目的: 建立青白通痹胶囊的指纹图谱,结合化学计量学和网络药理学方法,分析预测青白通痹胶囊潜在的质量标志物（Q-Marker）,并对Q-Marker成分进行含量测定,为其质量控制提供参考。方法: 采用Agilent 5 TC-C18（250 mm×4.6 mm,5 μm）色谱柱,以乙腈（A）-0.1%磷酸水溶液（B）为流动相,梯度洗脱,流速1.0 mL · min-1,柱温30 ℃,进样体积10 µL,紫外检测波长210 nm（0~45 min）、260 nm（45~70 min）,建立指纹图谱并确定共有峰,通过与对照品相对保留时间及紫外光谱的比对进行色谱峰对应化合物的指认,经单煎液对各共有峰进行归属。采用化学计量法对10批青白通痹胶囊进行质量评价,借助正交偏最小二乘-判别分析（OPLS-DA）分析筛选青白通痹胶囊的主要差异性标志物;结合网络药理学,通过相应数据库筛选差异性标志物的核心靶点和关键通路,构建“成分-靶点-通路”网络图,结合上述结果筛选可用于预测青白通痹胶囊的Q-Marker,并建立对预测得到的标志性成分进行含量测定的HPLC方法。结果: 建立了青白通痹胶囊的HPLC指纹图谱,标记24个共有峰,对其进行峰归属,其中1~3、5~8、10~11、13、16号峰来自青风藤,4、9、12、14、16、18~19号峰来自白芍,15、17、20~24号峰来自炙甘草,并指认了其中的8个共有峰,分别为儿茶素、青藤碱、没食子酸、木兰花碱、芍药苷、甘草苷、1,2,3,4,6-O-五没食子酰葡萄糖、甘草酸。相似度评价显示,10批青白通痹胶囊样品的相似度为0.934~1.000。主成分分析（PCA）显示前4个主成分的累积方差贡献率为93.998%,OPLS-DA显示有8个成分变量重要性投影（VIP）值较大。采用网络药理学的方法分析得出儿茶素、青藤碱等活性成分可能通过37个核心靶点、10条主要通路发挥药效作用,初步筛选儿茶素、青藤碱、没食子酸、木兰花碱、芍药苷、甘草苷、1,2,3,4,6-O-五没食子酰葡萄糖、甘草酸为青白通痹胶囊潜在的Q-Marker。同时测定这8个成分的含量,方法学考察结果良好,平均加样回收率为96.81%~103.44%,RSD为0.6%~3.7%。10批样品中儿茶素、青藤碱、没食子酸、木兰花碱、芍药苷、甘草苷、1,2,3,4,6-O-五没食子酰葡萄糖、甘草酸的质量分数分别为0.907 1~1.189 3、2.183 3~3.118 6、 0.397 0~1.427 6、3.507 9~5.446 6、14.207 7~19.570 1、1.412 8~3.577 5、0.442 0~1.697 7、2.738 8~4.761 2 mg · g-1。结论: 本研究建立的青白通痹胶囊指纹图谱方法简单,重复性好,筛选的指标性成分将为青白通痹胶囊的质量控制提供依据。
Objective: To establish the fingerprint of Qingbai Tongbi capsules, and to screen out its indicative compounds for quality control combined with chemometrics methods and network pharmacology. Methods: Agilent 5 TC-C18(250 mm×4.6 mm, 5 μm) chromatographic column was used for separation. The mobile phase was acetonitrile-0.1% phosphoric acid solution for gradient elution at a flow rate of 1.0 mL · min-1, and the column temperature was 30 ℃. The injection volume was 10 μL, and the wavelength was 210 nm (0-45 min), 260 nm (45-70 min). The fingerprint was established and the common peaks were determined. By comparing with the relative retention time and UV spectra of the reference substances, the corresponding compounds of the chromatographic peak were identified. Then the common peaks were identified by single decoction. Chemometrics methods was used to evaluate quality of 10 batches of Qingbai Tongbi capsules, and OPLS-DA analysis was used to screen out the main marker components of Qingbai Tongbi capsules. Combined with network pharmacology, the core targets and key pathways were constructed a “component-target-pathway” network map through corresponding databases. Combined with the above results, indicative compounds for quality control of Qingbai Tongbi capsules were screened out, and an HPLC method was established to determine the content of the Q-Markers. Results: An HPLC fingerprint of Qingbai Tongbi capsules was established, identifying 24 common peaks, and assigning them to different peaks. Among them, peaks 1-3, 5-8, 10-11, 13 and 16 came from Qingfengteng. Peaks 4, 9, 12, 14, 16, 18-19 came from Baishao. Peaks 15, 17, 20-24 came from Zhigancao. Eight common peaks were identified, including catechin, sinomenine, gallic acid, magnoflorine, paeoniflorin, glycyrrhizin, 1, 2, 3, 4, 6-O-pentagalloylglucose, glycyrrhizic acid. The similarity evaluation showed that the similarity of 10 batches of Qingbai Tongbi capsules samples ranged from 0.934-1.000. Principal component analysis (PCA) showed that the cumulative variance contribution rate of the first four principal components was 93.998%, while orthogonal partial least squares-discriminant analysis (OPLS-DA) showed that 8 components had higher variable importance projection values. On this basis, the network pharmacology method was used to analyze and conclude that catechin, sinomenine, gallic acid, magnoflorine, paeoniflorin, glycyrrhizin, 1, 2, 3, 4, 6-O-pentagalloylglucose and glycyrrhizic acid may be the potential Q-Marker of Qingbai Tongbi capsules. The contents of the above eight components were determined simultaneously, and the methodological investigation results were good. The average sample recovery rate was 96.81%-103.44%, and the RSD was 0.6%-3.7%. The mass fractions of catechin, sinomenine, gallic acid, magnoflorine, paeoniflorin, glycyrrhizin, 1, 2, 3, 4, 6-O-pentagalloylglucose and glycyrrhizic acid in 10 batches of samples were 0.907 1-1.189 3 mg · g-1, 2.183 3-3.118 6 mg · g-1, 0.397 0-1.427 6 mg · g-1, 3.507 9-5.446 6 mg · g-1,14.207 7-19.570 1 mg · g-1, 1.412 8-3.577 5 mg · g-1, 0.442 0-1.697 7 mg · g-1, 2.738 8-4.761 2 mg · g-1. Conclusion: The established HPLC fingerprint method is simple and good repeatability. The quality control indicative compounds of Qingbai Tongbi capsules can provide a basis for its quality control.
[1] ALIVERNINI S,FIRESTEIN GS,MCINNES IB.The pathogenesis of rheumatoid arthritis[J].Immunity,2022,55(12):2255
[2] XU Y,ZHAO M,CAO J,et al.Applications and recent advances in transdermal drug delivery systems for the treatment of rheumatoid arthritis[J].Acta Pharm Sin B,2023,13(11):4417
[3] 罗丰,袁雪梅,杨孝余,等.侗族医学治疗类风湿关节炎的研究进展[J].中医药导报,2023,29(2):79
LUO F,YUAN XM,YANG XY,et al.Research progress on the treatment of rheumatoid arthritis by dong medicine[J].Guid J Tradit Chin Med Pharm,2023,29(2):79
[4] 常岑,张润润,时一鸣,等.中医疗法治疗类风湿性关节炎的研究进展[J].中国中药杂志,2023,48(2):329
CHANG C,ZHANG RR,SHI YM,et al.Traditional Chinese medicine therapy for rheumatoid arthritis:a review[J].China J Chin Mater Med,2023,48(2):329
[5] 天津中医学院《素问》整理研究课题组.《黄帝内经素问》校注[J].天津中医学院学报,1985,(3):35
Tianjin University of Traditional Chinese Medicine’s “Su Wen” Compilation and Research Group.Annotated on the《Inner Canon of Huangdi Note》[J].J Tianjin Coll Tradit Chin Med,1985,(3):35
[6] 李晶,张华东,王振兴,等.从《金匮要略 · 中风历节病脉证并治》论痹病发病机制[J].北京中医药,2013,32(6):448
LI J,ZHANG HD,WANG ZX,et al.Discussion on the pathogenesis of Bi disease from the perspective of Synopsis of Jingui:Treatment of Stroke Meridian Syndrome[J].Beijing J Tradit Chin Med,2013,32(6):448
[7] 陈霞,关宏峰,赵巍,等.《中药新药质量标准研究技术指导原则(试行)》解读[J].中国食品药品监管,2021(9):70
CHEN X,GUAN HF,ZHAO W,et al.Interpretation of Guidance on Specifications for New TCM Drugs(Interim)[J].China Food Drug Admin Mag,2021(9):70
[8] 刘昌孝,陈士林,肖小河,等.中药质量标志物(Q-Marker):中药产品质量控制的新概念[J].中草药,2016,47(9):1443
LIU CX,CHEN SL,XIAO XH,et al.A new concept on quality marker of Chinese materia medica:quality control for Chinese medicinal products[J].Chin Tradit Herb Drugs,2016,47(9):1443
[9] LIU C,GUO D A,LIU L.Quality transitivity and traceability system of herbal medicine products based on quality markers[J].Phytomedicine,2018,44:247
[10] 杨慧珍,和润莹,李映,等.沉香的质量标志物预测与指纹图谱分析验证[J].中国现代应用药学,2022,39(3):285
YANG HZ,HE RY,LI Y,et al.Prediction of quality markers and fingerprint analysis of Aquilariae Lignum Resinatum[J].Chin J Mod Appl Pharm,2022,39(3):285
[11] 李佳涵,王慧,刘佳星,等.整合指纹图谱与多成分含量测定的酸枣仁汤质量评价研究[J].中草药,2022,53(15):4698
LI JH,WANG H,LIU JX,et al.Quality evaluation of Suanzaoren decoction by integrating fingerprint and quantitative analysis of multi-component[J].Chin Tradit Herb Drugs,2022,53(15):4698
[12] 王堯,张小飞,邹俊波,等.当归挥发油治疗高血压药理机制的网络分析[J].天然产物研究与开发,2021,33(4):657
WANG Y,ZHANG XF,ZOU JB,et al.Network analysis of the pharmacological mechanism of Angelica sinensis volatile oil in the treatment of hypertension[J].Nat Prod Res Dev,2021,33(4):657
[13] 岳超,张文婷,赵维良,等.化学模式分析联合指纹图谱评价不同基原枳壳药材的质量[J].中国现代应用药学,2021,38(23):3002
YUE C,ZHANG WT,ZHAO WL,et al.Quality evaluation for different original plant species of Aurantii Fructus based on HPLC fingerprint with chemical pattern analysis[J].Chin J Mod Appl Pharm,2021,38(23):3002
[14] 李春霞,何巧玉,刘静,等.基于网络药理学和多元统计分析的黄连质量标志物预测分析[J].中国中药杂志,2021,46(11):2718
LI CX,HE QY,LIU J,et al.Predictive analysis of quality markers of Coptidis Rhizoma based on network pharmacology and multivariate statistical analysis[J].China J Chin Mater Med,2021,46(11):2718
[15] LI S,ZHANG B.Traditional Chinese medicine network pharmacology:theory,methodology and application[J].Chin J Nat Med,2013,11(2):110
[16] 肖卓然,张海萍,何瑜芳,等.丝裂原激活蛋白激酶信号转导通路在类风湿关节炎中的研究进展[J].华西医学,2022,37(3):453
XIAO ZR,ZHANG HP,HE YF,et al.Research progress of mitogen activation protein kinase signaling pathway in rheumatoid arthritis[J].West China Med J,2022,37(3):453
[17] 杨炀,黄鹤,龙成燕,等.基于MAPK通路探讨蒿藤胶囊对Ⅱ型胶原诱导性关节炎大鼠的免疫抑制机制[J].中国医院药学杂志,2022,42(13):1326
YANG Y,HUANG H,LONG CY,et al.Immunosuppressive mechanism of Haoteng capsules in type Ⅱ collagen-induced arthritis rats[J].Chin J Hosp Pharm,2022,42(13):1326
[18] 吴丽,王丽丽,费文婷,等.芍药苷和芍药内酯苷对小鼠疼痛模型的镇痛作用及对β-EP、PGE2的影响[J].中华中医药杂志,2018,33(3):915
WU L,WANG LL,FEI WT,et al.Analgesic effect of paeoniflorin and albiflorin on acetic acid induced writhing mice and the expression of β-EP and PGE2[J].China J Tradit Chin Med Pharm,2018,33(3):915
[19] 张燕丽,田园,付起凤,等.白芍的化学成分和药理作用研究进展[J].中医药学报,2021,49(2):104
ZHANG YL,TIAN Y,FU QF,et al.Research progress of chemical constituents and pharmacological action of Paeonia tactilora Pall.[J].Acta Chin Med Pharmacol,2021,49(2):104
[20] 熊哲,谢家璇,朱鲲博,等.芍药苷调控酸敏感离子通道发挥镇痛作用[J].医药导报,2019,38(11):1403
XIONG Z,XIE JX,ZHU KB,et al.Anti-nociceptive effects of paeoniflorin in formalin-induced pain by regulating acid-sensing ion channels[J].Her Med,2019,38(11):1403
[21] CHEN L,WANG HJ,JI TF,et al.Chemoproteomics-based target profiling of sinomenine reveals multiple protein regulators of inflammation[J].Chem Commun(Camb),2021,57(48):5981
[22] ZHI YK,LI J,YI L,et al.Sinomenine inhibits macrophage M1 polarization by downregulating α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1[J].Phytomedicine,2022,100:154050
[23] IŞIK S,KARAMAN M,MICILI SÇ,et al.Sinomenine ameliorates the airway remodelling,apoptosis of airway epithelial cells,and Th2 immune response in a murine model of chronic asthma[J].Allergol Immunopathol(Madr),2018,46(1):67
[24] YI L,KE J,LIU J,et al.Sinomenine increases adenosine A(2A)receptor and inhibits NF-κB to inhibit arthritis in adjuvant-induced-arthritis rats and fibroblast-like synoviocytes through α7nAChR[J].
J Leukoc Biol,2021,110(6):1113
[25] LIU W,ZHANG Y,ZHU W,et al.Sinomenine inhibits the progression of rheumatoid arthritis by regulating the secretion of inflammatory cytokines and monocyte/macrophage subsets[J].Front Immunol,2018,9:2228
[26] 王钧楠,周永峰,崔园园,等.基于抗炎生物效价的甘草传统感官评价科学性研究[J].中国现代中药,2022,24(6):1018
WANG JN,ZHOU YF,CUI YY,et al.Scientificity of traditional sensory evaluation of licorice based on anti-inflammatory potency[J].Mod Chin Med,2022,24(6):1018
[27] 龚雪. 甘草三萜类化合物甘草酸通过免疫系统影响衰老的作用机制[D].石河子:石河子大学,2023
GONG X.The Mechanism by Which Glycyrrhetinic Acid,A Triterpenoid Compound of Licorice,Affects Aging through the Immune System[D].Shihezi:Shihezi University,2023
[28] KAO TC,SHYU MH,YEN GC.Glycyrrhizic acid and 18 beta-glycyrrhetinic acid inhibit inflammation via PI3K/Akt/GSK3beta signaling and glucocorticoid receptor activation[J].J Agric Food Chem,2010,58(15):8623
[29] MOLLICA L,DE MF,SPITALERI A,et al.Glycyrrhizin binds to high-mobility group box 1 protein and inhibits its cytokine activities[J].Chem Biol,2007,14(4):431
