439 −0.9 126.1265,100.0787,96.0807,72.0814,55.0573
    20 Cyclo(Pro-Ala) C8H12N2O2 7.00 [M+H]+ 169.0972 0.5 169.0958,141.1026,98.0609,72.0456,70.0673
    21 Cyclo(Pro-Thr) C9H14N2O3 5.82 [M+H]+ 199.1075 −1 181.0982,153.1010,125.0706,98.0608,70.0673,56.0532
    22 Cyclo(Ala-Tyr) C12H14N2O3 11.15 [M+H]+ 235.1075 −1.1 235.1204,207.1121,162.0886,136.0871
    23 Cyclo(Pro-Met) C10H16N2O2S 14.95 [M+H]+ 229.1003 −0.8 181.0959,153.0990,125.0688,84.0456,70.0669
    24 Cyclo(Tyr-Tyr) C18H18N2O4 13.75 [M+H]+ 327.1339 −0.2 164.0774,146.0596,136.0765,107.0501,98.9848
    25 Cyclo(His-Gly) C8H10N4O2 1.01 [M+H]+ 195.0871 −2.9 138.0675,122.0701,110.0714,82.0530,58.0320
    26 Cyclo(His-Phe) C15H16N4O2 11.13 [M+H]+ 285.1345 −0.3 138.0707,110.0709,82.0534
    27 Cyclo(His-Pro) C11H14N4O2 2.19 [M+H]+ 235.119 0 162.1044,110.0729,95.0596,82.0553
    28 Ala-Ile C9H18N2O3 7.52 [M+H]+ 203.1388 −0.9 203.1215,157.1411,140.0710,132.1009,130.0604
    29 Ala-Phe C12H16N2O3 10.87 [M+H]+ 237.1233 −0.2 237.1096,191.1215,166.0910,120.0809
    30 Val-Leu C11H22N2O3 11.98 [M+H]+ 231.1701 −0.8 132.1017,86.0973,72.0824
    31 Thr-Ile C10H20N2O4 8.04 [M+H]+ 233.1494 −0.6 132.0985,86.1021,69.0575,57.0711
    32 Leu-Tyr C15H22N2O4 11.84 [M+H]+ 295.1651 −0.6 182.0837,136.0806,86.0960,69.0750
    33 Val-Val C10H20N2O3 4.73 [M+H]+ 217.1547 0.2 118.0892,72.0817,55.0573
    34 Glu-Val C10H18N2O5 8.99 [M+H]+ 247.1287 −0.4 130.0641,103.0540,86.0977,74.0325
    35 Phe-Pro C14H18N2O3 12.48 [M+H]+ 263.139 0.1 217.1452,120.0809,116.0893,70.0675
    36 PyroGlu-Pro C10H14N2O4 8.60 [M+H]+ 227.1026 −0.1 209.0925,181.0964,116.0704,84.0459,70.0670
    37 PyroGlu-Val C10H16N2O4 10.96 [M+H]+ 229.1182 −0.5 183.1111,155.1209,138.0894,118.0879,84.0448,70.0829
    38 Gly-Ala-Phe C14H19N3O4 13.55 [M+H]+ 294.1449 0.3 276.1220,147.0905,114.0596,84.0453,68.0517
    39 Gly-Pro-Ala C10H17N3O4 1.00 [M+H]+ 244.129 −0.6 185.0894,147.0751,130.0488,112.0498,84.0444,70.0667
    40 Leu-Gly-Leu C14H27N3O4 18.41 [M+H]+ 302.2075 0.3 198.0965,189.1354,171.1067,143.1177,132.1012,86.0973
    41 Ile-Pro-Ile C17H31N3O4 19.20 [M+H]+ 342.2387 −0.1 229.1573,211.1425,183.1487,70.0667
    下载: 导出CSV
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