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Volume 40 Issue 5
Sep.  2022
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WU Jin, ZHAO Jing, PANG Tao. Identification of chemical constituents and blood-absorbed ingredients of FeiPi QiXu decoction by UHPLC-Q-TOF/MS[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(5): 446-453. doi: 10.12206/j.issn.2097-2024.202204079
Citation: WU Jin, ZHAO Jing, PANG Tao. Identification of chemical constituents and blood-absorbed ingredients of FeiPi QiXu decoction by UHPLC-Q-TOF/MS[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(5): 446-453. doi: 10.12206/j.issn.2097-2024.202204079

Identification of chemical constituents and blood-absorbed ingredients of FeiPi QiXu decoction by UHPLC-Q-TOF/MS

doi: 10.12206/j.issn.2097-2024.202204079
  • Received Date: 2022-04-19
  • Rev Recd Date: 2022-08-04
  • Available Online: 2022-09-29
  • Publish Date: 2022-09-25
  •   Objective   To identify the chemical constituents and blood-absorbed ingredients of FeiPi QiXu decoction, and provide reference for clarifying the material basis of its efficacy.   Methods  An ultrahigh performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UHPLC-Q-TOF/MS) method was applied to analyze the data of FeiPi QiXu decoction extraction and the serum samples of SD rats after oral administration. The chemical constituents and blood-absorbed ingredients were quickly identified by chemical reference substance, self-built mass spectrometry database, MSFINDER database and the relevant literature.   Results  A total of 56 chemical constituents were identified from Feiqi Deficiency Decoction, mainly were flavonoids, triterpenoids and phenols. Most of the compounds came from Radix Astragali praeparata cum melle, Citri Reticulatae Pericarpium, Poria and Codonopsis Radix. 11 prototype compounds were identified in the serum, including Formononetin, Astragaloside IV and Neohesperidin.   Conclusion  This study comprehensively clarified the chemical composition and source of medicinal materials, and preliminarily analyzed its blood-absorbed ingredients, which could provide a scientific basis for further study on the pharmacodynamic material basis and quality control of FeiPi QiXu decoction.
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Identification of chemical constituents and blood-absorbed ingredients of FeiPi QiXu decoction by UHPLC-Q-TOF/MS

doi: 10.12206/j.issn.2097-2024.202204079

Abstract:   Objective   To identify the chemical constituents and blood-absorbed ingredients of FeiPi QiXu decoction, and provide reference for clarifying the material basis of its efficacy.   Methods  An ultrahigh performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UHPLC-Q-TOF/MS) method was applied to analyze the data of FeiPi QiXu decoction extraction and the serum samples of SD rats after oral administration. The chemical constituents and blood-absorbed ingredients were quickly identified by chemical reference substance, self-built mass spectrometry database, MSFINDER database and the relevant literature.   Results  A total of 56 chemical constituents were identified from Feiqi Deficiency Decoction, mainly were flavonoids, triterpenoids and phenols. Most of the compounds came from Radix Astragali praeparata cum melle, Citri Reticulatae Pericarpium, Poria and Codonopsis Radix. 11 prototype compounds were identified in the serum, including Formononetin, Astragaloside IV and Neohesperidin.   Conclusion  This study comprehensively clarified the chemical composition and source of medicinal materials, and preliminarily analyzed its blood-absorbed ingredients, which could provide a scientific basis for further study on the pharmacodynamic material basis and quality control of FeiPi QiXu decoction.

WU Jin, ZHAO Jing, PANG Tao. Identification of chemical constituents and blood-absorbed ingredients of FeiPi QiXu decoction by UHPLC-Q-TOF/MS[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(5): 446-453. doi: 10.12206/j.issn.2097-2024.202204079
Citation: WU Jin, ZHAO Jing, PANG Tao. Identification of chemical constituents and blood-absorbed ingredients of FeiPi QiXu decoction by UHPLC-Q-TOF/MS[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(5): 446-453. doi: 10.12206/j.issn.2097-2024.202204079
  • 肺脾气虚汤由法半夏,陈皮,党参,炙黄芪,茯苓,藿香,砂仁等七味中药组成,其中,炙黄芪、党参共为君药,补肺脾之气,益卫固表,法半夏、陈皮、茯苓,健脾祛湿,化痰利肺是为臣药,藿香芳香化浊,辟秽和中,是为佐药,砂仁行气调中,和胃醒脾,为使药。作为从第4版新型冠状病毒感染肺炎诊疗方案便被纳入中医治疗方案的方剂之一,该方主治新冠患者恢复期出现的肺脾气虚证,对气短、倦怠乏力、纳差呕恶、痞满、大便无力、便溏不爽、舌淡胖、苔白腻等症状具有良好疗效[1]。但治疗新冠的中药方剂均由多味中药组成,化学成分复杂,目前诸多文献均侧重于基于公开数据库查询方剂组成的各个药味所含的化合物开展网络药理学研究[2-5],而没有对方剂的化学成分群进行系统考察。其次,中药复方体内与体外之间化学成分具有重要关联,针对复方进入机体内的化学成分群进行分析是有助于发现活性物质的有效手段[6]

    目前尚未见肺脾气虚汤的化学成分及入血成分研究的报道,因此本研究通过UHPLC-Q-TOF/MS技术首次对肺脾气虚汤所含化学成分进行快速分析鉴定,并对化学成分的药材来源进行归属,在此基础上开展其血中药源性成分的辨识,以期为进一步阐明肺脾气虚汤的活性成分,并为今后质量控制提供科学依据。

    • 超高效液相色谱-四极杆-飞行时间质谱仪(UHPLC-Q-TOF/MS)购自美国安捷伦公司,包含1290 Infinity型UHPLC及6530型Q-TOF/MS系统;CPA255D型1/10万电子天平购自德国Sartorius公司;Lyovapor L-200型冷冻干燥机购自瑞士Buchi公司;Centrifuge 5810R 高速台式冷冻离心机购自德国Eppendorf公司;SK7200H型超声波清洗器购自上海科导超声仪器有限公司;Milli-Q型纯化水系统购自美国Millipore公司;HC-800Y高速多功能粉碎机购自武义海纳电器有限公司。

    • 法半夏(批号191115042)、陈皮(批号19090603)、党参(批号19102901)、炙黄芪(批号19650710)、茯苓(批号19122304)、藿香(批号19051410)、砂仁(批号19102211)等饮片均购自吴江上海蔡同德堂中药饮片有限公司,以上样品均经上海中医药大学陈万生教授鉴定为正品。

      琥珀酸(批号A0727AS)、绿原酸(批号J0120AS)、芦丁(批号O0714AS)、芒柄花素(批号N0301AS)等对照品均购自大连美仑生物技术有限公司(纯度>97.0 %)。甲醇、乙腈为质谱纯(德国 Merck 公司);甲酸为质谱纯(美国 Thermo Fisher公司);超纯水由MILI-Q净水系统制备(美国Millipore公司);其他均为分析纯试剂。

    • 本研究所用动物为8周龄雄性SD大鼠,SPF级,购自上海市计划生育科学研究所实验动物经营部(合格证编号20180006021172),大鼠饲养于温度(22~26 ℃)、湿度(40%~70%)、12 h明暗循环可控的室内环境,可自由进食饮水。

    • 精密称取琥珀酸,加入超纯水配置成浓度为1.0 mg/ml的对照品储备液,分别精密称取绿原酸、芦丁、芒柄花素各对照品适量,加入含5% DMSO的甲醇分别配置成浓度为1.0 mg/ml的对照品储备液,所有储备液配制完成后均放于−20 ℃冰箱保存备用。精密吸取上述储备溶液各10 μl,置于1.5 ml离心管中,加入甲醇,涡旋混匀,配制成各对照品浓度均为10 μg/ml 的混合对照品溶液。

    • 将方剂所有药材冷冻干燥,用高速粉碎机分别打粉并过四号筛,并按处方比例混合,精密称取约0.500 g,置于100 ml具塞锥形瓶中,精密加入10倍体积70 %甲醇,密塞、称定重量,超声波提取法提取(760 W,50 kHz)30 min,冷却,70 %甲醇补足失重,滤过,3 000 r/min离心10 min,取1.0 ml上清液于1.5 ml EP管中,12 000 r/min离心 10 min,取200 μl上清液于进样小瓶中,待进样分析。另取方剂10倍体积纯净水浸泡过夜,回流提取1.5 h后用纱布过滤,药渣用8倍体积水继续回流提取1 h,过滤并离心,合并上清液,减压浓缩,得到浸膏供入血成分分析研究。

    • SD大鼠给药前适应性喂养1周后,随机分为2组,即空白对照组与肺脾气虚汤给药组,每组6只。给药组大鼠灌胃给予方剂提取物(10倍临床给药剂量),空白对照组大鼠灌胃给予等体积水。给药1 h后眼眶静脉丛取血1.5 ml于5 ml促凝管中, 室温下静置2 h,4 °C下3 000 r/min离心10 min,取100 μl血清于1.5 ml EP管中,加入4倍体积甲醇,涡旋混匀1 min,4 ℃ 12 000 r/min离心15 min,取上清液,真空冷冻干燥后,加入100 μl 80% 甲醇复溶,涡旋混匀1 min,4 ℃ 12 000 r/min离心15 min,取上清液进行检测。

    • 色谱柱:Waters ACQUITY UPLC BEH C18 色谱柱(2.1 mm×100 mm,1.7 μm);流动相:0.1 %甲酸水(A)-乙腈(B),色谱洗脱梯度见表1,后运行时间为 3 min;流速0.3 ml/min;柱温 30 ℃;进样体积 2 μl,检测波长 254 nm。

      时间(t/min)A(%)B(%)
      0955
      3955
      156535
      29595
      30595

      采用电喷雾离子源(ESI),分别在正、负离子模式下采集数据,数据采集范围 m/z 100~1 700,离子源温度 350 ℃,毛细管电压正离子为 4.0 kV,负离子为 3.5 kV,雾化气压力 45 Psi,干燥气流速 11 L/min,鞘气流速 11 L/min,鞘气温度 350 ℃,碎片电压 140 V,碰撞能量 30 V。

    • 基于中国知网、Pubmed、TCMSP等中英文在线数据库网站,查询肺脾气虚汤各味药材所含化学成分,并汇总其化学成分信息,导入Agilent PCDL Manager B.08 软件构建方剂化学成分数据库。

    • 将采集得到的质谱数据导入到 Agilent MassHunter Qualitative Analysis 10.0软件中,利用前期建立的肺脾气虚汤化学成分数据库初步识别其所含化学成分(绝对峰高≥2 000,匹配容差<±5 ppm),接下来对初步识别的化学成分进行进一步确认。采用对照品及MS DIAL4.70软件结合MS FINDER 3.5.2软件对数据库及离子碎片比对功能确认化合物信息[7]

      肺脾气虚汤在“2.2”及“2.4”项条件下得到的总离子流色谱图见图1。通过导入自建数据库得到肺脾气虚汤的初步鉴定结果,并进一步通过与对照品、MS FINDER数据库比对及查阅文献等方法最终共从肺脾气虚汤鉴定出56种化学成分,见表2。其中正离子模式下识别出8种化学成分,负离子模式下识别出46种化学成分。此外,大豆皂苷 I和茯苓酸在正、负两种模式下均被识别到。这些成分中有4种成分来源于法半夏[13-14],9种成分来源于陈皮,7种成分来源于党参[15-16],10种成分来源于茯苓[17-18],13种成分来源于炙黄芪[19-20],1种成分来源于藿香[21],3种成分来源于砂仁[22]。此外,也有化学成分为多个药材的共有成分,均在表格中标出。化合物来源占比最高的为炙黄芪、党参等药材,与其配伍占比相似。

      编号RT (t/min)m/z离子模式分子式理论分子量实测分子量
      (Tgt)
      误差
      (Tgt, ppm)
      名称类别来源
      10.702175.1183[M+H]+C6H14N4O2174.1111174.1117−3.57L-精氨酸氨基酸类党参
      20.766195.0522[M+Hac-H]、[M-H]C5H4N4O136.0384136.0385−1.06次黄嘌呤生物碱类党参,法半夏
      31.073282.0835[M-H]C10H13N5O5283.0918283.09170.45鸟苷生物碱类法半夏,炙黄芪
      4*#1.153117.0193[M-H]C4H6O4118.0266118.0266−0.37琥珀酸有机酸类党参,法半夏
      51.807124.0391[M+H]+C6H5NO2123.0319123.032−1.2烟酸生物碱类党参
      61.875284.1484[M+H]+C14H21NO5283.141283.142−3.36党参醇生物碱类党参
      72.41268.1534[M+Na]+ , [M+H]+C14H21NO4267.1462267.1471−3.31党参碱生物碱类党参
      8*5.723355.1023[M+K]+C16 H18 O9354.0954354.09510.28绿原酸苯丙素类党参,藿香,砂仁
      95.905169.0495[M+K]+、[M+H]+C8H8O4168.0422168.0423−0.6香草酸酚类党参,砂仁,法半夏
      106.15375.1294[M-H]C17H20N4O6376.1366376.1383−4.53核黄素维生素类炙黄芪
      11#7.141153.0194[M-H]C7H6O4154.0266154.02660.25原儿茶酸酚类藿香,砂仁
      127.653593.1518[M-H]C27H30O15594.159594.15850.87忍冬苷黄酮类陈皮
      138.211741.2245[M+FA-H]、[M-H]C33H42O19742.2316742.232−0.54柚皮苷4’-葡萄糖苷黄酮类陈皮
      148.86193.0508[M-H]C10H10O4194.0581194.05791.05阿魏酸酚酸类党参,法半夏,炙黄芪,砂仁
      158.917163.0399[M-H]C9H8O3164.0474164.04730.324-羟基肉桂酸酚类炙黄芪
      16*9.247609.146[M-H]C27H30O16610.1531610.1534−0.49芦丁黄酮类陈皮
      179.35595.1666[M-H]C27H32O15596.174596.1741−0.24圣草苷黄酮类陈皮
      189.73743.274[M+Na]+ , [M+K]+C32H48O18720.2848720.28411.05铜锤玉带草炔苷B聚乙炔苷党参
      1910.033521.13[M+FA-H]、[M-H]C23H24O11476.1313476.1319−1.21奥刀拉亭-7-O-β-D-葡萄吡喃糖苷黄酮类炙黄芪
      20#10.294137.0246[M-H]C7H6O3138.0319138.03171.67对羟基苯甲酸酚类砂仁
      2110.357579.1721[M-H]C27H32O14580.1796580.17920.64柚皮芸香苷黄酮类陈皮
      2210.454447.0946[M-H]C21H20O11448.1022448.10063.56异红草素黄酮类陈皮
      2310.704431.0988[M-H]C21H20O10432.1063432.10561.52染料木苷黄酮类炙黄芪
      24#11.023609.1824[M-H]C28H34O15610.1899610.18980.19新橙皮苷黄酮类陈皮
      2511.125359.0775[M+FA-H]、[M-H]C17H14O6314.0793314.0790.76熊竹素黄酮类炙黄芪
      2612.833285.041[M-H]C15H10O6286.0484286.04772.16木犀草素黄酮类党参
      2713.334593.1879[M+FA-H]、[M-H]C28H34O14594.1949594.19490.15香蜂草苷黄酮类陈皮
      2813.345285.077[M-H]C16H14O5286.0843286.08410.6甘草查尔酮B黄酮类炙黄芪
      2914.677315.0875[M-H]C17H16O6316.0949316.09470.53垂崖豆藤异黄烷醌黄酮类炙黄芪
      3015.144163.0766[M-H]C10H12O2164.084164.08371.44覆盆子酮酚类砂仁
      3115.896255.0668[M-H]C15H12O4256.0739256.07361.26异甘草素黄酮类炙黄芪,法半夏
      32*#16.761313.072[M+FA-H]C16H12O4268.0743268.07380.72芒柄花素黄酮类炙黄芪
      3316.396991.5086[M+FA-H]、[M-H]C47H78O19946.5111946.5137−2.82黄芪甲苷 VII三萜类炙黄芪
      3416.476283.0613[M-H]C16H12O5284.0686284.06850.34汉黄芩素黄酮类党参,炙黄芪
      3516.647837.3905[M-H]C42H62O17838.3977838.3987−1.2甘草皂苷G2三萜类法半夏
      3616.966297.0403[M-H]C16H10O6298.0476298.0477−0.46sophorophenolone黄酮类炙黄芪
      37#17.102829.4583[M+FA-H]、[M-H]C41H68O14784.4599784.4609−1.33黄芪甲苷 IV三萜类炙黄芪
      3818.241941.5103[M-H]C48H78O18942.5174942.5188−1.46大豆皂苷I三萜类炙黄芪
      18.246943.5236[M+H]+C48 H78 O18942.5149942.5188−4.13大豆皂苷 I炙黄芪
      3919.049499.3421[M-H]C31H48O5500.3495500.3502−1.29茯苓酸H三萜类茯苓
      4019.908891.4684[M+Na]+ , [M+K]+C45 H72 O16868.4793868.482−3.12黄芪甲苷 I三萜类炙黄芪
      41#21.531469.3314[M-H]C30H46O4470.3392470.3396−0.86甘草次酸三萜类法半夏
      4221.793471.3474[M-H]C30H48O4472.3545472.3553−1.67山楂酸三萜类藿香
      4321.963541.3524[M+Hac-H]、[M-H]C31H46O4482.3382482.3396−2.83猪苓酸C三萜类茯苓
      4422.214483.3133[M-H]C30H44O5484.3202484.31892.81茯苓酸B三萜类茯苓
      4522.339483.348[M+Hac-H]、[M-H]C31H48O4484.3551484.3553−0.273-表去氢土莫酸三萜类茯苓
      4622.601485.3635[M-H]C31H50O4486.3706486.3709−0.58块苓酸酚类茯苓
      22.606525.3319[M+K]+、[M+H]+C31 H50 O4486.3693486.3709−3.31茯苓
      4722.817389.1231[M-H]C20H22O8390.1306390.1315−2.23云杉新苷糖苷类砂仁
      4825.242513.3582[M-H]C32H50O5514.3655514.3658−0.68茯苓新酸 HM三萜类茯苓
      4925.527525.3588[M-H]C33H50O5526.3659526.36580.153-表去氢茯苓酸三萜类茯苓
      50#25.902527.3743[M-H]-C33H52O5528.3814528.3815−0.19茯苓酸三萜类茯苓
      5125.982455.3525[M-H]C30H48O3456.3597456.3603−1.47齐墩果酸三萜类党参
      52#27.189279.2333[M+FA-H]、[M-H]C18H32O2280.2404280.24020.62亚油酸脂肪酸类党参,炙黄芪,法半夏
      53#27.394241.2172[M-H]C15H30O2242.2245242.2246−0.22十五烷酸脂肪酸类法半夏
      5427.394485.3265[M-H]C30H46O5486.3334486.3345−2.31茯苓酸G三萜类茯苓
      5528.339255.2329[M-H]C16H32O2256.2402256.2402−0.09棕榈酸脂肪酸类法半夏
      56#28.532281.2487[M-H]C18H34O2282.256282.25590.28顺-10-十七碳烯酸甲酯脂肪酸类陈皮
      注:*为通过与对照品比对确定,#为入血原形成分。

      肺脾气虚汤含有多种有机酸类成分,如原儿茶酸、阿魏酸等,该类成分在质谱中易在能量的撞击下丢失CO2、CH2、CO等分子。如化合物11在7.141 min产生m/z 153.019 4[M-H]峰,判断该化合物的分子式为C7H6O4,在二级质谱扫描下产生 m/z 109.028 8,推测其为m/z 153.019 4丢失一分子CO2产生的碎片离子,通过与对照品及MS FINDER数据库及文献综合比对,鉴定该化合物为原儿茶酸[23],裂解规律见图2。化合物14在8.86 min产生m/z 193.050 8[M-H]的准分子离子峰,判断其分子式为C10H10O4,而二级质谱图中可观察到m/z 178.031 0、m/z 149.060 3、m/z 134.037 6等碎片离子,推测它们分别为准分子离子峰产生的[M-CH2-H]峰、[M-CO2-H]峰、[M-CO2-CO2-H]峰,根据参考文献及MS FINDER数据库推测该化合物为阿魏酸[24]

      黄酮类成分为肺脾气虚汤中检测到的主要类别,该类成分易在质谱扫描下发生糖苷键的断裂、脱羰基、脱甲基及RDA裂解等[25]。如化合物13可在8.211 min观察到m/z 741.2245[M-H]峰,判断其分子式为C33H42O19,在其质谱碎片中观察到m/z 433.1143[M-C12H20O9-H]m/z 271.0632[M-C12H20O9-C6H10O5-H],为其逐步丢失糖苷键产生的碎片离子,结合文献及MS FINDER数据库推测该化合物为柚皮苷4’-葡萄糖苷。化合物32可在16.761 min观察到其准分子离子峰m/z 313.0720,判断其分子式为C16H12O4,并观察到m/z 253.053 6,为该化合物脱去甲基产生的碎片离子[M-CH2-H],与对照品及MS FINDER数据库比对,鉴定该化合物为芒柄花素, 柚皮苷4’-葡萄糖苷及芒柄花素裂解规律见图3

    • 将血清样本数据导入Agilent MassHunter Qualitative Analysis 10.0软件中,并将3.1项中识别得到的化合物构建为带有保留时间的肺脾气虚汤入血成分数据库,根据化合物的保留时间及分子特征共筛选得到肺脾气虚汤的11种入血原形成分(图4表2)。其中,茯苓酸来源于茯苓,琥珀酸来源于党参、法半夏,十五烷酸、棕榈酸来源于法半夏,原儿茶酸来源于藿香、砂仁,对羟基苯甲酸来源于砂仁,芒柄花素和黄芪甲苷IV来源于炙黄芪,新橙皮苷及顺-10-十七碳烯酸甲酯来源于陈皮,亚油酸来源于党参、炙黄芪和法半夏。

    • 新冠肺炎患者恢复期常见的中医证候有肺脾气虚、气阴两虚和余邪未尽。另外,由于患者的脾胃失和气阴两虚,容易出现情志失调。肺脾气虚汤含有黄芪、党参、茯苓、陈皮、半夏、藿香、砂仁等,具有扶正祛邪、补肺固表、益气健脾、燥湿化痰、化浊除湿之功,为香砂六君子汤、参苓白术散、补中益气汤的主要组成药物[7-10]。从现代医学的角度来说,使用上述药物,可以有效防止恢复期患者出现凝血、炎症、免疫和器官功能障碍[11],以及稳定患者情绪防止病情演变的作用[12]

      根据我们的分析结果发现,入血原形成分中原儿茶酸、新橙皮苷等是具有代表性的有效成分,在现代药理学研究中均被证实有助于新冠肺炎患者的“扶正祛邪”。例如,来自于藿香的原儿茶酸由于具有多个酚羟基,已经被实验证实具有抑制蛋白酶3CLpro的功能[26],发挥抗炎活性[27];还可以通过改善海马和大脑皮层中的神经递质、内分泌因子改善抑郁大鼠的行为和神经生化改变[28]。新橙皮苷是黄酮类化合物的主要活性成分,具有很强的抗炎、抗氧化特性,以及神经保护和安神作用[29],近期研究表明其由于具有与人类AAK1蛋白和SARS-CoV-2 NSP16蛋白结合的潜力,被作为一种潜在的双重活性成分而受到关注[30-31]。黄芪甲苷 IV是黄芪的主要活性成分,在机体免疫系统、循环系统、泌尿系统、内分泌系统等多种系统具有正向调节作用[32-33]。上述成分经口服被吸收入血在体内进行循环,证实了方剂多成分多途径发挥功效的特点,是肺脾气虚汤发挥药效的物质基础。

    • 本研究首次运用UHPLC-Q-TOF/MS 对肺脾气虚汤的化学成分和入血成分进行分析,所鉴别出的化合物为其发挥药效的物质基础,该方法准确性高、稳定性好、灵敏度高、分析时间短,证实了基于入血成分的活性成分识别是一种筛选中药活性物质的快速有效方法。

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