Exploring the effective components and potential mechanisms of Zukamu granules against acute upper respiratory tract infections by UHPLC-Q-Exactive Orbitrap-MS and network pharmacology analysis

Kailin Li; Qian Yao; Min Zhang; Qing Li; Lilan Guo; Jing Li; Jianbo Yang; Wei Cai

doi:10.1016/j.arabjc.2023.104875

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Original article

08 2023

:16;

104875

doi:

10.1016/j.arabjc.2023.104875

Exploring the effective components and potential mechanisms of Zukamu granules against acute upper respiratory tract infections by UHPLC-Q-Exactive Orbitrap-MS and network pharmacology analysis

Kailin Li^{^a,^b,1}, Qian Yao^{^a,1}, Min Zhang^{^b}, Qing Li^{^b}, Lilan Guo^{^b}, Jing Li^{^b}, Jianbo Yang^{^c,^d,⁎}, Wei Cai^{^a,^b,⁎}

a Weifang Medical University, Weifang 261053, China

b Hunan Province Key Laboratory for Antibody-based Drug and Intelligent Delivery System, School of Pharmaceutical Sciences, Hunan University of Medicine, Huaihua 418000, China

c National Institutes for Food and Drug Control, Beijing 100050, China

d Xinjiang Uygur Autonomous Region Institute for Drug Control, Urumqi 830054, China

⁎Corresponding authors at: Hunan Province Key Laboratory for Antibody-based Drug and Intelligent Delivery System, School of Pharmaceutical Sciences, Hunan University of Medicine, Huaihua 418000, China (W. Cai), National Institutes for Food and Drug Control, Beijing 100050, China; Xinjiang Uygur Autonomous Region Institute for Drug Control, Urumqi 830054, China (J.B. Yang). yangjianbo@nifdc.org.cn (Jianbo Yang), 20120941161@bucm.edu.cn (Wei Cai)

Received: 2023-2-6, Accepted: 2023-3-28,

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

1 These authors contributed equally to this work.

Abstract

Acute upper respiratory tract infections (AURTIs) are common diseases of respiratory system, which are caused by adenoviruses and generate the mix of severe clinical presentation. Zukamu granules (ZKMG), a traditional Chinese medicine (TCM) prescription within Health Commission of Xinjiang Uygur Autonomous Region possesses anti-influenza virus, antibacterial and anti-inflammatory effects that exerts therapeutic effects in treatment of AURTIs. However, the main effective chemical components and their corresponding action mechanisms have not been clarified. Therefore, ultra-performance liquid chromatography coupled with quadrupole Exactive orbitrap tandem mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) and network pharmacology were used to detect and identify potentially effective components in ZKMG as well as uncover their pharmacological mechanisms against AURTIs. As a result, a total of 265 components from all 12 composed herbal medicines were characterized based on self-built database and fragmentation patterns, of which 38 compounds were unambiguously confirmed using reference standards. Then, the compound-target-pathway network was constructed that implied potential therapeutic mechanisms of ZKMG on AURTIs. Compounds noscapine, cryptopine, steviol-19-O-glucoside, N-methylnarcotine, allocryptopine, naringenin, boldine, methyl rosmarinate with related targets EGFR, PTGS2, IL2, MMP9, TNF, AKT1, PIK3CA, F3 were considered as the key components and targets. Besides, the results also indicated that PI3K-Akt, AGE-RAGE, PD-L1, HIF-1 signaling pathways contributed significantly to the therapeutic effects of ZKMG on AURTIs. Overall, ZKMG could have an effect on AURTIs based on multicomponent, multitarget, and multichannel mechanisms of action as well as this method provides guiding significance for the further development of TCM treatment.

Keywords

Zukamu granules

Acute upper respiratory tract infections

Effective components

Mechanisms

UHPLC-Q-Exactive Orbitrap-MS

Network pharmacology

1 Introduction

Acute upper respiratory tract infections (AURTIs) are one of the most common respiratory infectious diseases characterized by tonsillitis, pharyngitis, laryngitis, rhinitis, angina and the common cold, which are primarily caused by viruses rather than bacteria, fungi, and helminths (Huang et al., 2019; Xu et al., 2022). It was reported that the global incidence of AURTIs was 17.1 billion worldwide in 2017, which has brought about tremendous socioeconomic burden to public health (James et al., 2018). Although, the majority of AURTIs are slight and self-limited, they could result in undesirable clinical outcomes, as well as their complications might develop into serious diseases and threaten the security of the infected (Huang et al., 2019). The conventional treatment for AURTIs are preliminary medicines that relieve symptoms but do not treat disease infections, such as antipyretic analgesics and antihistamines (Pelzman and Tung, 2021). Antibiotics only for specific AURTIs and validated clinical indications are recommended, but most of antibiotics for common AURTIs are needless and ineffective (Wang et al., 2014; Harris et al., 2016; Zhang et al., 2021 ). Additionally, the inappropriate utilization of antibiotics could lead to the emergence of resistant bacteria and increase treatment difficulties in clinical practice (Alrafiaah et al., 2017). Therefore, it is necessary that new therapeutic drugs are explored for AURTIs treatment.

Traditional Chinese medicine (TCM) has the advantages of low toxicity and less adverse reactions, reversing multidrug resistance and reducing drug dosage, which has been widely considered to be effective method for various diseases because of its characteristics of multicomponents and multitargets (Chen et al., 2021; Kong et al., 2021). It is by using TCM that AURTIs have been cured for more than 1800 years in China and TCM plays a significant role in disease prevention (Zeng et al., 2022). Zukamu granules (ZKMG) as a famous classical formula was first recorded in the Uyghur medical book karibatin kader over 1500 years ago, which was known as the first choice for the treatment of colds in Uighur (Hou et al., 2019; Zeng et al., 2021). ZKMG is composed of Kaempferia galanga (KG), Pericarpium papaveris (Papaver somniferum) (PP), Matricaria recutita (MR), Ziziphus jujuba (ZJ), Mentha canadensis (MC), Glycyrrhiza uralensis (GU), Althaea rosea (AL), Nymphaea tetragona (NT), Rheum palmatum (RP), Co rdia dicholoma (CRD) (Zeng et al., 2021). Modern pharmacological studies have revealed that ZKMG exhibits preferable anti-inflammatory anti-oxidant stress, regulation of apoptosis, and analgesic activities, as well as it is widely used for treating AURTIs and lung diseases in clinical studies (Li et al., 2022). Alkaloids in PP, flavonoids in ZJ, saponins in GU and quinones in RP have been primarily regarded as the main components of ZKMG prescription herbs. Besides, these components have been found to possess pharmacological activities against AURTIs (Fiore et al., 2008; Song et al., 2014; Liu et al., 2015; Parsaei et al., 2016 ). However, as a complex TCM prescription, the therapeutic effect of ZKMG is not a function of a single herb as well as active compounds and action mechanism in the treatment of AURTIs remain still unclear because of the multichemical components, multi-pharmacological effects, and multi-action targets of ZKMG, so it is quite necessary to explore the potential pharmacodynamic substances and mechanism, which are related to its therapeutic function.

Apparently, the diversity of the chemical constituents of TCM possibly results in complex interactions between active ingredients and multiple targets in diseases. It is a great challenge that how to comprehensively explore potential effective constituents and mechanisms of action (Huang et al., 2021). Ultra-high performance liquid chromatography coupled with quadrupole-Exactive orbitrap mass spectrometry (UHPLC-Q Exactive Orbitrap-MS) is powerful qualitative analysis technique, which has been developed for rapid characterization of unknown trace components in herbal medicines due to its high sensitivity, high resolution and high selectivity (Wang et al., 2021). Network pharmacology could systematically illustrate complex interactions among drugs, chemical constituents, targets, diseases and pathways, which has been considered to be a reliable approach to predict the pharmacological mechanism of drug treatments for diseases based on the development of systems biology and bioinformatics (Lu et al., 2021).

Therefore, it is a powerful method that UHPLC-MS combined with network pharmacology was used to explore the active ingredients and their potential mechanisms of TCM formula. In this study, a rapid and sensitive UHPLC-Q Exactive Orbitrap-MS method was established to investigate complex chemical compounds of ZKMG and their related mechanisms were explored by network pharmacology analysis. The study has certain guiding significance for clinical use and exploration mechanisms of action of ZKMG in treatment of AURTIs.

2 Materials and methods

2.1

2.1 Materials and reagents

Zukamu granules (ZKMG) were provided by Xinjiang Uygur Pharmaceutical Co., Ltd. (220252, Xinjiang, China). The detailed information of reference standards is listed in Supplementary Table S1. Chromatographic grade methanol and acetonitrile were purchased from Merck (New Jersey, USA). Ultrapure water was prepared with a Milli-Q system (Millipore, Milford, MA, USA). LC-MS grade formic acid was obtained from Fisher Scientific (New Jersey, USA). All other reagents were of analytical grade.

2.2

2.2 Sample preparation

Zukamu granules (1 g) were extracted in 50 mL 70% (v/v) aqueous methanol with ultrasonic treatment for 1 h at 30 °C and 40 kHz. The extracting solution was filtered and evaporated (50 °C) using the rotary vacuum evaporator to obtain the residue of ZKMG. Then, 100 mg ZKMG extracts were approximately weighted and dissolved in 1 mL methanol. After centrifugation at 12000 rpm for 20 min, an aliquot (2 μL) of supernatant was injected into the UHPLC-MS system for analysis.

Individual stock solutions of standards (1 mg/mL) were respectively accurately weighted and prepared by dissolving in methanol. Then, the solutions of 38 standards were mixed and serially diluted at a concentration of 10 µg/mL stored at −20 °C before UHPLC-MS analysis.

2.3

2.3 UHPLC-MS conditions

Each sample was performed on an Ultimate 3000 system (Thermo Fisher Scientific, California, USA) equipped with an Thermo Scientific Syncronis C18 (100 mm × 2.1 mm, 1.7 μm) at a temperature of 45 °C. The mobile phases consisted of 0.1% formic acid in water (A) and acetonitrile (B), delivering at a flow rate of 0.3 mL/min with the following solvent gradient: 0–2 min, 95–92% A; 2–5 min, 92–85% A; 5–10 min, 85–78% A; 10–12 min, 78–50% A; 12–20 min, 50–35% A; 20–25 min, 35–20% A; 25–26 min, 20–95% A; 26–30 min, 95% A.

The UHPLC system was coupled to a Q-Exactive Orbitrap MS (Thermo Fisher Scientific, Bremen, Germany), equipped with a heated electrospray ionization source (HESI) in the negative and positive modes for detection with the mass range of m/z 120–1200. For the spray voltage, the positive ion mode was 3.5 kV and the negative ion mode was 3.0 kV. The MS¹ spectra were acquired with full MS mode with a resolution of 35,000, and the MS² spectra resolution was 17,500 conducted in the data-dependent acquisition (DDA) mode. The sheath gas flow rate of 30 arbitrary units and auxiliary gas flow rate of 10 arbitrary units; and the temperatures of the capillary and auxiliary gas heaters were kept at 320 and 350 °C, respectively. The acquisition mode of the stepped normalized collision energy was set to 30, 40, and 60 %. The mass data were analyzed by Xcalibur 4.2 software (Thermo Fisher Scientific, California, USA).

2.4

2.4 The UHPLC-MS data analysis of ZKMG

The approaches for identifying chemical composition of ZKMG were divided into three steps. (1) The self-built database based on searching compounds in domestic and international databases or related natural products research literatures, such as GoogleScholar, PubMed and CNKI, was established for consisted herbs of ZKMG including compounds names, molecular formulars, fragment ions and classifications. Besides, the fragmentation pathways of reference standards obtained were investigated to be beneficial for the characterization. (2) The Xcalibur 4.2 software was applied to acquire extracted ion chromatograms (EICs) of possible compounds, theoretical mass, experimental mass, retention times, mass error and detailed MS/MS fragment ions in both negative and positive modes. (3) The data pre-obtained from (1) was extracted in experimental row data in Xcalibur 4.2 software to identify chemical profiles by matching MS/MS fragments. Furthermore, the fragmentation patterns of reference standards were used to characterize unknown components, which possess similar skeleton structures or fragment ions based on the principle of structural similarity (Fig. 1).

The analytical strategy based on UHPLC-MS and network pharmacology for exploring potential pharmacodynamic substance and mechanisms of action of ZKMG on AURTIs.

2.5

2.5 Network pharmacology analysis

Firstly, all the identified compounds based on UHPLC-MS were transformed into canonical SMILES through the PubChem (https://pubchem.ncbi.nlm.nih.gov/), most recently updated in March 2019. However, a number of compounds were not found in the PubChem, then their structures were imported in SwissTargetPrediction (http://www.swisstargetprediction.ch/, updated in 2019) to obtain SMILES, and the canonical SMILES information was uploaded into the SwissTargetPrediction database in “homo sapiens” species to predict all potential targets of compounds with probability ≥ 0.1 as the screening condition (Daina et al., 2019). Then the target genes obtained were verified by the UniProt protein database (https://www.uniprot.org/, updated in 2022) and converted into corresponding standard gene names (UniProt. 2018). Secondly, the keywords about “AURTIs” as well as relevant diseases “acute pharyngitis”, “acute tonsillitis”, “acute rhinitis”, “acute laryngitis” and “acute angina” were searched to obtain AURTIs related gene targets from Online Mendelian Inheritance in Man database (OMIM http://omim.org/, updated in 2018) and GeneCards database (https://www.genecards.org/, version 5.0), and screen genes with “relevance score” of disease ≥ 30, and the repeated targets were deleted (Amberger et al., 2015; Stelzer et al., 2016). Thirdly, the chemical compounds targets and the diseases targets were imported in bioinformatics website (http://bioinformatics.psb.ugent.be/webtools/venn/, updated in 2022) to draw a Venn diagram and obtain their common gene targets. Then, the chemical components of ZKMG and its therapeutic targets in AURTIs were introduced into Cytoscape (https://cytoscape.org/, version 3.9.1) to construct the compound-target network (Shannon et al., 2003). Afterwards, the overlapping targets between the compounds targets and the AURTIs related targets were inputted into STRING database (http://string-db.org/, version 11.0) with a high confidence score ≥ 0.9 to construct the protein–protein interaction (PPI) network. The Cytoscape software (version 3.9.1) was used to visualize and analysis interactions in this network according to the network node topological parameter “degree”, it was used for evaluating the importance in the PPI network of a protein (Szklarczyk et al., 2018; Zhuang et al., 2020). Finally, the Database for Annotation, Visualization and Integrated Discovery (DAVID https://david.ncifcrf.gov/, version 6.8) was used for GO enrichment including molecular function (MF), biological process (BP), cellular components (CC), and KEGG pathway analysis (https://www.bioinformatics.com.cn/, updated in 2022), which provided systematic and comprehensive biological function annotation information. Meanwhile, top 20 enriched pathways of three GO terms (MF, BP, and CC) and KEGG were visualized in a bubble chart as well as three GO terms were drawn together with a bar chart and represented by a box line partition (p < 0.01) (Huang da et al., 2009; Li et al., 2022). Compound-target-pathway networks were constructed by using Cytoscape software (Fig. 1).

3 Results and discussion

3.1

3.1 Characterization of chemical components of ZKMG

The chemical ingredients of ZKMG were detected and identified by UHPLC-Q-Exactive Orbitrap MS in both positive and negative modes to obtain as much information as possible. Totally, 265 compounds were identified from ZKMG based on accurate precursor and product ions and comparing their fragmentation patterns with standards or reported in the literatures, including 46 alkaloids, 92 flavonoids, 28 triterpenoid saponins, 27 phenolic acids, 24 phenylpropanoids, 21 quinones, 13 tannins, 4 amino acids, 4 nucleosides, 2 naphthols, 2 phenols, 2 terpenoids in Table 1 and Supplementary Table S2. The extracted ion chromatograms (EICs) of these compounds in both positive mode and negative ion modes were showed in Fig. 2. And 38 compounds in ZKMG were unambiguously identified by comparison with reference standards.

Table 1 Identification of chemical components in ZKMG by UHPLC-Q-Exactive Orbitrap MS.

peak	t_R	Theoretical Mass m/z	Experimental Mass m/z	Error (ppm)	Formula	Identification	peak	t_R	Theoretical Mass m/z	Experimental Mass m/z	Error (ppm)	Formula	Identification
1*	0.84	136.0618	136.0617	−0.68	C₅H₅N₅	adenine	134	10.37	463.0882	463.0888	1.31	C₂₁H₂₀O₁₂	quercetin-7-O-glucoside
2*	0.84	191.0561	191.0553	−4.19	C₇H₁₂O₆	quinic acid	135	10.41	477.0675	477.0680	1.04	C₂₁H₁₈O₁₃	quercetin-3-O-glucuronide
3	0.90	191.0197	191.0190	−3.80	C₆H₈O₇	citric acid isomer	136*	10.47	463.0882	463.0888	1.32	C₂₁H₂₀O₁₂	isoquercitrin
4*	0.92	133.0142	133.0132	−8.24	C₄H₆O₅	malic acid	137	10.52	447.0933	447.0931	−0.45	C₂₁H₂₀O₁₁	luteolin‐5‐O‐glucoside
5	1.05	191.0197	191.0190	−3.80	C₆H₈O₇	isocitric acid	138	10.55	445.0776	445.0781	1.02	C₂₁H₁₈O₁₁	rhein-8-O-β-D-glucoside
6*	1.18	191.0197	191.0190	−3.75	C₆H₈O₇	citric acid	139*	10.66	447.0933	447.0937	0.91	C₂₁H₂₀O₁₁	cynaroside
7	1.28	331.0671	331.0674	1.03	C₁₃H₁₆O₁₀	1-O-galloylglucose	140	10.66	461.0725	461.0730	0.91	C₂₁H₁₈O₁₂	kaempferol-3-O-β-D-glucuronide
8	1.31	268.1040	268.1038	−1.01	C₁₀H₁₃N₅O₄	adenosine	141*	10.69	623.1981	623.1988	1.04	C₂₉H₃₆O₁₅	acteoside
9	1.32	132.1019	132.1019	−0.19	C₆H₁₃NO₂	isoleucine	142	10.87	615.0992	615.1002	1.63	C₂₈H₂₄O₁₆	quercetin-O-galloyl-glucopyranoside
10	1.43	284.0989	284.0987	−0.90	C₁₀H₁₃N₅O₅	guanosine	143	11.08	623.1618	623.1622	0.76	C₂₈H₃₂O₁₆	isorhamnetin-3-O-nehesperidine
11	1.45	132.1019	132.1019	−0.27	C₆H₁₃NO₂	leucine	144	11.12	370.1649	370.1646	−0.94	C₂₁H₂₃NO₅	allocryptopine
12	1.48	331.0671	331.0674	1.03	C₁₃H₁₆O₁₀	gallic acid-4-O-β-D-glucopyranoside	145*	11.26	515.1195	515.1198	0.49	C₂₅H₂₄O₁₂	isochlorogenic acid B
13*	1.49	330.0598	330.0592	−1.72	C₁₀H₁₂N₅O₆P	adenosine cyclophosphate	146	11.29	340.1543	340.1540	−1.10	C₂₀H₂₁NO₄	papaverine
14	1.51	302.1387	302.1383	−1.24	C₁₇H₁₉NO₄	morphine N-oxide	147	11.33	579.1719	579.1726	1.07	C₂₇H₃₂O₁₄	naringin
15	1.69	331.0671	331.0674	1.03	C₁₃H₁₆O₁₀	gallic acid-3-O-β-D-glucopyranoside	148*	11.42	593.1512	593.1517	0.91	C₂₇H₃₀O₁₅	kaempferol-3-O-rutinoside
16	1.80	286.1438	286.1433	−1.61	C₁₇H₁₉NO₃	morphine	149	11.44	414.1547	414.1541	−1.57	C₂₂H₂₃NO₇	noscapine isomer 1
17*	1.98	169.0142	169.0134	−4.95	C₇H₆O₅	gallic acid	150*	11.55	515.1195	515.1201	1.09	C₂₅H₂₄O₁₂	1,5-dicaffeoylquinic acid
18	2.44	166.0863	166.0862	−0.27	C₉H₁₁NO₂	phenylalanine	151*	11.56	137.0244	137.0235	−7.06	C₇H₆O₃	salicylic acid
19	3.23	179.0350	179.0553	−4.81	C₉H₈O₄	caffeic acid isomer	152	11.63	623.1981	623.1992	1.73	C₂₉H₃₆O₁₅	isoacteoside
20	3.32	197.0455	197.0448	−3.64	C₉H₁₀O₅	danshensu	153	11.65	400.1391	400.1387	−1.02	C₂₁H₂₁NO₇	narcotoline
21	3.51	329.0878	329.0882	1.29	C₁₄H₁₈O₉	pseudolaroside B	154*	11.70	515.1195	515.1199	0.72	C₂₅H₂₄O₁₂	isochlorogenic acid A
22	3.75	153.0193	153.0185	−5.76	C₇H₆O₄	protocatechuic acid	155	11.73	491.0831	491.0836	0.97	C₂₂H₂₀O₁₃	isorhamnetin-7-O-glucuronide
23	3.88	153.0557	153.0548	−5.93	C₈H₁₀O₃	3,4-dihydroxyphenylethanol	156	11.73	519.1872	519.1877	1.07	C₂₆H₃₂O₁₁	pinoresinol 4-O-β-D-glucopyranoside
24	4.15	451.1246	451.1249	0.78	C₂₁H₂₄O₁₁	catechin‐5‐O‐glucoside	157	11.78	577.1563	577.1569	1.11	C₂₇H₃₀O₁₄	apigenin-7-O-rutinoside
25	4.16	205.0972	205.0971	−0.07	C₁₁H₁₂N₂O₂	tryptophan	158	11.78	623.1618	623.1619	0.18	C₂₈H₃₂O₁₆	isorhamnetin-3-O-rutinoside
26	4.25	483.0780	483.0784	0.83	C₂₀H₂₀O₁₄	gallic acid-O-galloyl-glucoside	159	11.85	414.1547	414.1541	−1.42	C₂₂H₂₃NO₇	noscapine isomer 2
27	4.33	448.1966	448.1965	−0.14	C₂₃H₂₉NO₈	N-methylnorcoclaurine-7-O-glucoside	160	11.90	565.1563	565.1571	1.45	C₂₆H₃₀O₁₄	hydroxyliquiritin apioside
28	4.33	515.1406	515.1411	0.82	C₂₂H₂₈O₁₄	chlorogenic acid-hexoside	161	11.93	447.0933	447.0936	0.64	C₂₁H₂₀O₁₁	luteolin-4′-O-glucoside
29	4.43	311.0409	311.0412	1.21	C₁₃H₁₂O₉	caftaric acid	162	12.11	446.1809	446.1802	−1.71	C₂₃H₂₇NO₈	narceine
30	4.47	272.1281	272.1277	−1.47	C₁₆H₁₇NO₃	DL-demethylcoclaurine	163	12.17	282.1489	282.1485	−1.26	C₁₈H₁₉NO₂	O-nornuciferine
31*	4.51	353.0878	353.0880	0.67	C₁₆H₁₈O₉	neochlorogenic acid	164	12.17	433.1140	433.1143	0.55	C₂₁H₂₂O₁₀	naringenin-7-O-glucoside
32	4.52	462.2122	462.2115	−1.70	C₂₄H₃₁NO₈	N-methylcoclaurine-7-O-glucoside	165	12.28	431.0984	431.0985	0.19	C₂₁H₂₀O₁₀	emodin-1-O-D-glucoside
33	4.54	300.1594	300.1589	−1.83	C₁₈H₂₁NO₃	codeine	166	12.36	445.0776	445.0779	0.62	C₂₁H₁₈O₁₁	apigenin-7-O-glucuronide
34	4.58	285.0616	285.0619	1.16	C₁₂H₁₄O₈	uralenneoside isomer1	167*	12.37	609.1825	609.1832	1.21	C₂₈H₃₄O₁₅	hesperidin
35	4.71	163.0401	163.0392	−5.63	C₉H₈O₃	coumaric acid	168*	12.43	515.1195	515.1197	0.37	C₂₅H₂₄O₁₂	isochlorogenic acid C
36	4.80	483.0780	483.0784	0.83	C₂₀H₂₀O₁₄	gallic acid-O-galloyl-glucoside	169	12.43	607.1668	607.1674	0.95	C₂₈H₃₂O₁₅	diosmin
37	4.81	448.1966	448.1962	−0.94	C₂₃H₂₉NO₈	N-methylnorcoclaurine-4′-O-glucoside	170*	12.48	359.0772	359.0772	−0.09	C₁₈H₁₆O₈	rosmarinic acid
38	4.89	285.0616	285.0619	1.05	C₁₂H₁₄O₈	uralenneoside isomer 2	171	12.53	491.0831	491.0834	0.66	C₂₂H₂₀O₁₃	isorhamnetin-3-O-glucuronide
39	5.04	451.1246	451.1249	0.72	C₂₁H₂₄O₁₁	catechin‐7‐O‐glucoside	172	12.56	463.1235	463.1233	−0.49	C₂₂H₂₂O₁₁	tectoridin
40	5.21	339.0722	339.0727	1.58	C₁₅H₁₆O₉	esculin	173	12.66	639.1920	639.1920	0.09	C₂₉H₃₄O₁₆	aurantio-obtusin-6-O-rutinoside
41	5.27	137.0244	137.0234	−7.28	C₇H₆O₃	4-hydroxybenzoic acid	174	12.69	463.1235	463.1231	−0.82	C₂₂H₂₂O₁₁	homoplantaginin
42	5.40	483.0780	483.0783	0.58	C₂₀H₂₀O₁₄	gallic acid-O-galloyl-glucoside	175	12.71	463.1246	463.1250	0.90	C₂₂H₂₄O₁₁	hesperetin-7-O-β-D-glucoside
43	5.50	515.1406	515.1412	1.05	C₂₂H₂₈O₁₄	chlorogenic acid-hexoside	176	12.74	549.1614	549.1621	1.36	C₂₆H₃₀O₁₃	licuraside
44	5.63	451.1246	451.1249	0.65	C₂₁H₂₄O₁₁	catechin‐4′‐O‐glucoside	177	12.79	431.0984	431.0986	0.53	C₂₁H₂₀O₁₀	aloe-emodin-3-(hydroxymethyl)-O-β-D-glucopyranoside
45	5.65	483.0780	483.0785	0.96	C₂₀H₂₀O₁₄	gallic acid-O-galloyl-glucoside	178	12.79	591.1719	591.1727	1.25	C₂₈H₃₂O₁₄	liquiritigenin-4′-O-(β-D-3-O-acetyl-apiofuranosyl-(1 → 2)-β-d-glucopyranoside
46	5.71	314.1751	314.1747	−1.15	C₁₉H₂₃NO₃	lotusine	179	12.80	463.1235	463.1234	−0.10	C₂₂H₂₂O₁₁	diosmetin-7-O-β-D-glucopyranoside
47	5.74	337.0929	337.0933	1.27	C₁₆H₁₈O₈	5-p-coumaroylquinic acid	180	12.85	447.0933	447.0933	0.01	C₂₁H₂₀O₁₁	luteolin-3′-O-glucoside
48	5.86	316.1543	316.1540	−1.00	C₁₈H₂₁NO₄	norreticuline	181	12.86	475.0882	475.0885	0.57	C₂₂H₂₀O₁₂	diosmetin-7-O-glucuronide
49	5.92	515.1406	515.1411	0.93	C₂₂H₂₈O₁₄	chlorogenic acid-hexoside	182	12.89	459.1297	459.1302	1.13	C₂₃H₂₄O₁₀	6′-acetylliquiritin
50	6.02	483.0780	483.0786	1.14	C₂₀H₂₀O₁₄	gallic acid-O-galloyl-glucoside	183	12.90	417.1191	417.1195	0.90	C₂₁H₂₂O₉	isoliquiritin
51	6.04	451.1246	451.2189	0.85	C₂₁H₂₄O₁₁	catechin-3′-O-glucoside	184	12.92	493.1341	493.1340	−0.17	C₂₃H₂₄O₁₂	aurantio-obtusin-6-O-glucoside
53*	6.14	353.0878	353.0881	0.75	C₁₆H₁₈O₉	chlorogenic acid	185	12.96	431.1337	431.1334	−0.69	C₂₂H₂₂O₉	ononin
52	6.14	325.0929	325.0931	0.77	C₁₅H₁₈O₈	coumaric acid-O-glucoside	186	12.99	417.1191	417.1195	0.97	C₂₁H₂₂O₉	neoisoliquiritin
54	6.16	635.0890	635.0895	0.78	C₂₇H₂₄O₁₈	tri-O-galloyl-glucoside	187	13.03	433.0776	433.0779	0.50	C₂₀H₁₈O₁₁	quercetin 3-O-arabinoside
55*	6.37	353.0878	353.0881	0.84	C₁₆H₁₈O₉	cryptochlorogenic acid	188	13.04	695.1981	695.2002	−2.09	C₃₅H₃₆O₁₅	licorice-glycoside B
56	6.40	286.1438	286.1436	−0.77	C₁₇H₁₉NO₃	coclaurine	189	13.09	725.2087	725.2099	1.64	C₃₆H₃₈O₁₆	licorice-glycoside A
57	6.48	300.1594	300.1592	−0.80	C₁₈H₂₁NO₃	N-methylisococlaurine	190	13.09	459.0933	459.0938	1.21	C₂₂H₂₀O₁₁	carboxyl-chrysophanol-O-glucose
58	6.52	291.0146	291.0148	0.55	C₁₃H₈O₈	brervifolincaboxylic acid	191	13.21	283.0612	283.0607	−1.76	C₁₆H₁₂O₅	glycitein
59	6.55	367.1035	367.1041	1.84	C₁₇H₂₀O₉	4-feruloylquinic acid	192	13.21	447.0933	447.0935	0.44	C₂₁H₂₀O₁₁	isorhamnetin-3-O-arabinoside
60	6.63	483.0780	483.0785	0.96	C₂₀H₂₀O₁₄	gallic acid-O-galloyl-glucoside	193	13.25	593.1876	593.1881	0.90	C₂₈H₃₄O₁₄	didymin
61	6.63	625.1410	625.1420	1.61	C₂₇H₃₀O₁₇	quercetin-3-O-diglucoside	194	13.27	255.0663	255.0663	0.19	C₁₅H₁₂O₄	liquiritigenin
62	6.67	325.0929	325.0933	1.14	C₁₅H₁₈O₈	coumaric acid-O-glucoside	195	13.27	373.0929	373.0931	0.59	C₁₉H₁₈O₈	methyl rosmarinate
63	6.67	635.0890	635.0898	1.34	C₂₇H₂₄O₁₈	tri-O-galloyl-glucoside	196	13.29	473.1089	473.1090	0.16	C₂₃H₂₂O₁₁	aloe-emodin-8-O-(6-O-acetyl)-glucoside
64	6.67	771.1989	771.2005	2.03	C₃₃H₄₀O₂₁	quercetin-3-O-sophoroside-7-O-rhamnoside	197	13.31	999.4442	999.4453	1.00	C₄₈H₇₂O₂₂	24-hydroxy-licoricesaponin A3
65*	6.71	179.0350	179.0343	−3.87	C₉H₈O₄	caffeic acid	198	13.35	837.3914	837.3927	1.52	C₄₂H₆₂O₁₇	yunganoside K2
66	6.76	635.0890	635.0897	1.06	C₂₇H₂₄O₁₈	tri-O-galloyl-glucoside	199	13.40	489.1038	489.1059	4.25	C₂₃H₂₂O₁₂	luteolin-7-O-6″-ocetylglucoside
67	6.81	328.1543	328.1541	−0.87	C₁₉H₂₁NO₄	boldine	200	13.41	407.1348	407.1350	0.48	C₂₀H₂₄O₉	torachrysone-8-O-glucoside
68	6.82	344.1856 [M]⁺	344.1853	−0.88	C₂₀H₂₆NO₄⁺	N-methylreticuline	201*	13.42	285.0405	285.0406	0.45	C₁₅H₁₀O₆	luteolin
69	6.85	579.1719	579.1729	1.59	C₂₇H₃₂O₁₄	liquiritigenin-O-diglucuronide	202	13.42	431.0984	431.0985	0.26	C₂₁H₂₀O₁₀	emodin-8-O-D-glucoside
70	7.09	386.1598	386.1597	−0.37	C₂₁H₂₃NO₆	glaucamine	203*	13.44	301.0354	301.0356	0.78	C₁₅H₁₀O₇	quercetin
71	7.09	711.2141	711.2150	1.10	C₃₂H₄₀O₁₈	glucoliquirtin asioside	204	13.45	415.1035	415.1037	0.66	C₂₁H₂₀O₉	chrysophanol-1-O-β-D-glucoside
72*	7.11	342.1699 [M]⁺	342.1694	−1.62	C₂₀H₂₄NO₄⁺	magnoflorine	205	13.45	253.0506	253.0505	−0.48	C₁₅H₁₀O₄	chrysophanol
73	7.15	515.1194	515.1196	0.25	C₂₅H₂₄O₁₂	dicaffeylquinic acid	206	13.46	447.1286	447.1282	−0.77	C₂₂H₂₂O₁₀	calycosin-7-O-β-D-glucoside
74	7.16	314.1750 [M]⁺	314.1748	−0.86	C₁₉H₂₄NO₃⁺	isolotusine	207	13.46	983.4493	983.4506	1.25	C₄₈H₇₂O₂₁	licoricesaponin A3
75	7.17	337.0928	337.0931	0.56	C₁₆H₁₈O₈	3-p-coumaroylquinic acid	208*	13.48	283.0612	283.0611	−0.34	C₁₆H₁₂O₅	physcion
76	7.19	325.0928	325.0932	0.95	C₁₅H₁₈O₈	coumaric acid-O-glucoside	209	13.50	853.3863	853.3876	1.44	C₄₂H₆₂O₁₈	22-hydroxy-licoricesaponin G2 isomer 1
77	7.24	328.1543	328.1541	−0.59	C₁₉H₂₁NO₄	corytuberine	210	13.52	315.0510	315.0513	0.97	C₁₆H₁₂O₇	isorhamnetin isomer
78	7.42	153.1273	153.1273	−0.80	C₁₀H₁₆O	camphor	211	13.52	895.3969	895.3982	1.44	C₄₄H₆₄O₁₉	hydroxy acetoxyglycyrrhizin
79	7.42	330.1699	330.1696	−1.29	C₁₉H₂₃NO₄	reticuline	212	13.53	415.1035	415.1037	0.59	C₂₁H₂₀O₉	chrysophanol-8-O-glucoside
80	7.46	300.1594	300.1592	−0.70	C₁₈H₂₁NO₃	N-methylcoclaurine	213	13.62	301.0707	301.0704	−0.88	C₁₆H₁₂O₆	tectorigenin
81	7.48	635.0889	635.0896	0.97	C₂₇H₂₄O₁₈	tri-O-galloyl-glucoside	214	13.64	315.0510	315.0515	1.35	C₁₆H₁₂O₇	isorhamnetin isomer
82	7.55	337.0928	337.0932	0.92	C₁₆H₁₈O₈	4-p-coumaroylquinic acid	215	13.64	853.3863	853.3878	1.74	C₄₂H₆₂O₁₈	22-hydroxy-licoricesaponin G2 isomer 2
83	7.58	593.1511	593.1517	0.91	C₂₇H₃₀O₁₅	vicenin Ⅱ	216	13.70	879.4020	879.4031	1.24	C₄₄H₆₄O₁₈	acetoxy-glycyrrhizic acid
84	7.64	635.0889	635.0895	0.78	C₂₇H₂₄O₁₈	tri-O-galloyl-glucoside	217	13.72	837.3914	837.3924	1.17	C₄₂H₆₂O₁₇	licoricesaponin P2
85	7.67	314.1750	314.1746	−1.43	C₁₉H₂₃NO₃	4′-methyl-N-methylcoclaurine	218	13.72	859.3758	859.3742	−1.82	C₄₄H₆₀O₁₇	methyllicorice-saponin Q2 isomer 1
86	7.67	314.1750 [M]⁺	314.1746	−1.43	C₁₉H₂₄NO₃⁺	magnocurarine	219	13.77	445.1140	445.1145	1.15	C₂₂H₂₂O₁₀	physcion-8-O-β-D-glucoside
87	7.67	639.1567	639.1574	1.06	C₂₈H₃₂O₁₇	isorhamnetin-3,7-O-diglucoside	220	13.78	329.0667	329.0671	1.32	C₁₇H₁₄O₇	jaceosidin
88	7.98	328.1543	328.1541	−0.87	C₁₉H₂₁NO₄	scoulerine	221*	13.86	271.0612	271.0614	0.75	C₁₅H₁₂O₅	naringenin
89	8.02	298.1438	298.1436	−0.74	C₁₈H₁₉NO₃	codeinone	222	13.86	835.3758	835.3762	0.46	C₄₂H₆₀O₁₇	yunganoside M
90	8.08	635.0890	635.0898	1.25	C₂₇H₂₄O₁₈	tri-O-galloyl-glucoside	223	13.89	283.0612	283.0613	0.51	C₁₆H₁₂O₅	acacetin
91	8.39	356.1492	356.1489	−0.98	C₂₀H₂₁NO₅	amurensinine N-oxide A	224	13.89	445.1140	445.1144	0.94	C₂₂H₂₂O₁₀	physcion-1-O-β-D-glucoside
92	8.39	367.1035	367.1045	2.90	C₁₇H₂₀O₉	5-feruloylquinic acid	225	13.91	269.0455	269.0457	0.64	C₁₅H₁₀O₅	apigenin
93	8.43	635.0890	635.0886	−0.58	C₂₇H₂₄O₁₈	tri-O-galloyl-glucoside	226	13.93	329.0667	329.0669	0.56	C₁₇H₁₄O₇	iristectorigenin B
94	8.45	300.1594	300.1591	−1.20	C₁₈H₂₁NO₃	6-demethyl-4′-methyl-N-methylcoclaurine	227	13.98	879.4020	879.4040	−1.72	C₄₄H₆₄O₁₈	acetoxy-glycyrrhizic acid
95	8.45	344.1856	344.1852	−1.23	C₂₀H₂₅NO₄	codamine	228*	13.99	285.0405	285.0407	0.87	C₁₅H₁₀O₆	kaempferol
96	8.60	268.1332	268.1329	−1.03	C₁₇H₁₇NO₂	caaverine	229	14.10	301.0707	301.0702	−1.68	C₁₆H₁₂O₆	hispidulin
97	8.60	337.0929	337.0935	1.75	C₁₆H₁₈O₈	1-p-coumaroylquinic acid	230	14.10	301.0718	301.0721	1.16	C₁₆H₁₄O₆	hesperetin
98	8.62	625.1410	625.1417	1.12	C₂₇H₃₀O₁₇	myricetin-3-O-rutinoside	231	14.11	819.3809	819.3823	1.78	C₄₂H₆₀O₁₆	licoricesaponin E2
99*	8.63	163.0401	163.0392	−5.20	C₉H₈O₃	4-coumaric acid	232	14.11	837.3914	837.3922	0.94	C₄₂H₆₂O₁₇	licoricesaponin Q2
100	8.67	579.1719	579.1727	1.28	C₂₇H₃₂O₁₄	liquiritigenin-O-diglucuronide	233	14.11	859.3758	859.3737	−2.39	C₄₄H₆₀O₁₇	methyllicorice-saponin Q2 isomer 2
101	8.68	563.1406	563.1412	0.96	C₂₆H₂₈O₁₄	schaftoside	234	14.11	967.4544	967.4556	1.19	C₄₈H₇₂O₂₀	haoglycyrrhizin isomer 1
102	8.83	459.0933	459.0937	0.94	C₂₂H₂₀O₁₁	carboxyl-chrysophanol-O-glucose	235*	14.14	315.0510	315.0514	1.16	C₁₆H₁₂O₇	isorhamnetin
103	8.84	625.1410	625.1417	1.12	C₂₇H₃₀O₁₇	quercetin-7-O-diglucoside	236	14.22	301.0707	301.0705	−0.65	C₁₆H₁₂O₆	diosmetin
104	8.87	344.1856	344.1853	−0.97	C₂₀H₂₅NO₄	laudanine	237	14.22	837.3914	837.3923	1.08	C₄₂H₆₂O₁₇	uralsaponin N
105	8.89	312.1594	312.1589	−1.54	C₁₉H₂₁NO₃	thebaine	238	14.24	863.4071	863.4081	1.17	C₄₄H₆₄O₁₇	acetoxyglycyrrhaldehyde
106	8.91	326.1387	326.1382	−1.45	C₁₉H₁₉NO₄	pacodine (7-O-demethylpapaverine)	239	14.27	449.1453	449.1456	0.69	C₂₂H₂₆O₁₀	torachrysone-O-acetylglucoside
107	8.96	367.1035	367.1043	2.24	C₁₇H₂₀O₉	3-feruloylquinic acid	240	14.35	255.0663	255.0661	−0.64	C₁₅H₁₂O₄	isoliquiritigenin
108	9.08	463.0882	463.0887	0.97	C₂₁H₂₀O₁₂	quercetin-5-O-glucoside	242*	14.36	821.3965	821.3973	0.94	C₄₂H₆₂O₁₆	glycyrrhizic acid
109	9.36	433.1140	433.1143	0.55	C₂₁H₂₂O₁₀	naringenin-4′-O-glucoside	241	14.36	329.0667	329.0668	0.47	C₁₇H₁₄O₇	aurantio-obtusin
110	9.37	314.1751	314.1749	−0.45	C₁₉H₂₃NO₃	armepavine	243	14.38	837.3914	837.3920	0.72	C₄₂H₆₂O₁₇	hydroxyglycyrrhizin
111	9.40	417.1180	417.1178	−0.43	C₂₁H₂₀O₉	daidzin	244	14.48	457.1140	457.1146	1.18	C₂₃H₂₂O₁₀	chrysophanol-O-acetylglucoside
112	9.41	342.1700	342.1698	−0.45	C₂₀H₂₃NO₄	tetrahydrocolumbamine	245	14.55	354.1336	354.1335	−0.17	C₂₀H₁₉NO₅	pseudoprotopine
113	9.59	579.1719	579.1726	1.07	C₂₇H₃₂O₁₄	liquiritigenin-O-diglucuronide	246	14.55	967.4544	967.4561	1.76	C₄₈H₇₂O₂₀	haoglycyrrhizin isomer 2
114	9.59	621.1097	621.1104	1.01	C₂₇H₂₆O₁₇	apigenin-7-O-diglucuronide	247	14.60	837.3914	837.3924	1.17	C₄₂H₆₂O₁₇	hydroxyglycyrrhizin
115	9.63	326.1387	326.1384	−0.87	C₁₉H₁₉NO₄	palaudine (3′-O-demethylpapaverine)	248	14.73	821.3965	821.3975	1.23	C₄₂H₆₂O₁₆	licoricesaponin H2
116	9.67	549.1614	549.1619	1.03	C₂₆H₃₀O₁₃	liquiritin apioside	249	14.76	807.4172	807.4185	1.51	C₄₂H₆₄O₁₅	licoricesaponin B2
117	9.69	595.1668	595.1679	1.79	C₂₇H₃₂O₁₅	eriocitrin	250	14.80	955.4908	955.4923	1.58	C₄₈H₇₆O₁₉	yunganoside C1
118	9.72	417.1191	417.1195	0.83	C₂₁H₂₂O₉	neoliquiritin	251	14.82	369.1333	369.1329	−1.07	C₂₁H₂₀O₆	gancaonin N
119	9.81	433.1140	433.1144	0.97	C₂₁H₂₂O₁₀	naringenin-5-O-glucoside	252	14.94	955.4908	955.4924	1.70	C₄₈H₇₆O₁₉	yunganoside A1
120	9.94	428.1704	428.1699	−1.21	C₂₃H₂₅NO₇	N-methylnarcotine	253	15.03	479.2650	479.2654	0.83	C₂₆H₄₀O₈	steviol-19-O-glucoside
121	9.95	549.1614	549.1617	0.69	C₂₆H₃₀O₁₃	isoliquiritin apioside	254	15.10	297.0405	297.0406	0.33	C₁₆H₁₀O₆	6-methyl-rhein
122	9.97	517.0412	517.0438	4.86	C₂₆H₁₄O₁₂	1,1,3,4,5,6,8,8′-octahydroxy-9H,9′H-2,2′-bixanthene-9,9′-dione	255	15.17	953.4752	953.4742	−0.96	C₄₈H₇₄O₁₉	yunganoside D1
123	9.98	417.1191	417.1193	0.51	C₂₁H₂₂O₉	liquiritin	256*	15.44	283.0248	283.0249	0.31	C₁₅H₈O₆	rheic acid
124	10.00	370.1649	370.1643	−1.75	C₂₁H₂₃NO₅	cryptopine	257	15.54	343.0823	343.0825	0.57	C₁₈H₁₆O₇	eupatilin
125*	10.01	609.1461	609.1467	1.02	C₂₇H₃₀O₁₆	rutin	258	15.67	283.0612	283.0613	0.40	C₁₆H₁₂O₅	biochanin A
126	10.03	537.1038	537.1040	0.28	C₂₇H₂₂O₁₂	lithospermic acid isomer	259	15.72	369.1333	369.1329	−1.07	C₂₁H₂₀O₆	glicoricone
127	10.06	354.1336	354.1336	−0.05	C₂₀H₁₉NO₅	protopine	260	15.72	807.4172	807.4184	1.43	C₄₂H₆₄O₁₅	22-dehydrouralsaponin C
128*	10.09	300.9990	300.9991	0.40	C₁₄H₆O₈	ellagic acid	261	15.83	355.1187	355.1193	1.63	C₂₀H₂₀O₆	uralenin
129	10.10	431.0984	431.0985	0.33	C₂₁H₂₀O₁₀	apigenin-7-O-β-D-glucoside	262	15.98	589.1351	589.1359	1.21	C₃₁H₂₆O₁₂	1-methyl-8-hydroxy-9,10-anthraquinone-3-O-(6′-O-cinnamoyl)-glucoside
130	10.12	358.2013	358.2008	−1.24	C₂₁H₂₇NO₄	laudanosine	263*	16.27	593.1301	593.1309	1.34	C₃₀H₂₆O₁₃	procyanidin
131*	10.23	463.0882	463.0888	1.32	C₂₁H₂₀O₁₂	hyperoside	264*	17.72	269.0455	269.0458	0.87	C₁₅H₁₀O₅	emodin
132	10.29	593.1512	593.1520	1.43	C₂₇H₃₀O₁₅	luteolin-7-O-rutinoside	265*	23.15	469.3323	469.3333	1.95	C₃₀H₄₆O₄	18 β-glycyrrhetintic acid
133	10.34	344.1856	344.1855	−0.51	C₂₀H₂₅NO₄	tetrahydropapaverine

* Compared with standard compounds.

The high resolution extracted ion chromatograms (EICs) of ZKMG in the positive (P) and negative ion mode (N). P1. m/z 153.0557, 271.0611, 301.0353, 339.0721, 355.1187, 367.1034, 433.0776, 449.1453, 457.1140, 459.1296, 469.3323, 473.1089, 479.2650, 489.1038, 515.1406, 517.0412, 519.1871, 593.1300, 595.1668, 615.0991, 621.1097, 623.1617, 625.1410, 635.0889, 639.1566, 695.1981, 711.2141, 725.2087, 771.1989, 835.3757, 859.3757, 863.4070, 895.3969, 953.4751, 955.4908, 999.4442; P2. m/z 255.0662, 283.0248, 285.0615, 301.0717, 311.0408, 325.0928, 329.0878, 343.0823, 373.0928, 433.1140, 445.0776, 445.1140, 451.1245, 463.0881, 463.1245, 477.0674, 483.0780, 563.1406, 565.1562, 589.1351, 591.1719, 623.1981, 807.4172, 819.3808, 879.4019, 967.4544, 983.4493; P3. m/z 153.0193, 163.0400, 191.0561, 253.0506, 269.0455, 283.0611, 285.0404, 297.0404, 300.9989, 315.0510, 329.0666, 331.0670, 337.0928, 407.1347, 415.1034, 431.0983, 447.0932, 459.0932, 461.0725, 475.0881, 491.0831, 515.1194, 577.1562, 579.1719, 593.1875, 607.1668, 609.1461, 853.3863; P4. m/z 133.0142, 137.0244, 169.0142, 179.0349, 191.0197, 197.0455, 291.0146, 353.0878, 359.0772, 417.1191, 537.1038, 549.1613, 593.1511, 609.1824, 821.3965, 837.3914; N1. m/z136.0617, 153.1273, 205.0971, 268.1332, 272.1281, 282.1488, 284.0989, 298.1437, 301.0706, 302.1386, 314.1750, 314.1761, 316.1543, 326.1386, 328.1543, 330.0597, 354.1335, 356.1492, 358.2012, 369.1332, 370.1648, 386.1598, 400.1390, 448.1965, 462.2122, 463.1234, 493.1340, 639.1919; N2. m/z 132.1019, 166.0862, 268.1040, 286.1437, 300.1594, 312.1594, 330.1699, 340.1543, 342.1699, 344.1856, 414.1547, 417.1180, 428.1703, 431.1336, 446.1809, 447.1285.

3.1.1

3.1.1 Identification of alkaloids

In this study, a total of 46 alkaloids mainly derived from PP were identified, which could be further divided into different alkaloids, including 2 tetrahydroprotoberberines, 5 aporphines, 20 benzyltetrahydroisoquinolines, 5 phthalideisoquinolines, 6 protopines, 5 morphinans, 3 benzylisoquinolines. The proposed fragmentation pathways of each-type representative alkaloids (peaks 88, 72, 56, 153, 144, 16, 146) were observed in Supplementary Figure S3.

3.1.1.1

3.1.1.1 Identification of tetrahydroprotoberberine alkaloids

Tetrahydroprotoberberine is a four-ring structure, which derived from two isoquinoline rings connected by sharing one nitrogen atom, and its C2, C3, C9, C10 contain oxygen groups (Yang et al., 2016). The peaks 88 and 112 were deemed to be tetrahydroprotoberberine alkaloids because of their MS/MS spectral patterns, which produced characteristic ions by Retro Diels-Alder (RDA) cleavage. They were tentatively identified as scoulerine and tetrahydrocolumbamine based on MS/MS fragmentation pattern of tetrahydroprotoberberine alkaloids. The fragmentation ions of scoulerine and tetrahydrocolumbamine were observed at m/z 178.086 [C₁₀H₁₂O₂N]⁺ and 151.075 [C₉H₁₁O₂]⁺ due to RDA fragmentation and cleavage of the B-ring, respectively. Another characteristic ion at m/z 163.060 was indicated by the loss of the methyl radical from the ion at m/z 178.086 (Jeong et al., 2012).

3.1.1.2

3.1.1.2 Identification of aporphine alkaloids

For aporphine alkaloids, the elimination of CH₃NR group as well as lose CH₃OH moiety and CO were the characteristic fragmentation patterns. Peak 72 showed precursor ion [M]⁺ at m/z 342.1699, which produced characteristic ions at m/z 297.111 [M-(CH₃)₂NH]⁺, 282.088 [M-(CH₃)₂NH-CH₃]⁺, 265.085 [M-(CH₃)₂NH-CH₃OH]⁺. Comparing with reference standards, peak 72 was accurately identified as magnoflorine. Similarly, peaks 67, 77 were pairs of isomers with [M + H]⁺ ion at m/z 328.1543 and exhibited three major diagnostic fragment ions at m/z 297.111 [M + H-CH₃NH₂]⁺, 265.085 [M + H-CH₃NH₂-CH₃OH]⁺, 237.090 [M + H-CH₃NH₂-CH₃OH-CO]⁺. Therefore, peaks 67, 77 were respectively determined as boldine and corytuberine according to their retention behavior on the chromatographic column (Conceição et al., 2020). Similarly, peaks 96 and 163 were considered as caaverine and O-nornuciferine.

3.1.1.3

3.1.1.3 Identification of benzyltetrahydroisoquinoline alkaloids

For benzyltetrahydroisoquinoline alkaloids, they were easy to observe the elimination of NRH₂, CH₃OH groups and other characteristic ions at m/z 175.075 [C₁₁H₁₁O₂]⁺, 161.059 [C₁₀H₉O₂]⁺, 121.064 [C₈H₉O]⁺, 107.049 [C₇H₇O]⁺. Peak 30 with the parent ion [M + H]⁺ at m/z 272.12811, was easy to produce fragment ions at m/z 255.101 [M + H-NH₃]⁺, 161.059 [M + H-NH₃-C₆H₅O]⁺, and 107.049 [M + H-C₉H₁₁N₂O]⁺. Consequently, it was tentatively determined to be DL-demethylcoclaurine based on the related literature (Oh et al., 2018). Peak 56 showed the [M + H]⁺ ion at m/z 286.1437, which yielded characteristic ions at m/z 269.117 [M + H-NH₃]⁺, 237.090 [M + H-NH₃-CH₃OH]⁺, 175.075 [M + H-NH₃-C₆H₆O]⁺, so peak 56 was tentatively named coclaurine (Menéndez-Perdomo et al., 2021). Similarly, peaks 27, 32, 37, 46, 57, 74, 80, 85, 86, 94, 110 were successfully identified as N-methylnorcoclaurine-7-O-glucoside, N-methylcoclaurine-7-O-glucoside, N-methylnorcoclaurine-4′-O-glucoside, lotusine, N-methylisococlaurine, isolotusine, N-methylcoclaurine, 4′-methyl-N-methylcoclaurine, magnocurarine, 6-demethyl-4′-methyl-N-methylcoclaurine, armepavine. Peak 130 had a retention time of 10.12 min and the [M]⁺ ion at m/z 358.2012. The fragmentation ions at m/z 327.158 [M + H-CH₃NH₂]⁺, 206.117 [C₁₂H₁₆NO₂]⁺, 189.090 [C₁₂H₁₆NO₂-NH₃]⁺, and 151.075[C₉H₁₁O₂]⁺ suggested that peak 130 was tentatively inferred to be laudanosine. On the basis of this method, peaks 48, 79, 68, 95, 104, 133 were deduced to be norreticuline, reticuline, N-methylreticuline, codamine, laudanine, tetrahydropapaverine.

3.1.1.4

3.1.1.4 Identification of phthalideisoquinoline alkaloids

For phthalideisoquinolines, the fragment ions were obtained by the isoquinone after bond cleavage with the phthalide ring as well as the elimination of H₂O and OCH₃ groups (Menéndez-Perdomo et al., 2021). Peak 153 showed a protonated adduct ion at m/z 400.1390 [M + H]⁺, which fragmented to [M + H-C₁₀H₁₀O₄]⁺ at m/z 206.081, [M + H-H₂O]⁺ at m/z 382.127 and [M + H-H₂O-OCH₃]⁺ at m/z 351.110. Therefore, peak 153 could be considered as narcotoline (Menéndez-Perdomo et al., 2021). Similarly, peaks 120, 149, 159, 162 were identified as N-methylnarcotine, noscapine isomer 1, noscapine isomer 2, narceine.

3.1.1.5

3.1.1.5 Identification of protopine alkaloids

For protopines, it produced characteristic ions by RDA fragmentation, and subsequent loss of water from the isoquinoline fragment (Jeong et al., 2012). Peak 144 showed the parent ion at m/z 370.1648 [M + H]⁺, and fragment ions at m/z 352.154 [M + H-H₂O]⁺, 206.081 [C₁₁H₁₂NO₃]⁺, and 188.070 [C₁₁H₁₂NO₃-H₂O]⁺. Then, peak 144 was characterized as allocryptopine based on the Orbitrap Traditional Chinese Medicine Library (OTCML). Likewise, peaks 70, 91, 124, 127, 245 were assigned as glaucamine, amurensinine N-oxide A, cryptopine, protopine, pseudoprotopine according to the OTCML database and relevant literature (Oh et al., 2018).

3.1.1.6

3.1.1.6 Identification of morphinan alkaloids

Peak 16 exhibited a protonated molecular ion at m/z 286.1437 [M + H]⁺, which fragmented to [M + H-CH₂CHNHCH₃-CO]⁺ at m/z 201.090 and [M + H-CH₂CHNHCH₃-H₂O]⁺ at m/z 211.075. Therefore, this information led to the tentative conclusion that peak 16 was identified as morphine (Menéndez-Perdomo et al., 2021). Peak 33 with [M + H]⁺ ion at m/z 300.1594, was 14.015 Da (CH₂) higher than the mass of the [M + H]⁺ ion of peak 16 and generated fragment ions at m/z 215.106 [M + H-CH₂CHNHCH₃-CO]⁺, 225.090 [M + H-CH₂CHNHCH₃-H₂O]⁺. Peak 33 was characterized as codeine (Menéndez-Perdomo et al., 2021). Moreover, peaks 14, 89, 105 were respectively considered as morphine N-oxide, codeinone and thebaine (Oh et al., 2018; Menéndez-Perdomo et al., 2021).

3.1.1.7

3.1.1.7 Identification of benzylisoquinoline alkaloids

The precursor ion of peak 146 at m/z 340.1543 and the fragment ions at m/z 202.085 [C₁₂H₁₂O₂N]⁺ and 324.122 [M + H-CH₄]⁺ were formed by rearrangement of the C3′ and C4′ methoxy groups to a methylenedioxy bridge. Hence, peak 146 was identified as papaverine (Menéndez-Perdomo et al., 2021). Similarly, peaks 106, 115 were considered as pacodine and palaudine, respectively.

3.1.2

3.1.2 Identification of flavonoids

Flavonoids usually consist of the framework C6-C3-C6 that are formed when two phenyl rings (A and B) bind with C3, most of them undergo RDA cleavage (Luo et al., 2019). Totally, 92 flavonoids were characterized including 7 chalcones, 5 flavan-3-ols, 24 flavones, 25 flavonols, 21 flavonones, and 10 isoflavones in ZKMG. The proposed fragmentation pathways of each-type representative flavonoids (peaks 183, 24, 129, 235, 221, 213) were observed in Supplementary Figure S2.

3.1.2.1

3.1.2.1 Identification of chalcone flavonoids

The chalcones (1,3-diaryl-2-propen-1-ones) mainly derived from GU, which are open chain flavonoids. Peaks 183 and 186 showed the same [M−H]^- ion at m/z 417.1191, which further yielded fragment ions at m/z 255.066 [M−H−C₆H₁₀O₅]^- by the elimination of glucosyl group, 135.007 [C₇H₃O₃]^- and 119.048 [C₈H₇O]^- obtained due to RDA cleavage of the C-ring. Based on literature, peaks 183 and 186 were respectively identified as isoliquiritin and neoisoliquiritin (Xue et al., 2021). Peak 240 exhibited [M−H]^- ion at m/z 255.0662, which generated characteristic ions at m/z 135.007 [C₇H₃O₃]^- and 91.017 [C₇H₃O₃-CO₂]^-, so it was deduced as isoliquiritigenin based on OTCML database. Similarly, peaks 121, 176, 188, 189 were identified as lsoliquiritin apioside, licuraside, licorice-glycoside B, licorice-glycoside A according to the OTCML database and literature (Xue et al., 2021).

3.1.2.2

3.1.2.2 Identification of flavan-3-ol flavonoids

Peaks 24, 39, 44, 51 eluted at different time with the same precursor ion [M−H]^- at m/z 451.1245, and the MS² spectrum showed fragment ions at m/z 289.072 [M−H−C₆H₁₀O₅]^- by a loss of glucose, 245.081 [M−H−C₆H₁₀O₅−CO₂]^-, 137.023 [M−H−C₆H₁₀O₅−C₈H₈O₃]^-. Thus, peaks 24, 39, 44, 51 were tentatively identified as catechin-5-O-glucoside, catechin-7-O-glucoside, catechin-4′-O-glucoside, catechin-3′-O-glucoside based on ClogP values. Peak 263 was unambiguously identified as procyanidin in accordance with the reference standard by comparing with retention time and MS/MS fragmentations.

3.1.2.3

3.1.2.3 Identification of flavone flavonoids

Peak 225 displayed the protonated molecule ion [M−H]^- at m/z 269.0455, which subsequent fragment ions at m/z 227.034 [M−H−C₂H₂O]^-, 225.055 [M−H−CO₂]^-, 201.055 [M−H−C₃O₂]^-, and 151.002 [C₇H₃O₄]^- due to RDA cleavage, so peak 225 was assigned as apigenin based on OTCML database. Peak 129 exhibited molecular ion [M−H]^- at m/z 431.0983 and the main fragment ion at m/z 269.045 [M−H−C₆H₁₀O₅]^-, which indicated peak 225 as aglycone of peak 129. Therefore, peak 129 was tentatively identified as apigenin-7-O-β-D-glucoside. Likewise, peaks 114, 157, 166 were tentatively characterized as apigenin-7-O-diglucuronide, apigenin 7-O-rutinoside, apigenin-7-O-glucuronide. Peak 201 showed [M−H]^- ion at m/z 285.0404, and it could form the fragment ions m/z 241.049 [M−H−CO₂]^-, 217.049 [M−H−C₃O₂]^-, 199.039 [M−H−C₂H₂O−CO₂]^-, 243.029 [M−H−C₂H₂O]^-, 175.039 [M−H−C₂H₂O−C₃O₂]^-, 133.028 [C₈H₅O₂]^- and 151.002 [C₇H₃O₄]^- due to RDA cleavage. Thus, peak 201 was accurately identified as luteolin by comparing with the reference standard. Peaks 137, 139, 161, 180 displayed the same precursor ion [M−H]^- at m/z 447.0932, which produced the fragment ion at m/z 285.040 [M−H−C₆H₁₀O₅]^-. Based on the standard and literature, peaks 137, 139, 161, 180 were respectively assigned as luteolin‐5‐O‐glucoside, cymaroside, luteolin-4′-O-glucoside, luteolin-3′-O-glucoside (Zhao et al., 2019). Similarly, peaks 83, 199, 132, 101, 261, 236, 181, 179, 169, 257, 229, 174, 220, 223 were tentatively characterized as vicenin II, luteolin-7-O-6″-ocetylglucoside, luteolin-7-O-rutinoside, schaftoside, uralenin, diosmetin, diosmetin-7-O-glucuronide, diosmetin-7-O-β-D-glucopyranoside, diosmin, eupatilin, hispidulin, homoplantaginin, jaceosidin, acacetin based on OTCML database.

3.1.2.4

3.1.2.4 Identification of flavonol flavonoids

Peak 235 exhibited [M−H]^- ion at m/z 315.0510, yielded fragment ions at m/z 300.027 [M−H−CH₃]^-, 271.024 [M−H−CH₃−CHO]^- and 151.002 [C₇H₃O₄]^- by RDA cleavage. Therefore, peak 235 was identified as isorhamnetin according to the retention time of isorhamnetin reference standard and the comparison with MS² fragment ions. The protonated molecular ion [M−H]^- of peaks 155 and 171 was m/z 491.0831, which could form MS² fragments at m/z 315.051 [M−H−C₆H₈O₆]^- by loss of glucuronide unit and subsequent loss of CH₃ at m/z 300.027 [M−H−C₆H₈O₆−CH₃]^-, so they were respectively assigned as isorhamnetin-7-O-glucuronide and isorhamnetin-3-O-glucuronide by comparing with their structures and chromatographic elution order (Nakamura et al., 2018). Correspondingly, peaks 87, 143, 158, 192, 210, 214 were respectively characterized as isorhamnetin-3,7-O-diglucoside, isorhamnetin-3-O-nehesperidine, isorhamnetin-3-O-rutinoside, isorhamnetin-3-O-arabinoside, isorhamnetin isomer, isorhamnetin isomer. Peak 203 displayed deprotonated molecular ion [M−H]^- at m/z 301.0353, which produced characteristic ions at m/z 151.002 [C₇H₃O₄]^-, 178.997 [C₈H₃O₅]^- due to RDA cleavage. Based on reference standard and MS² data, peak 203 was identified as quercetin. Likewise, peaks 61, 64, 98, 103, 108, 125, 131, 134, 135, 136, 140, 142, 148, 187, 228 were respectively identified as quercetin-3-O-diglucoside, quercetin-3-O-sophoroside-7-O-rhamnoside, myricetin-3-O-rutinoside, quercetin-7-O-diglucoside, quercetin-5-O-glucoside, rutin, hyperoside, quercetin-7-O-glucoside, quercetin-3-O-glucuronide, isoquercitrin, kaempferol-3-O-β-D-glucuronide, quercetin-O-galloyl-glucopyranoside, kaempferol-3-O-rutinoside, quercetin 3-O-arabinoside and kaempferol.

3.1.2.5

3.1.2.5 Identification of flavonone flavonoids

Peak 194 exhibited the precursor [M−H]^- ion at m/z 255.0662 and yielded characteristic ions at m/z 135.007 [C₇H₃O₃]^-, 91.017 [C₇H₃O₃-CO₂]^-, and 119.048 [C₈H₇O]^- obtained due to RDA cleavage, suggesting that it was characterized as liquiritigenin by comparison with OTCML database. Moreover, peaks 69, 71, 100, 113, 116, 118, 122, 123, 160, 178, 182 were considered as liquiritigenin-O-diglucuronide, glucoliquirtin asioside, liquiritigenin-O-diglucuronide, liquiritigenin-O-diglucuronide, liquiritin apioside, neoliquiritin, 1,1,3,4,5,6,8,8′-Octahydroxy-9H,9′H-2,2′-bixanthene-9,9′-dione, liquiritin, hydroxyliquiritin apioside, liquiritigenin-4′-O-(β-D-3-O-acetyl-apiofuranosyl-(1 → 2)-β-D-glucopyranoside, 6′-acetylliquiritin in accordance with the reference literatures (Wang et al., 2020; Xue et al., 2021). Peak 221 showed protonated molecular ion at m/z 271.0611, which produced characteristic fragment ions at m/z 151.002 [C₇H₃O₄]^-, 119.049 [C₈H₇O]^- due to RDA cleavage. Therefore, peak 221 was identified as naringenin by comparison with reference standard along with the retention and the characteristic product ions. Meanwhile, based on similar fragmentation patterns, peaks 109, 117, 119, 147, 164, 167, 175, 230 were identified as naringenin-4′-O-glucoside, eriocitrin, naringenin-5-O-glucoside, naringin, naringenin-7-O-glucoside, hesperidin, hesperetin-7-O-β-D-glucosidehesperetin.

3.1.2.6

3.1.2.6 Identification of isoflavone flavonoids

Peak 213 generated the [M + H]⁺ ion at m/z 301.0706, and the MS² spectrum showed fragment ions at m/z 286.046 [M + H-CH₃]⁺, 258.054 [M + H-CH₃-CO]⁺, 168.005 [C₇H₄O₅]⁺, which allowed its identification as tectorigenin by comparison with the OTCML database. Based on similar fragmentation patterns, peaks 111, 172, 185, 191, 206, 226, 251, 258, 259 were respectively deduced as daidzin, tectoridin, ononin, glycitein, calycosin-7-O-β-D-glucoside, iristectorigenin B, Gancaonin N, biochanin A and glicoricone according to OTCML database.

3.1.3

3.1.3 Identification of triterpenoid saponins

In this study, a total of 28 saponins primarily derived from GU were characterized in ZKMG. Saponins are composed of a sapogenin of 3α-hydroxy oleanolic acid and sugar residues, such as glucose (Glc), glucuronic acid (GluA), rhamnose (Rha), and xylose (Xyl), which is mainly regarded as structure of 11-oxo-12-ene, 12-ene skeleton (Cheng et al., 2021). The proposed fragmentation pathway of glycyrrhizic acid (peaks 242) was observed in Supplementary Figure S3.

Peak 242 showed precursor ion [M−H]^- at m/z 821.3965, and its primary characteristic ions appeared at m/z 351.057 [2GluA-H]^-, 193.034 [GluA-H]^- and 175.024 [GluA-H₂O-H]^-. Therefore, it was exactly identified as glycyrrhizic acid by comparing the retention time and fragmentation patterns with reference standard. Peaks 198, 217, 232, 237, 243, 247, which eluting at retention time of 13.35, 13.72, 14.11, 14.22, 14.38, 14.60 min respectively, exhibited the same molecular ion at m/z 837.39142 and the main fragment ions at m/z 351.057 [2GluA-H]-, 193.034 [GluA-H]^- and 175.024 [GluA-H₂O-H]^-. Thus, they were respectively deduced as yunganoside K2, licoricesaponin P2, licoricesaponin Q2, uralsaponin N, hydroxyglycyrrhizin, hydroxyglycyrrhizin based on their MS² fragmentation behavior, chromatographic retention time, and comparison with the similar known compounds and other reference evidence (Wang et al., 2020). Likewise, peaks 197, 207, 209, 211, 215, 216, 218, 222, 227, 231, 233, 238, 242, 248, 249, 260, 265 were respectively as 24-hydroxy-licoricesaponin A3, licoricesaponin A3, 22-hydroxy-licoricesaponin G2 isomer 1, hydroxy acetoxyglycyrrhizin, 22-hydroxy-licoricesaponin G2 isomer 2, acetoxy-glycyrrhizic acid, methyllicorice-saponin Q2 isomer 1, yunganoside M, acetoxy-glycyrrhizic acid, licoricesaponin E2, methyllicorice-saponin Q2 isomer 2, acetoxyglycyrrhaldehyde, glycyrrhizic acid, licoricesaponin H2, licoricesaponin B2, 22-dehydro uralsaponin C, 18 β-glycyrrhetintic acid according to their similar fragmentation patterns, standard, OTCML database. Peak 255 gave a precursor ion [M−H]^- at m/z 953.4751, which yielded characteristic fragment ions at m/z 497.115 [2GluA + Rha-H]^-, 435.114 [2GluA + Rha-H₂O-CO₂-H]^-, 339.093 [GluA + Rha + H₂O-H]^-, 321.082 [GluA + Rha-H]^-. Compared with the data in literatures, peak 255 was assigned as yunganoside D1 (Ji et al., 2014). Similarly, peak 234, 246, 250, 252 were respectively characterized as haoglycyrrhizin isomer 1, haoglycyrrhizin isomer 2, yunganoside C1, yunganoside A1 based on their analogous fragmentation pathway and published data (Ji et al., 2014; Xue et al., 2021).

3.1.4

3.1.4 Identification of phenolic acids

In this research, a total of 27 phenolic acids were characterized. The proposed fragmentation pathway of ellagic acid (peaks 128) was observed in Supplementary Figure S3. Peak 2, 4, 6, 17, 65, 99, 128, 151 were exactly and respectively identified as quinic acid, malic acid, citric acid, gallic acid, caffeic acid, 4-coumaric acid, ellagic acid and salicylic acid with the reference standards. Peaks 3, 5, 6 indicated the same parent [M−H]^- ion at m/z 191.0197, could give main product ions at m/z 173.008 [M−H−H₂O]^-, 129.018 [M−H−H₂O−CO₂]^-, 111.007 [M−H−2H₂O−CO₂]^-. Based on their chromatographic elution orders, further MS² fragmentation patterns and reported literature (Al Kadhi et al., 2017). Peaks 7, 12, 15 exhibited the same precursor ion [M−H]^- at m/z 331.0670, which produced daughter ions at m/z 271.046 [M−H−C₂H₄O₂]^-, 211.024 [M−H−2C₂H₄O₂]^- and 169.013 [M−H−C₆H₁₀O₅]^- by loss of a glucose moiety. Based on similar fragmentation patterns and chromatographic elution orders, they were respectively identified as 1-O-galloylglucose, gallic acid-4-O-β-D-glucopyranoside, gallic acid-3-O-β-D-glucopyranoside (Jin et al., 2007). Peak 19 showed the similar protonated molecular ion at m/z 179.0349 with peak 65, and yielded characteristic ion at m/z 135.044 [M−H−CO₂]^-, so it was presumed to be caffeic acid isomer. Peaks 20, 22, 35, 41 were tentatively identified as danshensu, protocatechuic acid, coumaric acid, 4-hydroxybenzoic acid, respectively according to the OTCML database. Peaks 52, 62, 76 with the same protonated molecular ion [M−H]^- at m/z 325.0928, were glucose moiety more than peak 35, which produced characteristic ions at m/z 163.039 [M−H−C₆H₁₀O₅]^-, 145.028 [M−H−C₆H₁₀O₅−H₂O]^- and 119.049 [M−H−C₆H₁₀O₅−CO₂]^-. Their fragment patterns were similar with peak 35, so they were tentatively assigned as coumaric acid-O-glucoside. Peak 126 exhibited [M−H]^- ion at m/z 537.1038 and fragment behaviors are similar with lithospermic acid while retention time could not bring into correspondence with standard. Thus, it was tentatively assigned to lithospermic acid isomer. Peak 156 showed precursor ion [M−H]^- at m/z 519.1871 and yielded characteristic ions at m/z 357.134 [M−H−C₆H₁₀O₅]^-, 151.039 [C₈H₇O₃]^-, indicating that peak 156 was inferred as pinoresinol 4-O-β-D-glucopyranoside. Peak 193 generated the quasi-molecular ion [M−H]^- at m/z 593.1875 and fragment ions at m/z 309.077 and 285.076. Thus, it was tentatively characterized as didymin. Peak 21 showed protonated molecular ion at m/z 329.0878, and produced fragment ions at m/z 167.034 [M−H−C₆H₁₀O₅]^-, 152.010 [M−H−C₆H₁₀O₅−CH₃]^- and 123.044 [M−H−C₆H₁₀O₅−CO₂]^-, suggesting that it was pseudolaroside B. Peaks 34 and 38 exhibited the same precursor ion [M−H]^- at m/z 285.0615, and it could form the fragment ions m/z 153.018 [M−H−C₅H₈O₄]^- by loss of an arabinose group and 109.028 [M−H−C₅H₈O₄−CO₂]^-, suggesting that they were uralenneoside isomers.

3.1.5

3.1.5 Identification of phenylpropanoids

A total of 24 phenylpropanoids comprising of 4 coumaroylquinic acids, 3 feruloylquinic acids, 11 caffeylquinic acids and 6 other type acids were identified in ZKMG extract. The proposed fragmentation pathway of rosmarinic acid (peaks 170) was observed in Supplementary Figure S3. Peaks 31, 53, 55, 141, 145, 150, 154, 168, 170 were exactly identified as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, acteoside, isochlorogenic acid B, 1,5-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid C, rosmarinic acid, respectively. Likewise, peak 73 gave precursor ion [M−H]^- at m/z 515.1194, with the fragment ions at m/z 353.088 [M−H−caffeoyl]^-, 191.055 [quinic acid-H]^-, 179.034 [caffeic acid-H]^-, 135.044 [caffeic acid-H-CO₂]^-, which were consistent with the corresponding ions of dicaffeoylquinic acids. Therefore, peak 73 was inferred as dicaffeylquinic acid. Peaks 28, 43, 49 with the same parent ion [M−H]^- at m/z 515.1406 were a glucose group (C₆H₁₀O₅ 162.052 Da) more than peaks 31, 53, 55 and possessed similar characteristic fragment ions at m/z 173.044 [quinic acid-H-H₂O]^-, 191.055 [quinic acid-H]^-, 179.034 [caffeic acid-H]^- and 135.044 [caffeic acid-H-CO₂]^-. Thus, they were tentatively identified as chlorogenic acid-hexosides. Peaks 47, 75, 82, 97 were found to elute at 5.74, 7.17, 7.55, 8.60 min, with [M−H]^- ion at m/z 337.0928, and they could yield characteristic fragment ions at m/z 163.039 [coumaric acid-H]^-, 119.048 [coumaric acid-H-CO₂]^-, 191.055 [quinic acid-H]^-, 173.044 [quinic acid-H-H₂O]^-, suggesting that these compounds might be coumarylquinic acid. Therefore, Peaks 47, 75, 82, 97 were respectively identified as 5-p-coumaroylquinic acid, 3-p-coumaroylquinic acid, 4-p-coumaroylquinic acid, 1-p-coumaroylquinic acid according to their chromatographic elution behavior (Zhao et al., 2014). Peaks 59, 92, 107 were respectively eluted at 6.55, 8.39, 8.96 min with the parent ion [M−H]^- at m/z 367.1034 and displayed characteristic secondary fragments at m/z 193.050 [ferulic acid-H]^-, 149.059 [ferulic acid-H-CO₂]^-, 134.036 [ferulic acid-H-CO₂-CH₃]^-, 173.044 [quinic acid-H-H₂O]^-, suggesting that these compounds might be feruloylquinic acid. Hence, they were tentatively identified as 4-feruloylquinic acid, 5-feruloylquinic acid, 3-feruloylquinic acid based on their chromatographic elution orders, further MS² fragmentation patterns and reported literature (Zheleva-Dimitrova et al., 2017). Peak 29 with the precursor ion [M−H]^- at m/z 311.0408, gave characteristic product ions at m/z 149.008 [tartaric acid-H]^-, 179.034 [caffeic acid-H]^-, 135.044 [caffeic acid-H-CO₂]^-, suggesting that it was tentatively assigned to caftaric acid according to the OTCML database. Peak 40 was tentatively identified as esculin based on the OTCML database. Peak 195 with the parent ion [M−H]^- at m/z 373.0928, was methyl (CH₂ 14.015 Da) more than peak 170, and produced characteristic ions at m/z 197.045 [M−H−C₉H₇O₃]^-, 179.034 [M−H−C₉H₉O₄]^-, 161.023 [M−H−C₉H₉O₄−H₂O]^-, 135.044 [M−H−C₉H₉O₄−CO₂]^-, which were the same fragment patterns as peak 170. Therefore, it was identified as methyl rosmarinate based on the OTCML database. Peaks 141, 152 both gave precursor ion [M−H]^- at m/z 623.1981, and yielded fragment ions at m/z 461.166 [M−H−C₉H₆O₃]^-, 179.034 [C₉H₇O₄]^-, 161.023 [C₉H₇O₄-H₂O]^-, 135.043 [C₉H₇O₄-CO₂]^-, suggesting that they were a group of isomers. Peak 141 was confirmed as acteoside by standard, so peak 152 was identified as isoacteoside based on their chromatographic retention behavior.

3.1.6

3.1.6 Identification of anthraquinones

A total of 21 quinones were identified in ZKMG, including 3 rheic acid-types, 3 physcion-types, 3 emodin-types, 6 chrysophanol-types, 3 aurantio-obtusin-types, 2 aloe-emodin-types and 1other compound, which are all derived from RP. The proposed fragmentation pathway of rhein (peak 256) was observed in Supplementary Figure S3.

Peaks 208, 256, 264 were unambiguously attributed to physcion, rheic acid, emodin by comparison with the authentic standards. Rhein as the main anthraquinone in ZKMG, was used to characterize the fragmentation pathways. It exhibited a parent ion [M−H]^- at m/z 283.0248, and yielded characteristic product ions at m/z 255.030 [M−H−CO]^-, 239.034 [M−H−CO₂]^-, 211.039 [M−H−CO₂−CO]^-, 183.044 [M−H−CO₂−2CO]^-. Based on these fragmentation patterns, peaks 138, 254 were identified as rhein-8-O-β-D-glucoside and 6-methyl-rhein. Physcion showed a precursor ion [M−H]^- at m/z 283.0611, which produced characteristic ions at m/z 268.037 [M−H−CH₃]^-, 240.042 [M−H−CH₃−CO]^-. Therefore, peaks 219, 224 were respectively identified as physcion-8-O-β-D-glucoside and physcion-1-O-β-D-glucoside according to the chromatographic elution orders, similar fragment patterns. Emodin indicated the parent ion [M−H]^- at m/z 269.0455, and produced product ions at m/z 241.050 [M−H−CO]^-, 225.054 [M−H−CO₂]^-. Based on these fragmentation patterns, peaks 165, 202 were identified as emodin-1-O-D-glucoside and emodin-8-O-D-glucoside. Likewise, peaks 102, 173, 177, 184, 190, 196, 204, 205, 212, 241, 244, 262 were assigned to carboxyl-chrysophanol-O-glucose, aurantio-obtusin-6-O-rutinoside, aloe-emodin-3-(hydroxymethyl)-O-β-D-glucopyranoside, aurantio-obtusin-6-O-glucoside, carboxyl-chrysophanol-O-glucose, aloe-emodin-8-O-(6-O-acetyl)-glucoside, chrysophanol-1-O-β-D-glucoside, chrysophanol, chrysophanol-8-O-glucoside, aurantio-obtusin, chrysophanol-O-acetylglucoside, 1-Methyl-8-hydroxy-9,10-anthraquinone-3-O-(6′-O-cinnamoyl)-glucoside, respectively, according to the similar fragment patterns.

3.1.7

3.1.7 Identification of tannins

Tannins might exist in ZKMG as they are important compounds found in the crude drug RP. Totally, 13 tannins were identified in ZKMG extract. The proposed fragmentation pathway of tri-O-galloyl-glucoside (peaks 54) was observed in Supplementary Figure S3. Peaks 54, 63, 66, 81, 84, 90, 93 gave the precursor [M−H]- ion at m/z 635.0889, and produced characteristic ions at m/z 483.077 [M−H−C₇H₄O₄]^-, 465.067 [M−H−C₇H₄O₄−H₂O]^-, 313.057 [M−H−2C₇H₄O₄−H₂O]^-, 169.013 [C₇H₅O₅]^-, and 125.023 [C₇H₅O₅-CO₂]^-, thus they were assigned as tri-O-galloyl-glucoside isomers. According to this method, peaks 26, 36, 42, 45, 50, 60 were identified as gallic acid-O-galloyl-glucoside isomers.

3.1.8

3.1.8 Identification of other compounds

The other compounds including 4 amino acids, 2 naphthols, 2 phenols, 2 terpenoids were detected in ZKMG. The proposed fragmentation pathway of brervifolincaboxylic acid (peak 58) was observed in Supplementary Figure S3. Peaks 1 and 13 were confirmed as adenine and adenosine cyclophosphate, respectively, compared with known reference compounds. Peaks 9 and 11 both showed [M + H]⁺ at m/z 132.1019, and gave the same MS² fragmentation ion at m/z 86.096 [M + H-CO-H₂O]⁺, indicating that they were isomers. Consequently, peaks 9 and 11 were respectively characterized as isoleucine, leucine based on chromatographic elution orders. Peak 200 exhibited a precursor ion [M−H]^- at m/z 407.1347, and showed the product ions at m/z 245.081[M-H-C₆H₁₀O₅]^- by loss of a glucosyl group, 230.058 [M−H−C₆H₁₀O₅−CH₃]^- by elimination of a CH₃ radical. Hence, it was identified as torachrysone-8-O-glucoside. Peak 239 with the parent ion [M−H]^- at m/z 449.1453, was acetyl (C₂H₂O 42.010 Da) more than peak 200, and yielded the same fragment ions. Thus, it was tentatively characterized as torachrysone-O-acetylglucoside. Peak 58 showed [M−H]^- ion at m/z 291.0146, and gave MS² fragmentation ions at m/z 247.024 [M−H−CO₂]^-, 219.029 [M−H−CO₂−CO]^-, 191.034 [M−H−CO₂−2CO]^-, 173.023 [M−H−CO₂−2CO−H₂O]^-, suggesting that it was identified as brervifolincaboxylic acid (Chen et al., 2022). Besides, peaks 8, 10, 18, 23, 25, 78, 253 were respectively characterized as adenosine, guanosine, phenylalanine, 3,4-dihydroxyphenylethanol, tryptophan, camphor, steviol-19-O-glucoside based on the OTCML database.

3.2

3.2 Network pharmacology analysis

3.2.1

3.2.1 Potential bioactive compounds and targets of ZKMG in the treatment of AURTIs

In this study, UHPLC-MS was used to detect a total of 265 chemical components of ZKMG. By searching the Swiss Target Prediction, 836 targets were obtained from identified compounds, and 1317 AURTIs related targets based on OMIM and GeneCards database (Supplementary Table S3). Finally, 120 overlapping targets were obtained by precisely matching the potential targets of the above two steps through the online tool Venny 2.1, suggested that ZKMG would play a role in treating AURTIs associated with these 120 common targets (Supplementary Figure S4).

3.2.2

3.2.2 Compound‑target network analysis

The active ingredient potential target network of ZKMG in (Fig. 3). There are 271 nodes and 1117 edges in the network, among which the 155 pink nodes represent the main components of ZKMG, the 116 purple nodes represent the targets of AURTIs, and 1117 edges represent the interactions between the components and the targets of AURTIs. The size of the compounds in the network increases with the number of edges (degree of targets). The fact that the same active ingredient can act on multiple targets and the same target also corresponds to different chemical components were observed from the network, which fully reflect the multicomponent and multitarget characteristics of ZKMG in the treatment of AURTIs. The compounds were screened with a degree and betweenness centrality greater than the mean, such as 18 β-Glycyrrhetintic acid, noscapine, n-methylnarcotine, adenosine, methyl rosmarinate, thebaine, 6-methyl-rhein, which were possibly potential active ingredients of ZKMG in the treatment of AURTIs.

Compound-target network. pink rhombus nodes represent compounds, and purple rectangular nodes represent targets.

3.2.3

3.2.3 PPI network analysis

STRING analysis was used to compare 120 overlapping targets and produce a PPI network, as well as the visualization was realized by Cytoscape software (Fig. 4). There were 117 nodes and 1562 edges were observed with a combined score of greater than 0.4 (Supplementary Table S4). The size and color of the node reflected the importance of the degree. The larger the degree, the more important the node is in the network, suggesting that it may be a key target of ZKMG in the treatment of AURTIs. The top 10 nodes were selected as the major genes, including TNF, TP53, IL6, AKT1, EGFR, VEGFA, STAT3, HRAS, JUN, ERBB2, which were likely to be the critical genes in the development of AURTIs.

Protein-protein interaction (PPI) network.

3.2.4

3.2.4 GO analysis and KEGG pathway analysis

GO function and KEGG pathway analysis of the 120 candidate target genes were posted on the DAVID database to explore the molecular mechanism of ZKMG in treating AURTIs. GO evaluations were illustrated by using biological process (BP), cell component (CC), and molecular function (MF) terms. The results of GO analysis showed that potential target genes were enriched, which involved with 638 pathways, including 505 BPs, 52 CCs and 81 MFs (P＜0.05). The top 20 pathways of BP, CC, MF with the highest number of genes involved were shown in the Fig. 5. In BP, the targets mainly involved peptidyl-tyrosine phosphorylation, protein phosphorylation, inflammatory response. In CC, the targets mainly involved plasma membrane, receptor complex, macromolecular complex. In MF, the targets mainly involved transmembrane receptor protein tyrosine kinase activity, protein tyrosine kinase activity, identical protein binding, protein kinase activity (Supplementary Table S5). KEGG pathway annotation indicated that potential target genes were involved in 148 pathways (P＜0.05). The top 20 KEGG pathways with the highest number of genes were shown in the Fig. 6, including PI3K-Akt signaling pathway, AGE-RAGE signaling pathway in diabetic complications, PD-L1 expression and PD-1 checkpoint pathway in cancer, HIF-1 signaling pathway (Supplementary Table S6).

3.2.5

3.2.5 Compound‑target‑pathway network analysis

In order to explore the key compounds of ZKMG in treating AURTIs, the top 20 KEGG pathways, corresponding targets and compounds were constructed to the component-target-pathways network as shown in Fig. 7. The network contained 227 nodes with 134 representative components, 73 representative targets, 20 representative pathways, and 1193 edges. The results indicated that alkaloids mainly derived from PP played an important role in the treatment of ZKMG in treating AURTIs due to their higher degrees, such as noscapine (degree = 24), cryptopine (degree = 17), N-methylnarcotine (degree = 16), allocryptopine (degree = 15). In addition, the targets and active components were distributed in different pathways and played a key role in the treatment of AURTIs, which profoundly reflected the multicomponent, multitarget, and multipathway features of TCM.

Compound-target-pathway network. green circular nodes represent chemical compounds, red V-shaped nodes represent targets, and blue rectangular nodes represent pathways.

4 Conclusion

In this study, a rapid and sensitive UHPLC-Q-Exactive Orbitrap-MS method combined with network pharmacology was established to characterize pharmacodynamic Substance and predict potential molecular mechanisms of ZKMG in treating AURTIs. Finally, a total of 265 were identified or tentatively characterized including 46 alkaloids, 92 flavonoids, 28 triterpenoid saponins, 27 phenolic acids, 24 phenylpropanoids, 21 quinones, 13 tannins, 4 amino acids, 4 nucleosides, 2 naphthols, 2 phenols and 2 terpenoids. Based on the pharmacological network analysis of compound‑target‑pathway, EGFR, PTGS2, IL2, MMP9, TNF, AKT1, PIK3CA and F3 were regarded as key targets for ZKMG to exhibit its effects against AURTIs. Alkaloids, flavonoids, phenylpropanoids and terpenoids such as noscapine, cryptopine, steviol-19-O-glucoside, N-methylnarcotine, methyl rosmarinate, allocryptopine, naringenin and boldine indicated that multiple compounds possessed comprehensive activities by interacting with the above targets against AURTIs. In conclusion, the result revealed that the relationship between compounds and effects of ZKMG treated the AURTIs as well as could lay the foundation of quality control research and clinical application of ZKMG in the future. However, the effective constituents, active targets, and signaling pathways obtained based on UHPLC-MS and network pharmacology need to be confirmed and validated by animal experiment in further studies.

Funding

This study was funded by the Science and Technology Innovation Program of Hunan Province (no. 2022RC1228), Xinjiang Uygur Autonomous Region Institute for Drug Control, and Hunan Province Social Science Innovation Research Base (Ethnic medicine and ethnic culture research base).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Appendix A

Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2023.104875.

Appendix A

Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1

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Introduction

Materials and methods

Materials and reagents
Sample preparation
UHPLC-MS conditions
The UHPLC-MS data analysis of ZKMG
Network pharmacology analysis

Results and discussion

Characterization of chemical components of ZKMG
Network pharmacology analysis

Conclusion

Funding

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Exploring the effective components and potential mechanisms of Zukamu granules against acute upper respiratory tract infections by UHPLC-Q-Exactive Orbitrap-MS and network pharmacology analysis

Abstract

Keywords

Zukamu granules

Acute upper respiratory tract infections

Effective components

Mechanisms

UHPLC-Q-Exactive Orbitrap-MS

Network pharmacology

1 Introduction

2 Materials and methods

2.1 Materials and reagents

2.2 Sample preparation

2.3 UHPLC-MS conditions

2.4 The UHPLC-MS data analysis of ZKMG

2.5 Network pharmacology analysis

3 Results and discussion

3.1 Characterization of chemical components of ZKMG

3.1.1 Identification of alkaloids

3.1.1.1 Identification of tetrahydroprotoberberine alkaloids

3.1.1.2 Identification of aporphine alkaloids

3.1.1.3 Identification of benzyltetrahydroisoquinoline alkaloids

3.1.1.4 Identification of phthalideisoquinoline alkaloids

3.1.1.5 Identification of protopine alkaloids

3.1.1.6 Identification of morphinan alkaloids

3.1.1.7 Identification of benzylisoquinoline alkaloids

3.1.2 Identification of flavonoids

3.1.2.1 Identification of chalcone flavonoids

3.1.2.2 Identification of flavan-3-ol flavonoids

3.1.2.3 Identification of flavone flavonoids

3.1.2.4 Identification of flavonol flavonoids

3.1.2.5 Identification of flavonone flavonoids

3.1.2.6 Identification of isoflavone flavonoids

3.1.3 Identification of triterpenoid saponins

3.1.4 Identification of phenolic acids

3.1.5 Identification of phenylpropanoids

3.1.6 Identification of anthraquinones

3.1.7 Identification of tannins

3.1.8 Identification of other compounds

3.2 Network pharmacology analysis

3.2.1 Potential bioactive compounds and targets of ZKMG in the treatment of AURTIs

3.2.2 Compound‑target network analysis

3.2.3 PPI network analysis

3.2.4 GO analysis and KEGG pathway analysis

3.2.5 Compound‑target‑pathway network analysis

4 Conclusion

Funding

Declaration of Competing Interest

References

Appendix A

Supplementary material

Appendix A

Supplementary material

Supplementary data 1

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