Pharmacological Material Basis of Chushi Weiling Decoction and Its Mechanism in Eczema and Herpes Zoster Based on UPLC-Q-TOF-MS, GC-MS, and Network Pharmacology

Keywords

classic prescription - Chushi Weiling Decoction - network pharmacology - molecular docking

Introduction

Chushi Weiling Decoction (CWD) is one of the first batches of 100 classic prescriptions released by the State Administration of Traditional Chinese Medicine.[1] It is a classic prescription recorded in Chen Shigong's “Orthodox Manual of External Diseases” of the Ming Dynasty and Wu Qian's “The Golden Mirror of Medicine” of the Qing Dynasty, which is used to treat dampness-heat skin diseases such as “damp sore,” “fire erysipelas,” and “shingles.”[2] [3] [4] Modern Chinese medicine believes that “damp sore” refers to eczema, while “fire erysipelas” and “shingles” refer to skin diseases such as herpes zoster.[5] [6] Eczema and herpes zoster are common skin lesions. Eczema is an inflammatory skin lesion caused by various internal and external factors, while herpes zoster is an infectious skin disease caused by the varicella-zoster virus. These two diseases have different pathologies with similar characteristics such as causing rash, blisters, erosion, exudation, itching, and pain in the affected area. Modern traditional Chinese medicine (TCM) regards CWD as the main prescription for treating eczema and herpes zoster in clinical practice, which can achieve good therapeutic effects.[7] [8] [9]

The entire recipe of CWD consists of 14 Chinese medicinal materials, including Atractylodis Rhizoma (Cangzhu), Magnoliae Officinalis Cortex (Houpo), Citri Reticulatae Pericarpium (Chenpi), Glycyrrhizae Radix et Rhizoma (Gancao), Alismatis Rhizoma (Zexie), Poria (Fuling), Polyporus (Zhuling), Cinnamomi Cortex (Rougui), Atractylodis Macrocephalae Rhizoma (Baizhu), Gardeniae Fructus (Zhizi), Akebiae Caulis (Mutong), Saposhnikoviae Radix (Fangfeng), Talcum (Huashi), and Junci Medulla (Dengxincao). The information and abbreviations of herbs are provided in [Table 1]. Among them, Cangzhu is the monarch drug, which can strengthen the spleen and dry dampness; Houpo, Chenpi, and other medicinal herbs are used as ministerial drugs to promote diuresis, promote spleen function, and remove dampness; Rougui is used as an adjuvant drug to promote Yang and Qi circulation; Gancao is a conductant drug that can clear heat and detoxify.[10] [11] [12]

Table 1 Medical information of CWD

TCM name	Product name	Abbreviation	Origin	Lot number	Place of production	Manufacturer
Atractylodis Rhizoma	Cangzhu (stir fried with bran)	CZ	Atractylodes Lancea (Thunb.) DC.	220361341	Jiangsu	Beijing Kangmei Pharmaceutical Co., Ltd.
Magnoliae Officinalis Cortex	Houpo (processed with ginger juice)	HP	Magnolia officinalis Rehd. et Wils.	220301171	Sichuan	Kangmei Pharmaceutical Co., Ltd.
Citri Reticulatae Pericarpium	Chenpi	CP	Citrus reticulata cv. Chachiensis	220403921	Guangdong	Kangmei Pharmaceutical Co., Ltd.
Glycyrrhizae Radix et Rhizoma	Gancao	GC	Glycyrrhiza uralensis Fisch	210731	Xinjiang	Shanghai Kangqiao Traditional Chinese Medicine Slices Co., Ltd.
Alismatis Rhizoma	Zexie	ZX	Alisma orientale (Samuel) Juz.	220400501	Fujian	Kangmei Pharmaceutical Co., Ltd.
Poria	Fuling	FL	Poria cocos (Schw.) Wolf	210428	Anhui	Shanghai Kangqiao Traditional Chinese Medicine Slices Co., Ltd.
Polyporus	Zhuling	ZL	Polyporus umbellatus (Pers.) Fr.	220107	Shaanxi	Shanghai Hongqiao Traditional Chinese Medicine Slices Co., Ltd.
Cinnamomi Cortex	Rougui	RG	Cinnamomum cassia Presl	210203	Guangxi	Shanghai Kangqiao Traditional Chinese Medicine Slices Co., Ltd.
Atractylodis Macrocephalae Rhizoma	Baizhu (stir fried with soil)	BZ	Atractylodes macrocephala Koidz.	211003181	Zhejiang	Kangmei Pharmaceutical Co., Ltd.
Gardeniae Fructus	Zhizi (ground)	ZZ	Gardenia jasminoides Ellis.	211028	Jiangxi	Shanghai Hongqiao Traditional Chinese Medicine Slices Co., Ltd.
Akebiae Caulis	Mutong	MT	Akebia quinata (Houtt.) Decne.	20220701	Anhui	Guangdong Huiqun Traditional Chinese Medicine Slices Co., Ltd.
Saposhnikoviae Radix	Fangfeng	FF	Saposhnikovia divaricata (Trucz.) Schischk.	211117	Inner Mongolia	Shanghai Hongqiao Traditional Chinese Medicine Slices Co., Ltd.
Talcum	Huashi	HS	[Mg3(Si4O10)(OH)2]	1711213	Hebei	China Shineway Pharmaceutical Group Co., Ltd.
Junci Medulla	Dengxincao	DXC	Juncus effusus L.	220304	Jiangsu	Shanghai Hongqiao Traditional Chinese Medicine Slices Co., Ltd.

Abbreviation: CWD, Chushi Weiling Decoction.

Modern pharmacology and medical research have shown that Chinese medicinal herbs such as Cangzhu, Houpo, and Chenpi have therapeutic effects on eczema and herpes zoster. The terpenoids, flavonoids, and phenolic compounds from these herbs have anti-inflammatory, analgesic, and antiviral effects.[13] [14] [15] However, the material composition and therapeutic mechanism of CWD were still unclear. This study used ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and gas chromatography-mass spectrometry (GC-MS) techniques to analyze the composition of CWD and identify its main components. In addition, based on network pharmacology, the possible mechanism of the treatment of eczema and herpes zoster with CWD was explored. The results of this study provide a theoretical basis for clinical medication and quality control of CWD.

Material and Methods

Materials and Reagents

All 14 Chinese medicinal materials were purchased from real estate areas or main production areas ([Table 1]), and all complied with the relevant regulations of the Chinese Pharmacopoeia 2020 Edition Part 1.[16]

The reference standards cinnamaldehyde (98.0%, lot:7713), hesperidin (95.3%, lot:110721-201115), naringin (98.0%, lot:13822), gardenoside (98.0%, lot:15277), calceolarioside B (98.0%, lot:9644), 5-O-methylvisammioside (98.0%, lot:14863), and prim-O-glucosylcimifugin (98.0%, lot:14585) were purchased from the China Institute for the Control of Food and Drug Products. Liquid chromatography-MS (LC-MS)-grade acetonitrile (ThermoFisher, United States), methanol (ThermoFisher, United States), formic acid (ThermoFisher, United States), and deionized water prepared by a Millipore Alpha-Q water purification system (Millipore, United States) were used as the mobile phase for the chromatographic separation. Other reagents were of analytical grade.

Preparation of Standards and Samples

Preparation of Standards and Samples of UPLC-Q-TOF-MS

All reference materials were dissolved in methanol to prepare solutions of cinnamaldehyde (12.4 μg/mL), hesperidin (198 μg/mL), naringin (26 μg/mL), gardenoside (31 μg/mL), calceolarioside B (45 μg/mL), 5-O-methylvisammioside (57 μg/mL), and prim-O-glucosylcimifugin (62 μg/mL).

According to the “History of Science and Technology in China: Volume of Weights and Measures,” and by comparing it with the “Key Information Table of Ancient Classic Prescriptions (7 Prescriptions),”[17] [18] 3.73 g of each of Cangzhu, Houpo, Chenpi, Zexie, Fuling, Zhuling, Baizhu, Zhizi, Mutong, Fangfeng, and Huashi were weighed, and 1.12 g of each of Rougui and Gancao were weighed. These materials were soaked in 400 mL of ultrapure water for 30 minutes, then 0.22 g of Dengxincao was added. All the materials were boiled over high fire and then simmered until the liquid amount was 320 mL, to obtain the complete decoction. After the above preparation, the decoction was frozen into freeze-dried powder at −55°C, 500 Pa using a Buchi Lyovapor L-200 (Buchi, Swiss).

All samples were dissolved in methanol and each was prepared into a solution of 10 mg/mL for UPLC-QTOF-MS. The sample solutions and standard solutions were filtered through 0.22 µm microporous filter membrane.

Preparation of Samples of GC-MS

The method for preparing CWD samples was the same as that of the UPLC-Q-TOF-MS mentioned above. After the preparation and lyophilization, approximately 1 g of freeze-dried powder was weighed for GC-MS of each decoction sample.

Instrumentation and Conditions

Instrumentation and Conditions of UPLC-Q-TOF-MS

The UPLC-Q-TOF MS analysis was performed using a Waters Acquity UPLC system coupled with a Xevo G2-XS QTOF mass spectrometer (Waters, United States) with an electrospray ionization ion source in MS^E mode.

The chromatographic separation process was performed on a Thermo-Accucore aQ C18 (100 mm × 2.1 mm, 2.6 μm; ThermoFisher, United States) at 25°C, with a mobile phase consisting of acetonitrile (A) and 0.1% formic acid aqueous solution (B). The gradient elution was as follows: 0–20 minutes, 5–25% eluent A; 20–30 minutes, 25–45% eluent A; 30–40 minutes, 45–70% eluent A; 40–45 minutes, 70–95% eluent A. The flow rate was 0.3 mL/min.

MS conditions were operated in both positive and negative ion modes and applied as follows: solvent gas temperature (nitrogen), 450°C; capillary voltage, 3.5 KV; an ion source temperature, 120°C; desolvation gas flow, 500 L/h; cone gas flow, 100 L/h; the low collision energy, 6 eV; the high collision energy, 25 to 60 eV.

Instrumentation and Conditions of GC-MS

The GC-MS analysis was performed using a 7890B GC-5977A MS combined instrument (Agilent, United States) in full spectrum scanning mode.

The chromatographic separation process was performed on an HP-5MS UI (0.25 mm × 30 m × 0.25 μm; Agilent, United States). The temperature gradient was as follows: 0–3 minutes, 40°C; 3–23 minutes, 40–240°C; 23–24 minutes, 240°C; 24–28 minutes, 240–280°C; 28–33 minutes, 280°C. The injection volume was 1 μL of headspace injection. The injection port temperature was 250°C, the flow rate was 1 mL/min, the split ratio was 10:1, the equilibrium temperature of the sample was 85°C, and the balance time was 30 minutes.

MS conditions were as follows: solvent gas temperature (nitrogen) was 450°C, quality scanning range was 40 to 600 Da; an ion source temperature was 230°C; and quadrupole temperature was 150°C. The solvent delay time was 4 minutes.

Data Processing and Compound Identification

Masslynx 4.1 software (Waters, United States) and UNIFI v1.8 Analysis Platform (Waters, United States) were used to analyze the mass spectra peaks of CWD in positive and negative ion modes. According to the comparison of reference standards or references, the compounds were identified by UV spectrum, retention time, excimer ion peak, molecular formula, fragment ions, and other information combined with the SciFinder database.

Methods of Research on Network Pharmacology

Active Component Collection of CWD

On the basis of confirming the ingredients of CWD, oral bioavailability (OB) and drug-likeness property (DL) parameters were analyzed using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP; https://old.tcmsp-e.com/index.php). The active ingredients of various medicinal herbs were screened with the set of OB ≥30% and DL ≥0.18 as standards based on the investigation of relevant literature. Gene names corresponding to targets were identified from the protein database Uniprot (https://www.uniprot.org). The potential targets of each active molecule were screened using the target prediction tool SwissTargetPrediction (http://swisstargetprediction.ch/).

Collection of Therapeutic Targets for Eczema and Herpes Zoster

The keywords “eczema” and “herpes zoster” were used to search through the Drugbank database (https://go.drugbank.com/), the Genecards database (https://www.genecards.org/), and the OMIM database (https://omim.org/).

Construction of Visual Network

The screened predicted targets were imported into the protein interaction analysis platform STRING 11.0 (https://version-11-0.string-db.org/). A “components-targets” network was established through Cytoscope 3.9.1 between the compound molecules and target proteins of CWD. In the network, the associations between nodes of components and targets were depicted by edges. The “degree” was used to calculate the edges linked to each node, which indicated the significance of the nodes in the network.

Gene Ontology Analysis and Pathway Enrichment (KEGG and Reactome) Analysis

Overlapping drug targets and diseases were imported into the DAVID (Database for Annotation, Visualization, and Integrated Discovery) web server (https://david.ncifcrf.gov/) and OmicShare Cloud Platform (https://www.omicshare.com/) for gene ontology (GO) biological processes and KEGG (Kyoto encyclopedia of genes and genomes) pathway enrichment analysis. The Metascape Platform (http://metascape.org/gp/index.html) was used for Reactome analysis, to explain the results of high-throughput genomics research.

Molecular Docking

The molecule structure (Mol2 structure) of the active compounds in CWD was downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The 3D structure of the core protein targets was extracted from the Protein Data Bank (https://www1.rcsb.org/). Molecular docking and calculation of the binding affinity were performed using AutoDock (https://ccsb.scripps.edu/projects/).

Results and Discussion

The Analysis of UPLC-Q-TOF-MS and GC-MS

Components Determined by UPLC-Q-TOF-MS

A total of 194 components were identified from the samples of CWD by UPLC-Q-TOF-MS, including 71 terpenes, 37 flavonoids, 11 steroids, 11 phenylpropanoids, 8 alkaloids, 11 aromatics, 8 organic acids, 13 alcohols and esters, 6 simple ketones and aldehydes, and 18 other compounds. The retention time, excimer ion peak, molecular formula, herb source (in abbreviation), and other information are shown in [Table 2]. The ion flow diagram corresponding to peaks 1 to 194 is shown in [Fig. 1].

Fig. 1 Total ion flow diagram of CWD components in (A) positive ion mode and (B) negative ion mode of UPLC-Q-TOF-MS. CWD, Chushi Weiling Decoction.

Table 2 Analysis and identification of components from CWD by UPLC-Q-TOF-MS

Compd.	Component name	Neutral mass (Da)	Observed m/z	Mass error (mDa)	Mass error (ppm)	Observed RT (min)	Adducts	Fragment ions (m/z, ESI−/ESI+)	Formula	Herb-source (in Abbreviation[a])	Ref.
1	3-Indole carboxylic acid	161.0477	162.0546	−0.3	−2.0	0.64	[M + H]+	162.1524, 131.1723	C9H7NO2	GC	[36]
2	Dehydroeffusal	252.0786	253.0848	−1.1	−4.3	0.66	[M + H]+	253.1445, 203.1294	C16H12O3	DXC	[40]
3	Butenolide B	234.1256	235.1312	−1.6	−6.9	0.66	[M + H]+	235.0317, 234.1720, 219.0972, 157.1694	C14H18O3	CZ	[19]
4	5,7,3′-Trimethoxyl-(−)-epicatechin	332.1260	333.1335	0.2	0.7	0.67	[M + H]+	333.1335, 265.1961, 175.1598	C18H20O6	ZZ	[28] [29]
5	Naringin[b]	580.1792	581.1889	2.5	4.2	0.67	[M + H]+	581.1134, 461.0730, 417.1751	C27H32O14	CP	[35]
6	Quercitrin	448.1006	449.1108	3.0	6.6	0.67	[M + H]+	449.1108, 303.9655, 285.0639, 275.1771, 180.9662, 165.1959, 127.1546, 109.0404	C21H20O11	ZZ	[28] [29]
7	(+)-Syringaresinol	418.1628	419.1694	−0.7	−1.6	0.68	[M + H]+	419.2348	C22H26O8	RG, HP	[31] [32]
8	Genipin-1-O-gentiobioside	550.1898	549.1810	−1.5	−2.8	0.69	[M − H]−	549.1810, 387.1237, 371.0946, 225.0650, 123.0331	C23H34O15	ZZ	[28] [29]
9	Picrasmalignan A	534.1890	533.1850	3.3	6.3	0.70	[M - H]−	533.1850, 403.1144	C30H30O9	RG	[31]
10	Liriodenine	275.0582	276.0640	−1.6	−5.6	0.72	[M + H]+	276.0640	C17H9NO3	HP	[32]
11	N-Methylisosalsoline	207.1259	208.1327	−0.5	−2.3	0.73	[M + H]+	208.1327	C12H17NO2	HP	[32]
12	2-Hydroxyisoxypropyl-3-hydroxy-7-isopentene-2,3-dihydrobenzofuran-5-carboxylic	306.1467	307.1526	−1.4	−4.5	0.76	[M + H]+	307.1526, 291.1987, 263.2010	C17H22O5	CZ	[19]
13	Aristolochic acid A	340.0583	341.0635	−2.1	−6.1	0.77	[M + H]+	341.0635, 313.1517	C17H11NO7	MT	[21] [22]
14	Cassiferaldehyde	178.0630	179.0694	−0.9	−5.1	0.77	[M + H]+	179.0694, 163.9995, 145.1784	C10H10O3	RG	[31]
15	Gardenoside_qt[b]	242.1154	243.1217	−1.0	−4.1	0.77	[M + H]+	243.0620	C12H18O5	ZZ	[28] [29]
16	Genipin	226.0841	227.0916	0.2	1.1	0.78	[M + H]+	227.0916	C11H14O5	ZZ	[28] [29]
17	Erthro-guaiacy lglycerol	214.0841	215.0927	1.3	5.9	0.82	[M + H]+	215.0927, 151.1683	C10H14O5	RG	[31]
18	5′-Methoxylariciresinol	390.1679	391.1759	0.7	1.9	0.84	[M + H]+	391.1759, 353.1975, 289.0502	C21H26O7	RG	[31]
19	Sinapaldehyde 4-O-β-D-glucopyranoside	370.1264	371.1339	0.2	0.6	0.91	[M + H]+	371.1339, 197.0718	C17H22O9	HP	[32]
20	Magnoloside R	478.1686	479.1780	2.1	4.4	0.92	[M + H]+	479.1780, 457.2520, 441.1270	C20H30O13	HP	[32]
21	3-(3,4-Dimethoxyphenyl)-2-propenal	192.0786	193.0847	−1.2	−6.3	0.96	[M + H]+	193.0847, 175.1160, 149.1337	C11H12O3	RG	[31]
22	Sulfoorientalol C	300.1395	301.1455	−1.3	−4.5	1.07	[M + H]+	301.1455, 243.0157	C15H24O4S	ZX	[27]
23	Licorice glycoside A	726.2160	727.2266	3.4	4.6	1.50	[M + H]+	727.2266, 711.1833, 527.1888	C36H38O16	GC	[36]
24	11-Hydroxy-sec-O-β-D-glucosylhamaudol	472.1581	473.1638	−1.5	−3.2	1.59	[M + H]+	473.1638, 429.1854, 297.3093	C21H28O12	FF	[37]
25	Gancaonin Q	406.1780	407.1821	−3.2	−7.9	1.72	[M + H]+	407.1821, 385.2041, 305.3676	C25H26O5	GC	[36]
26	Prim-O-glucosylcimifugin[b]	468.1737	469.1790	−2.0	−4.2	1.75	[M + H]+	469.1790, 443.1633, 415.1733, 385.2041	C22H28O11	FF	[37]
27	8β-Methoxyatractylenolide I	262.1569	263.1629	−1.3	−4.8	1.92	[M + H]+	263.1629, 199.1105	C16H22O3	CZ, BZ	[19] [20]
28	Cinncassiol A	381.1913	382.2007	2.1	5.4	2.21	[M + H]+	381.1721, 339.1682, 325.1788	C20H30O7	RG	[31]
29	Anomalin	426.1679	427.1717	−3.4	−8.1	2.35	[M + H]+	427.1710, 263.1408, 245.0156, 217.0547	C24H26O7	FF	[38]
30	Epianhydrocinnzeylanol	366.2042	367.2080	−3.5	−9.5	2.43	[M + H]+	367.2080, 349.2000, 305.2243	C20H30O6	RG	[31]
31	(2S)-2-[4-Hydroxy-3-(3-methylbut-2-enyl)phenyl]-8,8-dimethyl-2,3-dihydropyrano[2,3-f]chromen-4-one	390.1831	391.1897	−0.6	−1.6	2.49	[M + H]+	391.1897, 369.2114	C25H26O4	GC	[36]
32	Isochlorogenic acid A[b]	516.1268	515.1217	2.2	4.3	2.49	[M − H]−	515.1217, 497.1316	C25H24O12	ZZ	[28] [29]
33	Deacetylasperulosidic acid methyl ester	404.1319	403.1231	−1.5	−3.6	2.50	[M − H]−	403.1231	C17H24O11	ZZ	[28] [29]
34	Tembetarine	344.1862	345.1925	−1.0	−2.8	2.81	[M + H]+	642.1540, 619.1677, 589.1520	C20H26NO4 +	HP	[32]
35	Fangfengalpyrimidine	296.1372	297.1444	−0.1	−0.4	2.95	[M + H]+	297.1444, 281.1784, 211.1701	C14H20O5N2	FF	[37]
36	Glycyrin	382.1416	383.1472	−1.7	−4.6	3.06	[M + H]+	383.1472, 309.1640, 265.1397	C22H22O6	GC	[36]
37	Magnoligan H	562.2355	561.2280	−0.3	−0.5	3.11	[M − H]−	561.2280, 519.9194, 475.0555	C36H34O6	HP	[32]
38	Paeonolide	460.1581	461.1668	1.5	3.1	3.17	[M + H]+	460.9483, 297.1050, 167.1323, 137.1349	C20H28O12	CZ	[19]
39	(4E,6E,12E)-4,6,12-Tetradecatriene-8,10-diyne-1,3,14-triol	232.1099	233.1169	−0.4	−1.5	3.26	[M + H]+	233.0911, 215.0813, 193.1007, 91.1242	C14H16O3	BZ	[20]
40	Nobiletin	402.1315	403.1381	−0.6	−1.5	3.45	[M + H]+	413.1381, 317.1877, 301.2659	C21H22O8	CP	[35]
41	Neocnidilide	194.1307	195.1392	1.3	6.6	3.50	[M + H]+	195.1392, 177.1313	C12H18O2	FF	[38]
42	1-Methoxyficifolinol	422.2093	423.2130	−3.6	−8.5	3.93	[M + H]+	423.2130, 365.1625	C26H30O5	GC	[36]
43	1,1'-Dibenzene-6',8',9'-trihydroxy-3-allyl-4-O-β-D-glucopyranoside	462.1890	463.2001	3.9	8.4	3.96	[M + H]+	463.2001, 293.1686, 241.1755	C24H30O9	HP	[32]
44	[(3R)-3,7-Dimethyloct-6-enyl] butanoate	226.1933	227.2000	−0.6	−2.7	4.31	[M + H]+	227.0265, 143.0355	C14H26O2	CP	[35]
45	Atractyloyne	314.1882	315.1982	2.7	8.6	4.38	[M + H]+	315.1982, 261.1326	C19H24O4	CZ	[19]
46	β-Hydroxyacteoside	640.2003	639.1923	−0.7	−1.2	4.51	[M − H]−	639.1923, 595.2032	C29H36O16	HP	[32]
47	Paeonioflorin	482.1788	483.1859	−0.2	−0.3	4.52	[M + H]+	483.1859, 397.2006, 343.2326	C23H30O11	CZ	[19]
48	Orientanone	348.1065	349.1138	0.0	0.1	4.60	[M + H]+	349.1138, 297.4816	C15H24O5S2	ZX	[27]
49	10-epi-Atractyloside A	448.2309	449.2390	0.9	2.0	4.68	[M + H]+	449.2390, 403.2103, 297.3092	C21H36O10	CZ	[19]
50	Kanzonol Y	410.2093	411.2162	−0.4	−0.9	4.70	[M + H]+	411.2162, 395.1499, 297.3092	C25H30O5	GC	[36]
51	Atractylenolide III	248.1412	249.1469	−1.7	−6.7	4.81	[M + H]+	249.1469, 223.1396	C15H20O3	CZ, BZ	[19] [20]
52	Gardenone	226.1569	227.1648	0.6	2.7	5.51	[M + H]+	227.1648, 209.1573, 191.1473, 177.1319	C12H20O3	ZZ	[28] [29]
53	(+)-Dehydrovomifoliol	222.1256	223.1333	0.4	1.9	5.51	[M + H]+	223.1333, 209.1573, 191.1473, 177.1319, 149.1367	C13H18O3	HP	[32]
54	Vitexin	432.1057	433.1114	−1.5	−3.5	5.51	[M + H]+	433.1114, 281.0650	C21H20O10	GC	[36]
55	Calceolarioside B[b]	478.1475	479.1549	0.2	0.3	5.51	[M + H]+	479.1550, 411.1791	C23H26O11	MT	[21] [22]
56	(E)-3-(3-Methoxyphenyl)acrylaldehyde	162.0681	163.0749	−0.4	−2.5	5.67	[M + H]+	163.0750, 143.0357, 127.0617	C10H10O2	RG	[31]
57	(± )-9-Hydroxy-10E,12Z-octadecadienoic acid	296.2351	297.2406	−1.8	−6.2	5.84	[M + H]+	297.2406, 269.1846, 211.1473, 146.1472	C18H32O3	HP	[32]
58	Oxypaeoniflorin	496.1581	497.1629	−2.4	−4.9	6.18	[M + H]+	497.1629, 425.1340	C23H28O12	CZ	[19]
59	Methyl 3,4,5-trimethoxycinnamate	252.0998	251.0949	2.4	9.4	6.88	[M − H]−	251.0949, 229.1132, 183.1062	C13H16O5	CZ	[19]
60	Gancaonin T	398.2093	399.2145	−2.1	−5.3	7.03	[M + H]+	399.2145, 297.3094	C24H30O5	GC	[36]
61	Pachyman	500.2105	501.2156	−2.2	−4.3	7.24	[M + H]+	501.2156, 485.2381, 439.2048	C20H30O14	FL	[24]
62	Coniferin	342.1315	343.1411	2.3	6.8	7.2	[M + H]+	343.1411, 185.1562	C16H22O8	HP	[32]
63	Albiflorin	480.1632	481.1725	2.1	4.3	7.49	[M + H]+	481.1725, 467.1944, 413.1339	C23H28O11	CZ	[19]
64	Euchrenone	406.2144	407.2199	−1.8	−4.4	8.1	[M + H]+	407.2199, 355.2335, 301.1431, 203.2675	C25H26O5	GC	[36]
65	Xambioona	388.1675	389.1761	1.4	3.5	8.85	[M + H]+	389.1761, 341.2324, 211.1702	C25H24O4	GC	[36]
66	Houpulin H	436.2614	437.2673	−1.3	−3.0	9.22	[M + H]+	427.2306, 355.2334	C28H36O4	HP	[32]
67	Magnoflorine	342.1705	342.1773	−2.1	−6.3	9.68	[M]+	342.1773, 311.2000, 297.2154, 237.2061	C20H24NO4 +	HP	[32]
68	(S)-Falcarinol	244.1827	245.1906	0.6	2.4	9.69	[M + H]+	245.1906, 221.1561, 203.1452	C17H24O	FF	[38]
69	Gancaonin R	382.2144	383.2219	0.2	0.5	9.75	[M + H]+	383.2219, 307.2192, 185.1916	C24H30O4	GC	[36]
70	Houpulin C	398.1882	399.1950	−0.5	−1.2	9.97	[M + H]+	399.1950, 373.1039, 331.1639	C27H26O3	HP	[32]
71	(4aS,6aR,6aS,6bR,8aR,12aS,14bS)-2,2,6a,6b,9,9,12a-Heptamethyl-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid	440.3654	441.3723	−0.4	−1.0	10.02	[M + H]+	441.1637, 419.1830	C30H48O2	MT	[21] [22]
72	Kanzonols X	394.2144	395.2200	−1.6	−4.2	10.34	[M + H]+	395.2200, 331.2170, 277.2113	C25H30O4	GC	[36]
73	Galloylpaeoniflorin	632.1741	633.1791	−2.3	−3.6	10.71	[M + H]+	633.1526, 611.1441, 529.2185, 477.1998	C30H32O15	CZ	[19]
74	Glyasperin A	422.1729	423.1781	−2.1	−4.9	10.72	[M + H]+	423.1781, 381.2039	C25H26O6	GC	[36]
75	Gancaonin H	420.1573	421.1615	−3.1	−7.4	10.88	[M + H]+	421.1615, 311.2493, 207.1408	C25H24O6	GC	[36]
76	(−)-Epicatechin-3-O-β-glucoside	464.1683	465.1780	2.5	5.4	11.35	[M + H]+	465.1780, 439.2774, 297.3092, 283.2953	C22H24O11	RG	[31]
77	Houpulin K	546.2406	547.2520	4.1	7.5	11.71	[M + H]+	403.2643, 385.2346, 339.2209	C36H34O5	HP	[32]
78	Blumenol A	224.1412	225.1495	1.0	4.4	11.94	[M + H]+	225.1495, 207.1414, 175.1156	C13H20O3	HP	[32]
79	3,5-Dimethoxy-4-glucosyloxyphenylallylalcohol_qt	210.0892	209.0835	1.5	7.3	12.05	[M − H]−	209.0835, 187.1010	C11H14O4	CZ	[19]
80	(+)-Leptolepisol C	498.1890	497.1770	−4.7	−9.4	12.57	[M − H]−	497.1770, 469.9197, 401.9299, 249.9649	C27H30O9	RG	[31]
81	Atractylenolide I	230.1307	231.1388	0.8	3.5	12.57	[M + H]+	231.1388, 215.1578, 189.1274, 177.1311, 145.1406	C15H18O2	CZ, BZ	[19] [20]
82	Isoschaftoside	564.1479	565.1540	−1.2	−2.2	12.82	[M + H]+	565.1540, 527.2766, 429.2011	C26H28O14	GC	[36]
83	(−)-Medicocarpin	432.1420	433.1498	0.5	1.1	13.09	[M + H]+	433.1498, 347.1408, 291.2258, 271.1308, 183.1919	C22H24O9	GC	[36]
84	Lactiflorin	462.1526	461.1465	1.2	2.6	13.64	[M − H]−	461.1638, 447.1721, 381.1842	C23H26O10	CZ	[19]
85	Poricoic acid A	498.3345	499.3412	−0.6	−1.2	15.05	[M + H]+	499.3412	C31H46O5	FL	[24]
86	Leonoside A	770.2633	771.2675	−3.1	−4.0	15.10	[M + H]+	771.2675, 745.2080	C35H46O19	HP	[32]
87	Cinnacasiol H	382.1992	383.2042	−2.3	−5.9	15.24	[M + H]+	383.2042, 335.1576, 275.2368	C20H30O7	RG	[31]
88	Decyl acetate	200.1776	201.1849	0.0	0.2	15.50	[M + H]+	201.1850, 187.1868	C12H24O2	FF	[37]
89	Magnoloside Y	626.2211	627.2296	1.2	2.0	16.77	[M + H]+	627.2296, 583.2188	C30H26O15	HP	[32]
90	2-Tetradecanone	212.2140	213.2227	1.4	6.7	17.41	[M + H]+	213.2227	C14H28O	GC	[36]
91	Poricoic acid C	482.3396	483.3489	2.0	4.2	17.59	[M + H]+	483.3489, 431.9511	C31H46O4	FL	[24]
92	8β-Ethoxy atractylenolide III	276.1725	277.1776	−2.2	−8.1	17.75	[M + H]+	277.1776, 259.1688, 205.1256	C18H28O2	CZ, BZ	[19] [20]
93	Dehydroabietic acid	300.2089	301.2173	1.0	3.5	18.33	[M + H]+	301.2173, 269.1532	C20H28O2	FL	[24]
94	Geniposide[b]	388.1370	389.1425	−1.8	−4.5	21.77	[M + H]+	389.1425, 365.1651	C17H24O10	ZZ	[28] [29]
95	Paeonin	660.1457	661.1529	−0.1	−0.2	21.78	[M + H]+	661.1529, 645.1854, 603.1822	C28H33ClO16	CZ	[19]
96	Magnoloside P	774.2582	775.2691	3.6	4.6	22.09	[M + H]+	775.7674, 757.3918	C34H46O20	HP	[32]
97	(−)-15-Hydroxy-T-muurolol	218.1671	219.1734	−1.0	−4.5	22.53	[M + H]+	219.1663, 207.1403, 147.1930, 123.1908	C15H22O	RG	[31]
98	Crocin I[b]	976.3788	977.3900	4.	4.1	22.57	[M + H]+	977.3900, 831.3745, 655.3856	C44H64O24	ZZ	[28] [29]
99	Dehydrotumulosic acid	484.3553	485.3640	1.4	3.0	23.01	[M + H]+	485.3640, 467.3574, 447.9464, 271.1655	C31H48O4	FL	[24]
100	Croceic acid	328.1675	327.1611	0.9	2.8	23.32	[M − H]−	327.1611, 309.1694	C20H24O4	ZZ	[28] [29]
101	(−)-Myrtenal	150.1045	151.1117	0.0	0.0	23.49	[M + H]+	151.1117, 121.1606	C10H14O	HP	[32]
102	Poricoic acid CE	510.3709	511.3764	−1.8	−3.6	23.55	[M + H]+	511.3764, 451.3655, 397.0895, 375.1113	C33H50O4	FL	[24]
103	Icariside F2	402.1526	403.1564	−3.5	−8.7	24.00	[M + H]+	403.1564, 315.1757	C18H26O10	CZ, BZ	[19] [20]
104	3-(2-Hydroxyacetoxy)-5α,8α-peroxydehydro-tumulosic acid	572.3349	573.3462	4.0	6.9	24.12	[M + H]+	573.3462, 555.1128, 469.1532	C33H48O8	FL	[24]
105	24-Methylene-3-oxolanost-8-en-21-oic acid	468.3604	469.3699	2.2	4.8	24.13	[M + H]+	469.1532, 429.1674	C31H48O3	FL	[24]
106	(−)-Epoxycaryophyllene	220.1827	221.1895	−0.5	−2.1	24.21	[M + H]+	221.1895, 191.0765	C15H24O	HP	[32]
107	Cinnamoid E	234.1620	235.1692	−0.1	−0.4	24.29	[M + H]+	235.1692, 185.1523	C15H22O2	RG	[31]
108	β-Eudesmol	224.2140	225.2226	1.3	6.0	24.40	[M + H]+	225.2226, 199.0994	C15H28O	CZ, BZ, HP, FF	[19] [20] [32] [38]
109	23-O-Methylalisol A	504.3815	505.3870	−1.8	−3.5	24.42	[M + H]+	505.3870, 487.3789, 469.3716	C31H52O5	ZX	[27]
110	5-O-Methylvisamminol[b]	290.1154	289.1097	1.6	5.4	24.55	[M − H]−	289.1097, 243.1042, 221.1214	C16H18O5	FF	[37]
111	Geranylacetone	194.1671	195.1763	1.9	9.8	24.60	[M + H]+	195.1734, 179.2052	C13H22O	FF	[38]
112	Tumulosic acid	486.3709	487.3803	2.1	4.4	24.66	[M + H]+	487.3803, 469.3732	C31H50O4	FL	[24]
113	Shanzhiside	392.1319	393.1405	1.4	3.5	24.79	[M + H]+	393.1405, 225.0653	C16H24O11	ZZ	[28] [29]
114	Eudesma-4(14)-en-1,6-diol	240.2089	241.2153	−0.9	−3.8	26.27	[M + H]+	241.2153, 197.2770	C15H28O2	BZ	[20]
115	Kanzonol H	424.2250	425.2340	1.8	4.1	26.41	[M + H]+	425.2282, 371.2043	C26H32O5	GC	[36]
116	Syringin	372.1420	373.1502	0.9	2.4	27.12	[M + H]+	373.1502, 357.1291	C17H24O9	CZ	[19]
117	Undecyl acetate	214.1933	215.2013	0.8	3.7	27.26	[M + H]+	215.2014, 159.1566	C13H26O2	FL	[24]
118	2,5,5-Trimethylhepta-1,6-diene	138.1409	139.1489	0.7	5.2	27.45	[M + H]+	139.1489, 103.1270	C10H18	CP	[35]
119	4-Keto-Magnoflorine	356.1498	357.1576	0.5	1.4	27.71	[M + H]+	357.1576, 343.3559	C20H22NO5 +	HP	[32]
120	16-Deoxyporicoic acid B	468.3240	469.3291	−2.1	−4.5	27.87	[M + H]+	469.3291, 365.1646, 259.2386	C30H44O4	FL	[24]
121	(22E)-Ergosta-7,22 -dien-3β,5α,6β-ol	430.3447	431.3530	1.0	2.4	28.00	[M + H]+	431.3530, 413.1703, 387.2134, 343.1938	C28H46O3	FL, ZL	[24] [26]
122	Cedrol	222.1984	223.2059	0.2	1.0	28.12	[M + H]+	223.2059, 197.1910	C15H28O	CZ	[19]
123	Cinnamoid D	236.1776	237.1842	−0.7	−2.9	28.16	[M + H]+	237.2576, 219.1752, 165.0047	C15H24O2	RG	[31]
124	Glyasperin D	370.1780	371.1829	−2.4	−6.5	28.17	[M + H]+	371.1829	C22H26O5	GC	[36]
125	Asperuloside_qt	252.0634	253.0694	−1.2	−4.9	28.25	[M + H]+	253.0694, 225.9753	C12H12O6	ZZ	[28] [29]
126	Magnocurarine	314.1756	315.1817	−1.2	−3.7	28.97	[M + H]+	315.1817	C19H24NO3 +	HP	[32]
127	(2R)-2-[3,4-Dihydroxy-5-(3-methylbut-2-enyl)phenyl]-5,7-dihydroxy-8-(3-methylbut-2-enyl)chroman-4-one	424.1886	425.1949	−0.9	−2.2	29.57	[M + H]+	425.1949, 409.2286, 355.2137, 299.2554	C25H28O6	GC	[36]
128	15-Hydroxy-7-oxoabieta-8,11,13-trien-18-oic acid	330.1831	331.1916	1.2	3.7	29.57	[M + H]+	331.1916, 299.2554	C20H26O4	FL	[24]
129	10-O-Methyl-alismoxide	252.2089	253.214	−1.5	−5.8	29.85	[M + H]+	253.2147, 179.2194	C16H28O2	ZX	[27]
130	Houpulin F	420.2665	421.2731	−0.7	−1.6	30.44	[M + H]+	421.2731, 341.3293, 271.3299	C28H36O3	HP	[32]
131	Dauricine	624.3199	625.3290	1.8	2.9	30.85	[M + H]+	625.3290, 205.2191, 189.1632, 161.1524	C38H44N2O6	MT	[21] [22]
132	16-Oxo-alisol A	504.3451	505.3518	−0.6	−1.1	30.88	[M + H]+	505.3518, 483.1921, 467.2456	C30H48O6	ZX	[27]
133	Caryolane-1,9β-diol	238.1933	239.2011	0.5	2.2	31.07	[M + H]+	239.2011, 193.1995	C15H26O2	RG	[31]
134	Houpulin J	402.2559	403.2641	1.0	2.4	31.51	[M + H]+	403.2641, 385.2346, 371.2216, 355.3386, 337.3595	C28H34O2	HP	[32]
135	Cinncassiol D1	352.2250	353.2333	1.0	2.9	32.02	[M + H]+	353.2333, 335.3303	C20H32O5	RG	[31]
136	Akebonic acid	440.3291	441.3379	1.5	3.5	33.10	[M + H]+	441.3379, 409.2314	C29H44O3	MT	[21] [22]
137	Poricoic acid D	514.3294	513.3193	−2.9	−5.6	33.33	[M − H]−	513.3193	C32H48O7	FL	[24]
138	Ergosta-7-en-3,5,6-triol	432.3604	433.3671	−0.5	−1.2	33.51	[M + H]+	433.3671, 417.3971, 313.2673	C28H48O3	ZL	[26]
139	Uralsaponin B	822.4038	823.4070	−4.0	−4.9	33.67	[M + H]+	803.4070, 779.3923, 765.5055	C42H62O16	GC	[36]
140	Kanzonols L	490.2355	491.2425	−0.3	−0.7	33.75	[M + H]+	491.2425, 475.2702, 327.1432	C30H34O6	GC	[36]
141	Cinncassiol D4-2-O-monoacetate	366.2406	367.2471	−0.8	−2.3	33.88	[M + H]+	367.2471, 319.3628	C21H34O5	RG	[31]
142	Hydroxytetracosanoic acid	384.3604	385.3677	0.1	0.2	34.33	[M + H]+	385.3677, 299.2268	C24H48O3	ZL	[26]
143	Cinncassiol D3	368.2199	369.2283	1.1	2.9	34.37	[M + H]+	369.2283, 319.1751	C20H32O6	RG	[31]
144	(22E)-Ergosta-6,8(14),22-trien-3β-ol	396.3392	397.3451	−1.4	−3.6	34.61	[M + H]+	397.3451, 381.3475, 365.3563, 279.1637	C28H44O	FL	[24]
145	2-Lauroleic acid	198.1620	199.1701	0.8	4.2	35.10	[M + H]+	199.1701, 185.1708, 161.1730	C12H22O2	FL	[24]
146	Poricoic acid DM	528.3451	529.3472	−5.2	−9.8	35.42	[M + H]+	529.3472	C32H48O6	FL	[24]
147	Poricoic acid B	484.3189	485.3290	2.8	5.9	35.56	[M + H]+	485.3290, 467.3383, 411.1554, 325.2855	C30H44O5	FL	[24]
148	(22E)-Er-gosta-5,7,9(11),22 -tetraen-3β-ol	394.3236	395.3306	−0.3	−0.7	35.61	[M + H]+	395.3306, 327.3144, 305.2249	C28H42O	FL	[24]
149	Oleanolic acid-28-O-beta-D-glucopyranoside	618.4132	619.4167	−3.8	−6.1	35.75	[M + H]+	619.4167, 535.3676, 475.3616	C36H58O8	HP	[32]
150	Alisol A 23,24-diacetate	574.3870	575.3991	4.9	8.5	35.85	[M + H]+	575.3984, 553.3695, 493.3624	C34H54O7	ZX	[27]
151	Crepenynic acid	278.2246	279.2295	−2.3	−8.3	35.92	[M + H]+	279.2296, 237.2577	C18H30O2	BZ	[20]
152	Citromitin	404.1471	403.1393	−0.5	−1.3	36.64	[M − H]−	403.1393	C20H20O7	CP	[35]
153	Polyporusterone F	462.3345	463.3445	2.7	5.8	36.68	[M + H]+	463.3445, 413.3189, 319.2897	C28H46O5	ZL	[26]
154	11,25-Anhydroalisol F	452.3291	453.3365	0.2	0.5	36.90	[M + H]+	453.3365, 429.3682, 413.2441, 302.3736	C30H44O3	ZX	[27]
155	Daedaleanic acid B	488.3502	489.3558	−1.6	−3.4	37.76	[M + H]+	489.3558, 473.3861, 341.3287	C30H48O5	FL	[24]
156	24-Hydroxy-11-deoxyglycyrrhetic acid	458.3396	459.3494	2.5	5.5	37.81	[M + H]+	459.3494, 421.3847	C29H46O4	GC	[36]
157	Alisol J 23-acetate	526.3294	527.3362	−0.5	−1.0	38.10	[M + H]+	527.3362, 487.3979, 475.3038	C32H46O6	ZX	[27]
158	Stigmasterol 3-O-beta-D-glucopyranoside	574.4233	575.4328	2.2	3.8	38.56	[M + H]+	575.4328, 545.4279, 537.3829, 343.2842	C35H58O6	CZ	[19]
159	16,23-Oxido-alisol B	470.3396	471.3465	−0.4	−0.8	39.07	[M + H]+	471.3456, 399.3606	C30H46O4	ZX	[27]
160	26-Hydroxyporicoic acid DM	544.3400	545.3496	2.3	4.3	39.08	[M + H]+	545.3496, 499.3533, 461.3603	C32H48O7	FL	[24]
161	Alisol B diacetate	556.3764	557.3873	3.6	6.5	39.51	[M + H]+	557.3918, 531.4154	C34H52O6	ZX	[27]
162	Stigmast-4-ene-3,6-dione	426.3498	427.3558	−1.3	−2.9	39.56	[M + H]+	429.3558, 349.3443, 299.3168	C29H46O2	ZZ	[28] [29]
163	(22E)-Ergosta-7,22 -dien-3β-ol	398.3549	399.3621	0.0	0.0	40.24	[M + H]+	399.3621, 345.3486, 301.3576	C28H46O	FL, ZL	[24] [25]
164	Glyasperin E	444.1573	445.1657	1.1	2.5	40.40	[M + H]+	445.1657, 429.3067, 301.2096	C27H24O6	GC	[36]
165	Polyporoid C	494.3244	495.3276	−4.0	−8.1	40.99	[M + H]+	495.3276, 439.3856	C28H46O7	ZL	[26]
166	3β-Hydroxystigmasta-5,22-dien-7-one	424.3341	425.3378	−3.6	−8.5	41.79	[M + H]+	425.3378, 399.4010, 257.2334	C29H44O2	HP	[32]
167	Stigmasterol	412.3705	413.3751	−2.7	−6.6	42.15	[M + H]+	413.3751, 399.3317, 313.2665	C29H48O	MT, ZZ	[21] [22] [28] [29]
168	Hesperidin[b]	610.1898	611.1940	−3.1	−5.0	42.17	[M + H]+	611.1940, 441.3571, 297.3090	C28H34O15	CP	[35]
169	Heptadecane	240.2817	241.2897	0.7	3.0	42.38	[M + H]+	241.2897	C17H36	BZ	[20]
170	Polyporusterone A	478.3294	479.3336	−3.1	−6.5	42.87	[M + H]+	479.3336, 441.3643	C28H46O6	ZL	[25] [26]
171	4,22-Stigmastadiene-3-one	410.3549	411.3608	−1.4	−3.3	43.49	[M + H]+	411.3608, 387.3042, 297.3090	C29H46O	HP	[32]
172	3α-Pachymic acid	528.3815	529.3891	0.3	0.6	43.51	[M + H]+	529.3891, 485.3752, 441.3631	C33H52O5	FL	[24]
173	Poricoic acid ZG	502.3294	503.3363	−0.4	−0.8	43.81	[M + H]+	503.3363, 419.3841	C30H46O6	FL	[24]
174	11-Deoxy 13,17-epoxy-alisol A	490.3658	491.3706	−2.5	−5.0	43.82	[M + H]+	491.3707, 463.3458, 439.2510, 333.1403	C30H50O5	ZX	[27]
175	Eburicoic acid	470.3760	471.3858	2.5	5.4	44.19	[M + H]+	471.3858, 447.4211, 433.2019	C31H50O3	FL	[24]
176	Cinnacaslol glucoside	544.2520	545.2608	1.6	2.9	44.42	[M + H]+	545.2608, 523.5031, 441.3846	C26H40O12	RG	[31]
177	13,17-Epoxy-alisol A	506.3607	507.3688	0.8	1.5	44.65	[M + H]+	507.3688, 493.3894, 365.4323, 283.2955	C30H50O6	ZX	[27]
178	Kaempferol[b]	286.0477	287.0556	0.6	2.2	44.82	[M + H]+	287.0557, 269.9386	C15H10O6	ZZ	[28] [29]
179	25-O-Ethylalisol A	518.3971	519.4084	4.0	7.7	44.85	[M + H]+	519.4084, 467.4148	C32H54O5	ZX	[27]
180	Oplopanane	192.1878	193.1960	0.9	4.5	45.02	[M + H]+	193.2343, 177.2408	C14H24	HP	[32]
181	beta-Sitosterol-3-O-β-D-xylopyranoside	546.4284	547.4303	−5.4	−9.9	45.05	[M + H]+	547.4303, 519.3270, 505.3836	C34H58O5	MT	[21] [22]
182	(4E,6E,12E)-Tetradecatriene-8,10-diyne-1,3-diyl diacetate	300.1362	301.1437	0.2	0.8	45.82	[M + H]+	301.1437, 261.2044, 217.1814, 173.1560	C18H20O4	BZ	[20]
183	8-Methylheptadecane	254.2974	255.3038	−0.	−3.4	45.98	[M + H]+	255.3051, 241.1753	C18H38	RG	[31]
184	2,4-Di-t-butylphenol	206.1671	207.1749	0.6	2.8	45.99	[M + H]+	207.1749, 189.0352, 147.0085	C14H22O	CP	[35]
185	5-Allyl-5′-(1″-hydroxyallyloxy)biphenyl-2,2'-diol	298.1205	299.1284	0.6	1.9	46.00	[M + H]+	297.3135, 283.2956, 255.2339	C18H18O4	HP	[32]
186	Squalene	410.3913	411.4005	2.0	4.9	46.00	[M + H]+	411.3562	C30H50	ZZ	[28] [29]
187	Myristic acid	228.2089	229.2164	0.2	1.0	46.00	[M + H]+	229.2164, 215.2060, 201.1866	C14H28O2	ZZ	[28] [29]
188	Palmitoleic acid	254.2246	255.2304	−1.5	−5.8	46.00	[M + H]+	255.2304	C16H30O2	ZZ	[28] [29]
189	Acetyl Eburicoic Acid	512.3866	513.3924	−1.4	−2.7	46.01	[M + H]+	513.4417, 495.4774, 359.3011	C33H52O4	FL	[24]
190	Heneicosane	296.3443	297.3526	1.0	3.5	46.01	[M + H]+	297.3095, 283.2956	C21H44	ZZ	[28] [29]
191	Licorisoflavan A	438.2406	439.2451	−2.8	−6.3	46.02	[M + H]+	439.2524, 383.1993, 311.3788	C27H34O5	GC	[36]
192	Procyanidin B2	578.1424	579.1471	−2.6	−4.5	46.07	[M + H]+	579.1022, 551.3563, 495.3051	C30H26O12	RG	[31]
193	(2E)-1-Butoxy-2-hexene	156.1514	157.1593	0.6	3.8	46.07	[M + H]+	157.1593	C10H20O	GC	[36]
194	Gancaonin C	354.1103	355.1191	1.5	4.2	46.19	[M + H]+	355.1191	C20H18O6	GC	[36]

Abbreviation: CWD, Chushi Weiling Decoction.

^a Abbreviations: CZ, Cangzhu; HP, Houpo; CP, Chenpi; GC, Gancao; ZX, Zexie; FL, Fuling; ZL, Zhuling; RG, Rougui; BZ, Baizhu; ZZ, Zhizi; MT, Mutong; FF, Fangfeng; DXC, Dengxincao.

^b Compared with reference substance.

Components Determined by GC-MS

A total of 92 components were identified from the samples of CWD by GC-MS, including 22 terpenes, 1 phenylpropanoid, 6 aromatics, 8 organic acids, 21 alcohols and esters, 20 simple ketones and aldehydes, and 14 other compounds. The results indicated that these compounds mainly come from Cangzhu, Mutong, Chenpi, etc. The retention time, ion peak, molecular formula, herb source (in abbreviation), and other information are shown in [Table 3]. The ion flow diagram corresponding to peaks 195 to 286 is shown in [Fig. 2].

Fig. 2 Total ion flow diagram of CWD components in GC-MS. CWD, Chushi Weiling Decoction.

Table 3 Analysis and identification of components from CWD by GC-MS

Compd.	Component Name	Observed RT (min)	Observed m/z	Fragment Ions(m/z)	Formula	Herb-source (in Abbreviation* )
195	2-Hydroxyethyl acrylate	5.0	116.047	88.030, 72.990, 55.020	C5H8O3	FF
196	2,3-Butanediol	5.2	90.068	75.020, 57.040	C4H10O2	CZ, HP, ZX
197	3-Furaldehyde	5.7	96.021	96.000, 66.990	C5H4O2	FF, FL, CP, CZ
198	3-Methylvaleric Acid	6.2	116.084	87.000, 60.020	C6H12O2	BZ
199	2-Methylidenecyclopropane-1-carboxylic acid	6.3	98.037	98.010, 86.970, 74.020	C5H6O2	HP
200	Pentanoic acid	7.0	102.068	86.960, 73.000, 60.010	C5H10O2	RG
201	3,3-Dimethylacrylic acid	7.1	100.052	100.030, 82.010, 60.020	C5H8O2	MT
202	2-Valerylfuran	7.3	152.084	133.010, 109.990, 94.980, 59.980	C9H12O2	HP
203	Tetrahydropyran	7.5	86.073	108.050, 86.000, 72.990, 56.020	C5H10O	HP, RG
204	Carene	7.7	136.234	136.080, 122.020	C10H16	MT
205	4-Methylanisole	7.8	122.073	122.030, 107.010, 79.000, 55.020	C8H10O	HP
206	Benzaldehyde	8.3	106.042	106.010, 94.990, 77.010	C7H6O	FF, RG, MT
207	5-Methyl-2-furaldehyde	8.3	110.037	109.990, 81.010	C6H6O2	GC, CP, ZL
208	5-Ethyl-2-methyl-2,3-dihydro-furan	8.4	112.089	111.930, 72.000, 54.980	C7H12O	CP
209	Methylal	8.5	76.094	76.020, 30.120	C3H8O2	BZ
210	2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one	8.7	144.042	144.000, 100.990, 87.010, 73.000, 55.020	C6H8O4	FF
211	2-Amylfuran	8.8	138.207	138.040, 108.980, 82.040, 68.100	C9H14O	ZL
212	Hexanoic acid	9.0	116.084	87.020, 73.020, 60.020	C6H12O2	FF, ZX, ZL
213	α-Phellandrene	9.1	136.234	136.050, 122.010, 107.980	C10H16	MT
214	α-Terpinene	9.3	136.234	136.000, 121.000, 93.200	C10H16	FL
215	Pyrrole-2-carboxaldehyde	9.3	95.037	94.990, 72.960, 60.020	C5H5NO	FF
216	Cymene	9.4	134.110	123.050, 119.040, 104.970, 91.000, 72.930	C10H14	HP, FL
217	D-Limonene	9.5	136.125	136.080, 107.050, 93.050, 79.050	C10H16	FL, CP, MT
218	Eucalyptol	9.6	154.249	154.100, 139.000	C10H18O	FL, HP
219	2-Ethyl-5-propylcyclopentanone	9.6	154.136	154.120, 123.920, 112.000, 84.050	C10H18O	FF
220	1-Phenylpropane-1,2-diol	9.6	152.190	152.070, 136.020, 119.980	C9H12O2	CP
221	m-Cresol	9.7	108.058	108.030, 79.020	C7H8O	GC
222	3,5,5-Trimethylcyclohex-3-en-1-one	9.7	138.207	138.040, 95.980	C9H14O	ZZ
223	Phenylacetaldehyde	9.8	120.058	120.000, 91.030, 65.020	C8H8O	RG, FF, CP, HP, ZZ, ZL
224	Salicylaldehyde	9.8	122.121	122.030, 93.010, 76.000	C7H6O2	FL
225	γ-Caprolactone	10.0	114.068	85.000, 55.030	C6H10O2	CP
226	Ethyl 4-ethyloxy-2-oxobut-3-enoate	10.0	172.074	136.000, 99.050, 71.080	C8H12O4	CP
227	γ-Terpinene	10.0	136.234	136.020, 121.100, 93.050	C10H16	FL
228	2-Acetylpyrrole	10.1	109.053	109.930, 94.010, 66.020	C6H7NO	FF, HP
229	n-Heptanoic acid	10.4	130.099	127.920, 87.020, 73.010, 60.020	C7H14O2	RG
230	Terpinolene	10.5	136.234	136.080, 121.050	C10H16	FL
231	Linalool	10.7	154.136	121.010, 93.030, 71.030	C10H18O	ZZ
232	Isophorone	11.1	138.104	123.070, 126.030, 82.030	C9H14O	GC, ZZ
233	3-Phenylpropanal	11.7	134.073	134.040, 115.020, 103.050	C9H10O	RG, CP, ZZ
234	Menthol	11.9	156.151	134.020, 123.080, 109.050, 95.050, 85.080	C10H20O	GC, RG, ZZ, MT, BZ, CZ, FL, ZL
235	Octanoic acid	12.0	144.211	144.010, 99.030	C8H16O2	FF
236	Thymol	12.1	150.104	135.040, 122.000	C10H14O	RG, CP, HP
237	Terpineol	12.2	154.136	136.080, 121.020, 84.980	C10H18O	CP
238	Safranal	12.3	150.104	135.030, 121.060, 107.040, 79.030	C10H14O	ZZ
239	3,5,5-Trimethyl-4-methylen-2-cyclohexen-1-on	12.6	150.218	150.120, 135.010, 107.960	C10H14O	ZZ
240	5-Hydroxymethylfurfural	12.7	126.032	135.040, 108.960, 69.020	C6H6O3	CP, MT, ZX
241	3-Phenylpropanol	12.8	136.089	117.040, 103.010, 72.940	C9H12O	RG
242	5-Indanol	13.1	134.175	134.010, 118.970	C9H10O	HP
243	p-Allylphenol	13.1	134.073	134.030, 118.990, 105.010, 72.960	C9H10O	RG, HP
244	R-γ-Decalactone	13.2	170.131	133.970, 109.960, 95.030	C10H18O2	FF
245	Nonanoic acid	13.2	158.131	127.030, 115.020, 98.020	C9H18O2	FF, RG
246	Cinnamaldehyde**	13.4	132.058	131.030, 114.980, 77.030	C9H8O	RG, GC, FL, HP, CP, BZ, ZZ
247	1,3,3-Trimethyl-2-vinyl-1-cyclohexene	13.7	150.141	135.010, 121.000, 106.980, 76.920	C11H18	GC, ZZ
248	Cinnamyl alcohol	13.8	134.073	115.010, 92.030, 78.000	C9H10O	RG
249	o-Acetyl-p-cresol	13.9	150.068	135.010, 107.020	C9H10O2	ZZ
250	2-Methoxy-4-vinylphenol	13.9	150.174	135.070, 118.990, 88.970	C9H10O2	CP
251	4′-Hydroxy-2′-methylacetophenone	13.9	150.174	150.120, 136.010, 117.960, 89.960	C9H10O2	ZZ
252	1-Butyl-3-methylcyclohex-2-en-1-ol	14.0	168.151	134.970, 120.930, 77.000	C11H20O	HP
253	4,4,6-Trimethylcyclohex-2-en-1-ol	14.3	140.120	125.070, 84.010	C9H16O	ZZ
254	Apricolin	14.6	156.115	128.010, 85.000	C9H16O2	CP
255	2-Methoxyphenylacetone	14.7	164.084	135.0500, 121.020, 91.050	C10H12O2	RG
256	Modhephene	15.0	204.351	204.160, 189.960, 161.960	C15H24	MT
257	Berkheyaradulene	15.0	204.350	204.120, 190.020, 175.980	C15H24	MT
258	Vanillin	15.1	152.047	136.920, 122.940, 78.980	C8H8O3	CP, DXC, HP
259	trans-Caryophyllene	15.5	204.351	204.120, 192.000, 134.020	C15H24	MT
260	γ-Elemene	15.6	204.351	204.140, 191.980, 135.960	C15H24	MT
261	Coumarin	15.7	146.037	134.010, 118.010,	C9H6O2	FF, RG, HP
262	Paeonol	15.7	166.174	166.060, 148.980, 134.000, 106.090	C9H10O3	ZL
263	Massoia lactone	16.1	168.115	123.000, 97.010, 67.990	C10H16O2	RG, HP
264	γ-Selinene	16.1	204.351	204.120, 190.000, 175.960	C15H24	MT
265	α-Curcumene	16.2	202.335	202.070, 175.990, 118.020, 89.950	C15H22	MT
266	Pentanoic acid, 5-hydroxy-, 2,4-bis(1,1-dimethylethyl)phenyl ester	16.5	306.219	252.910, 191.090, 109.080	C19H30O3	GC
267	β-Sesquiphellandrene	16.7	204.351	204.120, 192.020	C15H24	MT
268	Dihydroactinidiolide	16.9	180.115	179.990, 137.070, 111.020	C11H16O2	CP
269	Valencene	16.9	204.351	204.160, 189.980, 161.960, 136.010	C15H24	MT
270	γ-Eudesmol	18.0	222.366	222.130, 206.020, 177.980	C15H26O	HP
271	trans-Isoelemicin	18.1	208.254	208.050, 178.030, 147.980	C12H16O3	HP
272	Agarospirol	18.1	222.366	222.100, 206.010, 178.980, 126.020	C15H26O	MT, BZ, CZ
273	β-Eudesmol	18.3	222.198	204.130, 189.120, 149.080	C15H26O	BZ, CZ, FF, HP, MT
274	α-Eudesmol	18.3	222.366	222.100, 206.010, 178.980	C15H26O	HP
275	Atractylon	18.4	216.319	202.100, 178.020, 136.100	C15H20O	CZ, BZ, MT
276	Sandacanol	18.7	208.183	176.120, 161.100, 90.950, 69.010	C14H24O	RG
277	1-[(1S,3aR,4R,7S,7aS)-4-Hydroxy-4-methyl-7-propan-2-yl-1,2,3,3a,5,6,7,7a-octahydroinden-1-yl]ethanone	19.2	238.193	238.140, 205.070, 153.050, 135.050	C15H26O2	ZX, CZ
278	Longifolenaldehyde	19.5	220.183	220.160, 206.950, 121.070, 104.990, 95.020	C15H24O	ZX
279	Senkyunolide J	19.5	226.121	182.060, 152.030, 125.980, 111.040	C12H18O4	FF
280	Isospathulenol	19.6	220.183	220.150, 205.120, 162.110, 119.070	C15H24O	ZX
281	1-epi-Cubenol	19.8	222.198	206.990, 179.080, 162.070, 147.090, 135.020	C15H26O	RG
282	Tetridamine	19.9	165.127	165.020, 149.100, 119.990	C9H15N3	MT
283	Cryptomeridiol	20.1	240.209	204.140, 149.090	C15H28O2	CZ, HP
284	Diisobutyl phthalate	20.5	278.344	278.120, 223.010, 149.030, 103.960	C16H22O4	CP
285	7,9-Ditert-butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione	21.0	276.173	261.050, 232.080, 217.100, 205.060, 175.050	C17H24O3	FL, ZL
286	β-Cyclocostunolide	22.6	232.318	232.140, 217.990, 204.000	C15H20O2	BZ

Abbreviation: CWD, Chushi Weiling Decoction; RT, retention time.

*Abbreviation: CZ, Cangzhu; HP, Houpo; CP, Chenpi; GC, Gancao, ZX, Zexie; FL, Fuling; ZL, Zhuling; RG, Rougui; BZ, Baizhu; ZZ, Zhizi; MT, Mutong; FF, Fangfeng; DXC, Dengxincao.

**Compared with reference substance.

Total Components Determined of CWD

The total of 286 components from the samples of CWD included 93 terpenes, 37 flavonoids, 11 steroids, 12 phenylpropanoids, 8 alkaloids, 17 aromatics, 16 organic acids, 34 alcohols and esters, 26 simple ketones and aldehydes, and 32 other compounds.

From the perspective of medicinal herbs, there are 30 compounds in Cangzhu, 54 compounds in Houpo, 25 compounds in Chenpi, 35 compounds in Gancao, 19 compounds in Zexie, 37 compounds in Fuling, 14 compounds in Zhuling, 40 compounds in Rougui, 19 compounds in Baizhu, 33 compounds in Zhizi, 26 compounds in Mutong, 25 compounds in Fangfeng, and 2 compounds in Dengxincao in CWD.

Research of Cracking Rules

To systematically and qualitatively analyze the chemical components in CWD, the MS behaviors of the samples were studied to summarize their cracking rules and characteristic fragment ions based on relevant literature.[19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40]

Mass Spectrometric Cracking Rules of Terpenoids

Terpene compounds are the general term for compounds and their derivatives with a molecular formula multiple of isoprene. Based on relevant literature, the terpenoids in CWD were preliminarily classified: in the CWD, the terpenoids in Cangzhu (compounds 3, 27, 45, 47, 49, 51, 58, 63, 73, 81, 84, 92, 158, and 122), Baizhu (compounds 27, 51, 81, 92, and 114), and Mutong (compounds 71, 136, and 167) were mainly sesquiterpenoids.[19] [20] [21] [22] The terpenoids in Fuling (compounds 85, 91, 99, 102, 104, 105, 112, 120, 128, 137, 146, 147, 155, 160, 172, 173, 175, and 189) and Zhuling (compounds 153, 165, 170) were mainly lanostelane type triterpenes, while the terpenoids in Zexie (compounds 22, 109, 129, 132, 150, 154, 157, 159, 161, 174, 177, and 179) were mainly prototerpenane type tetracyclic triterpenes.[23] [24] [25] [26] [27] The terpenoids in Zhizi (compounds 8, 15, 16, 33, 94, 100, 113, 125, 167, and 186) were mainly iridoids and their glycosides.[28] [29] [30] In addition, there are also terpenoids (compounds 28, 30, 53, 78, 135, 139, 141, 143, 156, 176, and 180) in other medicinal herbs.[31] [32] [33]

There are three main rules for the cleavage of terpenoids: (1) when a compound forms a glycoside, it can lose all saccharides first, to obtain fragment ions. For example, genipin-1-O-gentiobioside (8; m/z 549.18096 [M − H]⁻) of Zhizi is an iridoid glycoside compound containing one group of gentian disaccharide (i.e., two molecules of glucose). In its secondary mass spectrometry, genipin-1-O-gentiobioside sequentially lost two glucose groups, generating fragment ions of m/z 387.12365 [M – H - Glc]⁻ and 225.06502 [M – H - 2Glc]⁻. (2) Terpene skeletons are prone to lose neutral groups such as CO, CO₂, and H₂O. (3) If the terpenoid skeleton forms a six-membered ring with unsaturated double bonds during mass spectrometry cleavage, it is prone to RDA cleavage. During the cracking process of genipin-1-O-gentiobioside, a six-membered ring containing unsaturated double bonds was generated, to obtain fragment ions of m/z 123.03313 ([Fig. 3]) through RDA cracking. This is consistent with the reference.[29] [30]

Fig. 3 Mass fragmentation pathways and secondary mass spectra of genipin-1-O-gentiobioside.

Atractylenolide I (81; m/z 231.13876 [M + H]⁺) in Cangzhu and Baizhu is a sesquiterpene lactone. In the positive ion mode, the ester bond broke on the five-membered lactone ring, to generate fragment ions of m/z 189.12743. Then, fragment ions of m/z 163.11331 or 145.14062 were generated through the cracking progress of the six-membered ring ([Fig. 4]).

Fig. 4 Mass fragmentation pathways and secondary mass spectra of atractylenolide I.

Mass Spectrometric Cracking Rules of Flavonoids

Flavonoids are widely distributed in the plant kingdom, often forming glycosides through O-glycosidic bonds. Based on relevant literature,[28] [29] [30] [31] [32] [33] [34] [35] [36] the flavonoids in CWD were preliminarily classified. There were a total of 37 confirmed flavonoids in CWD (compounds 4–6, 23–26, 31, 32, 36, 40, 50, 54, 60, 64, 65, 69, 72, 74-76, 80, 82, 95, 98, 110, 115, 124, 127, 140, 152, 164, 168, 178, 191, 192, and 194), mainly from medicinal herbs such as Chenpi, Fangfeng, Gancao, Rougui, and Zhizi. Through research on the cleavage patterns of flavonoids in CWD, we found that: (1) loss of saccharide groups tends to occur in flavonoid glycosides. (2) RDA cleavage reaction tends to occur on the C-ring of flavonoids. (3) Neutrality loss of CO, CO₂, and H₂O often occurs. These rules are consistent with reference.[34] [35]

Taking quercitrin (6; m/z 449.11080 [M + H]⁺) contained in Zhizi as an example, in the positive ion mode, the loss of rhamnose (m/z 146) occurred, generating fragment ions of m/z 303.96551 [M + H − Rha]⁺. Quercetin fragment ions continued to have RDA cleavage at positions ^1,3A of the C-ring, generating ^1,3A ions at m/z 153.06728. In addition, RDA cleavage could also occur at positions ^1,2A; ^0,2A; ^1,4A; or ^0,4A of the C-ring ([Fig. 5]). This cleavage pathway was believed to be reliable by comparing with reference.[34]

Fig. 5 Mass fragmentation pathways and secondary mass spectra of quercetin.

Mass Spectrometric Cracking Rules of Phenylpropanoids

Phenylpropanoid compounds include phenylpropanoic acids, coumarins, and lignans. Based on relevant literature,[31] [32] [33] [37] the phenylpropanoids in CWD were preliminarily classified. The phenylpropanoid compounds (compounds 7, 17, 29, 55, 62, 66, 70, 77, 86, 130, 134, and 261) in CWD mainly came from Houpo, Fangfeng, and Rougui. Phenylpropanoic acid ester bonds are prone to cleavage to generate phenylpropanoic acid fragment ions. Different characteristic skeleton fragment ions are generated due to different mother nucleus structures: fragment ions of m/z 179, 161, and 135 can be inferred to contain caffeoyl fragment ions, and m/z 193, 175, and 160 can be inferred to contain ferulic acid fragments ions, and m/z 163 and 119 can be inferred to contain para-coumarin acid fragment ions, which was consistent with the pyrolysis rule of phenylpropanoids in the positive ion mode described in the literature.[36] [38] The fragment ions often have a neutral loss of CO, H₂O, and CO₂.

Anomalin (29; m/z 427.17103 [M + H]⁺) contained in Fangfeng is a derivative of pyranocoumarin with a total of three ester bonds. In the positive ion mode, anomalin was prone to ester bond cleavage and neutral loss of 2-methyl-2-butenoic acid groups ([Fig. 6]), which can be referred to in the literature.[38]

Fig. 6 Mass fragmentation pathways and secondary mass spectra of anomalin.

Mass Spectrometric Cracking Rules of Phenols, Acids, and Esters

Under the conditions of dissociation, the main mass spectrometry cleavage pathway of phenolic compounds is the loss of substituents in the structure. Based on relevant literature,[26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] the phenols, acids, and esters in CWD were preliminarily classified. There were a total of 44 confirmed phenolic, acid, and ester compounds in CWD (compounds 1, 18, 21, 37, 38, 41, 44, 56, 57, 59, 83, 87–89, 93, 96, 116, 117, 142, 145, 151, 182, 184, 187, 195, 198-201, 212, 221, 225, 226, 228, 230, 236, 244, 245, 250, 254, 266, 270, 279, and 284). In the secondary mass spectrometry of phenolic glycosides, high-abundance fragment ions often originate from the loss of saccharides. Carboxylic acids and their ester compounds are prone to α-cracking, neutral loss of R or OR' groups (depending on which bond of the O atom breaks), and loss of CO, generating [R + H]⁺ and [OR' + H]⁺ fragment ions in positive ion mode. Generally speaking, the ion peak intensity generated by aromatic compounds and their esters is stronger than that of fatty acids and their esters.[20] [21] [22] [23] [24]

Taking paeonolide (38; m/z 460.94828 [M + H]⁺), a component in Cangzhu, as an example, it has paeonol as a aglycone and contains a nonreducing terminal 1-α-arabinopyranoside. During the dissociation process, paeonolide gradually removed saccharide groups and generated fragment ions of m/z 167.13227. Afterward, fragment ions of m/z 137.13492 and phenol fragments were generated ([Fig. 7]).[30]

Fig. 7 Mass fragmentation pathways and secondary mass spectra of paeonolide.

Mass Spectrometric Cracking Rules of Alkaloids

Alkaloids are a class of natural compounds containing basic nitrogen atoms, often with nitrogen heterocyclic structures. Based on relevant literature,[21] [22] [33] [37] [38] [39] the alkaloids in CWD were preliminarily classified and the cracking rules of alkaloids were summarized. The alkaloid components (compounds 10, 11, 34, 35, 67, 119, 126, and 131) in CWD mainly included aporphine alkaloids, isoquinoline alkaloids, and other alkaloids, which are mainly from Houpo, Mutong, and Fangfeng. Alkaloids have various cleavage patterns based on the different C–N skeleton structures, among which the most important fragmentation patterns are four: (1) the groups connected to N atoms are prone to loss, generating fragments such as CH₂, CH₄, NH₂, NH₄, etc. (2) When the alkaloid contains hydroxyl substitutions, it can cause neutral loss of H₂O and methylene. When the alkaloid contains carboxyl substituents, it can cause a loss of CO₂. The alkaloid skeleton with multiple hydroxyl groups in the side chain is prone to breakage and dehydration rearrangement. (3) When the alkaloid has a tetrahydroisoquinoline structure, an RDA cleavage reaction can occur, producing complementary fragment ions. (4) After the cleavage of benzyl isoquinoline alkaloids, benzyl fragment ions will be produced, resulting in typical peaks that are different from other types of alkaloids.

Dauricine (131; m/z 625.32900 [M + H]⁺) in Mutong is a type of bis benzyl tetrahydroisoquinoline alkaloid. In the positive ion mode, the cleavage at positions C-1 and C-1a would produce benzyl fragment ions at m/z 107.12712, which was a typical fragment ion different from the aporphine alkaloids mentioned above.[39] In the secondary mass spectrometry, after the loss of benzyl fragment ions, the mother nucleus fragment ions of dauricine, m/z 205.21913, continued to generate fragments ions of m/z 189.16320 and 161.15241 ([Fig. 8]).

Fig. 8 Mass fragmentation pathways and secondary mass spectra of dauricine.

Results on Network Pharmacology

Active Component and Targets Collection in CWD

After the screening (OB ≥ 30%, DL ≥ 0.18) and searching in relevant literature data, 143 chemical components for CWD were obtained from TCMSP. Furthermore, 1,051 targets for CWD were predicted by SwissTargetPrediction. A total of 4,174 related targets of “eczema” and “herpes zoster” were selected from the Drugbank database, the Genecards database, and the OMIM database. Finally, 1,051 targets of CWD and 4,174 disease-related targets were mapped to the Venn. A total of 362 overlapping targets were obtained.

Analysis of the PPI Network and “Compounds–Targets” Network

A total of 362 intersection targets were inputted into STRING 11.0, and the results show that the network consists of 6,280 edges, with an average degree value of 34.7 and an average local clustering coefficient of 0.501, p < 1.0E-16.

Nodes with a degree value less than 25 were deleted, and 48 core target proteins were used to form the protein–protein interaction (PPI) core network, then isolated targets without interaction were removed, as shown in [Fig. 9]. In the PPI network diagram, different colored lines between targets represent different evidence, with green representing adjacent genes, red representing fusion genes, and blue representing co-occurrence genes. The thicker the connecting lines, the stronger the interaction between proteins, indicating more interactions between proteins rather than the expected interactions of a random set of proteins extracted from the genome. The top 10 core targets for degree ranking were CYP19A1 (cytochrome P450 family 19 subfamily A member 1), AR (androgen receptor), HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase), ESR1 (estrogen receptor 1), PTGS2 (prostaglandin-endoperoxide synthase 2), ALOX5 (arachidonate 5-lipoxygenase), SHBG (sex hormone-binding globulin), NOS2 (nitric oxide synthase 2), ADORA3 (adenosine A3 receptor), and NR3C1 (nuclear receptor subfamily 3 group C member 1).

Fig. 9 The PPI network of protein interaction relationships. PPI, protein–protein interaction.

Screening of Active Ingredients

According to the “compounds–targets” network, the compounds with the highest degree ranking indicated that they were more likely to participate in a certain treatment process and related signaling pathways, and had stronger interactions with target proteins. By intersecting 143 core components in network pharmacology with 287 components of CWD, 25 overlapping components were obtained, with their numbers and degree values shown in [Table 4]. This indicates that these components may have therapeutic effects on eczema and herpes zoster.

Table 4 Overlapping components of material basis and network pharmacology

Compd.	Component name	Herb-source (in Abbreviation[a])	Average shortest path length	Closeness centrality	Stress	Degree
157	Alisol J 23-acetate	ZX	2.547	0.393	1026334	50
170	Polyporusterone A	ZL	2.567	0.390	991250	48
178	Kaempferol	ZZ	2.555	0.391	887930	47
29	Anomalin	FF	2.575	0.388	992448	47
99	Dehydrotumulosic acid	FL	2.594	0.385	737238	46
151	Crepenynic acid	BZ	2.650	0.377	677680	40
120	16-Deoxyporicoic acid B	FL	2.626	0.381	463502	39
12	2-Hydroxyisoxypropyl-3-hydroxy-7-isopentene-2,3-dihydrobenzofuran-5-carboxylic	CZ	2.746	0.364	808492	34
162	Stigmast-4-ene-3,6-dione	RG	2.642	0.378	500454	33
167	Stigmasterol	MT, ZZ	2.757	0.363	172222	22
94	Geniposide	ZZ	2.813	0.355	210508	17
17	Erthro-guaiacy lglycerol	RG	2.813	0.355	243830	16
103	Icariside F2	CZ, BZ	2.944	0.340	304602	15
92	8β-Ethoxy atractylenolide III	CZ, BZ	2.905	0.344	227614	15
168	Hesperidin	CP	2.781	0.360	157740	13
149	Oleanolic acid-28-O-β-D-glucopyranoside	HP	2.920	0.342	51986	12
40	Nobiletin	CP	2.765	0.362	72624	11
173	Poricoic acid ZG	FL	2.940	0.340	34882	10
152	Citromitin	CP	2.797	0.357	47464	9
80	(+)-Leptolepisol C	RG	3.091	0.323	35944	9
58	Oxypaeoniflorin	CZ	3.131	0.319	25476	7
19	Sinapaldehyde 4-O-β-D-glucopyranoside	HP	3.258	0.307	27060	4
45	Atractyloyne	CZ	3.469	0.288	3016	4
116	Syringin	CZ	3.183	0.314	1620	3
65	Xambioona	GC	4.010	0.249	0	1

^a Abbreviation: CZ, Cangzhu; HP, Houpo; CP, Chenpi; GC, Gancao; ZX, Zexie; FL, Fuling; ZL, Zhuling; RG, Rougui; BZ, Baizhu; ZZ, Zhizi; MT, Mutong; FF, Fangfeng.

Gene Ontology and KEGG Pathway Enrichment Analysis

GO and KEGG pathway enrichment analysis was performed on key intersection target genes to obtain the top 25 GO and KEGG signaling pathways, and they were annotated separately. Based on the PPI network, pathway enrichment analysis used protein interaction and metabolic pathway information to predict the therapeutic mechanism of CWD. This is a method beneficial for studying the holistic nature of CWD from a multi-pathway and multi-target perspective and can transform the overall effects of TCM decoction into descriptions used in modern pharmacology.

As shown in [Fig. 10], GO enrichment analysis revealed a total of 52 pathways, and the enrichment results showed that the biological process mainly included pathways such as biological regulation, single organism process, cellular process, response to stimuli, and regulation of biological process; cellular component mainly included pathways such as cell, cell part, organelle, membrane and organelle part; molecular function mainly included pathways such as binding, catalytic activity, signal transducer activity, molecular transducer activity, and nucleic acid binding transcription factor activity.

Fig. 10 Visualization and annotation of GO pathway enrichment analysis (top 25).

KEGG enrichment analysis resulted in a total of 194 pathways ([Fig. 11] ), covering multiple aspects such as metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases. The top 25 mainly included the pathways in cancer, prostate cancer, proteoglycans in cancer, the vascular endothelial growth factor signaling pathway, the C-type lectin receptor signaling pathway, and human cytomegalovirus infection.

Fig. 11 Visualization and annotation of KEGG pathway enrichment analysis (top 25).

Reactome Enrichment Analysis

Using the Metascape platform, Reactome analysis covered a total of 15 pathways ([Fig. 12]), including signaling by interleukins, nuclear receptor transcription pathway, metabolism of lipids, signaling by receptor tyrosine kinases, and metabolism of steroids ([Table 5]).

Table 5 Reactome pathways (top 5)

Category	GO ID	Description	Count	%	Log10(P)	Log10(q)
Reactome Gene Sets	R-HSA-449147	Signaling by Interleukins	14	29.17	−13.89	−10.7
Reactome Gene Sets	R-HSA-383280	Nuclear receptor transcription pathway	8	16.67	−13.81	−10.7
Reactome Gene Sets	R-HSA-556833	Metabolism of lipids	15	31.25	−12.49	−9.78
Reactome Gene Sets	R-HSA-9006934	Signaling by receptor tyrosine kinases	11	22.92	−9.32	−6.86
Reactome Gene Sets	R-HSA-8957322	Metabolism of steroids	7	14.58	−8.32	−5.91

Note: Log10(P) describes the significant level of gene enrichment, the smaller the value, the higher the significance; Log10(q) describes corrected Log10(P) value.

Fig. 12 Visualization and annotation of Reactome enrichment analysis.

Molecular Docking Analysis

Based on the above research, alisol J 23-acetate (157), kaempferol (178), anomalin (29), icariside F2 (103), and cinnamaldehyde (246) ([Fig. 13]) with high degree values were selected. These five components belong to naturally occurring major active constituents in the monarch drug Cangzhu, ministerial drugs Zhizi, Zexie, Fangfeng, and adjuvant drug Rougui of CWD, which have representative structures as terpene, flavonoid, phenylpropanoid, aromatic glycoside, and aldehyde.[27] [28] [29] [30] [31] Therefore, they were used to molecularly dock with core targets CYP19A1, AR, and HMGCR (HMG-CoA) ([Fig. 14]) using autodock software. The mode with the lowest binding energy was selected for plotting. The dark part represents the 3D conformation of the target protein, while the highlighted part represents the ligand molecular structure in [Fig. 15]. The results showed that the molecular docking binding energies of CYP19A1, AR, HMGCR with alisol J 23-acetate (157), kaempferol (178), anomalin (29), cinnamaldehyde (246) were the lowest, mostly lower than −5.00 kcal/mol, indicating strong binding activity between these active ingredients and the targets ([Table 6]).

Table 6 The lowest binding energy of molecular docking between CYP19A1, AR, HMGCR, and different components

The lowest binding energy (kcal·mol−1)	CYP19A1	AR	HMGCR
Alisol J 23-acetate	−7.19	−6.16	−5.77
Kaempferol	−5.95	−5.00	−5.17
Anomalin	−7.08	−4.60	−5.38
Icariside F2	−2.78	−1.87	−1.83
Cinnamaldehyde	−4.38	−4.81	−5.32

Fig. 13 Chemical structures of alisol J 23-acetate (157), kaempferol (178), anomalin (29), icariside F2 (103), and cinnamaldehyde (246).

Fig. 14 Target protein conformation of CYP19A1, AR, and HMGCR (HMG-CoA).

Fig. 15 Molecular docking results. The 3D conformation of target protein CYP19A1, AR, and HMGCR was presented from left to right, respectively.

Discussion

CWD is commonly used in the treatment of eczema and herpes zoster. It clears heat removes dampness, and strengthens the spleen and diuresis. Nevertheless, there is still insufficient research on the material basis and pharmacology of CWD. This study integrates the research results of UPLC-Q-TOF-MS, GC-MS, network pharmacology, and molecular docking to provide a basis for further research.

Through analysis from the PPI network and pathway enrichment (KEGG, GO, and Reactome) of CWD, it was found that the main target proteins of CWD in the treatment of eczema and herpes zoster were CYP19A1, AR, HMGCR, ESR1, PTGS2, etc. Based on the interactions and metabolic pathway information involved in these proteins, enrichment analysis can be summarized as follows: CWD may regulate C-type lectin receptor signaling pathway, human cytomegalovirus infection, interleukin-17 signaling pathway, inflammatory mediators of TRP channel, serotonergic synapses, arachidonic acid metabolism, and Fc-ε-biological pathways such as the RI signaling pathway, to act on anti-inflammatory and antiviral mechanisms. The target proteins above have been proven to be key enzymes in metabolic pathways such as the synthesis of estrogen, synthesis of cholesterol, prostaglandin biosynthesis, and arachidonic acid metabolism.[41] [42] [43] [44] This result indicates CWD may have the potential to regulate immune response mechanisms, which are usually the most important in the treatment of eczema and herpes zoster diseases.

From the perspective of active ingredients, some researchers have confirmed that natural products from 14 Chinese medicinal materials in CWD such as oxypaeoniflorin (58), kaempferol (178), geniposide (94), icariside F2 (103), and hesperidin (168), have anti-inflammatory, antibacterial, and anti-infective effects.[45] [46] [47] [48] [49] These components can reduce the expression level of inflammatory factors and reduce vascular permeability. Molecular docking also showed good binding activity of the above natural products with target proteins CYP19A1, AR, and HMGCR (HMG-CoA). These results to some extent mutually verified the analysis of the PPI network and pathway enrichment.

Besides, research has shown that natural steroid compounds in Chinese herbal medicine can exert therapeutic effects through these receptor signaling pathways, meanwhile, steroid hormone-like regulatory effects are also an important way to treat immune diseases.[50] [51] [52] Atractylodin and atractylone (atractyloyne, 45) contained in Cangzhu also have diuretic effects.[45] [46] Alisol (Alisol J 23-acetate, 157) in Zexie can significantly increase liver tissue SOD content, inhibit leukotriene production and β-hexosaminase release, reduce oxidative damage, and inhibit delayed allergic reactions.[53] Polyporusterone (polyporusterone A, 170) and poricoic acid (16-deoxyporicoic acid B, 120; poricoic acid ZG, 173) in Fuling and Zhuling can regulate blood lipids and reduce sodium and water retention. The sterones and sterols (ergone, cerevisterol) in Fuling have been proven to have diuretic functions, while increasing urine output, they can also increase the excretion of electrolytes such as K⁺, Na⁺, and Cl⁻. Fuling extract poricoic acid can play a similar role as an aldosterone antagonist.[54] [55] The pharmacological effects of these compounds are consistent with the “dehumidification” and “diuretic” effects of CWD and can reflect the possible steroid hormone-like regulatory effects to adjust the water-electrolyte metabolism.

In summary, all the analyses and examples indicate that CWD may have therapeutic effects on eczema and herpes zoster through the above core proteins, pathways, and ingredients from Chinese medicinal materials.

Conclusion

This study conducted a systematic chemical composition analysis of the classic prescription, CWD. The material basis of CWD was preliminarily characterized by UPLC-Q-TOF-MS and GC-MS techniques. A total of 286 chemical components were identified. The mass spectrometry fragmentation patterns of some terpenoids, flavonoids, phenylpropanoids, phenolic esters, and alkaloids in CWD were summarized. Through subsequent research, 25 overlapping components in material basis and network pharmacology were selected, to provide a basis for further research on the quality standards of CWD.

At the same time, network pharmacology, GO, KEGG, and Reactome enrichment analysis reveal that potential therapeutic mechanisms of action of CWD might be: (1) anti-inflammatory, antiviral, and mediated immune response; (2) regulating steroid metabolism. Meanwhile, molecular docking indicated that alisol J 23-acetate, kaempferol, anomalin, and cinnamaldehyde of CWD tend to combine with core target proteins at a low level of binding energy.

This study provides ideas and methods for the basic research of CWD and gives evidence support for clinical medication.

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