Skip to main content
NCBI home page
Springer Nature - PMC COVID-19 Collection logoLink to Springer Nature - PMC COVID-19 Collection
. 2019 Sep 28;82(12):1801–1825. doi: 10.1007/s10337-019-03806-w

Qualitative and Quantitative Analysis of 24 Components in Jinlianhua Decoction by UPLC–MS/MS

Mingyue Fang 1, Shuangyue Liu 1, Qingqing Wang 1, Xuan Gu 1, Pengmin Ding 1, Weihua Wang 2, Yi Ding 2, Junxiu Liu 3,✉, Rufeng Wang 1,✉
PMCID: PMC7088050  PMID: 32214429

Abstract

Jinlianhua Decoction (JD), composed of Flos Trollii, Herba Taraxaci, Folium Isatidis, Radix Puerariae Lobatae, and Folium Perillae in a ratio of 6:15:10:10:6, is a prescription for Fengwen which is a group of febrile diseases due to wind in Chinese medicine. It was originally used for the prevention and treatment of severe acute respiratory syndrome (SARS), and could also be used to treat influenza due to their common pathomechanism. To elucidate the unclear pharmacodynamic basis of JD, the LC-QExactive-MS system was used to qualitatively analyze its main components in this study. As a result, 89 compounds were identified and 24 important ones were selected thereby to further perform the simultaneous quantification in 8 batches of JD samples using LC-QTrap-MS with multiple reaction monitoring (MRM). Based on the qualitative and quantitative results in combination with the bioactivities reported, 16 compounds including orientin, 2″-O-β-l-galactopyranosylorientin, puerarin, trollisin I, rosmarinic acid, 2″-O-(2′″-methylbutanoyl) isoswertisin, daidzin, scutellarin, 3′-methoxy puerarin, vitexin, 3′-hydroxy puerarin, 2″-O-(2′″-methylbutanoyl) vitexin, kaempferol, caffeic acid, 3,4-dimethoxybenzoic acid, and cynaroside were determined as the major components of JD. This study provides a useful combinational method for analyzing the major pharmacodynamic substances of JD and lays a foundation for the quality control research of the decoction.

Keywords: Jinlianhua decoction, Qualitatively and quantitatively analysis, UPLC–MS/MS, Flavonoid, Phenolic acid, Alkaloid

Introduction

Jinlianhua decoction (JD) is a prescription for the prevention and treatment of severe acute respiratory syndrome (SARS) issued by State Administration of Traditional Chinese Medicine in 2003. It is an aqueous decoction made of Flos Trollii (the flowers of Trollius chinensis), Herba Taraxaci (the whole plant of Taraxacum mongolicum), Folium Isatidis (the leaves of Isatis indigotica), Radix Puerariae Lobatae (the roots of Pueraria lobata), and Folium Perillae (the leaves of Perilla frutescens) in a ratio of 6:15:10:10:6. The entire prescription has efficacies of clearing away heat and toxic material, and dispelling wind and pathogen, and thus can be used for the treatment of Fengwen disease. According to traditional Chinese medicine (TCM), Fengwen is a group of warm diseases caused by exogenous evils of wind and heat, which occurs in the warm and windy spring or in the late winter. It starts with fever, cold intolerance, headache, cough and other mild pulmonary symptoms. In addition to SARS, influenza in western medicine or so-called seasonal cold in Chinese medicine also belongs to the category of warm diseases [1]. In Chinese medicine, the treatment of warm diseases not only focuses on direct elimination of influenza virus, but also highlights the relationship among virus, organism and drug. Thus, dialectical treatment through improving the state of human body and regulating its capacity of disease resistance is preferred. Studies have shown that the abnormal increase of various inflammatory cytokines as the influenza virus infects the body is the basis of immune damage [2]. TCM not only directly inhibits the replication of influenza virus, but also protects the human body from the damage caused by the virus through regulating the expression of inflammatory factors and the immune network in the body [3]. It is well known that the pharmacological actions of TCM are closely related to its chemical composition. In the previous studies, we have confirmed the anti-influenza effect of JD [4], and have quantitatively analyzed a part of the components in this decoction by high-performance liquid chromatography (HPLC) [5]. However, this is not enough for elucidation of its effective substances because the efficacy of TCM is the result of synergistic effects of multiple effective components [6]. Consequently, the comprehensive study of the composition of JD is of great significance for revealing its acting mechanism.

Although there is still technical difficulty in fully understanding the composition of TCM, ultra-high-performance liquid chromatography (UHPLC) shows great advantages in the separation and analysis of complex systems such as TCM due to its ultra-high efficiency, resolution and sensitivity. UHPLC tandem quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-QExactive-MS) system with high-throughput scanning and multiple detection capabilities can provide high-quality mass spectra and accurate molecular weight of compounds. It has been applied to multi-component microanalysis, so as to realize the separation and qualitative detection of the compounds in complex systems [7]. Another technique quadrupole-linear ion trap mass spectrometry (QTrap), which uses a multiple reaction monitoring information-dependent acquisition-enhanced product ion scanning (MRM-IDA-EPI) detection mode [8], can obtain the high-quality MS/MS spectra of parent ions in the corresponding MRM channel to double characterize the unknown compounds and improve the accuracy of the qualitative analysis. Furthermore, it also can use the high-selectivity and high-sensitivity MRM scan to obtain the peak area of the compounds for quantitative analysis. In view of this, we used LC-QExactive-MS and LC-QTrap-MS to qualitatively and quantitatively analyze the constituents of JD, respectively, to provide a basis for the determination of pharmacodynamic substance basis. In addition, we used morphological identification in combination with DNA barcoding technique [9] to identify the crude drugs used in the preparation of JD decoction.

Materials and Methods

Crude Drugs

The five crude drugs including Flos Trollii, Herba Taraxaci, Folium Isatidis, Radix Puerariae Lobatae, and Folium Perillae were purchased from materia medica markets or drugstores in various regions of China (Table 1), and the voucher specimens have been deposited at the Herbarium of School of Life Sciences, Beijing University of Chinese Medicine.

Table 1.

Morphological and molecular identification of crude drugs

ID Crude drug Sample number Place of purchase Morphological identification Genenbank registration number Molecular identification
HB-2-20171123 Flos Trollii HB-2-FT Anguo, Hebei province Trollius chinensis KC004044.1 Trollius chinensis
Radix Puerariae Lobatae HB-2-RPL Anguo, Hebei province Pueraria lobata KY860933.1 Pueraria lobata
Folium Isatidis HB-2-FI Anguo, Hebei province Polygonum tinctorium MG730606.1 Polygonum tinctorium
Folium Perillae HB-2-FP Anguo, Hebei province Perilla frutescens KT220698.1 Perilla frutescens
Herba Taraxaci HB-2-HT Anguo, Hebei province Taraxacum alaskanum MG219040.1 Taraxacum alaskanum
SZ-1-20171128 Flos Trollii HB-1-FT Anguo, Hebei province Trollius chinensis KC004044.1 Trollius chinensis
Radix Puerariae Lobatae SZ-1-RPL Shenzhen, Guangdong province Pueraria lobata KY860933.1 Pueraria lobata
Folium Isatidis SZ-1-FI Shenzhen, Guangdong province Isatis indigotica MG730905.1 Isatis indigotica
Folium Perillae SZ-1-FP Shenzhen, Guangdong province Perilla frutescens KR082768.1 Perilla frutescens
Herba Taraxaci SZ-1-HT Shenzhen, Guangdong province Taraxacum mongolicum JN407433.1 Taraxacum mongolicum
CD-1-20171127 Flos Trollii CD-1-FT Chengdu, Sichuan province Trollius chinensis KC004044.1 Trollius chinensis
Radix Puerariae Lobatae CD-1-RPL Chengdu, Sichuan province Pueraria thomsonii MG236579.1 Pueraria thomsonii
Folium Isatidis CD-1-FI Chengdu, Sichuan province Isatis indigotica DQ813301.1 Isatis indigotica
Folium Perillae CD-1-FP Chengdu, Sichuan province Perilla frutescens MG731094.1 Perilla frutescens
Herba Taraxaci CD-1-HT Chengdu, Sichuan province Taraxacum mongolicum AY548210.1 Taraxacum mongolicum
HB-1-20171123 Flos Trollii HB-1-FT Anguo, Hebei province Trollius chinensis KC004044.1 Trollius chinensis
Radix Puerariae Lobatae HB-1-RPL Anguo, Hebei province Pueraria lobata KY860933.1 Pueraria lobata
Folium Isatidis HB-1-FI Anguo, Hebei province Isatis indigotica DQ813301.1 Isatis indigotica
Folium Perillae HB-1-FP Anguo, Hebei province Perilla frutescens MG731094.1 Perilla frutescens
Herba Taraxaci HB-1-HT Anguo, Hebei province Taraxacum mongolicum AY548210.1 Taraxacum mongolicum
CD-2-20171127 Flos Trollii HB-1-FT Anguo, Hebei province Trollius chinensis KC004044.1 Trollius chinensis
Radix Puerariae Lobatae CD-2-RPL Chengdu, Sichuan province Pueraria thomsonii MG236579.1 Pueraria thomsonii
Folium Isatidis CD-2-FI Chengdu, Sichuan province Isatis indigotica MG730905.1 Isatis indigotica
Folium Perillae CD-2-FP Chengdu, Sichuan province Perilla frutescens KT210247.1 Perilla frutescens
Herba Taraxaci CD-2-HT Chengdu, Sichuan province Taraxacum mongolicum AY548210.1 Taraxacum mongolicum
GD-1-20171202 Flos Trollii HB-1-FT Anguo, Hebei province Trollius chinensis KC004044.1 Trollius chinensis
Radix Puerariae Lobatae GD-1-RPL Guangzhou, Guangdong province Pueraria thomsonii MG236579.1 Pueraria thomsonii
Folium Isatidis GD-1-FI Guangzhou, Guangdong province Isatis indigotica MG730905.1 Isatis indigotica
Folium Perillae GD-1-FP Guangzhou, Guangdong province Perilla frutescens MG731094.1 Perilla frutescens
Herba Taraxaci GD-1-HT Guangzhou, Guangdong province Taraxacum mongolicum AY548210.1 Taraxacum mongolicum
HF-1-20171219 Flos Trollii HF-1-FT Hefei, Anhui province Trollius chinensis KC004044.1 Trollius chinensis
Radix Puerariae Lobatae HF-1-RPL Hefei, Anhui province Pueraria thomsonii MG236579.1 Pueraria thomsonii
Folium Isatidis HF-1-FI Hefei, Anhui province Isatis indigotica MG730905.1 Isatis indigotica
Folium Perillae HF-1-FP Hefei, Anhui province Perilla frutescens MG731094.1 Perilla frutescens
Herba Taraxaci HF-1-HT Hefei, Anhui province Taraxacum officinale KY860926.1 Taraxacum officinale
CD-3-20171127 Flos Trollii CD-3-FT Chengdu, Sichuan province Trollius chinensis KC004044.1 Trollius chinensis
Radix Puerariae Lobatae CD-3-RPL Chengdu, Sichuan province Pueraria thomsonii MG236579.1 Pueraria thomsonii
Folium Isatidis CD-3-FI Chengdu, Sichuan province Isatis indigotica MG730905.1 Isatis indigotica
Folium Perillae CD-3-FP Chengdu, Sichuan province Perilla frutescens MG731094.1 Perilla frutescens
Herba Taraxaci CD-3-HT Chengdu, Sichuan province Taraxacum mongolicum AY548210.1 Taraxacum mongolicum
Open in a new tab

Reagents and Materials

Thermo UHPLC series quadrupole-electrostatic field orbitrap high-resolution mass (Ultimate3000 QExactive plus LC–MS) spectrometer, the data processing software Xcalibur and Compound Discoverer (CD) 2.1 were the products of Thermo Fisher Scientific (Pittsburgh, PA, USA). Acquity ultra-performance liquid chromatograph (UPLC) was from Waters Corporation (Milford, MA, USA). AB Sciex QTrap 4500 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source and the data processing software Analyst 1.6.1 was manufactured by AB Sciex Pte. Ltd. (Framingham, MA, USA). Sigma 1–14 desktop high-speed centrifuge was the product of Sigma-Aldrich, Inc. (St. Louis, MO, USA). Milli-Q integral water purification system was produced by Millipore Corporation (Bedford, MA, USA). METTLER TOLEDO Xp26 one-millionth electronic analytical balance was from METTLER TOLEDO (Zurich, CH). IKA VORTEX GENIUS 3 vortex mixer was made by Janke & Kunkel KG.IKA-werk (Staufen, DE). LongGene thermal cycle analyzer was provided by Hangzhou Langji Scientific Instrument Co., Ltd. (Hangzhou, Zhejiang, China). BG-gdsAUTO510 gel imaging system was the product of Beijing Bay Gene Biotechnology Co., Ltd. (Beijing, China). DYY-6C electrophoresis apparatus was provided by Beijing Liuyi Device Factory (Beijing, China). KQ-500DE CNC ultrasonic cleaner was provided by Kunshan Ultrasonic Instrument Co. Ltd. (Kunshan, Jiangsu, China). Methanol and acetonitrile of LC–MS grade were manufactured by Fisher Scientific (Pittsburgh, PA, USA). Formic acid of chromatographic grade was the product of Tedia Company, Inc (Fairfield, Ohio, USA). DNA rapid extraction kit for broad spectrum plant genome, 2 × Taq PCR Master Mix enzyme, and BM2000 + DNA Marker were purchased from Beijing Bomaide Biotechnology Co., Ltd. (Beijing, China). Deionized water was purified by Milli-Q system. All other chemicals were available products of at least analytical grade.

Reference compounds including genistin, apigenin, daidzin, kaempferol, rutin, scutellarin, vitexin, ferulic acid, 3′-hydroxy puerarin, 2″-O-β-l-galactopyranosylorientin, indirubin and cynaroside were bought from Shanghai Yuanye Biotech Co., Ltd. (Shanghai, China). 3′-Methoxy puerarin was purchased from Chengdu Push Biotech Co., Ltd. (Chengdu, Sichuan, China). Caffeic acid was from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, Sichuan, China). Orientin was from Pharmacodia (Beijing) Co., Ltd. (Beijing, China). Puerarin and rosmarinic acid were purchased from National Institutes for Food and Drug Control (Beijing, China). Trollioside [10], 3,4-dimethoxybenzoic acid [10], trollisin I [11], proglobeflowery acid [10], 2″-O-(2′″-methylbutanoyl)isoswertisin [11], tecomin [12], and 2″-O-(2′″-methylbutanoyl)vitexin [11] were isolated from Flos Trollii in our lab. The individual purity of each reference was confirmed over 98% according to HPLC analysis.

Morphological and Molecular Identification of Crude Drugs

Morphological Identification

The five crude drugs were morphologically identified by comparing their morphological characteristics including shape, color, odor, size, texture and sectional properties with those recorded in Pharmacopoeia of the People’s Republic of China [13, 14].

Molecular Identification

The five crude drugs (0.1 g) were subject to total DNA extraction using DNA rapid extraction kit of broad spectra plant genome. The sequence of forward primer ITS2F was 5′-ATGCGATACTTGGTGTGAAT-3′, while that of the reverse primer ITS3R was 5′-GACGCTTCTCCAGACTACAAT-3′. The reaction system of PCR included 12.5 µL 2 × Taq PCR Master Mix enzyme, 1.0 µL of forward primer and 1.0 µL of reverse primer (5 μmol L−1), 1.0 µL of total DNA and 9.5 µL of ddH2O. The PCR amplification was carried out using the program compose of 94 °C for 5 min, 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 45 s (40 recycles), then 72 °C for 10 min again. The resultant samples were stored at 4 °C. The samples were sent to Beijing Bomaide Biotechnology Co., Ltd. for sequencing after test with 1% agarose gel electrophoresis. Contig Express 3.0 (Informax., Inc, USA) was used for the assembly and sequence checking. The checked ITS2 sequences were uploaded to the GenBank database for Basic Local Alignment Search Tool (BLAST) comparison to determine whether they were the target ones.

Qualitative Analysis of JD by LC-QExactive-MS

Preparation of Test Samples

In accordance with the proportion of the prescription, a quantity of five crude drugs was taken, and a volume of water was added to extract two times, each for 30 min. The extract was filtered and combined, and then concentrated to each 1 mL containing 0.1 g of crude drug. The concentrate was stored at − 20 °C, and centrifuged at 12,000 r min−1 for 10 min before use. The supernatant was taken as the test solution.

Apparatus and Analytical Conditions

Chromatographic separation was performed with a Waters BEH C18 column (100 mm × 2.1 mm i.d.; 1.7 μm). Gradient elution was performed in 60 min using the mobile phase consisting of acetonitrile with formic acid (0.1% v/v) (A) and 0.1% v/v aqueous formic acid (B) from 5 to 95% A at a flow rate of 0.2 mL min−1. The injection volume was 5 μL.

System used in positive and negative ion modes was coupled with heated electrospray source (HESI), and the spray voltages were 3.5 kV for positive and 2.8 kV for negative. The flow rate of sheath gas was 30 arbitrary units (a.u.), and that of auxiliary gas was 10 a.u.. Other conditions included capillary temperature of 320 °C and resolution of 70,000.

Statistical Analysis

In accordance with the precise molecular weight of the compound obtained from the total ion chromatogram (TIC) and the fragment ions generated in the characteristic mode, Xcalibur and CD 2.1 were used to calculate the possible composition (error less than 5 ppm). Then, based on the characteristic fragment ion information of compounds, the attribution information and the existing relevant composition reported, the possible structure was inferred. The compound designation referred to the ChemSpider database, the Pubmed database, the mzVault database, and the mzCloud database.

Quantitative Analysis of 24 Components by LC-QTrap-MS

Apparatus and Analytical Conditions

Chromatographic separation was performed with an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm i.d.; 1.8 μm). The mobile phase consisted of acetonitrile with formic acid (0.1% v/v) (A) and 0.1% v/v aqueous formic acid (B), and the flow rate was 0.2 mL min−1. Gradient elution was as follows: 0–1 min, 0% A; 1–3 min, 0–20% A; 3–11 min, 20% A; 11–14 min, 20–35% A; 14–17 min, 35–70% A; 17–19 min, 70–100% A; 19–21 min, 100% A; 21–21.1 min, 100–0% A. The injection volume was 5 μL.

System used in negative ion mode was coupled with electrospray ionization source (ESI). The ionspray voltage (IS) was 4500 V, and ionization temperature (TEM) was 500 °C. The nebulizer gas (GS1) and heater gas (GS2) were 50 and 40 psi, respectively. For MRM mode, the precursor ion, product ion, de-clustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) of the 24 measured components are shown in Table 2. Total ion MRM chromatogram of mixed references and samples are shown in Fig. 1.

Table 2.

Optimized structure parameters of 24 components in JD

ID Analyte Precursor ion Product ion DP (volts) EP (volts) CE (volts) CXP (volts)
1 Genistin 431.2 269.1 − 145.0 − 8.0 − 25.0 − 18.0
2 Apigenin 269.0 117.1 − 140.0 − 10.0 − 43.0 − 8.0
3 Trollioside 397.2 235.1 − 145.0 − 10.0 − 20.0 − 17.0
4 3′-Methoxy puerarin 445.2 325.1 − 170.0 − 5.0 − 33.0 − 20.0
5 Daidzin 415.1 252.2 − 170.0 − 5.0 − 36.0 − 15.0
6 Kaempferol 285.0 211.0 − 170.0 − 10.0 − 40.0 − 15.0
7 Caffeic acid 179.0 135.0 − 100.0 − 4.0 − 22.0 − 8.0
8 Puerarin 415.1 295.1 − 150.0 − 6.0 − 32.0 − 19.0
9 Rutin 609.2 300.0 − 220.0 − 10.0 − 50.0 − 20.0
10 Scutellarin 461.1 285.2 − 125.0 − 10.0 − 28.0 − 20.0
11 Vitexin 431.2 311.1 − 160.0 − 4.0 − 31.0 − 20.0
12 Rosmarinic acid 359.1 161.0 − 110.0 − 4.0 − 20.0 − 12.0
13 Ferulic acid 193.0 134.0 − 110.0 − 5.0 − 22.0 − 9.0
14 Orientin 447.2 327.1 − 140.0 − 7.0 − 30.0 − 21.0
15 3,4-Dimethoxybenzoic acid 181.0 137.1 − 130.0 − 9.0 − 16.0 − 9.0
16 3′-Hydroxy puerarin 431.2 311.1 − 165.0 − 6.0 − 32.0 − 20.0
17 Trollisin I 545.2 443.2 − 180.0 − 10.0 − 40.0 − 9.0
18 2″-O-β-l-Galactopyranosylorientin 609.2 327.1 − 170.0 − 10.0 − 42.0 − 20.0
19 Proglobeflowery acid 235.0 191.1 − 130.0 − 9.0 − 20.0 − 14.0
20 2″-O-(2′″-Methylbutanoyl)isoswertisin 529.2 427.2 − 140.0 − 6.0 − 37.0 − 12.0
21 Tecomin 343.2 181.0 − 105.0 − 9.0 − 15.0 − 13.0
22 2″-O-(2′″-Methylbutanoyl)vitexin 515.2 413.2 − 145.0 − 10.0 − 21.0 − 9.0
23 Cynaroside 447.2 285.1 − 170.0 − 10.0 − 35.0 − 17.0
24 Indirubin 261.0 157.1 − 160.0 − 10.0 − 40.0 − 11.0
Open in a new tab
Fig. 1.

Fig. 1

Open in a new tab

Total ion MRM chromatogram of mixed references (left) and samples (right)

Preparation of Reference Solutions

Stock solutions of 24 references (each 1.0 mg mL−1) were individually prepared by dissolving accurately weighed reference compounds in methanol. A mixed reference solution containing all 24 reference compounds was prepared and serially diluted with methanol to appropriate concentrations to produce working solutions for quantitative analysis (0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 ng mL−1). The solutions were stored at − 20 °C before use.

Results

Morphological and Molecular Identification of Crude Drugs

The results of morphological identification were consistent with those of molecular identification (Table 1), which showed that 34 samples out of the total of 40 samples of crude drugs used were from the original plant collected in Chinese pharmacopoeia and the remaining six samples including five samples of Radix Puerariae Lobatae and 1 sample of Folium Isatidis were from the alternative original plants Pueraria thomsonii and Polygonum tinctorium, respectively. All identification results were confirmed by Professor Rufeng Wang.

Qualitative Analysis of JD

The TIC of JD analyzed by LC-QExactive-MS system is shown in Fig. 2. Eighty-nine compounds were deduced and their structures are provided in Tables 3, 4 and Fig. 3, respectively.

Fig. 2.

Fig. 2

Open in a new tab

Total ion chromatogram (TIC) of JD in negative mode (left) and positive mode (right)

Table 3.

Characterization of constituents of JD by LC-QExactive-MS (negative mode)

No. tR(min) Identification Molecular formula [M − H]− Fragment Reference (ChemSpider) Source Bioactivity
1 16.32 Genistin C21H20O10 −/431.0986 311.1[M-H-C4H8O4], 270.0[M-H-C6H9O5], 269.0[M-H-C6H10O5], 268.0[M-H-C6H11O5], 211.0[M-H-C8H12O7] 23 Radix Puerariae Lobatae Antiviral, anti-inflammatory, immune-modulatory
2 23.83 Apigenin C15H10O5 −/269.0456 225.1[M-H-C2H4O], 197.1[M-H-C3H4O2], 159.0[M-H-C6H6O2], 151.0[M-H-C7H3O2], 117.0[M-H-C8H8O3] 1 Herba Taraxaci, Folium Perillae Antiviral, anti-inflammatory, immune-modulatory
4 17.45 3′-Methoxy puerarin C22H22O10 −/445.1140 325.1[M-H-C4H8O4], 310.0[M-H-C5H11O4], 297.1[M-H-C5H8O5], 282.1[M-H-C6H11O5] 10 Radix Puerariae Lobatae –
5 16.88 Daidzin C21H20O9 −/415.1035 – 11 Radix Puerariae Lobatae Anti-inflammatory, immune-modulatory
7 15.40 Caffeic acid C9H8O4 −/180.0423 135.1[M-H-CO2], 135.9[M-H-CHO2] 6 Herba Taraxaci, Folium Perillae Antiviral, anti-inflammatory, immune-modulatory
12 23.54 Rosmarinic acid C18H16O8 −/359.0772 197.0[M-H-C9H6O3], 179.0[M-H-C9H8O4], 161.0[M-H-C9H10O5], 151.0[M-H-C10H8O5], 135.0[M-H-C10H8O6], 133.0[M-H-C10H10O6], 123.0[M-H-C11H8O6], 109.0[M-H-C13H10O6] 12 Folium Perillae Antiviral, anti-inflammatory, immune-modulatory
13 19.79 Ferulic acid C10H10O4 −/194.0578 178.0[M-H–O], 149.1[M-H-CHO2], 134.0[M-H-C2H4O2] 8 Herba Taraxaci, Folium Perillae Antiviral, anti-inflammatory, immune-modulatory
15 12.38 3,4-Dimethoxybenzoic acid C9H10O4 −/181.0507 155.0[M-H-C2H2], 151.0[M-H-C2H6], 136.9[M-H-CO2], 137.0[M-H-CO2], 123.0[M-H-C2H2O2] 3 Flos Trollii Anti-inflammatory
23 22.05 Cynaroside C21H20O11 −/447.0934 285.0[M-H-C6H10O5], 255.0[M-H-C7H12O6], 227.0[M-H-C8H12O7], 151.0[M-H-C13H12O8], 149.0[M-H-C13H14O8], 133.0[M-H-C13H14O9] 8 Herba Taraxaci Antiviral, anti-inflammatory, immune-modulatory
25 24.38 Formononetin C16H12O4 −/267.0659 132.02[M-C8H7O2], 135.0[M-C8H4O2], 167.1[M-C5H8O2], 195.0[M-C4H8O], 196.1[M-C4H6O], 208.1[M-C2H3O2], 226.0[M-C3H5]252.06[M-CH3] 12 Radix Puerariae Lobatae Antiviral, anti-inflammatory
26 10.98 Chlorogenic acid C16H18O9 −/353.0879 59.0[M-C20H38O], 71.0[M-C19H38O], 85.0[M-C19H24O], 87.0[M-C19H22O], 93.0[M-C18H28O], 111.0[M-C16H34O] 4 Herba Taraxaci Anti-inflammatory, immune-modulatory
27 9.29 Protocatechuic acid C7H6O4 −/153.0194 108.0[M-C2H5O], 109.0[M-C2H5O], 111.0[M-], 123.0[M-CH2O] 8 Herba Taraxaci Antiviral, anti-inflammatory
28 1.54 Allantoin C4H6N4O3 −/157.0366 140.0[M-OH], 114.0[M-C2H3O], 113.1[M-C2H4O], 98.0[M-C3H7O], 71.0[M-C5H10O] 2 – Anti-inflammatory
29 1.47 D-Mannitol C6H14O6 −/181.0718 59.0[M-C3H10O4], 71.0[M-C7H10O], 73.0[M-C3H8O4], 87.0[M-C3H10O3], 89.0[M-C4H12O2], 163.1[M-OH] 5 – Anti-inflammatory
30 8.74 Ethyl gallate C9H10O5 −/197.0456 74.0[M-C3H7O5], 95.1[M-C4H6O3], 109.0[M-C3H4O3], 123.0[M-C3H6O2] 4 – Antiviral, anti-inflammatory
31 9.46 Syringol C8H10O3 −/153.0557 – 8 – –
32 2.77 Citric acid C6H8O7 −/191.0198 101.0[M-C2H2O4], 112.0[M-CH3O4], 129.0[M-CH2O3], 147.0[M-COOH] 4 Folium Isatidis Antiviral, anti-inflammatory, immune-modulatory
33 1.75 α-Trehalose C12H22O11 −/341.1088 59.0[M-C10H18O9], 71.0[M-C10H6O9], 85.0[M-C6H8O11], 89.0[M-C11H8O7], 101.0[M-C6H8O10], 119.0[M-C7H10O8], 131.0[M-C6H10O8], 143.0[M-C5H10O8], 149.0[M-C6H8O7], 281.1[M-C2H4O2] 17 – Anti-inflammatory
34 25.66 (±)-Abscisic acid C15H20O4 −/263.1289 83.0[M-C4H4O8], 111.0[M-C4H8O6], 151.1[M-C3H10O4], 153.1[M-C2H6O2] 46 Folium Isatidis Anti-inflammatory, immune-modulatory
35 19.73 Asperulosidic acid C10H24N6O9S −[M-C8O3 + N6S-H]/403.1247 59.01[M-C15H14O5N5], 89.0[M-C9H20O9N3], 121.0[M-C8H18O7N4], 165.0[M-C9H14O2N6], 371.1[M-H2ON] 3 – Anti-inflammatory
36 19.71 Carminic acid C22H16N4O7 −[M-O6H4 + N4-H]/447.0933 269.0[M-C10H12O2N3], 357.1[M-C4H8O4] 10 – Antiviral
37 1.75 D-Raffinose C18H32O16 −/503.1619 59.0[M-C15H24O15], 71.0[M-C14H24O15], 89.0[M-C14H22O14], 101.0[M-C13H22O14], 113.0[M-C12H2O14], 161.1[M-C12H6O12] 1 – –
38 12.38 Esculin C16H12N4O5 −[M-O4H4 + CN4-H]/339.0722 89.02[M-C13H6O2N4], 133.03[M-C2H10O9N2], 177.02[M-C6H12O6] 1 – Anti-inflammatory
39 24.62 Genistein C15H10O5 −/269.0456 91.0[M-C9H6O4], 107.0[M-C6H10O5], 132.0[M-C4H9O5], 180.0[M-C4H9O2], 183.0[M-C4H6O2] 4 Radix Puerariae Lobatae Antiviral, anti-inflammatory, immune-modulatory
40 10.84 l-Dopa C13H12O9 −[M-O5HN + C4-H]/311.0410 135.0[M-C6H8O6], 179.0[M-C5H8O4] 1 – Antiviral
41 20.55 Oxytetracycline C22H16N4O5 −[M-O4H8 + N2-H]/415.1036 117.0[M-C21H14O2], 135.0[M-C13H16O5N2], 253.0[M-C6H14O3N2], 267.1[M-C6H16O2N2] 3 – Antiviral, anti-inflammatory
42 1.74 α-Cyclodextrin C54H94O35 −/1303.3110 71.0[M-C52H64O34], 113.0[M-C53H58O31], 161.0[M-C46H62O33], 221.1[M-C48H58O28], 323.1[M-C42H44O27], 383.1[M-C30H48O32] 1 – –
43 12.96 3′,4′-Dihydroxyphenylacetone C9H10O3 −/165.0556 93.0[M-C4H8O], 95.0[M-C5H10], 122.0[M-C3H7] 1 – –
44 18.68 3-Coumaric acid C9H8O3 −/163.0401 93.0[M-C4H6O], 119.0[M-CO2] 2 – –
45 21.11 4-[4-(4-Hydroxy-3-methoxyphenyl)tetrahydro-1H, 3H-furo[3,4-c]furan-1-yl]-2-methoxyphenyl hexopyranoside C26H32O11 −/519.1874 136.0[M-C19H27O8], 151.0[M-C15H28O10], 161.0[M-C21H26O5], 342.1[M-C7H13O5], 357.1[M-C6H10O5] 1 – –
46 4.92 Adenine C5H5N5 −/134.0473 65.0[M-C2H3N3], 92.0[M-CH2N2], 107.0[M-CHN] 1 – –
47 4.92 Adenosine C10H13N5O4 −/266.0895 92.0[M-C6O2N5], 107.0[M-C2HO4N5], 134.0[M-C2H2O4N3] 1 – Anti-inflammatory, immune-modulatory
48 1.68 D-(-)-Quinic acid C7H12O6 −/191.0562 59.0[M-C4H4O5], 85.0[M-C6H2O2], 93.0[M-C4H2O3], 111.0[M-CH4O4] 2 – Antiviral, anti-inflammatory
49 11.18 Tryptophan C11H12N2O2 −/203.0827 74.0[M-C8HO2], 116.0[M-C6HN], 159.1[M-CH2ON] 1 Folium Perillae Immune-modulatory
50 1.93 Fumaric acid C4H4O4 −/115.0036 – 2 – Anti-inflammatory, immune-modulatory
51 12.35 Gentisic acid C7H6O4 −/153.0194 108.0[M-C H O2], 109.0[M-C2 H4 O] 2 – Anti-inflammatory
52 5.71 Guanosine C10H13N5O5 −/282.0844 108.0[M-C3H2O5N4], 133.0[M-C6HO3N2], 150.0[M-C2H2O4N3] 1 Folium Isatidis Antiviral
53 8.14 Homovanillic acid C9H10O4 −/181.0508 92.99[M-C3H4O3], 94.92[M-C5H10O], 121.03[M-C2H4O2], 136.98[M-C2H4O] 5 – –
54 1.95 Isocitric acid C6H8O7 −/191.0198 57.0, 73.0[M-C3H2O5], 85.0[M-C6H2O2], 111.0[M-CH4O4], 117.0[M-C2H2O3] 1 – –
55 1.43 l-Glutamic acid C5H9NO4 −/146.0459 74.0[M-C2H2O2N], 102.0[M-CH2ON], 128.0[M-H2O] 2 Folium Isatidis –
56 9.46 Pantothenic acid C9H17NO5 −/218.1034 71.0[M-C7H15O3], 88.0[M-C7H14O2], 99.0[M-C5H13O2N], 116.1[M-C6ON] 1 – Anti-inflammatory
57 2.15 Pseudouridine C9H12N2O6 −/243.0623 110.0[M-C3H3O5N], 111.0[M-C6O2N2], 153.0[M-C5ON], 183.0[M-CH2O2N] 1 – –
58 3.34 Uridine C9H12N2O6 −/243.0622 82.0[M-C7HO3N2], 110.0[M-C3H3O5N], 111.0[M-C6O2N2], 124.0[M-C6HO2N] 1 Folium Isatidis Anti-inflammatory
59 1.75 α-Lactose C12H22O11 −/387.1147 59.0[M-C11H20O11], 71.0[M-C10H20O11], 161.0[M-C8H18O7]179.0[M-C8H16O6] 1 – –
Open in a new tab

Table 4.

Characterization of constituents of JD by LC-QExactive-MS (positive mode)

No. tR (min) Identification Molecular formula [M + H]+ Fragment Reference (ChemSpider) Source Bioactivity
3 21.22 Trollioside C19H26O9 +/237.1122([M-C6H11O5 + H]+) 181.0[M + H-C10H19O5], 169.1[M + H-C11H19O5], 137.1[M + H-C12H21O6] 0 Flos Trollii Anti-inflammatory
6 14.78 Kaempferol C15H10O6 +/287.05515 287.1[M + H], 241.1[M + H-CH2O2], 91.1[M + H-C8H4O6] 5 Folium Isatidis Antiviral, anti-inflammatory, immune-modulatory
8 9.43 Puerarin C21H20O9 +/417.1176 399.1[M + H-H2O], 381.1[M + H-H4O2], 281.1[M + H-C4H8O5], 267.1[M + H-C4H6O6], 145.0[M + H-C12H16O7] 4 Radix Puerariae Lobatae Anti-inflammatory, immune-modulatory
9 11.71 Rutin C27H30O16 +/611.16138 303.0[M + H-C11H16O10], 301.1[M + H-C11H18O10], 257.0[M + H-C13H22O11], 229.0[M + H-C14H22O12] 4 Radix Puerariae Lobatae, Folium Isatidis Antiviral, anti-inflammatory, immune-modulatory
10 14.02 Scutellarin C21H18O12 +/463.0873 – 1 Folium Perillae Antiviral, anti-inflammatory, immune-modulatory
11 11.24 Vitexin C21H20O10 +/433.1132 415.1[M + H-CH3], 397.1[M + H-H4O2], 367.1[M + H-CH6O3], 283.1[M + H-C5H10O5], 313.1[M + H-C4H8O4], 337.1[M + H-C5H4O2] 10 Flos Trollii Anti-inflammatory
14 11.01 Orientin C21H20O11 +/449.1081 413.1[M + H-H4O2], 353.1[M + H-C5H4O2], 339.1[M + H-C6H6O2], 329.1[M + H-C4H8O4] 2 Flos Trollii Anti-inflammatory, immune-modulatory
16 8.27 3′-Hydroxy puerarin C21H20O10 +/433.1127 415.1[M + H-H2O], 397.1[M + H-H4O2], 379.1[M + H-H6O3], 367.1[M + H-CH6O3], 351.1[M + H-CH6O4], 313.1[M + H-C4H8O4] 10 Radix Puerariae Lobatae –
17 18.23 Trollisin I C27H30O12 +/547.1815 – 0 Flos Trollii Anti-inflammatory
18 11.08 2″-O-β-l-Galactopyranosylorientin C27H30O16 +/611.1611 431.1[M + H-C6H12O6], 329.1[M + H-C10H18O9], 299.1[M + H-C11H20O10] 4 Flos Trollii Anti-inflammatory
19 21.21 Proglobeflowery acid C13H16O4 +/237.1122 181.0[M + H-C4H8], 169.0[M + H-C4H4O], 69.1[M + H-C9H12O3] 0 Flos Trollii –
20 19.24 2″-O-(2′″-Methylbutanoyl)isoswertisin C27H30O11 +/531.1862 429.1[M + H-C4H6O3], 369.1[M + H-C6H10O5], 313.1[M + H-C4H8], 297.1[M + H-C4H8O], 285.1[M + H-C11H18O6] 0 Flos Trollii Anti-inflammatory
21 7.85 Tecomin C15H20O9 +/327.1075 – 0 Flos Trollii Anti-inflammatory
22 16.98 2″-O-(2‴-Methylbutanoyl)vitexin C26H28O11 +/517.1709 415.1[M + H-C4H6O3], 397.1[M + H-C4H8O4], 313.1[M + H-C9H16O5], 297.1[M + H-C9H16O6], 284.1[M + H-C10H16O5] 1 Flos Trollii Anti-inflammatory
24 24.80 Indirubin C16H10N2O2 +/263.0818 234.1[M-C2H5], 235.1[M-C2H4], 219.1[M-C2H4O], 206.1[M-C2HO2] 1 Folium Isatidis Antiviral, anti-inflammatory, immune-modulatory
60 11.09 Isoquercetin C21H20O12 +/465.1032 137.0[M-C11H20O11], 153.0[M-C18H16O5], 303.1[M-CH6O9], 345.1[M-C3H4O5] 2 Flos Trollii Antiviral, anti-inflammatory
61 23.56 Biochanin A C16H12O5 +/285.0758 149.0[M-C10O], 153.0[M-C4H4O5], 213.1[M-C2O3], 229.1[M-C2O2], 269.0[M-CH4] 12 – Antiviral, anti-inflammatory
62 11.92 Sinapinic acid C11H12O5 +/225.0757 95.0[M-C8H2O2], 121.0[M-C3H4O4], 135.0[M-C2H2O4], 149.0[MC2H4O3] 6 – Anti-inflammatory
63 15.88 Glycitein C16H12O5 +/285.0757 89.0[M-C15O], 135.0[M-C7H2O4], 213.1[M-C3H4O2], 225.1[M-C2H4O2], 257.0[M-C2H4], 269.0[M-CH4] 12 – Anti-inflammatory, immune-modulatory
64 15.68 Daidzein C15H10O4 +/255.0651 65.0[M-C13H2O2], 69.0[M-C10H2O4], 81.0[M-C9H2O4], 105.0[MC7H2O4] 6 Radix Puerariae Lobatae Antiviral, anti-inflammatory
65 18.59 Epimedin C C27H30O11 +[M-C12H20O8 + H]/531.1866 105.1[M-C23H6O9], 313.1[M-C14H2O3] 0 – Anti-inflammatory
66 12.14 Morin C18H12N10O4 +[M + C3H2N10-O3 + H]/433.1131 153.0[M-C9H12O3N8] 10 – Antiviral, anti-inflammatory, immune-modulatory
67 7.65 Neohesperidin C29H28N4O11 +[M + CN4-H6O4 + H]/609.1821 85.0[M-C29H18O9N], 237.0[M-C24H12ON4], 267.1[M-C16H6O9], 449.1[M-C5H4O6] 1 – Antiviral, anti-inflammatory
68 11.03 4-Hydroxyindole C8H7NO +/134.0601 79.0[M-C2HON], 106.1[M-CH2N], 107.0[M-CHN], 116.0[M-CH6] 0 – –
69 14.69 4-O-Methylpinosylvic acid C16H14O4 +/271.0966 165.1[M-C6H2O2], 181.1[M-C2H2O4], 210.1[M-CHO3], 225.1[M-CH2O2], 253.1[M-H2O] 11 – –
70 16.90 5-O-Methylgenistein C16H12O5 +/285.0758 115.0[M-C10H2O3], 153.0[M-C10HO2], 270.0[M-CH3] 12 – –
71 11.61 Chrysin C15H10O4 +/255.0652 95.1[M-C8O4], 129.1 [M-C5H2O4], 153.1[M-C3H2O4] 6 – Antiviral, anti-inflammatory
72 8.58 Esculetin C9H6O4 +/179.0340 89.0[M-C2H2O4], 105.1[M-C2H2O3], 123.0[M-C2O2], 133.0[M-CH2O2], 151.0[M-CO] 0 Folium Perillae Antiviral, anti-inflammatory
73 12.51 Graveolide C15H20O3 +/249.1485 79.0[M-C9H14O3], 91.0[M-C8H14O3], 131.1[M-C7H2O2], 145.1[M-C6O2], 185.1[M-C4O], 213.1[M-H4O2], 231.1[M-H2O] 38 – –
74 15.61 Isoliquiritigenin C15H12O4 +/257.0807 91.0[M-C11H2O2], 163.0[M-C5H2O2] 8 – Antiviral, anti-inflammatory
75 23.68 Neobavaisoflavone C20H18O4 +/323.1280 69.1[M-C17H2O3], 137.0[M-C13H14O], 239.1[M-C3O3], 255.1[M-H4O4], 267.1[M-C2O2] 4 – Anti-inflammatory
76 10.15 N-Methylhernagine C20H23NO4 +/342.1701 191.1[M-C7H3O4], 247.1[M-C4HO2N], 265.1[M-C5HO] 3 – –
77 7.28 Normorphine C16H17NO3 +/272.1281 57.1[M-C14HO2N], 69.1[M-C13HO2N], 91.0[M-C14H13], 107.0[M-C10H13O2], 115.0[M-C9H13ON], 123.0[M-C10H13O], 143.0[M-C7H13O2], 145.1[M-C8HON], 209.1[M-HO3N], 211.1[M-CHO3], 227.1[M-CHO2] 7 – –
78 11.07 Quercetin C15H10O7 +/303.0500 111.0[M-C9H4O5], 137.0[M-C11H2O2], 153.0[M-C7H2O4], 201.0[M-C3H2O4], 229.0[M-C2H2O3], 257.0[M-CH2O2] 2 Flos Trollii, Herba Taraxaci, Folium Perillae Antiviral, anti-inflammatory
79 16.16 Rhoifolin C27H30O14 +/579.1716 153.0[M-C15H22O14], 242.0[M-C27H13] 7 – Anti-inflammatory
80 13.79 Robinetin C15H10O7 +/303.0500 137.0[M-C11H2O2], 229.0[M-C2H2O3], 257.0[M-CH2O2] 2 – Anti-inflammatory
81 28.31 Testosterone undecanoate C30H48O3 +/457.3674 95.1[M-C28H26], 253.2[M-C16H12] 29 – anti-inflammatory
82 1.75 Stachydrine C7H13NO2 +/144.1020 55.5[M-C6HO], 56.0[M-C5H12O], 68.0[M-C4H12O], 70.1[M-C5N], 72.1[M-C2H2O2N], 84.1[M-CH2O2N], 98.1[M-CH2O2], 102.1[M-CON] 1 – –
83 11.94 Keracyanin C27H30O15 +/595.1660 137.0[M-C26H18O8], 171.0[M-C19H20O11], 241.0[M-C9H22O14], 269.0[M-C15H18O8], 287.0[M-C19H16O4], 449.1[M-C8H2O3] 6 – –
84 11.08 Luteolin-3′,7-diglucoside C27H30O16 +/611.1611 137.0[M-C19H22O14], 153.0[M-C26H18O8], 241.0[M-C27H14O2], 287.0[M-C19H16O5], 288.0[M-C12H19O10], 299.0[M-C18H16O5], 449.1[M-CH6O9], 450.1[M-C12HO] 4 – –
85 11.46 Pelargonidin C15H10O5 +/271.0599 121.0[M-C7H2O4], 145.0[M-C5H2O4], 216.0[M-C3H3O] 10 – Antiviral, anti-inflammatory
86 15.76 Taxifolin C15H12O7 +/287.0553 69.0[M-C14H2O3], 123.0[M-C4H4O7], 149.0[M-C6H2O4], 153.0[M-C7H2O3], 157.1[M-C4H2O5], 185.0[M-C3H2O4], 197.0[M-C2H2O4], 203.1[M-C3O3], 213.0[M-C2H2O3], 231.1[M-C2O2], 241.0[M-CH2O2], 269.0[M-H2O] 5 – Antiviral, anti-inflammatory
87 18.39 Diosmetin C16H12O6 +/301.07077 245.1[M-C2O2], 229.0[M-C2O3], 241.0[M-CO3], 187.0[M-C4H2O4], 153.0[M-C4H4O6], 69.0[M-C11H4O6], 16 Herba Taraxaci Antiviral, anti-inflammatory
88 12.61 Luteolin C15H10O6 +/287.05515 153[M-C7H2O3], 241.0[M-CH2O2], 231.1[M-C2O2] 5 Folium Perillae, Flos Trollii Antiviral, anti-inflammatory, immune-modulatory
89 1.84 Trolline C12H13NO3 +/220.0968 202.1[M-H2O], 164.0[M-C2O2] 1 Flos Trollii Antiviral, anti-inflammatory
Open in a new tab

Fig. 3.

Fig. 3

Open in a new tab

The structures of 89 assigned compounds

Validation of Quantitative Analysis

Linearity, Lower Limit of Detection (LLOD), And lower Limit of Quantification (LLOQ)

For the calibration curve established, different concentrations of reference solution were taken for LC–MS/MS analysis in triplicate. Calibration curves for 24 analytes were generated by plotting the average peak areas versus the corresponding concentrations. Good linearity (R ≤ 0.9919) in the tested concentration ranges of the analytes except indirubin was observed. The minimum concentration of linearity solution was selected and then diluted to series of solution, respectively, and the values calculated at signal to noise (S/N) ratios of 3 and 10 were considered as LLOD and LLOQ, respectively (Table 5).

Table 5.

Calibration curves, correlation coefficient (R), linear range, lower limit of detection (LLOD), and lower limit of quantification (LLOQ) of 24 analytes

ID Analyte Calibration curve R Linear range (ng mL−1) LLOD (ng mL−1) LLOQ (ng mL−1)
1 Genistin Y = 2.06 × 104x + 5.93 × 103 0.9989 1–500 0.02 0.06
2 Apigenin Y = 3.14 × 104x − 1.91 × 104 0.9988 1–1000 0.08 0.27
3 Trollioside Y = 4.7 × 104x + 2.7 × 104 0.9985 1–500 0.02 0.08
4 3′-Methoxy puerarin Y = 2.82 × 104x + 6.44 × 103 0.9984 1–500 0.02 0.07
5 Daidzin Y = 1.08 × 104x + 6.35 × 103 0.9986 1–500 0.05 0.15
6 Kaempferol Y = 2.88 × 103x + 425 0.9979 2–500 0.46 1.55
7 Caffeic acid Y = 1.6 × 105x + 8.44 × 104 0.9990 1–100 0.22 0.73
8 Puerarin Y = 4.25 × 104x + 2.91 × 103 0.9994 1–200 0.04 0.12
9 Rutin Y = 1.2 × 104x + 429 0.9977 0.5–1000 0.06 0.20
10 Scutellarin Y = 2.34 × 104x + 2.32 × 103 0.9987 0.5–500 0.08 0.27
11 Vitexin Y = 4.11 × 104x + 1.63 × 103 0.9989 0.5–1000 0.02 0.05
12 Rosmarinic acid Y = 6.38 × 104x − 9.96 × 103 0.9987 0.5–500 0.03 0.11
13 Ferulic acid Y = 2.43 × 104x + 8.91 × 103 0.9977 1–500 0.14 0.45
14 Orientin Y = 3.66 × 104x + 9.17 × 104 0.9955 5–500 0.04 0.13
15 3,4-Dimethoxybenzoic acid Y = 3 × 103x + 4.58 × 103 0.9985 5–500 1.40 4.67
16 3′-Hydroxy puerarin Y = 2.49 × 104x + 1.06 × 104 0.9961 0.5–1000 0.03 0.09
17 Trollisin I Y = 3.66 × 104x − 1.77 × 106 0.9919 50–1000 0.07 0.22
18 2″-O-β-l-Galactopyranosylorientin Y = 1.4 × 104x − 5.99 × 103 0.9996 1–500 0.03 0.09
19 Proglobeflowery acid Y = 1.28 × 103x − 367 0.9992 1–1000 0.20 0.68
20 2″-O-(2′″-Methylbutanoyl)isoswertisin Y = 3.55 × 104x − 9.19 × 104 0.9987 5–500 0.05 0.17
21 Tecomin Y = 1.32 × 103x − 197 0.9981 5–500 0.96 3.19
22 2″-O-(2′″-Methylbutanoyl)vitexin Y = 3.28 × 104x − 5.12 × 103 0.9941 0.5–500 0.04 0.13
23 Cynaroside Y = 3.4 × 104x + 1.48 × 103 0.9980 0.5–500 0.04 0.12
24 Indirubin Y = 630 x − 1.77 × 103 0.9875 10–1000 1.50 5.27
Open in a new tab

Precision, Repeatability and Stability

The precision of the developed method was validated by determining intra-day and inter-day variations and was expressed in the form of relative standard deviations (RSD). Six replicates were analyzed within a day for the intra-day variation assessment, and the experiments were duplicated on three consecutive days for the inter-day variation assessment. The RSD values of the intra-day and inter-day variations were in the ranges of 2.17–4.79% and 0.91–4.95%, respectively. The repeatability was tested using six test solutions prepared according to the same method, and the RSD values were in the range of 1.20–4.95%. Stability was evaluated by analyzing the sample solutions at room temperature at 0, 2, 4, 8, 12, and 24 h, and the RSD values for 24 references were all less than 4.95%. Thus, the developed method exhibited good precision, repeatability, and stability (Table 6).

Table 6.

Precision, repeatability and stability of 24 analytes (n = 6)

ID Analyte Precision Repeatability Stability
Concentration (ng mL−1) Intra-day RSD (%) Concentration (ng mL−1) Inter-day RSD (%) Concentration (μg g−1) RSD (%) Concentration (μg g−1) RSD (%)
1 Genistin 100.88 ± 4.46 4.42 64.93 ± 1.66 2.56 134.92 ± 2.13 1.58 144.19 ± 6.72 4.66
2 Apigenin 99.83 ± 4.09 4.10 35.55 ± 1.47 4.14 2.79 ± 0.09 3.12 4.08 ± 0.12 2.92
3 Trollioside 100.51 ± 3.98 3.96 75.53 ± 3.40 4.50 16.46 ± 0.28 1.73 17.77 ± 0.57 3.18
4 3′-Methoxy puerarin 41.62 ± 1.27 3.04 77.33 ± 2.85 3.69 560.99 ± 15.76 2.81 572.52 ± 26.74 4.67
5 Daidzin 108.69 ± 3.30 3.04 73.62 ± 3.20 4.35 1146.50 ± 36.00 3.14 1220.13 ± 36.36 2.98
6 Kaempferol 104.91 ± 4.07 3.88 59.37 ± 2.84 4.78 30.08 ± 0.56 1.87 31.55 ± 1.56 4.95
7 Caffeic acid 84.94 ± 2.14 2.52 71.82 ± 1.22 1.70 4.57 ± 0.19 4.18 7.77 ± 0.10 1.26
8 Puerarin 87.06 ± 2.85 3.27 79.57 ± 3.94 4.95 1317.35 ± 15.81 1.20 1284.37 ± 30.82 2.40
9 Rutin 124.18 ± 5.77 4.65 70.18 ± 2.65 3.78 4.62 ± 0.13 2.89 4.09 ± 0.16 3.86
10 Scutellarin 91.71 ± 4.19 4.57 65.94 ± 1.15 1.75 36.68 ± 1.02 2.77 37.25 ± 1.08 2.91
11 Vitexin 96.75 ± 4.24 4.39 75.52 ± 2.79 3.69 425.43 ± 20.72 4.87 447.91 ± 19.17 4.28
12 Rosmarinic acid 93.92 ± 4.06 4.32 86.58 ± 2.77 3.20 5.00 ± 0.18 3.59 5.29 ± 0.09 1.69
13 Ferulic acid 91.79 ± 2.07 2.25 84.57 ± 3.86 4.56 3.66 ± 0.18 4.95 3.43 ± 0.09 2.54
14 Orientin 107.86 ± 5.17 4.79 58.38 ± 1.05 1.79 1163.50 ± 40.26 3.46 1185.66 ± 40.55 3.42
15 3,4-Dimethoxybenzoic acid 94.66 ± 2.92 3.09 147.08 ± 5.87 3.99 87.22 ± 3.39 3.89 87.76 ± 3.15 3.59
16 3′-Hydroxy puerarin 118.44 ± 4.82 4.07 78.20 ± 2.90 3.71 103.26 ± 4.09 3.96 106.83 ± 3.57 3.34
17 Trollisin I 103.71 ± 2.44 2.35 14.86 ± 0.53 3.60 105.76 ± 2.87 2.71 150.69 ± 5.06 3.36
18 2″-O-β-l-Galactopyranosylorientin 89.99 ± 3.37 3.74 57.46 ± 1.03 1.80 203.07 ± 4.45 2.19 200.71 ± 4.03 2.01
19 Proglobeflowery acid 81.13 ± 1.76 2.17 35.33 ± 0.32 0.91 13.78 ± 0.52 3.77 14.90 ± 0.54 3.63
20 2″-O-(2′″-Methylbutanoyl)isoswertisin 84.03 ± 3.86 4.59 14.11 ± 0.70 4.94 183.67 ± 5.29 2.88 2.70 ± 0.01 0.18
21 Tecomin 89.54 ± 3.95 4.41 43.27 ± 2.05 4.73 162.24 ± 6.78 4.18 163.73 ± 7.94 4.85
22 2″-O-(2′″-Methylbutanoyl)vitexin 102.21 ± 4.11 4.02 32.22 ± 1.42 4.40 123.50 ± 5.52 4.47 154.40 ± 7.04 4.56
23 Cynaroside 113.11 ± 4.26 3.77 60.04 ± 0.64 1.07 73.37 ± 2.37 3.23 76.69 ± 1.40 1.82
24 Indirubin 51.26 ± 2.88 5.62 22.22 ± 1.56 7.02 6.91 ± 0.90 13.02 17.44 ± 2.02 11.58
Open in a new tab

Recovery

The recovery test was conducted with three concentration levels (low, medium, and high) of the mixed references added to the known amounts of samples. The resulting samples were extracted and analyzed by the proposed method. The whole process was repeated, and the content of each analyte was determined by the corresponding calibration curve. The percentage recoveries were calculated according to the equation: (total detected amount − original amount)/added amount × 100%. The results showed that the recoveries were within the range of 80.35–119.68%, and the RSD value variations were in the range of 0.19–5.08%. Thus, the developed method exhibited good accuracy (Table 7).

Table 7.

Method recoveries for 23 analytes (n = 3)

ID Analyte Initial (μg) Spiked (μg) Detected (μg) Recovery (%) RSD (%)
1 Genistin 79.10 92.49 157.47 ± 3.86 84.73 2.45
92.25 159.5 ± 6.36 87.15 3.99
46.48 119.96 ± 4.39 87.90 3.66
2 Apigenin 6.27 8.27 13.40 ± 0.31 86.22 2.35
6.06 13.30 ± 0.48 112.04 3.62
4.40 11.26 ± 0.55 113.29 4.86
3 Trollioside 21.73 33.97 49.07 ± 2.22 80.48 4.52
22.53 43.12 ± 1.66 94.91 3.86
10.90 32.03 ± 1.46 94.52 4.57
4 3′-Methoxy puerarin 1726.70 2042.37 3822.73 ± 105.13 102.63 2.75
1616.70 3380.00 ± 55.77 102.27 1.65
945.82 2608.83 ± 90.79 93.27 3.48
5 Daidzin 2533.00 3743.00 6057.00 ± 261.66 94.15 4.32
3150.13 5775.24 ± 198.09 102.92 3.43
1431.61 3984.522 ± 137.47 101.39 3.45
6 Kaempferol 149.90 199.23 366.94 ± 16.81 108.94 4.58
148.00 293.00 ± 4.25 96.69 1.45
48.80 197.40 ± 6.53 97.32 3.31
7 Caffeic acid 208.60 302.08 502.54 ± 22.01 97.31 4.38
164.40 354.74 ± 16.11 88.89 4.54
112.97 343.12 ± 11.43 119.07 3.33
8 Puerarin 8983.33 12,733.33 24,150.00 ± 799.37 119.11 3.31
11,248.06 20,969.17 ± 993.94 106.56 4.74
3593.33 12,050.00 ± 506.10 85.34 4.20
9 Rutin 8.76 12.10 20.56 ± 0.73 97.49 3.57
11.10 20.92 ± 0.34 109.55 1.64
5.43 13.85 ± 0.31 93.80 2.27
10 Scutellarin 163.90 186.02 374.61 ± 13.79 113.27 3.68
166.90 298.00 ± 0.57 80.35 0.19
89.59 264.67 ± 11.17 112.48 4.22
11 Vitexin 1206.67 1711.45 2844.81 ± 126.59 95.72 4.45
1466.67 2466.67 ± 81.15 85.91 3.29
809.33 1999.22 ± 92.76 97.93 4.64
12 Rosmarinic acid 273.00 438.84 740.54 ± 28.51 106.54 3.85
290.30 539.16 ± 18.06 91.68 3.35
268.00 547.0 ± 1.42 102.24 0.26
13 Ferulic acid 22.03 50.52 63.98 ± 2.10 83.04 3.28
30.20 46.38 ± 2.00 80.63 4.31
16.52 40.66 ± 1.66 112.82 4.09
14 Orientin 5033.33 6290.00 10,592.00 ± 433.21 88.85 4.09
5421.90 11,385.99 ± 561.33 117.17 4.93
1589.47 6483.64 ± 243.14 91.24 3.75
15 3,4-Dimethoxybenzoic acid 1926.67 2377.63 4542.85 ± 131.29 110.03 2.89
1393.43 3154.21 ± 147.30 88.10 4.67
576.00 2616.00 ± 132.89 119.68 5.08
16 3′-Hydroxy puerarin 1206.67 1712.92 2851.39 ± 127.17 96.02 4.46
1466.67 2466.67 ± 81.15 85.91 3.29
809.33 1999.22 ± 92.76 97.93 4.64
17 Trollisin I 273.00 333.20 636.25 ± 25.00 109.02 3.93
251.00 562.00 ± 28.27 115.14 5.03
197.47 460.04 ± 6.72 94.72 1.46
18 2″-O-β-l-Galactopyranosylorientin 531.33 656.7 1148.23 ± 45.13 93.94 3.93
594.33 1175.00 ± 44.06 108.30 3.75
176.66 737.10 ± 33.61 116.48 4.56
19 Proglobeflowery acid 24.17 32.02 49.92 ± 2.39 80.42 4.79
22.93 45.64 ± 2.17 93.63 4.75
6.80 32.13 ± 1.13 117.05 3.51
20 2″-O-(2′″-Methylbutanoyl)isoswertisin 428.00 557.45 968.85 ± 38.08 97.02 3.93
351.00 816.00 ± 19.82 110.54 2.43
253.99 662.09 ± 16.88 92.16 2.55
21 Tecomin 300.67 376.67 616.00 ± 25.93 83.72 4.21
365.30 611.76 ± 21.41 85.16 3.50
153.55 466.78 ± 17.32 108.18 3.71
22 2″-O-(2′″-Methylbutanoyl)vitexin 221.00 278.57 488.57 ± 23.65 96.05 4.84
232.00 450.00 ± 16.97 98.71 3.77
118.80 348.03 ± 9.43 106.93 2.71
23 Cynaroside 119.80 157.60 283.00 ± 9.91 103.55 3.50
155.84 292.20 ± 9.73 110.63 3.33
102.39 202.48 ± 8.30 80.75 4.10
Open in a new tab

Sample Analysis

The validated UPLC–MS/MS analytical method was applied to simultaneous quantification of 24 components in eight batches of JD. The contents of the compounds (n = 3) were calculated with an external standard method based on their respective calibration curves. The results are listed in Table 8.

Table 8.

The contents (μg g−1 crude drug) of 24 analytes in samples

ID Analyte Batch Average
HB-2-20171123 SZ-1-20171128 CD-1-20171127 HB-1-20171123 CD-2-20171127 GD-1-20171202 HF-1-20171219 CD-3-20171127
1 Genistin 214.67 ± 15.01** 101.67 ± 0.58** 2.19 ± 0.22** 116.00 ± 8.19** 5.58 ± 0.10** 4.45 ± 0.35** 7.82 ± 0.06** 1.69 ± 1.62** 56.75875 ± 79.48
2 Apigenin 9.47 ± 0.29** 9.2 ± 0.21** 8.05 ± 0.71** 7.04 ± 0.73** 30.73 ± 0.64** 14.33 ± 0.21** 2.62 ± 0.07** 10.7 ± 0.44* 11.5175 ± 8.44
3 Trollioside 94.27 ± 5.87** 13.73 ± 0.85** 25.3 ± 1.68** 21.60 ± 0.70** 28.70 ± 0.52 21.53 ± 1.34** 48.83 ± 4.32** 4.38 ± 0.10** 32.2925 ± 28.11
4 3′-Methoxy puerarin 894.67 ± 31.72** 734.67 ± 16.26** 1.27 ± 0.07** 672.00 ± 36.29** 2.58 ± 0.13** 0.47 ± 0.03** 0.38 ± 0.05** 0.56 ± 0.06** 288.325 ± 401.20
5 Daidzin 1143.33 ± 45.09** 831.33 ± 41.02** 23.17 ± 0.57** 998.33 ± 39.27** 84.70 ± 6.68** 27.70 ± 0.62** 45.90 ± 2.55** 13.13 ± 0.21** 395.9488 ± 500.22
6 Kaempferol 364.67 ± 7.23** 245.00 ± 18.36** 5.24 ± 0.18** 97.70 ± 1.81** 58.83 ± 4.41** 482.00 ± 18.08** 34.30 ± 0.70** 471.00 ± 11.27** 219.8425 ± 198.11
7 Caffeic acid 134.67 ± 9.02 156.00 ± 2.00** 47.00 ± 1.01** 115.00 ± 8.19** 121.00 ± 2.65* 206.67 ± 6.66** 109.33 ± 8.96** 195.67 ± 5.51** 135.6675 ± 51.07
8 Puerarin 1283.33 ± 25.17** 1383.33 ± 57.74** 327.33 ± 10.60** 1343.33 ± 20.81** 467.00 ± 43.21** 94.03 ± 2.99** 209.33 ± 2.52** 266.33 ± 5.51** 671.7513 ± 561.11
9 Rutin 11.50 ± 0.87* 53.83 ± 1.76** 5.19 ± 0.24** 12.07 ± 0.40 6.64 ± 0.33** 6.29 ± 0.09** 0.80 ± 0.06** 6.44 ± 0.46** 12.845 ± 16.94
10 Scutellarin 558.33 ± 6.11** 455.67 ± 9.29** 16.67 ± 0.65** 151.67 ± 3.21** 96.70 ± 37.50** 818.00 ± 13.45** 60.20 ± 1.14** 775.33 ± 21.55** 366.5713 ± 327.52
11 Vitexin 823.67 ± 13.50** 599.67 ± 23.71** 4.27 ± 1.01** 631.00 ± 32.23** 4.83 ± 0.81** 0.41 ± 0.12** 2.37 ± 0.41** 2.66 ± 1.74** 258.61 ± 358.81
12 Rosmarinic acid 806.33 ± 28.54** 588.33 ± 17.10** 11.17 ± 0.40** 276.67 ± 7.09** 182.00 ± 6.56** 570.67 ± 16.04** 189.67 ± 9.07** 1066.00 ± 62.19** 461.355 ± 358.83
13 Ferulic acid 23.30 ± 0.52 35.83 ± 2.66** 24.23 ± 0.21 24.70 ± 0.61 22.60 ± 0.78** 22.47 ± 1.53** 16.93 ± 0.31** 27.63 ± 0.35** 24.71125 ± 5.40
14 Orientin 1420.00 ± 30.00** 1360.00 ± 55.68 1426.67 ± 30.55** 1346.67 ± 32.15 1190.00 ± 45.83* 1366.67 ± 41.63* 921.00 ± 7.55** 1263.33 ± 83.27 1286.793 ± 167.48
15 3,4-Dimethoxybenzoic acid 182.33 ± 38.37* 193.33 ± 7.51** 134.67 ± 14.36 191.67 ± 46.23** 73.30 ± 20.29** 120.67 ± 15.14 39.83 ± 4.09** 104.00 ± 18.73 129.975 ± 56.92
16 3′-Hydroxy puerarin 765.67 ± 12.50** 557.67 ± 21.73** 4.67 ± 0.94** 586.67 ± 29.94** 5.19 ± 0.75** 1.09 ± 0.11** 2.91 ± 0.38** 3.17 ± 1.62** 240.88 ± 333.23
17 Trollisin I 491.00 ± 14.73 677.33 ± 21.50** 494.00 ± 11.36 556.00 ± 13.08** 418.00 ± 24.43** 599.67 ± 23.69** 257.00 ± 8.72** 348.67 ± 19.55** 480.2088 ± 136.37
18 2″-O-β-l-Galactopyranosylorientin 1030.00 ± 0.00** 521.00 ± 38.35** 578.00 ± 7.00** 471.67 ± 22.19** 646.00 ± 31.51** 1033.33 ± 32.15** 1243.33 ± 50.33** 441.00 ± 20.07** 745.5413 ± 308.84
19 Proglobeflowery acid 81.27 ± 9.55** 6.80 ± 1.27** 9.00 ± 2.43** 9.41 ± 1.43* 9.00 ± 2.52** 7.87 ± 3.01** 19.53 ± 2.31 1.08 ± 0.40** 17.995 ± 26.06
20 2″-O-(2′″-Methylbutanoyl)isoswertisin 499.00 ± 15.72** 489.00 ± 14.18** 403.00 ± 25.00** 456.33 ± 16.01** 356.00 ± 8.72** 501.33 ± 12.66** 235.67 ± 8.50** 282.00 ± 11.36** 402.7913 ± 102.80
21 Tecomin 111.00 ± 8.72** 71.67 ± 4.83 38.87 ± 15.34 62.87 ± 3.76 38.20 ± 19.15 63.93 ± 11.51 29.77 ± 21.68* 34.53 ± 2.40* 56.36 ± 27.09
22 2″-O-(2‴-Methylbutanoyl)vitexin 244.00 ± 6.24** 264.33 ± 9.29** 268.33 ± 9.29** 250.67 ± 4.51** 187.67 ± 6.81** 257.33 ± 7.77** 142.33 ± 7.09** 164.00 ± 3.46** 222.3325 ± 49.83
23 Cynaroside 241.33 ± 9.50** 80.53 ± 2.83** 52.70 ± 2.00** 113.00 ± 5.29 54.27 ± 1.70** 160.67 ± 3.79** 106.67 ± 2.52 45.73 ± 2.50** 106.8625 ± 66.70
24 Indirubin 8.97 ± 0.84** 122.00 ± 8.89** 45.73 ± 5.52 107.00 ± 15.39** 25.80 ± 0.82** 6.90 ± 1.03** 22.53 ± 2.95** 50.10 ± 5.11 48.62875 ± 43.62
Open in a new tab

** and * indicate P < 0.01 and P < 0.05, respectively

Discussion

Unlike individual crude drugs, decoction of Chinese medicine contains diversified compounds. These compounds are difficult to be characterized by the common analytical procedure because of their complexity. We established a qualitative and quantitative method for analysis of the main components in JD by UHPLC–MS/MS for the first time. The qualitative analysis was performed using LC-QExactive-MS under high resolution and high sensitivity. Based on the resultant precise molecular weight, and mass spectrometry fragmentation of the compounds, the composition of JD has been comprehensively explained. The quantitative analysis was conducted by LC-QTrap-MS. This method is fast and efficient, and does not require complete separation of the chromatographic peaks of multiple components. The precision, repeatability, stability and recovery for the assigned compounds excluding indirubin are in well compliance with the measurement requirements. And the LLOQ is as low as 0.05–4.67 ng mL−1, which can be used for related studies with low component content.

Qualitative and quantitative analyses of JD prepared from eight batches of crude drugs obtained from various places of China were carried out by the above methods. The qualitative results showed that JD mainly contains 89 compounds, and most of them belong to flavonoids, phenolic acids and alkaloids. After these compounds were screened on the basis of their bioactivities related to the efficacy of JD, 24 compounds including 16 flavonoids, 7 phenolic acids and 1 alkaloid were selected to perform the quantitative analysis. The results showed that the compounds whose contents in the decoction were above 100 μg g−1 crude drug included orientin, 2″-O-β-l-galactopyranosylorientin, puerarin, trollisin I, rosmarinic acid, 2″-O-(2′″-methylbutanoyl) isoswertisin, daidzin, scutellarin, 3′-methoxy puerarin, vitexin, 3′-hydroxy puerarin, 2″-O-(2′″-methylbutanoyl) vitexin, kaempferol, caffeic acid, 3,4-dimethoxybenzoic acid, and cynaroside in descending order. These components are mainly from Flos Trollii, Radix Puerariae Lobatae, and Folium Perillae [15–17], and have related bioactivities such as antiviral, antibacterial and anti-inflammatory effects [12, 18–37]. Therefore, these compounds can be considered as the major components of JD.

It was found through statistical analysis that the contents of the 24 components differ greatly in JD prepared from different batches of crude drugs. This might be because the crude drugs of different batches were different in origin, growing environments, preparation process, storage, etc., which resulted in uneven drug quality. For example, based on the results of morphological and molecular identifications, some samples of Radix Puerariae Lobatae used in the experiments were from the roots of P. thomsonii rather than Pueraria lobata. Although the Chinese pharmacopoeia 2005 edition onward has excluded P. thomsonii as the original plant of Puerariae Lobatae, the crude drugs from both original plants are still used indiscriminately in the clinical practice. Thus, the content of puerarin in JD decoction prepared with the roots of Pueraria lobata was significantly higher than that prepared with the roots of P. thomsonii. Our results were also consistent with the findings reported previously [38]. Even for crude drugs of the same origin, their quality is also different due to the diversified growing environment, preparation process and storage condition. The difference of components in the crude drugs and decoctions inevitably affects the consistency of efficacy. The quality control of the chemical components is an important means to guarantee the pharmacological effects of crude drugs and the decoctions [39]. Therefore, the genuine regional crude drugs should be used as much as possible, and the preparation process and storage condition should also be strictly standardized. In addition, indirubin was detected as one of the main components of Folium Isatidis in qualitative analysis; however, it could not be quantified due to its poor ion response. In the methodological test of quantitative analysis, the precision, stability, and repeatability results for indirubin were not qualified. Consequently, the quantitative results for indirubin are for reference only, which may be related to the poor stability of this compound. It has been reported that the content of this compound decreased significantly with the increase of standing time under natural light and room temperature, and slightly reduced even under refrigeration conditions [40].

Conclusions

The qualitative and quantitative analysis methods established in this study are suitable for the analysis and monitoring of the main components in JD. The 16 compounds determined based on the qualitative and quantitative results are the major components of the decoction. This study provides a scientific basis for the determination of pharmacodynamic substances of JD, and lays a foundation for the quality control research of the decoction.

Acknowledgements

We would like to express our thanks to Ms. Lili Li, Meng Chen, Siqi Liu and Jingya Ai, and Mr Yuesheng Pang for their technical support in the experiments.

Funding

This work was supported by Beijing Municipal Natural Science Foundation No. 7172129.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Junxiu Liu, Email: liujunxiusanyuan@sina.com.

Rufeng Wang, Email: wangrufeng@tsinghua.org.cn.

References

  • 1.Ge ZY, Tong J, Wang XY, Na JJ, Zou L, Lu GF. Analysis of TCM about influenza. J Liaoning Univ Tradit Chin Med. 2015;17:77–79. [Google Scholar]
  • 2.Yu XP, Fu HT. Recent research of mechanism of immunoregulation on Chinese medicine anti-influenza-virus actions. Liaoning J Tradit Chin Med. 2006;33:1044–1046. [Google Scholar]
  • 3.Hao Y, Shi LQ, Duan LY, Wu J, Ren JG, Hao QY. Effect of Xuanfei Toujie prescription on lung inflammation and inflammation-related cytokines in mouse with pneumonia induced by influenza virus. Chin J TCM WM Crit Care. 2006;13:198–201. [Google Scholar]
  • 4.Liang YX, Liu XL, Zhang LJ, Wang RF, Hu XH. Experimental study on the effect of the soup of Trollius chinensis from different extraction methods on cell proliferation and antivirus activity in vitro. J Med Res. 2018;47:100–105. [Google Scholar]
  • 5.Wang TY, Li DL, Fang MY, Wang QQ, Liu SY, Geng D. Content assay of five components in Jinlianhua decoction by HPLC. Drug Eval Res. 2018;41:1053–1056. [Google Scholar]
  • 6.Ge H, Wang YF, Xu J, Gu Q, Liu HB, Xiao PG. Anti-influenza agents from traditional Chinese medicine. Nat Prod Rep. 2010;27:1758–1780. doi: 10.1039/c0np00005a. [DOI] [PubMed] [Google Scholar]
  • 7.Li R, Wei W, Qiao X, Liu J, Liang H, Ye M. Chemical profiling of scutellaria barbata by ultra high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry. J Chin Pharm Sci. 2015;24:635–646. [Google Scholar]
  • 8.Yang B, Liu Z, Wang Q, Xia P. Chemical interaction between Lilium Brownii and Rhizoma Anemarrhenae, the herbal constituents of Baihe Zhimu decoction, by liquid chromatography coupled to hybrid triple quadrupole linear ion trap mass spectrometer. Biomed Chromatogr. 2018;32:e4118. doi: 10.1002/bmc.4118. [DOI] [PubMed] [Google Scholar]
  • 9.Ma XY, Xie CX, Liu C, Song JY, Yao H, Luo K. Species identification of medicinal pteridophytes by a DNA barcode marker, the chloroplast psbA-trnH intergenic region. Biol Pharm Bull. 2010;33:1919–1924. doi: 10.1248/bpb.33.1919. [DOI] [PubMed] [Google Scholar]
  • 10.Wang RF, Yang XW, Ma CM, Liu HY, Shang MY, Cai SQ, Hee J, Zhang QY. Trollioside, a new compound from the flowers of Trollius chinensis. J Asian Nat Prod Res. 2004;6:139–144. doi: 10.1080/1028602031000147393. [DOI] [PubMed] [Google Scholar]
  • 11.Cai SQ, Wang RF, Yang XW, Shang MY, Ma CM, Shoyama Y. Antiviral flavonoid-type C-glycosides from the flowers of Trollius chinensis. Chem Biodivers. 2006;3:343–348. doi: 10.1002/cbdv.200690037. [DOI] [PubMed] [Google Scholar]
  • 12.Peng YS, Liu LJ, Zhao C, Guo LN, Wang RF. A phenolic acid from the flowers of Trollius chinensis and its anti-inflammatory and antibacterial activities. Chin Arch Tradit Chin Med. 2015;33:1349–1351. [Google Scholar]
  • 13.Chinese Pharmacopoeia Commission (1977) Pharmacopoeia of the People’s Republic of China, vol I. People’s Medical Publishing House, Beijing, pp 359–360
  • 14.Chinese Pharmacopoeia Commission . Pharmacopoeia of the People’s Republic of China. Beijing: China Medical Science Press; 2015. pp. 21–22. [Google Scholar]
  • 15.Qian Z, Zhou Z, Shi XJ, Han SY, Lv ZW. Advances in studies on the chemical constituents and the extraction and separation of Trollius chinensis Bunge. Chin J Pharm Econ. 2014;9:95–98. [Google Scholar]
  • 16.Li X, Pan JX, Chen SG, Xu WC, Jin MR, Ye XQ. Research progress of chemical components and pharmacological action of Pueraria lobata. J Chin Inst Food Sci Technol. 2017;17:189–195. [Google Scholar]
  • 17.Yang H, Ma P, Lin MB, Hou Q. Research progress in chemical constituents, anti-inflammatory effect and mechanism of Folium Perillae. Chin J Pharmacol Toxicol. 2017;31:279–286. [Google Scholar]
  • 18.Wang XR, An F, Wang SL, An ZX, Wang SH. Orientin attenuates cerebral ischemia/reperfusion injury in rat model through the AQP-4 and TLR4/NF-kappa B/TNF-alpha signaling pathway. J Stroke Cerebrovasc. 2017;26:2199–2214. doi: 10.1016/j.jstrokecerebrovasdis.2017.05.002. [DOI] [PubMed] [Google Scholar]
  • 19.Xing ZH, Ma YC, Li XP, Zhang B, Zhang MD. Research progress of puerarin and its derivatives on anti-inflammatory and anti-gout activities. China J Chin Mater Med. 2017;42:3703–3708. doi: 10.19540/j.cnki.cjcmm.20170907.003. [DOI] [PubMed] [Google Scholar]
  • 20.Li DZ, Hu YF, Yang KP. Protective effect of puerarin on endothelial dysfunction of heat shock protein 60 induced specific immunity in apolipoprotein E-null mice. Chin J Integr Trad West Med. 2006;26:4–6. [PubMed] [Google Scholar]
  • 21.Wu XA, Qin F, Du MQ. The QSAR study on anti-inflammatory activities of C-glycosyflavones. Lishizhen Med Mater Med Res. 2012;23:632–633. [Google Scholar]
  • 22.Jiang KF, Ma XF, Guo S, Zhang T, Zhao G, Wu HC. Anti-inflammatory effects of rosmarinic acid in lipopolysaccharide-induced mastitis in mice. Inflammation. 2018;41:437–448. doi: 10.1007/s10753-017-0700-8. [DOI] [PubMed] [Google Scholar]
  • 23.Zhou D, Li AL, Du GH. Advance in pharmacological research of rosmarinic acid. Chin J New Drugs. 2011;20:594–598. [Google Scholar]
  • 24.Choi JH, Chung MJ, Jeong DY, Oh DH. Immunostimulatory activity of isoflavone-glycosides and ethanol extract from a fermented soybean product in human primary immune cells. J Med Food. 2014;17:1113–1121. doi: 10.1089/jmf.2013.3040. [DOI] [PubMed] [Google Scholar]
  • 25.Liu Y, Jing YY, Zeng CY, Li CG, Xu LH, Yan L. Scutellarin suppresses NLRP3 inflammasome activation in macrophages and protects mice against bacterial sepsis. Front Pharmacol. 2018;8:975. doi: 10.3389/fphar.2017.00975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Wang S, Wang H, Guo H, Kang L, Gao X, Hu L. Neuroprotection of scutellarin is mediated by inhibition of microglial inflammatory activation. Neuroscience. 2011;185:150–160. doi: 10.1016/j.neuroscience.2011.04.005. [DOI] [PubMed] [Google Scholar]
  • 27.Zhang GH, Wang Q, Chen JJ, Zhang XM, Tam SC, Zheng YT. The anti-HIV-1 effect of scutellarin. Biochem Biophys Res Commun. 2005;334:812–816. doi: 10.1016/j.bbrc.2005.06.166. [DOI] [PubMed] [Google Scholar]
  • 28.Zhang S, Guo C, Chen Z, Zhang P, Li J, Li Y. Vitexin alleviates ox-LDL-mediated endothelial injury by inducing autophagy via AMPK signaling activation. Mol Immunol. 2017;85:214–221. doi: 10.1016/j.molimm.2017.02.020. [DOI] [PubMed] [Google Scholar]
  • 29.Lin MK, Yu YL, Chen KC, Chang WT, Lee MS, Yang MJ. Kaempferol from Semen Cuscutae attenuates the immune function of dendritic cells. Immunobiology. 2011;216:1103–1109. doi: 10.1016/j.imbio.2011.05.002. [DOI] [PubMed] [Google Scholar]
  • 30.Onat K, Janine N, Mohamed EMS, Barbara S, Thomas E. Kaempferol is an anti-inflammatory compound with activity towards NF-κB pathway proteins. Anticancer Res. 2015;35:2645–2650. [PubMed] [Google Scholar]
  • 31.Jeong HJ, Ryu YB, Park SJ, Kim JH, Kwon HJ, Kim JH. Neuraminidase inhibitory activities of flavonols isolated from Rhodiola rosea roots and their in vitro anti-influenza viral activities. Bioorgan Med Chem. 2009;17:6816–6823. doi: 10.1016/j.bmc.2009.08.036. [DOI] [PubMed] [Google Scholar]
  • 32.Wang GF, Shi LP, Ren YD, Liu QF, Liu HF, Zhang RJ. Anti-hepatitis B virus activity of chlorogenic acid, quinic acid and caffeic acid in vivo and in vitro. Antivir Res. 2009;83:186–190. doi: 10.1016/j.antiviral.2009.05.002. [DOI] [PubMed] [Google Scholar]
  • 33.Yang WS, Jeong D, Yi YS, Park JG, Hyohyun S, Sang HM. IRAK1/4-targeted anti-inflammatory action of caffeic acid. Mediat Inflamm. 2013;2013:1–12. doi: 10.1155/2013/518183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Qin P, Wei Y, Hou M, Zhao C, Shen Z. A multicenter clinical trial of caffeic acid tablet in treatment of 103 primary immune thrombocytopenia patients. Chin J Hematol. 2015;36:103–106. doi: 10.3760/cma.j.issn.0253-2727.2015.02.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Lee KE, Park JE, Jung E, Ryu J, Kim Y, Youm JK. A study of facial wrinkles improvement effect of veratric acid from cauliflower mushroom through photo-protective mechanisms against UVB irradiation. Arch Dermatol Res. 2016;308:183–192. doi: 10.1007/s00403-016-1633-z. [DOI] [PubMed] [Google Scholar]
  • 36.Li HX, Zhang Q, Liu YN, Liu P, Wang LY, Li DN. Comparative study on anti-inflammatory activity of luteolin and luteoloside. J Yantai Univ. 2018;31:114–120. [Google Scholar]
  • 37.Liu XT, Gu LG, Deng DY, Yu ZN, Wang YQ, Wu J. Effects of baicalin and luteolin-7-O-glucoside on NF-κB signaling pathway in A549 cells infected with influenza virus H1N1 in vitro. Chin J Tradit Chin Med Pharm. 2016;31:1937–1941. [Google Scholar]
  • 38.Wong KH, Razmovski-Naumovskia V, Li KM, Li GQ, Chana K. Comparing morphological, chemical and anti-diabetic characteristics of Puerariae Lobatae Radix and Puerariae Thomsonii Radix. J Ethnopharmacol. 2015;164:53–63. doi: 10.1016/j.jep.2014.12.050. [DOI] [PubMed] [Google Scholar]
  • 39.Tang SH, Chen JX, Wang YP, Wang L, Yang HJ. Research ideas and strategies on the dose-effect relationship of traditional Chinese medicine prescriptions and herbs. J Tradit Chin Med. 2012;32:110–113. doi: 10.1016/S0254-6272(12)60042-9. [DOI] [PubMed] [Google Scholar]
  • 40.Lv HT, Liu J, Shan H, Fang XL. The optimization of extracting indigo and indirubin from Dyers woad leaves by orthogonal experiment. Food Sci Technol. 2011;36:246–250. [Google Scholar]

Articles from Chromatographia are provided here courtesy of Nature Publishing Group

RESOURCES