Objective  To establish a HPLC method and fingerprints study for the simultaneous determination of loganic acid, gentiopicroside, paeoniflorin, vitexin, liquiritin, and ammonium glycyrrhizinatein, and other components in the alcohol extract of Mongolian medicine Qinggan Manu-4.

Methods  The Agilent Eclipse XDB-C₁₈ column (250 mm×4.6 mm, 5 μm) was used, with the detection wavelength of 230 nm for paeoniflorin, vitexin and liquiritin, 237 nm for loganic acid, 250 nm for glycyrrhizic acid, and 270 nm for gentiopicroside. The mobile phase was 0.1% phosphoric acid aqueous solution-acetonitrile with gradient elution. The flow rate was 1.0 mL/min, the column temperature was 30 ℃, and the injection volume was 10  μL.

Results  The mass concentrations of loganic acid, gentiopicroside, paeoniflorin, vitexin, liquiritin and ammonium gentiopicroside in the alcohol extract of Mongolian medicine Qinggan Manu-4 showed good linear relationships with the chromatographic peak area in the range of 0.020 3-0.121 0, 0.053 3-0.317 7, 0.021 3-0.127 0, 0.011 5-0.069 0, 0.014 1-0.083  8 and 0.035  1-0.209 1 mg/mL, respectively (r≥0.999 8). The average recovery rates of the six components were 94.75%, 99.49%, 92.32%, 95.82%, 101.29% and 98.04%, with the RSDs of 1.11%, 0.76%, 0.99%, 2.75%, 1.09% and 2.43%, respectively (n=6). The HPLC fingerprint of the alcoholic extract of Mongolian medicine Qinggan Manu-4 was established, using the chromatogram of sample S1 as the reference chromatogram, the control fingerprints were generated through multi-point correction and full-spectrum peak matching. A total of ten common peaks were identified, and after comparison with the reference substance, eight components were identified.

Conclusion  The established method and fingerprints are accurate, reliable, reproducible and specific, which provide a basis for the quality control and subsequent development of Mongolian medicine Qinggan Manu-4.

1.内蒙古自治区食品药品监督管理局. 内蒙古蒙药饮片炮制规范2020年版[S]. 2020: 104-105.

2.叶繁庆, 孙雨颉, 范春雨, 等. 龙胆化学成分、生物活性及临床应用研究进展[J]. 中草药, 2023, 54(19): 6497-6510. [Ye FQ, Sun YJ, Fan CY, et al. Research progress on chemical constituents, biological activities and clinical applications of Gentianae Radix et Rhizoma[J]. Chinese Traditional and Herbal Drugs, 2023, 54(19): 6497-6510.] DOI: 10.7501/j.issn.0253-2670.2023.19.030.

3.罗布桑, 主编. 蒙药学[M]. 呼和浩特: 内蒙古人民出版社, 2018: 190-191, 435-436.

4.杨山景, 封安杰, 孙越, 等. 白芍总苷的药理作用及机制研究进展[J]. 中国现代应用药学, 2021, 38(13): 1627-1633. [Yang SJ, Feng AJ, Sun Y, et al. Research progress on mechanism and pharmacological activities of total glucosides of paeony[J]. Chinese Journal of Modern Applied Pharmacy, 2021, 38(13): 1627-1633.] DOI: 10.13748/j.cnki.issn1007-7693. 2021.13.01.

5.邓桃妹, 彭灿, 彭代银, 等. 甘草化学成分和药理作用研究进展及质量标志物的探讨[J]. 中国中药杂志, 2021, 46(11): 2660-2676. [Deng TM, Peng C, Peng  DY, et al. Research progress on chemical constituents and pharmacological effects of Glycyrrhizae Radix et Rhizoma and discussion of Q-markers[J]. China Journal of Chinese Materia Medica, 2021, 46(11): 2660-2676.] DOI: 10.19540/j.cnki.cjcmm.20210304.201.

6.刘长河, 菅单单, 陈胜虎, 等. 基于HPLC指纹图谱与主成分分析相结合的四黄烧伤膏提取工艺研究[J]. 中国药师, 2023, 26(12): 374-381. [Liu CH, Jian  DD, Chen SH, et al. Study on extraction technology for Sihuang Shaoshang ointment based on HPLC fingerprint and principal component analysis[J]. China Pharmacist, 2023, 26(12): 374-381.] DOI: 10.12173/j.issn.1008-049X.202208130.

7.潘旭, 朱鹤云, 张昌浩, 等. 龙胆化学成分和药理作用研究进展[J]. 吉林医药学院学报, 2020, 41(2): 150-151. [Pan X, Zhu HY, Zhang CH, et al. The research progress of the chemical components and the pharmacology of Gentiana scabra Bge[J]. Journal of Jilin Medical University, 2020, 41(2): 150-151.] DOI: 10.13845/j.cnki.issn1673-2995. 2020.02.028.

8.沈涛, 金航, 王元忠, 等. 中药龙胆化学成分研究进展 [J]. 安徽农业科学, 2010, 38(30): 16868-16870, 16874. [Shen T, Jin H, Wang YZ, et al. Research progress in the chemical constituents of Chinese medication Gentiana scabra Bge[J]. Journal of Anhui Agricultural Sciences, 2010, 38(30): 16868-16870, 16874.] DOI: 10.13989/j.cnki.0517-6611.2010.30.185.

9.项昭保, 平雪丽, 屠大伟. 山奈的活性成分及在食品工业中应用的研究进展[J]. 食品工业科技, 2021, 42(17): 448-458. [Xiang SB, Ping XL, Tu DW. Active ingredient of Kaempferia galanga L. and its application in food industry[J]. Science and Technology of Food Industry, 2021, 42(17): 448-458.] DOI: 10.13386/j.issn1002-0306. 2020090040.

10.Kumar A. Phytochemistry, pharmacological activities and uses of traditional medicinal plant Kaempferia galanga L.-an overview[J]. J Ethnopharmacol, 2020, 253: 112667. DOI: 10.1016/j.jep.2020.112667.

11.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): 104-109. [Zhang YL, Tian Y, Fu QF, et al. Research progress of chemical constituents and pharmacological action of Paeonia tactilora Pall[J]. Acta Chinese Medicine and Pharmacology, 2021, 49(2): 104-109.] DOI: 10.19664/j.cnki.1002-2392.210047.

12.文怡欣, 鄂志野, 张珊, 等. 甘草苷对刀豆蛋白A诱导的实验性肝损伤的保护作用及机制[J]. 牡丹江医学院学报, 2022, 43(3): 16-19, 79. [Wen YX, E ZH, Zhang S, et al. Effect of glycyrrhizin on experimental liver injury induced by concanavalin A[J]. Journal of Mudanjiang Medical University, 2022, 43(3): 16-19, 79.] DOI: 10.13799/j.cnki.mdjyxyxb.2022.03.003.

13.张芬芬, 程梦迪, 周珊珊, 等. 甘草苷对急性灌酒小鼠肝损伤的保护作用[J]. 阜阳师范大学学报(自然科学版), 2021, 38(2): 69-72, 84. [Zhang FF, Cheng  MD, Zhou  SS, et al. Repair effect of liquiritin on liver injury in mice with acute alcohol inundation[J]. Journal of Fuyang Normal University (Natural Science), 2021, 38(2): 69-72, 84.] DOI: 10.14096/j.cnki.cn34-1069/n/2096-9341(2021) 02-0069-04.

14.陈云华, 万新, 孙建宁, 等. 甘草酸、甘草苷、异甘草素对醋氨酚人肝细胞损伤模型的保护作用比较[J]. 中国实验方剂学杂志, 2012, 18(4): 245-248. [Chen  YH, Wan X, Sun JN, et al. Hepato-protective activity of glycyrrhizin, liquiritin and isoliquiritigenin on HL-7702 cells injury induced by acetaminophen[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2012, 18(4): 245-248.] DOI: 10.13422/j.cnki.syfjx.2012.04.072.

15.Yang HX, Shang Y, Jin Q, et al. Gentiopicroside ameliorates the progression from hepatic steatosis to fibrosis induced by chronic alcohol intake[J]. Biomol Ther (Seoul), 2020, 28(4): 320-327. DOI: 10.4062/biomolther. 2020.008.

16.孙晓可, 杨飒, 孟祥波, 等. 不同配比18α-与18β-甘草酸对酒精性肝损伤大鼠肝功能的影响[J]. 江西师范大学学报(自然科学版), 2018, 42(2): 166-170. [Sun  XK, Yang S, Meng XB, et al. Effect of different proportion of 18α-, 18β- glycyrrhizic acid on the liver functions and inflammatory cytokines in alcoholic liver disease rat[J]. Journal of Jiangxi Normal University (Natural Science) 2018, 42(2): 166-170.] DOI: 10.16357/j.cnki.issn1000-5862.2018.02.09.

17.Tu CT, Li J, Wang FP, et al. Glycyrrhizin regulates CD4+ T cell response during liver fibrogenesis via JNK, ERK and PI3K/AKT pathway[J]. Int Immunopharmacol, 2012, 14(4): 410-421. DOI: 10.1016/j.intimp.2012.08.013.

18.张晔. 芍药苷对各类型肝损伤保护作用的实验研究进展[J]. 现代中西医结合杂志, 2019, 28(6): 681-684. [Zhang Y. Progress of experimental research on the protective effect of paeoniflorin on various types of liver injury[J]. Modern Journal of Integrated Traditional Chinese and Western Medicine, 2019, 28(6): 681-684.] DOI: 10.3969/j.issn.1008-8849.2019.06.032.

19.陈圆圆. 大孔树脂富集纯化蒙药查那-4中总苷、总环烯醚萜、总皂苷工艺研究及其质量控制[D]. 呼和浩特: 内蒙古医科大学, 2023.

20.卓静, 葛雪松, 孙桐, 等. HPLC法同时测定复方硫酸软骨素片中芍药苷甘草苷和甘草酸的含量[J]. 中国药品标准, 2022, 23(6): 643-647. [Zhuo J, Ge XS, Sun T, et al. Simultaneous determination of paeoniflorin, liquiritin andglycyrrhizic acid in compound chondroitinsulfate tablets by HPLC[J]. Drug Standards of China, 2022, 23(6): 643-647.] DOI: 10.19778/j.chp.2022.06.015.

21.眭荣春, 李金林, 周玉春, 等. HPLC法测定蕲蛇药酒中马钱苷酸和龙胆苦苷的含量[J]. 药品评价, 2022, 19(23): 1423-1426. [Sui RC, Li JL, Zhou, YC, et al. Determination of loganic acid and gentiopicroside in Agkistrodon medicinal liquor by HPLC[J]. Drug Evaluation, 2022, 19(23): 1423-1426.] DOI: 10.19939/j.cnki.1672-2809.2022.23.04.

22.陈晓玲. HPLC法同时测定十全大补丸中8个成分的含量[J]. 中国药师, 2021, 24(4): 770-773. [Chen  XL. Simultaneous determination of eight constituents in Shiquan Dabu pills by HPLC[J]. China Pharmacist, 2021, 24(4): 770-773.] DOI: 10.3969/j.issn.1008-049X.2021.04.035.

23.李维业, 刘学良, 刘安平, 等. 藏药抱茎獐牙菜HPLC指纹图谱及10个成分含量测定研究[J]. 药物分析杂志, 2023, 43(2): 209-218. [Li WY, Liu XL, Liu AP, et al. Study on the chromatographic fingerprint and 10 components of Tibetan medicine Swertia franchetiana H. Smith by HPLC[J]. Chinese Journal of Pharmaceutical Analysis, 2023, 43(2): 209-218.] DOI: 10.16155/j.0254-1793.2023.02.04.

24.马海生, 钱建波. 前列欣胶囊HPLC指纹图谱及多成分含量测定的研究[J]. 药物流行病学杂志, 2023, 32(5): 538-544. [Ma HS, Qian JB. HPLC fingerprint and multi-component content determination of Qianliexin capsules[J]. Chinese Journal of Pharmacoepidemiology, 2023, 32(5): 538-544.] DOI: 10.19960/j.issn.1005-0698.202305008.

25.姚晓璇, 张兰兰, 施文婷, 等. 一测多评法同时测定蔓荆子配方颗粒中5种化学成分的含量[J]. 药物流行病学杂志, 2023, 32(7): 788-795. [Yao XX, Zhang LL, Shi WT, et al. Simultaneous determination of the contents of 5 components in viticis fructus formula granules by QAMS method[J]. Chinese lournal of Pharmacoepidemiology, 2023, 32(7): 788-795.] DOI: 10.19960/j.issn.1005-0698.202307009.

26.中国药典2020年版. 四部[S]. 2020: 480-481.