Investigating the pharmaceutical substances and action mechanisms of Changmaxifeng granules against tic disorders

Abstract

BACKGROUND

Tic disorders (TDs) are a type of neurological and psychiatric disorder characterized by vocal or motor tics in the head, body, or limbs. Clinical studies have shown that Changmaxifeng granules (CG) can treat TDs. However, the pharmaceutical substances and mechanism of action of CG remain unclear.

AIM

To investigate the pharmaceutical substances and action mechanisms of CG against TDs, this study employs serum medicinal chemistry, network pharmacology, and molecular docking analysis.

METHODS

Ultrahigh-performance liquid chromatography with quadrupole time-of-flight mass spectrometry was used to identify the blood-absorbed constituents of CG; Network pharmacology was then used to integrate these compounds with disease targets, followed by protein-protein interaction (PPI) networks analysis to pinpoint key proteins. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses combined with molecular docking elucidated the underlying mechanism of action.

RESULTS

Overall, 187 chemical components, including terpenoids, sugars, phenolic acids, and flavonoids, were identified in vitro. In addition, 75 components, namely 49 prototype components and 26 metabolites, were identified in vivo. The PPI results revealed 225 overlapping targets, with TNF, IL-6, FOS, VEGFA, and ESR1 being the major targets. GO and KEGG analyses were performed to identify key signaling pathways and biological processes. Paeonol, evofolin B, aspalathin, and paeoniflorin were identified as potential pharmacodynamic substances based on the results of the “compound-target” network. The maximum binding energy between the core target and the active ingredient was less than -4.7 kcal/mol, indicating that the pharmacophore exhibited a strong affinity toward the core ingredient.

CONCLUSION

This study elucidated the in vitro and in vivo chemical components of CG and outlined their potential targets and action mechanisms. This study provides a basis for further research into the action mechanism and clinical application of CG.

Key Words: Changmaxifeng granule; Against tic disorders; Chemical components; Network pharmacology; Molecular docking

Core Tip: Clinical studies have shown that Changmaxifeng granules (CG) can treat tic disorders. However, the pharmaceutical substances and mechanism of action of CG remain unclear. In this study, we combined serum pharmacochemistry with network pharmacology to identify the chemical constituents of CG that enter the bloodstream and to predict their potential molecular targets, thereby laying the groundwork for elucidating the underlying mechanisms of action.

INTRODUCTION

Tic disorder (TD) is a mental developmental disorder that most commonly affects children between the ages of 5 and 10 years and is characterized by involuntary blinking, strange face-making, head twisting, eyebrow squeezing, and lower limb twitching[1]. Symptoms are aggravated by mental stress, diminished through concentration, and absent while sleeping. The duration of the condition usually ranges from a few months to a year, and the condition severely affects children’s learning, daily life, and physical and mental development[2]. The clinical manifestations of this disorder are diverse and often associated with various comorbidities, such as attention deficit hyperactivity disorder, obsessive compulsive disorder, anxiety, and depression[3]. The pathogenesis of TD is complex and currently remains unclear[4]. However, there has been a gradual increase in the incidence of TD, globally[5]. Most medications used to treat TD are psychotropic drugs, which can relieve symptoms but have been clinically shown to be ineffective in the long term. For example, the most common drugs used for TD treatment include dopamine receptor blockers, which can lead to adverse effects, such as cognitive impairment[6]. Modern studies have shown that traditional Chinese medicine (TCM) can help regulate neurotransmitter release and metabolism through dopaminergic synapses, neuroligand-receptor interactions, and other pathways, exerting a multicomponent, multitarget, and multipathway anti-TD effect with better clinical outcomes, fewer side effects, and a low relapse rate[7-9].

Jiren Pharmaceutical Co., Ltd. (Heilongjiang Province, China) developed Changmaxifeng Tables (CT; State Drug License Z20140013) based on a famous clinical prescription. CT comprises five types of Chinese medicines: Paeoniae Radix Alba [Baishao (BS)], Gastrodiae Rhizoma [Tianma (TM)], Polygalae Radix [Yuanzhi (YZ)], Acori Tatarinowii Rhizoma [Shichangpu (SCP)], and Margaritifera Concha [Zhenzhumu (ZZM)]. Modern pharmacological studies have found that Chinese medicines such as BS, TM, and YZ possess varying degrees of neuroprotective effects[10-13]. Gai[14] reported that CT exhibits higher clinical efficacy and safety than Thiabetic, and it was recommended as a first-line drug.

After experimental evaluation, it was found that the tablets were changed to granules could improve drug compliance; however, the pharmacodynamic composition and action mechanisms of Changmaxifeng granules (CG) remain unclear. This has become an obstacle to the marketing of CG as a new drug. Therefore, according to the concept of “effective, unique, transmission and traceability, measurable and prescription matching” in Chinese medicine quality markers, investigating the blood components of Chinese medicines is important to understand their pharmacodynamic composition and action mechanism in vivo. Thus, this study analyzed the composition of CG and its components that are absorbed into the blood via and ultrahigh-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) technology. Based on the blood components alongside network pharmacology and molecular docking, we initially discovered the pharmacodynamic composition and action mechanism of CG against TD. Therefore, this approach lays the foundation for understanding the pharmacodynamic composition and action mechanism of CG and development of new CG dosage forms. Meanwhile, it provides strong support for the completion of new drug application.

MATERIALS AND METHODS

Instruments

LE2O4E/O2 electronic analytical balances (Mettler-Toledo International Inc); Acquity high performance liquid chromatography, Xevo G2 Q TOF mass spectrometer (Waters Corporation, United States), chromatographic column (Waters ACQUITY UPLC BEH C₁₈ 2.1 mm × 100 mm, 1.7 μm), UC-250E ultrasonic cleaner (Shanghai Jingqi Instrument Co., Ltd., Shanghai, China), high-speed freezing centrifuge (Shanghai Lixin Scientific Instrument Co. Shanghai, China).

Reagents

CG was provided by Jiren Pharmaceutical Co., Ltd. (Heilongjiang Province, China), 1 g/bag; methanol, acetonitrile, and formic acid were of mass spectral grade (Thermo Fisher Scientific Ltd., Batch No. 20210323, 202100517, 20190312). Paeoniflorin, paeonilactone, asparagine, 3,6'-dierucoylsucrose, and 1,2,3,4,6-O-galloylglucose were obtained from Chengdu Push Biotechnology Co., LTD. (Batch No. PS000825, PS021057, PS012103, PS011608, and PS013974, respectively). Gallic acid was procured from Shanghai Yuanye Biotechnology Co., Ltd. (Batch No. AN1127SA14).

Animals

Twelve 4-week-old specific pathogen-free-grade Sprague-Dawley rats (six male and six female) were provided by Changchun Yisi Experimental Animals Technology Limited Co., Ltd. (Certificate No. 01021731571831465). The animals were housed in separate cages (n = 6) in a 12-hour alternating light and dark environment at a temperature of 18-21 °C and a relative humidity of 50% ± 5%. The experimental animals were fed standard chow and allowed to eat and drink freely throughout the experimental period. All animals were handled in accordance with the procedures described in the Guidelines for the Management and Use of Laboratory Animals (People’s Republic of China), which were reviewed by the Animal Ethics Committee of Harbin University of Commerce and found to be in accordance with the standards (Ethical Approval No. HSDYXY-2024055).

The animal protocol was designed to minimize pain or discomfort to the animals. Intragastric gavage administration was carried out with conscious animals, using straight gavage needles appropriate for the animal size. All animals were anesthetized by barbiturate overdose (intravenous injection, 150 mg/kg pentobarbital sodium) for plasma collection.

In vivo and in vitro chemical composition analysis of CG

Establishment of a chemical composition database of CG: By searching domestic and foreign literature (CNKI, PubMed, and Web of Science), TCM literature databases, and organizing the chemical composition of five types of TCMs in CG, a comprehensive database was established.

Chromatographic and mass spectrometric conditions: Chromatographic conditions: The time point with the most transitional components was selected to perform the UPLC-Q-TOF-MS analysis; an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm) was used. The binary gradient elution system comprised 0.1% formic acid aqueous solution and acetonitrile. The chromatogram was monitored at 275 nm and obtained using the following linear gradient elution program: B started at 2% and increased linearly from 2% to 10% (0-2 minute), 10%-16% (2-4 minute), 16%-31% (4-8 minute), 31%-46% (8-12 minute), 46%-68% (12-17 minute), 68%-80% (17-20 minute), and finally 80%-2% (20-23 minute). The injection volume of each sample was 2.0 μL. The column temperature was maintained at 30 °C, and the mobile phase was pumped at a flow rate of 0.3 mL/min.

MS conditions: MS was conducted on an Agilent-1100 HPLC/6520 Q-TOF-MS system (Waters Corp., United States) equipped with an electrospray ionization source in the negative ion mode. The collision energy was 20-45 mV, and the volume flow rate of the desolvation gas was 800 L/h. The temperatures of the desolvation gas and ion source were 400 °C and 100 °C, respectively. The spray voltage was 3.0 kV. The mass data were recorded within a scan range of 50-2000 Da.

Preparation of test and control solutions: Approximately 4 g of CG was added to a stoppered conical flask containing 25 mL of 50% methanol. The mixture was subjected to ultrasonic treatment for 10 minutes, cooled, shaken well, and centrifuged at 4000 r/min for 10 minutes. The supernatant was then passed through a 0.22-μm microporous membrane for filtration, and the filtrate was obtained.

Paeoniflorin, paeonilactone, asparagine, 1,2,3,4,6-O-galloylglucose, gallic acid, and 3,6'-dierucoylsucrose were added in appropriate amounts to a volumetric flask containing 10 mL of 50% methanol. The mixture was passed through a 0.22-μm microporous filtration membrane, and the filtrate was obtained.

Animal grouping and drug administration: The rats were randomly divided into blank and drug administration groups (n = 6), half of which were male and female. In the drug administration group, rats were gavaged with an aqueous solution of CG at a dose of 0.95 g/kg/day (The clinical pediatric doses of CG are 3 g/day for children aged 3-6 years and 6 g/day for children aged 7-11 years. Using the FDA body-surface-area conversion factor, the rat equivalent dose is calculated as: Human dose × 6.3/40 kg approximately 0.95 g/kg/day), by oral gavage once daily for 5 consecutive days. while the blank group was gavaged with an equal amount of saline. The drugs were administered once daily for 5 days. Blood was collected at 0.5-hour, 1-hour, 1.5-hour, and 2-hour after the last administration of the drugs and centrifuged in sodium heparin tubes for further preservation.

Plasma sample collection and pretreatment: Plasma sample collection: Rats were used as experimental subjects, and the aqueous solution of CG was gavaged. Blood was collected from the fundus venosus at 0.5-hour, 1-hour, 1.5-hour, and 2-hour, and all Eppendorf tubes and capillaries were infiltrated with heparin before blood collection. The collected blood was centrifuged at 3000 r/minute for 15 minutes, and the supernatant was removed with a pipette gun to obtain the plasma sample.

Plasma sample processing: A four-fold amount of methanol was added to 3 mL of plasma to precipitate proteins, and then vortexed and mixed for 2 minutes. After standing for 5 minutes, the plasma was centrifuged for 10 minutes at 13000 r/minute. The supernatant was aspirated and blow dried using a nitrogen blower. Two times the amount of methanol was added, and the process was repeated. The blow-dried solid was reconstituted with 0.5 mL of methanol, vortexed for 30 seconds, and centrifuged for 10 minutes at 13000 r/minute. The supernatant was aspirated and passed through a 0.22-μm microporous filter membrane to obtain a serum sample, which was retained for measurement.

Network pharmacology predicts the mechanism of the anti-TD activity of CG

Collection and screening of blood-entry components and TD targets: By inputting the names of components absorbed into blood from the TCMSP database (https://old.TCMsp-e.com/TCMsp.php), we retrieved information on the components of CG that were absorbed into blood and exported the corresponding effective targets of the screened active ingredients. Converting these targets into human genetic proteins using the UniProt database (https://www.uniprot.org/) using “TD” as a keyword, we searched the GeneCards (https://www.genecards.org/), OMIM (https://omim.org), CTD (http://ctdbase.org/), and DisGeNET (https://www.disgenet.org/) databases to obtain targets related to TD, removing duplicate targets. We compiled the circulating component-related and disease targets, creating a Venn diagram to illustrate the intersecting targets.

Active compounds and disease targets were retained only if they met the following thresholds: Oral bioavailability > 30% and drug-likeness > 0.18 in TCMSP; GeneCards (v5.15) and DisGeNET (v7.0): All entries with a relevance/association score > 0 were retained without further cut-off to avoid missing any potential targets.

Common target gene-protein interaction network construction and Hub gene screening: The intersecting target genes were imported into the STRING database (https://cn.string-db.org/), and the minimum required interaction score was set to the highest confidence (0.900). Protein species was selected as “Homo sapiens”, and the off-node was deleted. The interconnections between genes were predicted.

Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses: Cross-target genes were imported into the Metascape website, the species was set to human, the screening threshold for enrichment analysis was P < 0.01, and Gene Ontology (GO) biological process, cellular localization, and molecular function analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG)signaling pathway enrichment analyses were performed for the cross-targets, respectively. The results were integrated to draw GO enrichment analysis histograms and KEGG enrichment analysis bubble diagrams.

CG blood component-target-pathway-disease pull network analysis and construction: CG ingestion components were incorporated into Cytoscape 3.7.2 software to construct a network of “blood components-potential targets-associated pathways-anti-TD effects” and the analysis was visualized.

Molecular docking

To balance biological relevance with computational efficiency, we prioritized compounds for docking based on (1) Degree centrality in the compound-target network (degree value > 100); (2) Plasma abundance; and (3) Literature-documented activity against core targets. The three-dimensional structures of the core proteins were downloaded from the PDB database (https://www.rcsb.org/), and the protein molecules were imported into the software Discovery Studio 2019 to remove water molecules, add hydrogen atoms, and set the atom types. The structures of the compounds were downloaded through the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), and the small-molecule compounds were imported into Discovery Studio 2019 software to perform molecular docking. The results were visualized and analyzed.

RESULTS

In vitro chemical characterization

The chemical components of CG were analyzed using UPLC-Q-TOF-MS/MS, and the MS information was compared with the control, combined with the self-constructed library and UNIFI software. A total of 187 compounds were identified in the positive and negative ion modes, including phenolic acids, terpenes, flavonoids, and saccharides. Among them, 83 compounds were characterized in BS, 44 compounds in TM, 63 compounds in YZ, 9 compounds in SCP, and 1 compound in ZZM. The information and sources of each constituent and control quality profile are shown in Table 1 and Figure 1.

Figure 1 The results of in vitro chemical composition analysis. A: Total ion current diagram of negative ion mode; B: Total ion current diagram of positive ion mode; C: Total ion current diagram of reference in negative ion mode; D: Class of chemical composition of Changmaxifeng granules (CG); E: Source of chemical composition of CG. C1 for gallic acid; C2 for gastrodin; C3 for albiflorin; C4 for paeoniflorin; C5 for 1,2,3,4,6-pentagalloylglucose; C6 for 3, 6′-disinapoyl sucrose.

Table 1 Chemical composition information of Changmaxifeng granules.

No.	tR (minute)	Ion mode	Deviation (ppm)	Fragment; ions	Theoretical value	Molecular formula	Ingredient name	Source
1	0.81	[M-H]- 215.0348	1.86	149.0428	216.0423	C12H8O4	1-furan-2-yl-2-(4-hydroxy-phenyl)ethane-1,2-dione	TM
2	0.82	[M+H]+ 175.1197	1.14	116.0725; 158.0872	174.1117	C6H14N4O2	Arginine	BS
3	0.84	[M-H]- 165.039	-5.45	131.0469	166.0477	C5H10O6	Arabic acid	TM
4	0.85	[M-H]- 195.0492	-6.66	165.0399; 179.0583; 129.0202	195.0583	C6H12O7	Gluconic acid	BS/TM
5	1.03	[M-H]- 133.0139	1.5	71.0096; 115.0034	134.0215	C4H6O5	D-(+)-malic acid	TM
6	1.05	[M-H]- 115.0034	2.61	71.0133	116.011	C4H4O4	Fumaric acid	TM
7	1.37	[M-H]- 128.035	1.56	96.9606	129.0426	C5H7NO3	L-pyroglutamic acid	TM
8	1.49	[M-H]- 191.0206	7.33	111.0066	192.027	C6H8O7	Citric acid	BS/TM
9	1.49	[M-H]- 173.0096	5.78	111.0066; 129.0202	174.0164	C6H6O6	Trans-aconitic acid or its isomer	BS
10	1.71	[M-H]- 188.0566	3.72	128.035; 144.0627; 173.0038	189.0637	C7H11NO5	N-acetylglutamic acid	TM
11	1.72	[M-H]- 343.0668	0.87	191.0206	344.0743	C14H16O10	5-galloylquinic acid	BS
12	1.8	[M-H]- 243.0642	-6.17	107.0534	244.0736	C14H12O4	Gastrodibenzin B/C	TM
13	1.9	[M-H]- 375.1257	-9.06	341.1122; 300.9911	376.1369	C16H24O10	8-debenzoylpaeoniflorin	BS
14	1.95	[M-H]- 493.1178	-3.04	169.0121; 313.0574	494.1272	C19H26O15	6'-O-galloylsucrose or its isomer	BS
15	2.13	[M+H]+ 268.1019	-10.01	119.0341; 136.0614; 268.1090	267.0968	C10H13N5O4	Adenosine	BS
16	2.13	[M+H]+ 136.0614	-6.61	91.0423; 119.0389	135.0545	C5H5N5	AdenIne	TM
17	2.15	[M-H]- 331.0651	-4.23	316.1480; 271.4559	332.0743	C13H16O10	6-O-galloylglucose or its isomer	BS
18	2.28	[M-H]- 493.1178	-3.04	169.0121; 313.0574	494.1272	C19H26O15	6'-O-galloylsucrose or its isomer	BS
19	2.37	[M-H]- 282.0842	1.42	133.0139; 150.0412	283.0917	C10H13N5O5	Guanosine	BS/TM
201	2.46	[M-H]- 169.0121	-9.47	125.0238; 107.0082	170.0215	C7H6O5	Gallic acid	BS/TM
21	2.46	[M-H]- 125.0238	-0.8	81.0333	126.0317	C6H6O3	Maltol	BS
22	2.48	[M-H]- 125.0238	-0.8	107.7866; 95.0097; 77.0962	126.0317	C6H6O3	Pyrogallol	BS
23	2.62	[M-H]- 331.1049	6.04	313.0575	332.1107	C14H20O9	Mudanoside A	BS
241	2.62	[M-H]- 285.0993	6.66	268.0276	286.1053	C13H18O7	Gastrodin	TM
25	2.79	[M-H]- 359.1365	6.4	329.0024; 271.0459	360.142	C16H24O9	1-O-β-D-glucopyranosyl-paeoniflorin	BS
26	3.03	[M-H]- 493.1178	-3.04	169.0121; 313.0574	494.1272	C19H26O15	6'-O-galloylsucrose or its isomer	BS
27	3.14	[M-H]- 164.0698	-8.53	103.0476; 131.0419	165.079	C9H11NO2	L-phenylalanine	TM/ZZM
28	3.15	[M-H]- 331.0651	-4.23	313.0497; 271.0459	332.0743	C13H16O10	6-O-galloylglucose or its isomer	BS
29	3.24	[M-H]- 493.1178	-3.04	169.0121; 313.0574	494.1272	C19H26O15	6'-O-galloylsucrose or its isomer	BS
30	3.27	[M-H]- 313.0574	4.47	113.0234; 137.0263	314.0638	C13H14O9	2-carboxyphenyl-α-D-glucopyranuronic acid	TM
31	3.44	[M-H]- 447.1514	2.46	89.0203; 341.0718; 389.0217	448.1581	C19H28O12	Gastrodin A	TM
32	3.85	[M-H]- 527.1414	2.47	479.0424	528.1479	C23H28O14	Galloyl desbenzoylpaeoniflorin	BS
33	3.98	[M-H]- 589.1218	-1.53	167.0366; 259.0247	590.1305	C24H30O15S	Paeoniflorin E sulfite	BS
34	4	[M+H]+ 385.1481	-4.67	323.103	384.142	C18H24O9	Tenuifoliside D	YZ
35	4.01	[M-H]- 361.1504	1.38	315.0695	362.1577	C16H26O9	6-O-copyranosyl-lactinlide	BS
36	4.24	[M-H]- 343.1396	0.87	310.0282	343.1393	C16H24O8	Mudanpioside F/mudanpioside G	BS
37	4.24	[M-H]- 459.1151	2.61	401.1037	460.1217	C19H24O13	Parishin E/parishin G	TM
38	4.24	[M-H]- 173.0096	5.78	125.0287; 137.0071	174.0164	C6H6O6	Trans-aconitic acid or its isomer	TM
39	4.33	[M-H]- 633.0732	7.58	555.1772	634.0806	C27H22O18	Strictinin	BS
40	4.37	[M-H]- 461.129	-1.08	93.0323; 137.0225	462.1373	C19H26O13	Sibiricose A3	YZ
41	4.41	[M-H]- 705.1734	4.68	543.1223; 259.0247; 121.0279	706.1779	C29H38O18S	Isomaltopaeoniflorinsulfite or its isomer	BS
42	4.48	[M-H]- 451.1218	-4.88	169.0065; 245.0836; 289.0727	452.1319	C21H24O11	Catechin glucoside	BS
43	4.5	[M-H]- 203.0833	5.91	116.0501; 159.0521	204.0899	C11H12N2O2	Tryptophan	SCP
44	4.54	[M-H]- 459.1151	2.61	400.9901	460.1217	C19H24O13	Parishin E/parishin G	TM
45	4.54	[M-H]- 421.0773	0.47	259.0247	422.0849	C19H18O11	Isomangiferin	TM
46	4.55	[M-H]- 543.1121	-9.39	121.0279; 259.0247	544.1251	C23H28O13S	Paeoniflorin sulfite	BS
47	4.56	[M+H]+ 579.1458	-7.77	409.1026	578.1424	C30H26O12	Procyanidin B1/B2	BS
48	4.69	[M-H]- 483.0744	-6.42	125.0189; 151.0002; 169.0121	484.0853	C20H20O14	Paeoniflorin sulfite	BS
49	4.76	[M-H]- 577.1319	-4.68	289.0727	578.1424	C30H26O12	Procyanidin B1/B2	BS
50	4.79	[M-H]- 495.1466	-7.47	465.1419; 137.0225	496.1581	C23H28O12	Oxypaeoniflorin or its isomer	BS
51	4.81	[M+H]+ 414.1312	-5.55	162.0233; 308.0806	413.1257	C17H23N3O7S	Sulfur-(4-hydroxybenzyl)-glutathione	TM
52	4.81	[M+H]+ 576.1881	3.12	320.0902	575.1785	C23H33N3O12S	S-gastrodin-glutathione	TM
53	4.82	[M-H]- 175.0605	-0.57	161.0244	176.0685	C7H12O5	2-Isopropylmalic acid	TM
54	4.92	[M-H]- 635.0884	4.41	169.0121; 313.0574	636.0963	C27H24O18	1,3,6-Tri-O-galloyl-β-D-glucose	BS
55	4.95	[M-H]- 517.1526	-5.99	160.0174; 175.0432; 193.0479	518.1636	C22H30O14	Sibiricose A5	YZ
56	4.97	[M+H]+ 139.0400	3.6	77.0374	138.0317	C7H6O3	Salicylic acid	TM/YZ/SCP
57	4.98	[M+H]+ 291.0884	5.15	247.0267; 263.1024	290.079	C15H14O6	Catechin	BS
58	4.99	[M-H]- 245.0836	8.98	190.026	246.0892	C14H14O4	Peonol	BS
59	5	[M-H]- 289.0727	5.19	123.0444; 205.0483	290.079	C15H14O6	Epicatechin	BS
60	5	[M-H]- 183.0303	5.46	78.0115; 124.0151; 168.0370	184.0372	C8H8O5	Methyl gallate	BS
61	5	[M-H]- 547.1702	7.13	205.0483	548.1741	C23H32O15	Sibiricose A1	YZ
62	5.19	[M-H]- 205.0359	5.36	111.0112	206.0427	C7H10O7	3-Hydroxy-3-(methoxycarbonyl) pentan	TM
63	5.36	[M-H]- 785.0837	0	125.0279	786.0916	C34H26O22	Tellimagrandin I	BS
64	5.36	[M-H]- 889.2623	1.01	85.0282; 111.0066; 780.1116	890.2692	C38H50O24	Parishin V	TM
65	5.39	[M+H]+ 374.1462	-0.53	136.0614	373.1386	C17H19N5O5	N-(4-hydroxypheny) adenosine	TM
66	5.43	[M-H]- 727.2141	7.56	129.0202	728.2164	C32H40O19	Parishin B	TM
67	5.57	[M-H]- 757.2173	-2.38	71.0133; 453.0717	758.2269	C33H42O20	Parishin M	TM
68	5.63	[M-H]- 687.2103	-4.8	635.0635; 165.0502; 121.0279	688.2215	C30H40O18	6'-O-β-D-glucopyranosylalbiflorin	BS
69	5.66	[M+H]+ 342.1699	-1.75	237.0880; 297.0598	341.1627	C20H23NO4	N-Methylhernagine	BS/TM
701	5.71	[M-H]- 479.1568	2.09	525.1579	480.1632	C23H28O11	Albiflorin	BS
71	5.71	[M-H]- 525.158	-5.33	165.0558; 167.0309; 327.1120; 363.1698	526.1686	C24H30O13	Mudanpioside E	BS
72	5.72	[M-H]- 727.2141	7.56	111.0066; 161.0465	728.2164	C32H40O19	Parishin C	TM
73	5.72	[M+H]+ 319.1151	-9.71	301.1047	318.1103	C17H18O6	Evofolin B	SCP
74	5.73	[M+H]+ 197.0822	4.06	151.078; 179.0684	196.0736	C10H12O4	Paeonilactone B	BS
75	5.73	[M+H]+ 133.0648	-3.76	77.0374; 105.0681; 115.0545	132.0575	C9H8O	Cinnamaldehyde	BS/SCP
76	5.98	[M+H]+ 539.1373	-5.19	503.1316	538.1323	C24H26O14	Sibiricaxanthone A	YZ
77	6.00	[M-H]- 285.0404	1.75	257.0396; 268.0276	286.0477	C15H10O6	Kaempferol	BS/TM
78	6.00	[M-H]- 537.1243	-0.19	315.0464; 387.0604	538.1323	C24H26O14	Sibiricaxanthone B	YZ
79	6.08	[M+H]+ 463.1623	4.1	151.0780; 179.0684	462.1526	C23H26O10	Lactiflorin	BS
801	6.09	[M-H]- 479.1568	2.09	77.0387; 121.0329; 165.0607; 327.1063; 449.1483; 959.3233	480.1632	C23H28O11	Paeoniflorin	BS
81	6.09	[M-H]- 121.0279	-9.09	77.0387	122.0368	C7H6O2	4-hydroxybenzaldehyde	TM
82	6.1	[M-H]- 165.0558	3.64	121.0279; 77.0387	166.063	C9H10O3	Paeonol	BS
83	6.16	[M-H]- 431.1373	7.19	327.1041	432.142	C22H24O9	Heptemthoxyflavone	BS
84	6.23	[M-H]- 567.1356	1.06	345.0594; 315.0541; 272.0318	568.1428	C25H28O15	Polygalaxanthone VIII	YZ
85	6.42	[M-H]- 495.1466	-7.47	427.1026; 137.0225	496.1581	C23H28O12	Oxypaeoniflorin or its isomer	BS
86	6.48	[M-H]- 787.104	5.84	169.0121; 456.0666; 617.0786	788.1072	C34H28O22	1,3,4,6-tetragalloylglucose	BS
87	6.55	[M+H]+ 199.0596	-5.02	163.0358; 153.0205	198.0528	C9H10O5	Ethyl gallate	BS
88	6.64	[M-H]- 995.3086	5.43	423.0960; 728.2154	996.3111	C45H56O25	Parishin A	TM
89	6.69	[M-H]- 433.1162	6.23	111.0020; 271.0603; 397.1084	434.1213	C21H22O10	Dihydroxyflavone-glucoside	BS
90	6.73	[M-H]- 300.9986	0.66	271.0603; 245.9800	302.0063	C14H6O8	Ellagic acid	BS
91	6.89	[M+H]+ 271.0762	-0.37	147.0451; 153.0151; 273.0834	272.0685	C15H12O5	Naringenin	BS
92	6.9	[M-H]- 667.1871	-0.45	205.0483; 461.1384	668.1952	C30H36O17	Tenuifoliside B	YZ
93	6.92	[M-H]- 433.1162	6.23	119.0471; 151.0002; 271.0603	434.1213	C21H22O10	Isosalipurposide	BS
94	6.94	[M-H]- 223.0618	5.38	93.0365; 121.0279	224.0685	C11H12O5	Sinapinic acid	TM
95	7.18	[M-H]- 631.1628	-5.55	465.1419; 313.1419; 271.0459; 169.0121	632.1741	C30H32O15	Galloylpaeoniflorin or its isomer	BS
96	7.25	[M-H]- 631.1628	-5.55	465.1419; 313.1419; 271.0459; 169.0121	632.1741	C30H32O15	Galloylpaeoniflorin or its isomer	BS
971	7.44	[M-H]- 939.1152	5.11	769.0880; 617.0786; 447.0591; 169.0121	940.1182	C41H32O26	1,2,3,4,6-pentagalloylglucose	BS
98	7.71	[M-H]- 631.1628	-5.55	465.1419; 313.1419; 271.0459; 169.0121	632.1741	C30H32O15	Galloylpaeoniflorin or its isomer	BS
99	7.81	[M-H]- 611.1589	-3.76	465.1042; 287.0197	612.169	C27H32O16	Polygalaxanthone VII	YZ
1001	7.82	[M-H]- 753.2277	-4.65	205.0518; 223.0675; 367.1056; 529.1638; 547.1773	754.232	C34H42O19	3,6′-disinapoyl sucrose	YZ
101	7.97	[M-H]- 631.1628	-5.55	465.1419; 313.1419; 271.0459; 169.0121	632.1741	C30H32O15	Galloylpaeoniflorin or its isomer	BS
102	8.13	[M-H]- 631.1628	-5.55	465.1419; 313.1419; 271.0459; 169.0121	632.1741	C30H32O15	Galloylpaeoniflorin or its isomer	BS
103	8.2	[M-H]- 723.2156	2.77	631.1628	724.2215	C33H40O18	Arillanin A	YZ
104	8.5	[M+H]+ 481.1678	-6.65	105.0323; 436.1096	480.1632	C23H28O11	Mudanpioside I	BS
105	8.52	[M-H]- 503.1763	-0.4	209.0763; 485.1499	504.1843	C22H32O13	Polygalatenosides E	YZ
106	8.64	[M-H]- 651.1879	-7.06	137.0225; 281.0656; 443.1159	652.2003	C30H36O16	Tenuifoliside-652	YZ
107	8.7	[M-H]- 1453.4358	-6.81	145.0315; 1039.3169; 1119.3817; 1161.3540	1454.4535	C65H82O37	Tenuifoliose Q	YZ
108	8.74	[M-H]- 509.1694	6.87	479.1472; 169.0121; 121.0279	510.1737	C24H30O12	Mudanpioside D	BS
109	8.78	[M+H]+ 481.1678	-6.65	77.0374	480.1632	C23H28O11	Albiflorin R1	BS
110	8.79	[M-H]- 647.1489	8.34	121.0279; 213.0223; 259.0247; 525.0979	648.1513	C30H32O14S	Benzoylpaeoniflorin sulfonate	BS
111	8.81	[M-H]- 283.0826	2.83	255.8088	284.0894	C13H16O7	Benzyl glucoside	BS
112	9.18	[M-H]-783.1761	-1.53	169.0121; 465.1325; 631.1519	784.1851	C37H36O19	3’-6’-D-O-galloylpaeoniflorin	BS
113	9.25	[M-H]- 681.2019	-1.76	179.0349; 281.0656; 443.1159	682.2109	C31H38O17	Tenuifoliside A	YZ
114	9.61	[M+H]+ 271.0609	1.11	95.0152; 207.0684	270.0528	C15H10O5	Galangin	SCP
115	9.65	[M-H]- 269.0438	-4.46	251.0329	270.0528	C15H10O5	Baicalein	BS
116	9.66	[M-H]- 599.1808	7.18	137.0276	600.1843	C30H32O13	Benzoyloxypaeoniflorin	BS
117	9.71	[M-H]- 1525.4702	2.23	1379.4265	1526.4746	C68H86O39	Tenuifoliose F	YZ
118	9.74	[M-H]- 1495.4609	3.14	1161.3540; 1203.3713; 1349.4047	1496.4641	C67H84O38	Tenuifoliose L	YZ
119	9.82	[M-H]- 1253.3766	-0.48	753.2396; 809.2495; 955.3134; 1077.3359	1254.385	C56H70O32	Tenuifoliose T	YZ
120	9.85	[M+H]+ 314.1379	-4.14	93.0645	313.1314	C18H19NO4	N-trans-Feruloyltyramine	TM
121	9.88	[M-H]- 312.1232	-1.28	281.2414	313.1314	C18H19NO4	Feruloyl tyramine	SCP
122	10.01	[M-H]- 447.2253	5.14	59.0121; 89.0244	448.2308	C21H36O10	Geraniol-primeveroside	BS
123	10.12	[M-H]- 767.2444	5.87	223.0672; 237.0764; 529.1638	768.2477	C35H44O19	Tenuifoliside C	YZ
124	10.17	[M-H]- 1223.3766	8.17	145.0315; 307.0751; 955.2988; 1077.3217; 1101.3361	1224.3745	C55H68O31	Tenuifoliose S	YZ
125	10.43	[M-H]- 1295.3929	3.94	997.3134; 1119.3524	1296.3956	C58H72O33	Tenuifoliose C/tenuifoliose E	YZ
126	10.57	[M-H]- 237.0761	-0.84	206.0752; 222.0520	238.0841	C12H14O5	4-hydroxy-3,5-dimethoxylcinnamate	YZ
127	10.67	[M-H]- 1397.621	-1.07	-	1398.6303	C64H102O33	Arillatanoside C	YZ
128	10.79	[M-H]- 445.2051	-5.17	311.0902	446.2152	C21H34O10	Β-pinen-10-yl-β-vicianoside	BS
129	10.83	[M-H]- 1307.3984	8.11	307.0827; 653.1922; 1161.3689	1308.3956	C59H72O33	Tenuifoliose J/tenuifoliose I	YZ
130	10.88	[M-H]- 1265.5903	7.9	425.3125; 455.3217; 499.1701; 585.1967; 1235.5663	1266.5881	C59H94O29	Desacylsenegasaponin B	YZ
131	10.96	[M-H]- 1541.6667	-8.63	425.3125; 1317.3502	1542.6879	C74H110O34	Onjisaponin H	YZ
132	10.98	[M-H]- 1411.6322	-4.25	-	1412.646	C65H104O33	Desacylsenegasaponin III	YZ
133	11.2	[M-H]- 1235.5818	9.79	337.1186; 455.3217; 555.1955	1236.5775	C58H92O28	Arillatanoside A	YZ
134	11.29	[M-H]- 1381.6334	4.2	701.1796; 1157.5767; 1351.6196	1382.6354	C64H102O32	Polygalasaponin XIX	YZ
135	11.36	[M-H]- 711.2166	4.22	694.1769	712.2215	C32H40O18	Telephiose C	SCP
136	11.47	[M-H]- 1249.5913	4.8	425.3125; 455.3217; 1025.5084; 1219.5607	1250.5932	C59H94O28	Onjisaponin Tf	YZ
137	11.5	[M-H]- 1525.6748	3.21	1157.5618; 1351.6196; 1463.7104	1526.6777	C70H110O36	Onjisaponin Te	YZ
138	11.53	[M-H]- 1379.6213	6.81	425.3125; 455.3217; 1235.5817	1380.6198	C64H100O32	Onjisaponin TG	YZ
139	11.54	[M-H]- 1103.5229	-4.08	455.3146	1104.5353	C53H84O24	Polygalasaponin XXVIII	YZ
140	11.55	[M-H]- 1307.3984	8.11	307.0827; 653.1922; 1161.3689	1308.3956	C59H72O33	Tenuifoliose J/tenuifoliose I	YZ
141	11.71	[M-H]- 741.2241	-0.13	179.0583; 684.2599	742.232	C33H42O19	Parishin K	TM
142	11.9	[M-H]- 1367.4008	-5.92	1027.3135; 1191.3721	1368.4167	C61H76O35	Tenuifoliose O	YZ
143	11.91	[M-H]- 1265.3884	8.85	631.1901; 753.2276; 1143.3408; 1223.3461	1266.385	C57H70O32	Tenuifoliose K	YZ
144	11.97	[M-H]- 1349.4048	4.82	1039.3309; 1161.3689; 1203.3713	1350.4061	C61H74O34	Tenuifoliose H	YZ
145	12.18	[M-H]- 583.1800	-2.74	553.1725; 121.0279	584.1894	C30H32O12	Benzoylpaeoniflorin	BS
146	12.18	[M-H]- 629.1896	4.13	121.0279	630.1949	C31H34O14	Mudanpioside B/mudanpioside J	BS
147	12.19	[M-H]- 1379.4103	1.01	347.0917; 1161.3540	1380.4167	C62H76O35	Tenuifoliose A	YZ
148	12.22	[M-H]- 1409.418	-1.06	825.3434; 1069.3090; 1111.3448; 1173.3512; 1191.3871	1410.4273	C63H78O36	Tenuifoliose N	YZ
149	12.22	[M-H]- 1325.3993	0.75	661.2083; 1149.3740; 1203.3866	1326.4061	C59H74O34	Senegose B/C	YZ
150	12.38	[M+H]+ 585.1997	4.27	77.0375; 105.0323	584.1894	C30H32O12	Benzoylalbiflorin	BS
151	12.4	[M-H]- 629.1896	4.13	121.0279	630.1949	C31H34O14	Mudanpioside B/mudanpioside J	BS
152	12.46	[M-H]- 1307.3984	8.11	307.0827; 653.1922; 1161.3689	1308.3956	C59H72O33	Tenuifoliose J/tenuifoliose I	YZ
153	12.51	[M-H]- 327.2147	-7.33	171.1021	328.225	C18H32O5	(12Z, 15Z) -9,10,11-trihydroxy-12,15-octadecadienoic acid	TM
154	12.62	[M+H]+ 303.0843	-8.58	245.0417; 261.0185	302.079	C16H14O6	Onjixanthone I	YZ
155	12.76	[M-H]- 677.355	1.92	629.0362	678.3615	C36H54O12	Sibiricasaponin A	YZ
156	12.83	[M-H]- 679.3682	-1.77	425.3086; 455.3146	680.3772	C36H56O12	Tenuifolin	YZ
157	12.89	[M+H]+ 295.2275	0.68	221.0807	294.2195	C18H30O3	13(S)-HOTrE	BS
158	12.9	[M-H]- 329.2322	-1.82	171.1021; 211.1272; 229.1422	330.2406	C18H34O5	9,12,13-TriHOME	BS
159	13	[M-H]- 1703.7188	-8.22	157.1910; 1479.7279	1704.7407	C80H120O39	Onjisaponin A	YZ
160	13.09	[M-H]- 1631.7034	-5.09	425.3125; 455.3123; 567.1970; 1601.6403	1632.7195	C77H116O37	Onjisaponin O	YZ
161	13.18	[M-H]- 1571.6836	-4.45	567.1865; 1347.6085; 1541.6838	1572.6984	C75H112O35	Onjisaponin B/onjisaponin D	YZ
162	13.23	[M-H]- 1617.6907	-3.34	425.3035; 455.3123; 1393.8030	1618.7039	C76H114O37	Polygalasaponin XLIV	YZ
163	13.26	[M-H]- 1673.7109	-6.81	425.3035; 1617.6730	1674.7301	C79H118O38	Z-polygalasaponin XXXII	YZ
164	13.26	[M+H]+ 347.1114	-4.9	289.0669; 331.1214	346.1053	C18H18O7	1,2,3,6,7-pentamethoxyxanthone	YZ
165	13.33	[M-H]- 1469.6482	3.13	1439.6489	1470.6515	C67H106O35	Polygalasaponin XXXXII	YZ
166	13.34	[M-H]- 1469.6482	-7.28	405.1388	1470.6667	C71H106O32	Onjisaponin Z	YZ
167	13.42	[M-H]- 1485.6475	-4.24	425.3125; 1455.6677	1486.6616	C71H106O33	Onjisaponin E	YZ
168	13.45	[M-H]- 1409.6310	-4.82	425.3125; 455.3217; 1185.6506; 1379.6375	1410.6456	C69H102O30	Onjisaponin Y	YZ
169	13.52	[M-H]- 1587.6880	1.57	155.3035; 583.1805; 1455.6344	1588.6933	C75H112O36	Onjisaponin F	YZ
170	13.52	[M-H]- 1455.6344	-6.05	237.0832; 425.3125; 455.3123; 1425.36638	1456.6511	C70H104O32	Onjisaponin G/onjisaponin J	YZ
171	13.58	[M-H]- 1733.7567	7.67	455.3217; 669.2285	1734.7512	C81H122O40	Onjisaponin S	YZ
172	13.62	[M-H]- 1599.6833	-1.38	1455.6344; 1537.7080	1600.6933	C76H112O36	Onjisaponin Gg/onjisaponin K	YZ
173	13.66	[M-H]- 1425.6309	-1.26	425.3035; 455.3123; 1395.6298; 1426.6205	1426.6405	C69H102O31	Senegasaponin B	YZ
174	13.72	[M-H]- 1323.6033	1.74	425.3035; 455.3217; 1293.5858	1324.6088	C65H96O28	Onjisaponin TH	YZ
175	13.99	[M-H]- 1263.5869	5.54	425.3125; 455.3123; 1233.6174	1264.5877	C63H92O26	Onjisaponin MF	YZ
176	14.31	[M-H]- 317.0649	-3.78	259.0338	318.074	C16H14O7	6,8-dihydroxy-1,2,4-trimethyoxyxanthone	YZ
177	15.66	[M-H]- 455.3146	-3.29	441.2885; 427.2561	456.324	C29H44O4	30-demethylated hederagenin	BS
178	16.3	[M+H]+ 373.2014	-0.27	331.0904	372.1937	C22H28O5	Pyrethrin II	BS
179	16.57	[M-H]- 471.3503	6.15	393.3143	472.3553	C30H48O4	Hederagenin or its isomer	BS
180	16.65	[M+H]+ 520.3367	-6.92	86.0983; 184.0721	519.3325	C26H50NO7P	2-dioleoyl-sn-glycero-3-phosphocholine	TM
181	16.99	[M-H]- 471.3503	6.15	393.3143	472.3553	C30H48O4	23-hydroxybetulinic acid	BS
182	17.29	[M-H]- 233.1523	-8.15	218.8748; 197.9078	234.162	C15H22O2	Acoronene	SCP
183	18.5	[M+H]+ 301.1426	8.63	127.0094; 132.9692	300.1321	C13H20N2O6	5-butyluridine	SCP
184	18.69	[M-H]- 293.1782	9.89	245.8978	294.1831	C17H26O4	6-gingerol	TM
185	20.34	[M+H]+ 297.2404	-8.75	253.1753; 261.0256	296.2351	C18H32O3	13-HODE	TM
186	20.57	[M-H]- 455.3519	-1.32	441.7197; 355.7012	456.3603	C30H48O3	Oleanolic acid or its isomer	BS
187	21.12	[M-H]- 455.3519	-1.32	441.7197; 355.7012	456.3603	C30H48O3	Oleanolic acid or its isomer	BS

¹Identification by comparison with reference material.

TM: Tianma; BS: Baishao; ZZM: Zhenzhumu; YZ: Yuanzhi; SCP: Shichangpu.

Analysis of fragmentation patterns

Identification of terpenoids: In total, 63 terpenoids have been characterized in CG, mainly from BS and YZ. Most terpenes combine with sugars to form glycosides. The characterized monoterpenes include paeoniflorin, albiflorin, benzoylpaeoniflorin, etc. The characterized triterpenes include onjisaponin Y, arillatanoside A, desacylsenegasaponin III, etc. The molecular ion peak during cleavage usually begins with a glycosidic bond break, forming a genin. Subsequently, the genin continues to lose some substituents, including -OH and -CH₃.

The monoterpenes represented by paeoniflorin mostly contain a pinane skeleton. Fragment ion peaks at m/z 165 were observed. The quasi-molecular ion peaks of [M-H]^- are usually lower, and they mostly exist as [M+CH₃COOH]^-. Taking peak 80 (t_R = 6.09) as an example, the primary mass spectrogram in negative ion mode exists [M+CH₃COOH]^- and peaks at m/z 525, which has a lower [M-H]^- abundance at m/z 479. The secondary mass spectrometry showed the presence of m/z 449, which is presumed to be the [M-CH₂O]^- fragment ion peak; further loss of one molecule of C₇H₅O₂ yields the fragment ion peak at m/z 327. The m/z 165 fragment ion peak of pinane skeleton and the m/z 959 fragment ion peak of [2M-H]^- were also observed. After comprehensive analysis, peak 80 was presumed to be paeoniflorin. The cleavage pattern and secondary mass spectrometry data are shown in Figure 2A.

Figure 2 Possible cleavage pattern and secondary mass diagram. A: The degradation pattern and secondary mass diagram of paeoniflorin; B: The degradation pattern and secondary mass diagram of onjisaponin Y; C: The degradation pattern and secondary mass diagram of 3,6’-disinapoyl sucrose; D: The degradation pattern and secondary mass diagram of tenuifoliside A; E: The degradation pattern and secondary mass diagram of parishin B; F: The degradation pattern and secondary mass diagram of gallic acid; G: The degradation pattern and secondary mass diagram of polygalaxanthone VIII; H: The degradation pattern and secondary mass diagram of acetyl glutamic acid.

The triterpenoids represented by onjisaponin Y are mostly combined with sugar to form saponins. These saponins belong to the oleanolic acid type of pentacyclic triterpenes, and the C3 position is mostly replaced by glucose. The C28 position is also linked to rhamnose, galactose, and other monosaccharides. Therefore, the cleavage pattern of their mass spectra mostly showed glycosidic bond breaking, and one or more H₂O and CO₂ molecules might have been lost during the cleavage process. According to the cleavage characteristics of parent nucleus of onjisaponin Y, its characteristic fragment ions were m/z 455 and m/z 425. Taking peak 168 (t_R = 13.45) as an example, the peak with m/z 1409.6310 was detected as an [M-H]^- peak in the negative ion mode. In the secondary MS, there were peaks at m/z 1379, which were presumed to be [M-H-CH₂O]^-, peaks at m/z 1185 were presumed to be [M-H-Glc-H₂O-CO₂]^-, and peaks at m/z 1007 were presumed to be [M-H-Glc-H₂O-CO₂-C₁₀H₁₀O₃]^-, followed by CH₂OH removal to obtain the peaks at m/z 977. The peaks at m/z 455 and m/z 425 were attributed to breakage of sugar bonds, dehydration, decarboxylation, and CH₂OH cleavage of parent nucleus of onjisaponin Y. The cleavage pattern and secondary mass spectra are shown in Figure 2B.

Identification of carbohydrate components: Overall, 41 carbohydrate components were characterized in CG, and these components were mainly from TM and YZ. Sugar esters, which use sucrose as the parent nucleus and connect different monosaccharides with glycosidic bonds at different positions, are mainly present in YZ. Sugar ester components are mainly categorized into monosaccharide and oligosaccharide esters. Oligosaccharide esters are linked to different amounts of glucose and contain different acyl groups, mainly acyl, benzoyl, cinnamoyl, and feruloyl. Glycose esters are present in many plants, but oligosaccharides containing three or more glucose molecules in the parent nucleus are mainly found in YZ. Oligosaccharides are considered compounds unique to YZ, and Fargesia oligosaccharides may have some specific pharmacological activities[15].

Oligosaccharides were detected at peak 100 (t_R = 7.82) as an [M-H]^- peak with an ion peak at m/z 753 in the negative ion mode. m/z 547 was present in the secondary mass spectrogram, presumed to be [M-H-C₁₁H₁₀O₄]^-; m/z529 was presumed to be [M-H-C₁₁H₁₂O₅]^-, m/z 367 presumed to be [M-H-C₁₁H₁₂O₅-C₆H₁₀O₅]^-, and m/z 205 was presumed to be [M-H-C₁₂H₁₄O₅-C₆H₁₀O₅-C₄H₆O₃]^-. After comprehensive analysis and comparison with the control, peak 100 was confirmed to be 3,6'-dierucoyl sucrose. The cleavage pattern and secondary mass spectra are shown in Figure 2C.

Sugar esters are exemplified by peak 113 (t_R = 9.25), where an ion peak at m/z 681 was detected in the negative ion mode as the [M-H]^- peak. In the secondary mass spectrum, m/z 443 was presumed to be [M-H-C₁₂H₁₄O₅]^-, m/z 281 was presumed to be [M-H-C₁₂H₁₄O₅-C₆H₁₀O₅]^-, and m/z 179 was presumed to be [M-H-C₁₁H₁₂O₅-C₆H₁₀O₅-C₄H₆O₃]^-. The hydrolysis of one molecule of CO continued to drop off to form the peak of m/z 137. After comprehensive analysis, peak 113 was presumed to be tenuifoliside A. The cleavage pattern and secondary mass spectra are shown in Figure 2D.

Glycosides are mainly present in TM, and these components, represented by palisarin, undergo ester bond breaking during the cleavage process. Taking peak 66 (t_R = 5.43) as an example, the ion peak at m/z 727 was detected as [M-H]^- peak in the negative ion mode. In the secondary mass spectrum, m/z 459 was presumed to be [M-H-C₁₃H₁₇O₆]^-, m/z 423 was presumed to be [M-H-C₁₃H₁₇O₆-2H₂O]^-, m/z 161 was presumed to be [M-H-C₁₃H₁₇O₆-2H₂O-C₁₃H₁₀O₆]^-, m/z 441 was presumed to be [M-H-C₁₃H₁₇O₆-OH]^-, m/z 397 was presumed to be [M-H-C₁₃H₁₇O₆-OH-CO₂]^-, and m/z 205 was presumed to be [M-H-C₁₃H₁₇O₆-OH-CO₂-C₆H₈O₇]^-. After the comprehensive analysis, peak 66 was presumed to be palisarin B. The cleavage pattern and second-level mass spectra are shown in Figure 2E.

Identification of phenolic acid: A total of 36 phenolic acid components were characterized in CG, and these phenolic acid components were mainly found in BS and TM. The molecular weights of these analogs are relatively small, and they tend to -OH, -COOH, and other groups during the cleavage process, and the phenyl ring fragment ions of m/z 77 are generally characteristic fragments.

As an example, peak 21 (t_R = 2.46), an ion peak at m/z 169 was detected at the negative ion mode as the [M-H]^- peak. The presence of m/z 125 in the secondary mass spectrum was presumed to be [M-H-CO₂]^-, and m/z153 was presumed to be [M-H-OH]^-. After comprehensive analysis and comparison with the control, peak 21 was confirmed to be gallic acid. The cleavage pattern and secondary mass spectra are shown in Figure 2F.

Identification of flavonoid components: After UPLC-Q-TOF-MS/MS detection, the structural type of flavonoid components in CG was mainly xanthone. Xanthone, also known as benzochromanone and dibenzo-gamma-pyrone, is a general term for a class of secondary metabolites composed of tricyclic aromatic hydrocarbons. Xanthone exists mainly in the form of oxyketones and ketoglycosides, of which xanthone glycosides are the characteristic constituents of YZ.

For example, peak 84 (t_R = 5.43) was detected as an [M-H]^- peak at m/z 567 at the negative ion mode. In the secondary mass spectrum, m/z 345 was present, which was presumed to be [M-H-C₈H₁₄O₇]^-, m/z 315 was presumed to be [M-H-C₈H₁₄O₇-CH₂O]^-, and m/z 272 was presumed to be [M-H-C₈H₁₄O₇-2H₂O-C₂H₄O]^-. After comprehensive analysis, peak 84 was presumed to be polygalaxanthone VIII. The cleavage pattern and secondary mass spectra are shown in Figure 2G.

Identification of other components: In addition to the above components, alkaloids, peptides, and other components, such as amino acids and fatty acids, were detected in CG. Taking peak 10 (t_R = 1.71) as an example, the ion peak at m/z 188 was detected as [M-H]^- peak at the negative ion mode. The presence of m/z 145 in the secondary mass spectrum was presumed to be [M-H-CO₂]^-, m/z 128 was presumed to be [M-H-CO₂-OH]^-; m/z 172 was presumed to be [M-H-OH]^-; and peak 10 was presumed to be N-acetyl-L-glutamic acid after comprehensive analysis. The cleavage pattern and secondary mass spectra are shown in Figure 2H.

UPLC-Q-TOF-MS/MS analysis of blood component

We compared the plasma total ion current chromatograms of rats before and after CG administration, combined with the self-constructed libraries of in vivo and in vitro components, UNIFI software, and control quality spectra information. A total of 75 chemical components were characterized, namely 49 prototypical components, of which 16 were from BS, 11 from TM, 22 from YZ, and 2 from SCP. Twenty-six metabolites, including paeoniflorin, aspalathosin, gallic acid, 3,6'-dierucoyl sucrose, Siberian farnesose A5, onjisaponin F, and 23-hydroxybetulinic acid, were metabolized, and metabolic reactions such as hydrolysis, methylation, glucuronidation, and sulfate esterification mainly occurred. Specific compound information is presented in Table 2 and Figure 3, the structural formulas of the blood-entry prototype components are presented in Figure 4.

Figure 3 Characterization results of plasma components. A: Plasma total ion current diagram of negative ion mode; B: Plasma total ion current diagram of positive ion mode.

Figure 4 Structural formulas of the blood-entry prototype components. A: Structural formulas of the blood-entry prototype components from Baishao; B: Structural formulas of the blood-entry prototype components from Tianma; C: Structural formulas of the blood-entry prototype components from Shichangpu; D: Structural formulas of the blood-entry prototype components from Yuanzhi. Gal: Galactose; Api: Apiofuranosyl; Rha: Rhamnose; Ara: Arabinose; Xyl: Xylose; TC: 3,4,5-trimethoxy cinnamoyl; DC: 3,4-dimethoxy cinnamoyl.

Table 2 Blood component information of Changmaxifeng granules.

No.	tR/minute	Ion mode	Deviation (ppm)	Fragment ions	Theoretical value	Molecular formula	Ingredient name	Note
1	0.83	[M-H]- 215.0329	-6.98	149.0457	216.0423	C12H8O4	1-Furan-2-yl-2-(4-hydroxy-phenyl)ethane-1,2-dione	P
2	0.85	[M-H]- 195.0516	5.64	165.0439; 179.0590	195.0583	C6H12O7	Gluconic acid	P
3	1.46	[M-H]- 191.0206	7.33	111.0066	192.027	C6H8O7	Citric acid	P
4	1.75	[M-H]-243.0651	-2.47	107.9124	244.0736	C14H12O4	Gastrodibenzin B/C	P
5	1.9	[M-H]- 375.1257	-9.06	341.1122; 300.9911	376.1369	C16H24O10	8-Debenzoylpaeoniflorin	P
6	2.4	[M-H]- 169.0121	9.47	125.0244; 107.1245	170.0215	C7H6O5	Gallic acid	P
7	2.59	[M-H]- 285.0973	-0.35	268.0276	286.1053	C13H18O7	Gastrodin	P
8	2.81	[M-H]- 137.0243	2.92	109.0268	138.0317	C7H6O3	The product of gallic acid takes off two oxygen atoms or its isomer	M
9	2.81	[M-H]- 299.0766	-0.33	175.0088; 283.0656; 285.0088	300.0845	C13H16O8	Gastrodin hydrolysis of pyranose, glucuronidation	M
10	3.01	[M+H]+ 265.0752	2.26	218.0879	264.0668	C10H16O6S	C10H14O3 sulfate	M
11	3.12	[M-H]- 199.0981	5.52	137.0908; 162.8295; 181.0398	200.1049	C10H16O4	Paeonimetabolin II	M
12	3.18	[M-H]- 493.1193	0	169.0118; 313.0659	494.1272	C19H26O15	1'-O-Galloylsucrose	P
13	3.43	[M-H]- 389.1425	-5.91	375.0822	390.1526	C17H26O10	The product of paeoniflorin loses C7H4O and methylation	M
14	3.92	[M-H]- 262.9856	-2.28	125.0146; 168.0027; 183.0244	263.994	C8H8O8S	Methyl gallate sulfate	M
15	3.97	[M-H]- 183.0300	3.82	125.0287; 167.0704	184.0372	C8H8O5	Methyl gallate	M
16	4.34	[M-H]- 461.1273	-4.77	175.0088; 285.0088	462.1373	C19H26O13	Gastrodin glucuronidation	M
17	4.34	[M-H]- 461.1273	-4.77	93.0382; 137.0294	462.1373	C19H26O13	Sibiricose A3	P
18	4.39	[M-H]- 705.1751	7.09	259.0203; 121.0329	706.1779	C29H38O18S	Sulfitation of isomaltosyl paeoniflorin	M
19	4.51	[M-H]- 261.0062	-2.68	171.0163; 215.0649	262.0147	C9H10O7S	3,4-dihydroxyphenylpropionic acid sulfate	M
20	4.54	[M-H]- 459.1132	-1.52	400.9565	460.1217	C19H24O13	Parishin E/Parishin G	P
21	4.56	[M-H]- 543.1189	3.13	121.0281; 259.0273	544.1251	C23H28O13S	Paeoniflorin sulfite	P/M
22	4.74	[M-H]- 345.1527	-6.37	169.1254; 175.0088	346.1628	C16H26O8	C10H18O2-glucuronidation	M
23	4.8	[M-H]- 495.1483	-4.04	137.0345	496.1581	C23H28O12	Oxypaeoniflora	P/M
24	4.87	[M-H]- 223.0607	0.45	164.0299; 176.0824; 208.0458	224.0685	C11H12O5	Sinapinic acid	M
25	4.91	[M-H]- 517.1567	1.93	160.0218; 175.0377; 193.0743	518.1636	C22H30O14	Sibiricose A5	P
26	4.95	[M-H]- 289.071	-0.69	123.0399; 205.0580	290.079	C15H14O6	L-Epicatechin	P
27	5.16	[M-H]- 193.0500	-0.52	133.0153; 151.0411	194.0579	C10H10O4	Ferulic acid	M
28	5.39	[M-H]- 727.2063	-3.16	111.0058	728.2164	C32H40O19	Parishin B	P
29	5.44	[M-H]- 373.0803	8.58	197.0426; 359.0924	374.0849	C15H18O11	Dimethylgallic acid glucuronidation	M
30	5.44	[M-H]- 547.1671	1.46	175.0319; 190.0279; 205.0330; 223.0607	548.1741	C23H32O15	Sibiricose A1	P/M
31	5.67	[M-H]- 727.2062	-3.3	129.0162	728.2164	C32H40O19	Parishin C	P
32	5.68	[M+H]+ 197.0800	-7.1	151	196.0736	C10H12O4	Paeonilactone B	P
33	5.69	[M-H]- 525.1628	3.81	165.0551; 363.1675	526.1686	C24H30O13	Mudanpioside E	P
34	5.7	[M-H]- 479.1568	2.09	525.1579	480.1632	C23H28O11	Albiflorin	P
35	5.72	[M+H]+ 319.1173	-2.82	301.1047	318.1103	C17H18O6	Evofolin B	P
36	5.99	[M-H]- 537.1202	-7.82	315.0473 387.0947	538.1323	C24H26O14	Albiflorin B	P
37	6.03	[M-H]- 449.1454	1.34	77.0388; 197.0390	450.1526	C22H26O10	Paeoniflorin dehydroxymethylene	M
38	6.05	[M-H]- 479.1569	2.3	121.0281; 77.0388	480.1632	C23H28O11	Paeoniflorin	P
39	6.09	[M-H]- 165.0551	-0.61	121.0329	166.063	C9H10O3	Paeonol	P
40	6.16	[M-H]- 276.9997	-7.58	197.0451	278.0096	C9H10O8S	Sulfation of dimethylgallic acid	M
41	6.17	[M-H]- 567.1345	-0.88	315.055	568.1428	C25H28O15	Polygalaxanthone VIII	P
42	6.18	[M+H]+ 569.3103	-7.9	525.3032	568.307	C30H48O8S	Oxidized-23-hydroxybetulinic acid sulfate	M
43	6.5	[M-H]- 197.0451	0.51	151.0840; 167.0474	198.0528	C9H10O5	Dimethylgallic acid	M
44	6.56	[M-H]- 995.3008	-2.41	423.0817; 728.2076	996.3111	C45H56O25	Parishin A	P
45	6.88	[M-H]- 291.0198	7.9	197.0329; 211.0615	292.0253	C10H12O8S	Trimethyl gallate sulfate	M
46	7.15	[M-H]- 631.1572	-1.43	121.0281; 589.0381	632.1659	C30H33O13P	Benzoylpaeoniflorin lost oxygen and oxidative phosphorylation	M
47	7.18	[M-H]- 631.1682	3.01	465.0559; 313.0582; 271.0066; 169.0118	632.1741	C30H32O15	Galloylpaeoniflorin	P
48	7.43	[M-H]- 939.1154	5.32	769.0880; 617.0786; 447.0591; 169.0121	940.1182	C41H32O26	Pentagalloylglucose	P
49	7.91	[M-H]- 753.2276	4.51	205.0518; 223.0541; 529.1585; 547.1569	754.232	C34H42O19	3,6'-Disinapoyl sucrose	P
50	8.09	[M+H]+ 333.0979	1.5	303.0536	332.0896	C17H16O7	Oxypaeoniflorin loses C6H10O6 andketonisation	M
51	8.9	[M-H]- 137.0243	2.92	109.0268	138.0317	C7H6O3	The product of gallic acid takes off two oxygen atoms or its is isomer	M
52	9.12	[M+H]+ 241.1073	-1.24	227.1124	240.0998	C12H16O5	Sinapic acid methylation	M
53	10.52	[M-H]- 237.0764	0.42	103.0904; 163.0302; 193.0258; 222.0247	238.0841	C12H14O5	3,4,5-Trimethoxycinnamic acid	M
54	10.66	[M-H]- 1397.6211	-1	-	1398.6303	C64H102O33	Arillatanoside C	P
55	10.84	[M-H]- 1307.3827	-3.9	307.1561; 1161.3540	1308.3956	C59H72O33	Tenuifoliose J	P
56	10.9	[M-H]- 1265.5747	-4.42	425.3125; 455.3123; 499.1799; 585.2072; 1235.5510	1266.5881	C59H94O29	desacylsenegasaponin B	P
57	11.11	[M-H]- 1367.6119	0.88	425.3035; 687.2446; 1143.5181; 1137.5907	1368.6198	C63H100O32	Onjisaponin F-C12H12O4	M
58	11.23	[M-H]- 1235.5663	-2.75	337.2389; 455.3123; 555.1955	1236.5775	C58H92O28	Arillatanoside A	P
59	11.44	[M-H]- 1103.5256	-1.63	455.3123	1104.5353	C53H84O24	Polygalasaponin XXVIII	P
60	11.5	[M-H]- 1249.5913	4.8	425.3125; 455.3123; 1025.5084	1250.5932	C59H94O28	Onjisaponin Tf	P
61	11.54	[M-H]- 1525.6748	3.21	1157.4280; 1351.5072; 1463.5935	1526.6777	C70H110O36	Onjisaponin Te	P
62	11.56	[M-H]- 1307.3827	-3.9	307.1561; 1161.3540	1308.3956	C59H72O33	Tenuifoliose I	P
63	12.16	[M-H]- 1379.4103	1.01	1161.3391	1380.4167	C62H76O35	Tenuifoliose A	P
64	12.8	[M-H]- 679.3673	-3.09	425.3035; 455.3123	680.3772	C36H56O12	Tenuifolin	P
65	12.9	[M-H]- 329.2345	5.16	171.0906; 211.0615; 229.2078	330.2406	C18H34O5	9,12,13-TriHOME	P
66	13	[M-H]- 1703.7188	-8.22	157.0906; 1479.6775	1704.7407	C80H120O39	Onjisaponin A	P
67	13.1	[M-H]- 1631.7034	-5.09	425.3125; 455.3217; 567.2073; 1601.6753	1632.7195	C77H116O37	Onjisaponin O	P
68	13.21	[M-H]- 1571.6836	-4.45	567.1970; 1347.6245; 1541.6838	1572.6984	C75H112O35	Onjisaponin B	P
69	13.25	[M-H]- 1617.6938	-1.42	425.3035; 455.3123; 1393.6237	1618.7039	C76H114O37	Polygalasaponin XLIV	P
70	13.28	[M-H]- 1673.7109	-6.81	425.3125; 1617.6731	1674.7301	C79H118O38	Z-polygalasaponin XXXII	P
71	13.33	[M-H]- 1469.6482	-7.28	405.1388	1470.6667	C71H106O32	Onjisaponin Z	P
72	13.33	[M-H]- 1469.6482	3.13	1439.566	1470.6515	C67H106O35	Polygalasaponin XXXXII	P
73	13.48	[M-H]- 1409.6310	-4.82	425.3125; 455.3217; 1379.7347	1410.6456	C69H102O30	Onjisaponin Y	P
74	17.29	[M-H]- 233.1522	-8.58	218.8733; 197.8061	234.162	C15H22O2	Acoronene	P
75	19.22	[M-H]- 293.1770	5.8	245.899	294.1831	C17H26O4	6-Gingerol	P

P: Prototypical components ; M: Metabolites.

Pathway analysis

Metabolism is divided into one-phase and two-phase metabolism. One-phase metabolism involves the introduction of functional groups, and lipid-soluble compounds are hydrolyzed, oxidized, or subjected to other reactions to generate large polar substances. The two-phase metabolism is dominated by binding reactions in which polar groups are combined with endogenous substances, resulting in an increase in polarity and easy elimination[16].

The metabolic process analysis using gallic acid as an example. Peak at m/z 169, which was found at t_R = 2.40, analyzed, and confirmed to be gallic acid. Peak 16 (t_R = 3.97), m/z 183, which differed from gallic acid by 14 (CH₂), existed with the same fragment m/z 125 as gallic acid and m/z 167 was presumed to be [M-H-O]^-, which was analyzed and confirmed to be methyl gallic acid. Similarly, peak 43 (t_R = 3.97, m/z 197) was identified as dimethyl gallic acid. Peak 16 (t_R = 3.92, m/z 263), which had a molecular formula of C₈H₈O₈S, also showed the presence of the same fragments as gallic acid, m/z 125, and m/z 168, as [M-H-SO₃-CH₃]^-, which was presumed to be gallic acid sulfate glycoside. Similarly, peak 41 (t_R = 6.16, m/z 277) was presumed to be dimethyl gallic acid sulfate glycoside. Peak 32 (t_R = 5.44, m/z 373), which differed from gallic acid by 204, was the weight of two CH₃ and a glucuronide group, showing the presence of m/z 197 for [M-H-GlcA-2CH₃]^- and m/z 359 for [M-H-CH₃]^-, and this peak is presumed to be a dimethyl gallic acid glucuronide. Peak 45 (t_R = 6.88, m/z 291), differing from peak 41 by 14. m/z 197, a dimethyl gallic acid sulfate glycoside ion peak, was present. This peak is presumed to be trimethyl gallic acid glucuronide. Peak 50 (t_R = 8.90, m/z 137), which differs from gallic acid by 32; m/z 109 for [M-H-CO]^- was present. It is hypothesized that this peak represents the product of gallic acid degradation of 2 oxygen atoms. The above results show that gallic acid mainly undergoes methylation, sulfation, glucuronidation, and deoxygenation. The metabolic pathways of the components are illustrated in Figure 5.

Figure 5 Possible metabolic pathways of the components that enter the bloodstream. A: The metabolic pathways of 23-hydroxybetulinic acid; B: The metabolic pathways of gastrodin; C: The metabolic pathways of paeoniflorin; D: The metabolic pathways of gallic acid; E: The metabolic pathways of onjisaponin F, 3',6-disinapoylsucrose and sibiricose A5.

Target prediction based on network pharmacology

Target screening of CG for treating TD: UPLC-Q-TOF-MS/MS was used to characterize 49 prototype compounds and 26 metabolites. Metabolite backtracking yielded 7 prototype components, and comparison and de-emphasis yielded 50 incoming prototype components. Entering these 50 components into the TCMSP database yielded 684 targets. Screening yielded 1840 TD targets. After the intersection, 225 targets were obtained. The Venn diagram of the intersecting targets is shown in Figure 6A.

Figure 6 The results of network pharmacology. A: The Venn diagram of the intersecting targets; B: The protein interaction network diagram; C: Top 10 of protein interaction network diagram; D: The Gene Ontology enrichment results; E: The Kyoto Encyclopedia of Genes and Genomes enrichment results; F: The component-target-pathway-disease topological network. TD: Tic disorder; CG: Changmaxifeng granules.

Protein interaction network construction and Hub gene screening results: The common targets obtained were imported into the STRING database to construct a protein interaction network diagram (Figure 6B). The genes with the top 10 degree values were screened, which were AKT1, TNF, IL6, CASP3, FOS, VEGFA, JUN, HSP90AA1, and ESR1(Figure 6C).

GO functional enrichment and KEGG pathway enrichment analyses: A total of 5420 GO enrichment results were obtained by importing the crossed genes into the database, of which 4395 were biological processes, mainly including modulation of chemical synaptic transmission, response to xenobiotic stimulus, regulation of membrane potential, and cellular ion homeostasis. A total of 383 cellular compositions were enrichment, mainly including the synaptic membrane, neuronal cell body, membrane rafts, and membrane microdomain. Six hundred and forty-two were molecular functions, mainly involving neurotransmitter receptor activity, metal ion transmembrane transporter activity, and ion channel activity. A total of 273 signaling pathways were enriched by KEGG analysis, including neuroactive ligand-receptor interaction, pathways of neurodegeneration-multiple diseases, and cyclic AMP signaling pathway. The GO and KEGG enrichment results were imported into the network drawing platform and visualized. The results are shown in Figure 6D and E and Tables 3 and 4. The results indicated that CG exerted anti-TD effects through multiple targets and pathways.

Table 3 Significantly enriched Gene Ontology biological processes of the target genes.

GO term	Subgroup	-log10 (P value)	Count	-log10 (q value)
Modulation of chemical synaptic transmission	Biological process	31.3	47	28.23
Regulation of trans-synaptic signaling	Biological process	31.3	47	28.23
Response to xenobiotic stimulus	Biological process	28.8	44	26.4
Regulation of membrane potential	Biological process	27.1	43	24.8
Signal release	Biological process	23.0	40	21.0
Cellular divalent inorganic cation homeostasis	Biological process	21.6	40	19.7
Cellular calcium ion homeostasis	Biological process	21.9	39	19.9
Calcium ion homeostasis	Biological process	21.5	39	19.6
Calcium ion transport	Biological process	21.0	37	19.2
Muscle system process	Biological process	19.2	36	17.5
Synaptic membrane	Cellular component	31.4	44	29.0
Postsynaptic membrane	Cellular component	29.8	38	27.8
Presynapse	Cellular component	20.3	38	18.7
Neuronal cell body	Cellular component	19.7	37	18.2
Membrane raft	Cellular component	19.2	31	17.8
Membrane microdomain	Cellular component	19.2	31	17.8
Intrinsic component of synaptic membrane	Cellular component	19.6	24	18.1
Distal axon	Cellular component	14.2	24	13.0
Transmembrane transporter complex	Cellular component	11.1	24	10.0
Transporter complex	Cellular component	10.6	24	9.6
Neurotransmitter receptor activity	Molecular function	36.5	33	34.0
Metal ion transmembrane transporter activity	Molecular function	10.9	26	9.3
Ion channel activity	Molecular function	10.6	26	9.1
Channel activity	Molecular function	9.7	26	8.3
Passive transmembrane transporter activity	Molecular function	9.6	26	8.3
Gated channel activity	Molecular function	11.5	24	9.8
Amide binding	Molecular function	10.0	24	8.5
Cation channel activity	Molecular function	10.5	23	9.0
G protein-coupled amine receptor activity	Molecular function	25.0	20	22.8
Peptide binding	Molecular function	8.7	20	7.4

GO: Gene Ontology.

Table 4 Results of Kyoto Encyclopedia of Genes and Genomes enrichment.

Description	Gene ratio	Count	-log10 (P value)	-log10 (q value)
Neuroactive ligand-receptor interaction	52/203	52	25.8	23.78
Pathways of neurodegeneration - multiple diseases	43/203	43	13.7	12.38
Alzheimer disease	36/203	36	11.9	10.68
cAMP signaling pathway	32/203	32	15.7	14.18
Calcium signaling pathway	27/203	27	10.8	9.7
Serotonergic synapse	26/203	26	17.9	16.2
MAPK signaling pathway	24/203	24	6.6	6.1
Estrogen signaling pathway	21/203	21	10.9	9.8
Chemical carcinogenesis - reactive oxygen species	21/203	21	7.1	6.5
Ras signaling pathway	20/203	20	6	5.6
Cholinergic synapse	18/203	18	9.8	8.8
Chemokine signaling pathway	18/203	18	6.1	5.7
T cell receptor signaling pathway	17/203	17	9.47	8.5
Neurotrophin signaling pathway	17/203	17	8.5	7.7
Phospholipase D signaling pathway	17/203	17	7	6.5
Thyroid hormone signaling pathway	16/203	16	7.5	6.9
Dopaminergic synapse	16/203	16	7	6.4
Apoptosis	16/203	16	6.8	6.3
C-type lectin receptor signaling pathway	15/203	15	7.6	6.9
Sphingolipid signaling pathway	15/203	15	6.7	6.2
B cell receptor signaling pathway	14/203	14	8	7.3
TNF signaling pathway	14/203	14	6.3	5.8
Endocrine resistance	13/203	13	6.2	5.8
VEGF signaling pathway	12/203	12	7.9	7.2
Gap junction	12/203	12	5.9	5.5
IL-17 signaling pathway	12/203	12	5.6	5.3
Toll-like receptor signaling pathway	12/203	12	5.1	4.9
Fc epsilon RI signaling pathway	11/203	11	6.2	5.8
Longevity regulating pathway - multiple species	10/203	10	5.7	5.34

Construction results of the component-target-pathway-disease topological network of CG: The 49 blood-entry prototype components and corresponding targets were imported into Cytospace 3.10.0 software and visualized. The results are shown in Figure 6F, with red representing BS and its key components, yellow representing SCP and its key components, blue representing YZ and its key components, purple representing TM and its key components, and green representing the key targets of each component’s action. The results showed that the blood-entry components and the key targets acted closely with each other, indicating that CG could exert anti-TD effects through multicomponents and multitargets. According to the degree value, paeonol, evofolin B, gastrodin, paeoniflorin, and other components may be potential pharmacodynamic substances, and FOS, AKT1, ESR1, and TNF may be potential targets.

Molecular docking results

According to the degree value (degree value > 100) of the “component-target” network, Plasma abundance and the active compounds reported in the literature, paeonol, evofolin B, gallic acid, gastrodin, peoniflorin, and tenuifolin were selected as key components; IL-6, TNF, FOS, AKT1, ESR1, and VEGFA were selected as the core proteins; and molecular docking was performed on the key components and core proteins to verify the binding between the key components and the core targets. The smaller binding energies indicated easy binding of the components to the targets. The results showed that the binding energies of most of the key components and the core targets were less than -5 kcal/mol, indicating that the key components were tightly bound to the core targets with high affinity and might have strong pharmacodynamic activities. The binding process is dominated by hydrogen bonding. In addition to that, there are van der Waals forces, hydrophobic effects, and other forces. For example, during the binding process between tenuifolin and IL-6, SER-118 and LEU-19 form hydrogen bonds with tenuifolin, ARG-24 binds to tenuifolin via van der Waals forces, and VAL-121 and tenuifolin bind via hydrophobic interaction. The molecular docking results are shown in Figure 7A, and a part of the docking process is shown in Figure 7B-F. The positive control of the core protein and its inhibitor are shown in Table 5.

Figure 7 The results of molecular docking. A: Results of molecular docking; B: The binding mode of VEGFA with peoniflorin; C: The binding mode of AKT1 with evofolin, and the binding mode of VEGFA with peoniflorin; D: The binding mode of ESR1 with peoniflorin; E: The binding mode of FOS with tenuifolin; F: The binding mode of IL-6 with tenuifolin.

Table 5 The results of molecular docking of positive control molecules.

Receptor-ligand	VEGFA-Ginsenoside Rg3	ESR1-Tamoxifen	AKT1-MK2206	FOS-224	TNF-SPD304	IL-6-LMT-28
Binding energies (kcal/mol)	-9.3	-10.3	-10.0	-8.6	-9.8	-8.8

DISCUSSION

Herein, UPLC-Q-TOF-MS/MS was used to analyze the chemical composition of CG, mainly terpenoids, glycosides, flavonoids, and phenolic acid components. The plasma samples were examined for pretreatment methods and analyzed for composition, and the results of five methods of plasma treatment with methanol, acetonitrile, ethyl acetate, water-saturated n-butanol, and SPE solid-phase extraction were compared. The results showed that the methanol-treated plasma samples were well separated and had many components suitable for compositional analyses; and the results of the characterization of the compositions showed that a total of 49 prototype components were found, which were mainly terpenoids, saccharides, and phenolic acids. After screening the blood-entry components with TD targets and analyzing them via network pharmacology, a total of 225 intersecting targets were obtained. Through GO, KEGG, “component-target” network construction, and molecular biology analyses, alongside relevant literature reports, the active components and related targets of CG were initially identified. Molecular docking revealed that the binding affinities of Tenuifolin-FOS, Tenuifolin-IL-6, Paeoniflorin-ESR1, and Paeoniflorin-VEGFA are comparable to those of the positive control, indicating similar binding potency. Subsequent studies will focus on cell-based pharmacodynamic evaluation and target verification of Tenuifolin and Paeoniflorin, along with quantitative analysis and pharmacokinetic profiling, to elucidate their in vivo behavior.

A total of 26 metabolites were identified using UPLC-Q-TOF-MS/MS, primarily resulting from glucuronidation, methylation, sulfation, and hydrolysis. Oxidation and hydrolysis are indicative of phase-I metabolism, whereas glucuronidation and sulfation are characteristic of phase-II metabolism. Upon entry into the body, phase-I enzymes located in the liver and intestine are activated, facilitating hydrolysis and other reactions that modify the molecular structure, enhance polarity and water solubility, and ultimately alter the compound's biological activity. Onjisaponin F was not detected in plasma as the intact parent compound; rather, its hydrolysis product, ferulic acid, was identified. Ferulic acid has been documented to inhibit inflammatory pathways by reducing the production of pro-inflammatory mediators such as TNF-α, thereby mitigating inflammation[17]. This observation is consistent with our network-pharmacology findings. Furthermore, research has demonstrated that ferulic acid enhances the Akt/CRMP2 signaling pathway, promoting neuronal regeneration and synaptic remodeling, and exerting neuroprotective effects[18]. These observations suggest that hydrolytic reactions occurring in vivo have the potential to produce small-molecule metabolites with notable biological activity. During phase-II metabolism, xenobiotics undergo conjugation with glucuronic acid, activated sulfate, or other polar groups through the action of phase-II enzymes, resulting in the formation of more water-soluble metabolites that are easily excreted in urine. Typically, glucuronide conjugates exhibit reduced activity and toxicity compared to their parent compounds; however, there are instances where glucuronidation can lead to the formation of more potent metabolites, such as morphine-6-O-glucuronide[19,20]. Sulfation represents another critical phase-II metabolic pathway. Research indicates that sulfation not only enhances water solubility but may also impart bioactivity. Nonetheless, most evidence supporting this comes from studies on polysaccharides, with limited knowledge available regarding sulfated small molecules in TCMs. Regardless of whether these metabolites are glucuronidated or sulfated, they warrant further investigation, as certain phase-I or phase-II products may result in unexpected active compounds[21]. In summary, once a drug enters the body it undergoes metabolic reactions that may generate bioactive small molecules or simply increase its water solubility, thereby facilitating urinary excretion.

TD is a class of psychiatric disorders with arrhythmic tics as the main manifestation, and the drugs used to treat TD are mainly psychotropic, including dopamine receptor blockers, antiepileptic drugs, and α-adrenergic receptor agonists. However, these drugs had a high rate of relapse and obvious adverse effects[22,23]. Modern pharmacological studies have found that TD is associated with dysfunctional neuroimmune interactions, and it is possible that inflammatory neuronal components are involved in the pathogenesis of TD[24,25]. Microglia are immune cells present in neural tissues and are usually involved in inflammation, nerve injury and other diseases[26]. Microglia activity has been reported to be high in patients with TD[27]. Microglia may differentiate into proinflammatory microglia that secrete proinflammatory factors, such as IL-2, TNF-α, and other inflammatory factors or into anti-inflammatory microglia that secrete anti-inflammatory factors and upregulate neuroprotective factors when stimulated by the disease[28,29]. Kutuk et al[30] found that serum levels of IL-1β, TNF-α, IL-6, and IL-4 are significantly elevated in children with TD. Most cells in the nervous system can produce IL-6, which modulates dopaminergic and cholinergic neurons, thereby regulating neuronal excitability and sleep. TNF-α can modulate the activation of microglia and astrocytes and influence the blood-brain barrier. We therefore hypothesize that these inflammatory factors cross the immature blood-brain barrier, infiltrate the striatum, activate microglia, and lead to hyperactivity of the direct pathway and suppression of the indirect pathway, ultimately producing motor tics such as eye-blinking and shoulder-shrugging. The PI3K/AKT signaling pathway is also a common inflammatory pathway with various biological effects[31]. TNF serves as an upstream signal to activate PI3K/AKT, while PI3K/AKT in turn negatively regulates TNF-mediated inflammation. In our protein-protein interaction network, TNF/AKT1 ranked among the top 10 hub genes; therefore, we postulate that the PI3K/AKT pathway can influence the therapeutic efficacy of CG in TD[32]. Some studies have found that the use of PI3K inhibitors can alleviate TD symptoms and downregulate the levels of inflammatory factors, suggesting that the PI3K/AKT signaling pathway mediates the pathological process of TD. Tao et al[33] reported that the levels of serum inflammatory factors, such as IL-6 and TNF-α, in patients with TD were significantly higher than those in the control group. Therefore, the occurrence of TD is closely related to the inflammatory signaling pathway. In addition, other studies have found that the core targets of TD include JUN and FOS[34], which is consistent with the results of this study.

Currently, Chinese medicines, such as Tianmagoutengyin, CG, Ning Dong Granule, and Jing An Oral Liquid, are first-line medicines for the treatment of TD. Most of these Chinese medicines are based on BS, TM, and YZ, which are characterized by good therapeutic effects, fewer adverse effects, and simultaneous action on multiple targets. After UPLC-Q-TOF-MS/MS and network pharmacology screening, paeoniflorin could enter the bloodstream and is a potential medicinal substance. Modern pharmacological studies have found that paeoniflorin is widely found in plants of the family Paeoniaceae, and it has neuronal cell-protecting, anti-inflammatory, and other activities[35,36]. Zhao et al[37], found that paeoniflorin exerted neuroprotective effects by attenuating brain damage through the PI3K/AKT signaling pathway by means of transmission electron microscopy, immunofluorescence, liquid chromatography-mass spectrometry, and molecular docking. In addition, gastrodin and tenuifolin may be potential pharmacodynamic substances. Some studies have shown that gastrodin and tenuifolin can exert neuroprotective effects by acting on targets such as TNF-α and IL-6[38,39]. The molecular docking results also showed that paeoniflorin, gastrodin, and tenuifolin have strong binding power to the core targets.

This study is the first to integrate serum pharmacochemistry, network pharmacology, and molecular docking to characterize the in vitro and in vivo chemical constituents of CG in pediatric TDs. The fragmentation patterns and metabolic pathways of the relevant compounds were elucidated, providing a solid foundation for identifying the active substances of CG. The combined approach offers a comprehensive view of CG’s mechanism of action. Emphasizing CG’s potential as a safer alternative to psychotropic drugs addresses an unmet need in the treatment of TD. Moreover, it provides robust evidence for new-drug registration. To date, the marketing-authorization dossier for CG has been accepted by the National Medical Products Administration (acceptance No. CXZS2500020) and granted priority-review status.

In summary, CG effectively improves TD through its multicomponent and multitarget synergistic effects. The core mechanism may involve the inhibition of inflammatory factors to alleviate inflammation. These findings provide a theoretical basis for the treatment of TD with CG and lay the foundation for the subsequent interactions between components and targets. In future studies, we plan to employ a lipopolysaccharide-induced microglial inflammation model. Western blot, ELISA and RT-qPCR will be used to quantify TNF-α, IL-6, and so on protein levels, providing direct experimental validation of the predicted anti-inflammatory pathway.

CONCLUSION

Herein, serum pharmacology was analyzed using UPLC-Q-TOF-MS/MS, which provided a basis for the further investigation of the material basis and action mechanism of CG against TD. Overall, 187 components were characterized in CG, and 75 components were characterized in plasma, of which 49 were prototypes and 26 were metabolites. CG might exert its therapeutic effect on TD through the action of paeoniflorin, gastrodin, gallic acid, and tenuifolin on key targets such as TNF, IL-6, FOS, and VEGFA. So far, CG have been included in the priority approval of TCM new drugs, in China. However, this study has some limitations. It relies on network pharmacology to predict the key targets. To ensure the accuracy of the key targets, timely experimental verification is required. Therefore, to solve this problem, our group will perform in vivo and in vitro experiments for validation in subsequent studies to thoroughly investigate the mechanism by which CG exerts anti-TD effects.