Skip to main content
NCBI home page
Journal of Analytical Methods in Chemistry logoLink to Journal of Analytical Methods in Chemistry
. 2025 May 5;2025:2948965. doi: 10.1155/jamc/2948965

Characterization of the Active Ingredients and Prediction of the Potential Anticolitis Mechanism of the Feng-Liao-Chang-Wei-Kang Capsule via Mass Spectrometry and Network Pharmacology

Tingting Liu 1, Zhijiang He 2, Witiao Lv 3, Liyun Deng 3, Xizhe Sun 1,✉, Yanfei Chen 1,✉
PMCID: PMC12069849  PMID: 40365509

Abstract

The Feng-Liao-Chang-Wei-Kang (FLCWK) capsule is a nationally protected Chinese patent medicine for the treatment of colitis. However, the potential active components and the pharmacological mechanism underlying the anticolitis effect of the FLCWK capsule remain unclear. This study aimed to reveal the active ingredients and possible anticolitis mechanism of the FLCWK capsule using an integrated approach combining mass spectrometry and network pharmacology analysis. Ultra-performance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap MS) was applied to identify the components of the FLCWK capsule. A network pharmacology study, including target gene prediction and functional enrichment, was applied to screen the active ingredients of the FLCWK capsule and explore its potential mechanism for the treatment of colitis. A total of 115 components were identified in the FLCWK capsule. Network pharmacology results showed that 46 of these compounds with good bioavailability and drug-likeness, such as 4′,5-dihydroxyflavone, pinostrobin, naringenin chalcone, apigenin, and morin, were selected as active ingredients. The active ingredients may act on 352 core protein targets, including EGFR, AKT1, PIK3R1, PIK3CB, and MAPK1, thereby modulating relevant pathways, such as MAPK and PI3K-Akt signaling pathways, and thus alleviating inflammation and intestinal damage in colitis. This study provided a useful approach to identify active components and the anticolitis mechanism of the FLCWK capsule and built up a reliable foundation for its clinical treatment.

Keywords: anticolitis mechanism, characterization of active ingredients, Feng-Liao-Chang-Wei-Kang capsule, mass spectrometry, network pharmacology

1. Introduction

Colitis, an inflammatory bowel disease (IBD), is characterized by mucosal epithelial damage and disruption of intestinal homeostasis [1]. The clinical manifestations of colitis include bellyache, diarrhea, and bloody stool, which seriously compromise the life quality of patients [2]. Current therapeutic strategies primarily rely on aminosalicylic acid derivatives, immunosuppressive steroids, and biological agents [3]. However, the long-term administration of these medications is limited by drug resistance and toxicity, underscoring the need for safer and more effective complementary and adjuvant therapies [4]. Traditional Chinese herbal medicine has shown promise in colitis management, particularly the Feng-Liao-Chang-Wei-Kang (FLCWK) capsule, a nationally protected Chinese patent medicine (protection number: ZYB2072004057) [5–8]. The FLCWK capsule containing Daphniphyllum calycinum Benth. and Polygonum hydropiper Linn. has emerged as a particularly noteworthy therapeutic agent for colitis treatment [7, 8]. In clinical practice, the FLCWK capsule is commonly administered with mesalazine, with clinical studies demonstrating its capacity to enhance therapeutic outcomes [6, 9]. For example, a clinical study involving 120 patients with chronic colitis demonstrated that combination therapy with mesalazine and FLCWK significantly improved clinical outcomes compared to mesalazine monotherapy. The combination group exhibited superior clinical efficacy (p < 0.05), significantly reduced inflammatory markers (IL-6, CRP, TNF-α; p < 0.001), decreased mucosal lesions (p < 0.001), and enhanced quality of life (p < 0.001) [6]. Similarly, a study by Hou and Gan involving 52 patients with recurrent ulcerative colitis revealed that the combination of mesalazine and FLCWK resulted in a higher total effective rate (92.31% vs. 69.23%, p < 0.05) compared to mesalazine alone [9]. However, the potential active components and the pharmacological mechanism underlying the anticolitis effect of the FLCWK capsule remain unclear.

Orbitrap mass spectrometry (MS) is a powerful analytical platform for characterizing complex chemical compositions in Chinese herbal medicines, offering high sensitivity, resolution, and a broad dynamic range [10, 11]. Its ability to analyze MSn fragments enables precise structural elucidation of compounds, making it ideal for investigating the chemical profile of the FLCWK capsule [12, 13]. Network pharmacology is well-suited for studying Chinese medicine due to its multicomponent, multitarget, and multipathway nature [10, 14–16]. It has been widely used to investigate interactions between herbal compounds and disease mechanisms, providing a robust framework for predicting the FLCWK capsule's active components and anticolitis mechanisms [17–19].

In conclusion, in this study, the chemical constituents of the FLCWK capsule were analyzed efficiently and accurately by using ultra-performance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap MS). Then, network pharmacology was employed to illustrate the potential active components and anticolitis mechanisms of the FLCWK capsule. The obtained results could be helpful to build up reliable information on the clinical application of FLCWK capsules in the treatment of colitis.

2. Materials and Methods

2.1. Reagents and Materials

The FLCWK capsule (0.37 g per capsule, lot number: 221103) was produced by Haikou Qili Pharmacy Co., Ltd. (Haikou, China). The 99 reference compounds of the FLCWK capsule were purchased from Chemexpress Co., Ltd. (Shanghai, China) and Sigma-Aldrich (St. Louis, MO, USA). The purity of all standard compounds was determined to be higher than 98%. The name, molecular formula, batch number, and company of each reference compound are shown in Table S1. Acetonitrile, methanol, and formic acid were HPLC grade and purchased from Thermo Fisher Scientific (Fair Lawn, NJ, USA).

2.2. Analysis of the FLCWK Capsule Chemical Components

2.2.1. Preparation of the Sample Solution

Precisely 0.4 g of FLCWK capsule contents were weighed and transferred into a 50-mL volumetric flask. Subsequently, 40 mL of 80% methanol was added, followed by ultrasonication for 20 min. After cooling to ambient temperature, the solution was brought to volume with 80% methanol and homogenized by thorough mixing. A 2-mL aliquot of this solution was then quantitatively transferred and diluted to 10 mL with 80% methanol. The diluted solution was filtered through a 0.22-μm microporous membrane. For final preparation, 1 mL of the filtrate was mixed with an equivalent volume of 80% methanol to obtain the test sample solution. The prepared sample (5 μL) was subsequently injected into the UPLC-Q-Exactive Orbitrap system for analysis.

2.2.2. UPLC-Q-Exactive Orbitrap MS Conditions

UPLC analysis was carried out on an ACQUITY UPLC I-Class plus system (Waters Co., Milford, MA, USA). The separation was performed on an ACQUITY UPLC HSS T3 column (100 × 2.1 mm, 1.8 μm) maintaining a flow rate of 0.35 mL/min at 45°C with a 5-μL injection volume. The mobile phase consisted of A (water with 0.1% formic acid) and B (acetonitrile), with the following elution gradient program: 0.0 min A:B (95:5) ⟶ 2.0 min A:B (95:5) ⟶ 4.0 min A:B (70:30) ⟶ 8.0 min A:B (50:50) ⟶ 10.0 min A:B (20:80) ⟶ 14.0 min A:B (0:100) ⟶ 15.0 min A:B (0:100) ⟶ 15.1 min A: B (95:5) ⟶ 16.0 min A:B (95:5).

MS analysis was performed on a Q-Exactive Orbitrap MS (Thermo Fisher Scientific, Fair Lawn, NJ, USA) connected to an electrospray ionization (ESI) source operating in both positive and negative modes. The mass range was 100–1200 m/z, the nitrogen sheath gas flow rate was 35 Arb, the auxiliary gas was 8 Arb, the capillary temperature was 320°C, and the spray voltage in the positive and negative mode was 3800 and −3000 V, respectively.

2.2.3. Compound Identification

The raw data were acquired using the Xcalibur 4.1 software (Thermo Fisher Scientific, Fair Lawn, NJ, USA), and all obtained data were processed by the Compound Discoverer (CD) 3.0 (Thermo Fisher Scientific, Fair Lawn, NJ, USA) and Xcalibur 4.1 software packages. The compounds were identified by comparing the chromatographic feature, empirical molecular formulas, and characteristic fragment ions with those of reference compounds or published known compounds in the HERB database (https://herb.ac.cn/).

2.3. Network Pharmacology

2.3.1. Screening of Active Ingredients and Potential Targets of the FLCWK Capsule

The absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling of the identified components from the FLCWK capsule was estimated using the SwissADME online server (https://www.swissadme.ch/) [20]. The screening of the active ingredients was based on the following characteristics: (i) gastrointestinal absorption (GI absorption) was “High,” indicating good oral bioavailability and absorption of the ingredient; (ii) at least two of the five categories (Lipinski, Ghose, Veber, Egan, and Muegge) were set to “Yes”, indicating that the compound has good drug-like properties [20, 21]. Although some of the ingredients did not satisfy the above criteria, they were still included if their good pharmacological properties were confirmed through a literature review. Finally, the canonical SMILES of the collected active ingredients were entered into the SwissTargetPrediction database (https://www.swisstargetprediction.ch/) to obtain the corresponding target genes. The criterion for target screening was top 100 with a probability greater than 0.1.

2.3.2. Colitis Target Prediction and Intersection With the Targets of the FLCWK Capsule

Information regarding colitis-associated target genes was obtained from the DisGeNET (https://www.disgenet.org/) and GeneCards (https://www.genecards.org/) databases by entering the keyword “Colitis”. The targets of the FLCWK active ingredients and the colitis-related targets were intersected by the Venny 2.1.0 software (https://bioinfogp.cnb.csic.es/tools/venny/) to obtain the potential targets for FLCWK against colitis.

2.3.3. Protein-Protein Interaction (PPI) Network Construction

In order to clarify the interaction of therapeutic target genes and identify the central gene, the potential targets for FLCWK against colitis were imported into the STRING platform (https://string-db.org/) to obtain the PPI network. The species was set as homosapiens, and the parameter was set to the highest confidence (0.900). Then, the Cytoscape 3.10.1 software (https://cytoscape.org/) was used to visualize the PPI network structure and to analyze the topological characteristics.

2.3.4. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis

The potential targets for FLCWK against colitis were imported into the DAVID database (https://david.ncifcrf.gov/). Then, GO analysis was performed to demonstrate the roles of the potential targets in the biological process (BP), cell composition (CC), and molecular function (MF) of colitis. KEGG analysis was conducted to relate the targets to signaling pathways. The species was set to homosapiens.

2.3.5. Network Construction

The potential targets for FLCWK against colitis and pathways enriched by KEGG analysis were taken into the Cytoscape 3.10.1 software to construct a compound-target-pathway-disease network.

3. Results and Discussion

3.1. Analysis of the FLCWK Capsule Chemical Components

Based on the UPLC-Q-Exactive Orbitrap MS conditions of “2.2.2”, 115 compounds in the FLCWK capsule were identified under the positive and negative ion modes, including 37 flavonoids, 15 terpenes, 10 fatty acid and derivatives, 9 glycosides, 8 carbohydrates, 7 phenylpropanoids, 5 alkaloids, 5 amino acids, 4 phenols, 4 carboxylic acid and derivatives, four organic acids and derivatives, 3 nucleotides and derivatives, and four others. The total ion chromatograms of the FLCWK capsule are shown in Figure 1. The compound lists are shown in Table 1. The top 7 kinds of compounds with the highest content were flavonoids (54.7%), carbohydrates (22.1%), terpenes (9.3%), alkaloids (3.5%), glycosides (2.8%), phenylpropanoids (1.7%), and phenols (0.4%). Based on the content and anticolitis effects according to reference, flavonoids, terpenes, alkaloids, glycosides, phenylpropanoids, and phenols were chosen to explain their identifications in detail by the following illustrative examples.

Figure 1.

Figure 1

Open in a new tab

Total ion chromatogram (TIC) of the FLCWK capsule with positive (a) and negative (b) electrospray ionization (ESI).

Table 1.

Chemical components characterized from the FLCWK capsule.

No. Retention time (min) Compound Molecular formula Selective ion Theoretical mass (m/z) Measured mass (m/z) Error (ppm) Major fragments (m/z) Classification
1 0.77 Mannosamine C6H13NO5 [M + H − H2O]+ 162.0761 162.0759 −1.05 72.0451, 84.0449, 84.9602, 85.0289, 97.0288, 102.9706, 120.9810, 143.9967, 144.0654 Carbohydrates
2 0.77 Iminodiacetic acid C4H7NO4 [M − H]− 132.0302 132.0303 0.68 86.0248, 88.0405 Amino acids
3 0.78 L-Histidine C6H9N3O2 [M + H]+ 156.0768 156.0766 −1.23 56.9655, 74.0971, 95.0608, 110.0714, 130.0630 Amino acids
4 0.78 L-Arginine C6H14N4O2 [M + H]+ 175.1190 175.1188 −1.04 72.0814, 112.0857, 116.0708, 130.0500, 130.0974, 132.0211, 139.0389, 158.0921 Amino acids
5 0.82 6-(alpha-D-Glucosaminyl)-1D-myo-inositol C12H23NO10 [M + H]+ 342.1395 342.1390 −1.40 144.0656, 162.076, 174.076, 240.0862, 288.1073, 306.1182 Carbohydrates
6 0.85 3-Pyridineacetic acid C7H7NO2 [M + H]+ 138.0550 138.0547 −1.61 56.9656, 110.0603 Carboxylic acid and derivatives
7 0.87 Melezitose C18H32O16 [M + Na]+ 527.1583 527.1576 −1.39 185.0421, 203.0524, 347.0944, 365.1052 Carbohydrates
8 0.88 Turanose C12H22O11 [M + FA − H]− 387.1145 387.1141 −0.98 59.0139, 71.014, 89.0245, 101.0245, 113.0244, 119.035, 179.0557, 341.1085 Carbohydrates
9 0.88 Gluconic acid C6H12O7 [M − H]− 195.0510 195.0510 −0.12 59.0139, 75.0087, 85.0293, 87.0088, 89.0245, 99.0087, 129.0191, 159.0298, 177.0406 Carbohydrates
10 0.88 Gentianose C18H32O16 [M + FA − H]− 549.1673 549.1669 −0.65 143.0347, 161.0456, 179.0561, 191.0558, 207.0509, 221.0662, 323.0978, 341.1090, 503.1613 Carbohydrates
11 0.88 Threonic acid C4H8O5 [M − H]− 135.0299 135.0300 0.76 87.0087, 89.0245, 92.0255, 108.0453, 116.0074, 134.0176 Carbohydrates
12 0.89 Stachydrine C7H13NO2 [M + K]+ 182.0578 182.0576 −0.98 84.9602, 120.0808, 166.0836 Alkaloids
13 0.89 FAPy-adenine C5H7N5O [M + H − H2O]+ 136.0618 136.0617 −0.73 91.0547,109.0649,119.0355 Others
14 0.90 Glucose C6H12O6 [M − H]− 179.0561 179.0562 0.68 72.9932, 75.0087, 85.0294, 87.0087, 89.0245, 95.0140, 101.0251, 113.0247, 119.0350 Carbohydrates
15 0.90 Glycolaldehyde dimer C4H8O4 [M − H]− 119.0350 119.0351 0.74 71.0139, 72.0092, 72.9932, 73.0296, 74.0248, 75.0088, 89.0245, 101.0245, 118.0509 Others
16 0.92 Citric acid C6H8O7 [M − H]− 191.0197 191.0199 0.81 71.0136, 85.0296, 87.0088, 93.0346, 111.0088, 111.0452, 127.0403, 129.0194, 173.0457 Organic acids and derivatives
17 0.93 Malic acid C4H6O5 [M − H]− 133.0142 133.0144 1.17 71.0139, 74.0248, 114.934, 115.0037 Organic acids and derivatives
18 0.93 Uridine C9H12N2O6 [M − H]− 243.0623 243.0622 −0.41 99.9259, 110.0239, 153.0305 Nucleotides and derivatives
19 1.04 6-Methylnicotinamide C7H8N2O [M + H]+ 137.0709 137.0708 −1.34 94.0655 Alkaloids
20 1.32 L-Isoleucine C6H13NO2 [M + H]+ 132.1019 132.1017 −1.20 69.0705, 72.9378, 86.0969, 90.0554 Amino acids
21 1.39 Adenosine C10H13N5O4 [M + H]+ 268.1040 268.1035 −2.13 136.0617 Nucleotides and derivatives
22 1.41 Methylmalonic acid C4H6O4 [M − H]− 117.0193 117.0195 1.40 59.0139, 71.0503, 72.0092, 73.0295, 74.0248, 99.0089, 99.9258, 116.9286 Organic acids and derivatives
23 1.82 Gallic acid C7H6O5 [M − H]− 169.0142 169.0144 1.11 98.0247, 125.0245 Phenols
24 2.28 N-(1-Deoxy-1-fructosyl)phenylalanine C15H21NO7 [M + H]+ 328.1391 328.1383 −2.50 120.0809, 132.0807, 166.0863, 178.0865, 264.1225, 292.1174, 310.1281 Amino acids
25 3.29 Protocatechuic acid C7H6O4 [M − H]− 153.0193 153.0195 1.00 91.0301, 109.0296 Phenols
26 3.33 Geniposidic acid C16H22O10 [M − H]− 373.1140 373.1137 −0.77 59.0138, 71.0137, 89.0244, 93.0347, 101.0244, 123.0451, 149.0606, 167.0717, 211.0608, 373.1134 Terpenes
27 3.63 Mussaenosidic acid C16H24O10 [M − H]− 375.1297 375.1296 −0.23 89.0243, 101.0241, 102.9566, 121.0662, 125.0609, 149.0603, 151.0768, 169.0877, 213.0766 Terpenes
28 3.69 8-Epi-loganic acid-6′-O-beta-D-glucoside C22H34O15 [M − H]− 537.1825 537.1824 −0.09 250.1856, 307.1030, 310.4752, 327.3016, 382.9057, 454.2435, 459.8475, 491.1820, 491.2547 Terpenes
29 3.80 4-O-beta-glucopyranosyl-cis-coumaric acid C15H18O8 [M + FA − H]− 371.0984 371.0981 −0.90 96.9888, 110.0945, 119.0501, 134.0316, 134.2957, 136.3259, 147.0451, 163.0401, 342.3176 Glycosides
30 3.81 5′-Methylthioadenosine C11H15N5O3S [M + H]+ 298.0968 298.0962 −2.20 136.0617 Nucleotides and derivatives
31 3.95 Asperulosidic acid C18H24O12 [M − H]− 431.1195 431.1192 −0.71 165.0563, 269.0666 Terpenes
32 3.99 3,17-dihydroxy-4,4,8,10,14-pentamethyl-2,3,5,6,7,9-hexahydro-1H-cyclopenta[a]phenanthrene-15,16-dione C22H30O4 [M + NH4]+ 376.2483 376.2477 −1.41 225.1230,358.2383 Terpenes
33 4.05 Kaempferol 3-sophoroside-7-glucoside C33H40O21 [M − H]− 771.1989 771.1990 0.05 299.0192, 300.0272, 301.0349, 462.0822, 609.1448 Flavonoids
34 4.05 Trans-ferulic acid-4-beta-glucoside C16H20O9 [M − H2O − H]− 337.0929 337.0927 −0.56 119.0502, 163.0401, 173.0453, 191.0562 Glycosides
35 4.10 Crenulatin C11H20O6 [M + FA − H]− 293.1242 293.1241 −0.21 59.0139, 71.0138, 89.0246, 101.0245, 113.0243, 119.0347, 131.0714, 157.0119 Terpenes
36 4.12 Syringin C17H24O9 [M + Na]+ 395.1313 395.1307 −1.45 185.0424,232.0702,233.0772 Glycosides
37 4.17 Catechin C15H14O6 [M + H]+ 291.0863 291.0858 −1.93 111.0806, 113.0962, 123.0442, 127.0389, 129.0911, 139.0388, 145.0496, 147.0437, 165.0545 Flavonoids
38 4.23 Asperuloside C18H22O11 [M + FA − H]− 459.1144 459.1144 −0.03 59.0139, 119.0502, 147.0452, 191.035, 413.1062 Terpenes
39 4.23 2-[4,5-dihydroxy-2-(hydroxymethyl)-6-[(5-methyl-2-propan-2-yl-2H-furan-5-yl)oxy]oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol C20H34O12 [M + FA − H]− 511.2033 511.2029 −0.72 71.0139, 89.0245, 92.5903, 101.0243, 388.8512, 465.1975 Glycosides
40 4.31 Oxyresveratrol 2-O-beta-D-glucopyranoside C20H22O9 [M + H]+ 407.1337 407.1330 −1.51 149.0597, 163.0389, 205.0492, 235.0597, 253.0703, 257.0805, 271.0811, 275.0911, 406.2216 Phenylpropanoids
41 4.32 Afzelechin C15H14O5 [M + H]+ 275.0914 275.0908 −2.03 107.0494, 111.0807, 127.0756, 129.0908, 139.0389, 149.0598, 191.0704 Flavonoids
42 4.38 Roseoside C19H30O8 [M + FA − H]− 431.1923 431.1922 −0.26 223.1345, 385.1848 Glycosides
43 4.40 Epicatechin C15H14O6 [M − H]− 289.0718 289.0718 0.02 137.0245, 151.0412, 161.0609, 179.0364, 187.0404, 203.0710, 205.0509, 221.0823, 245.0814 Flavonoids
44 4.45 Manghaslin C33H40O20 [M + H]+ 757.2186 757.2172 −1.78 71.0499, 85.029, 129.0548, 287.0546, 303.0496, 449.1082, 465.1024, 611.1595 Flavonoids
45 4.45 2-[2-Hydroxy-4-(3-hydroxybut-1-enyl)-3,5,5-trimethylcyclohex-3-en-1-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol C19H32O8 [M + FA − H]− 433.2079 433.2080 0.14 89.0246, 92.5869, 101.0245, 113.0246, 119.0350, 179.0562, 225.1486, 366.1724, 387.2025 Glycosides
46 4.45 Isovanillic acid C8H8O4 [M + H]+ 169.0495 169.0493 −1.33 141.0546, 146.9613, 146.9804, 151.0390, 151.0755, 151.0864, 168.0641 Phenols
47 4.45 1,3,4-Trihydroxy-5-[3-(4-hydroxyphenyl)prop-2-enoyloxy]cyclohexane-1-carboxylic acid C16H18O8 [M − H]− 337.0929 337.0930 0.18 93.0347, 111.0452, 113.0245, 119.05, 162.0555, 163.0403, 173.0455, 191.0563 Organic acids and derivatives
48 4.52 Megastigm-7-ene-3,5,6,9-tetraol C13H24O4 [M + H − H2O]+ 227.1641 227.1638 −1.66 177.0549, 181.0489, 181.9503, 186.9560, 191.1428, 199.0868, 209.0795 Terpenes
49 4.54 Paeonolide C20H28O12 [M + Na]+ 483.1473 483.1465 −1.77 331.0988 Glycosides
50 4.57 Butin-7-O-β-D-glucopyranoside C21H22O10 [M − H2O − H]− 415.1035 415.1037 0.47 137.0249, 149.0244, 149.0605, 203.0721, 205.0511, 215.0726, 245.0822, 289.0715, 301.0698 Flavonoids
51 4.59 Mauritianin C33H40O19 [M + H]+ 741.2237 741.2222 −2.01 71.0498, 85.029, 129.0547, 287.0547, 449.1079, 595.1638 Flavonoids
52 4.59 Neridienone A C21H26O3 [M + NH4]+ 344.2220 344.2220 −0.14 308.2006, 326.211 Terpenes
53 4.66 Lyoniresinol 9′-O-glucoside C28H38O13 [M + FA − H]− 627.2294 627.2295 0.05 205.0710, 359.1140, 371.1140, 373.1273, 389.1249, 404.1474, 419.1715, 534.2642, 535.2736, 581.2214 Phenylpropanoids
54 4.70 Quercetin 3-O-neohesperidoside C27H30O16 [M − H]− 609.1461 609.1460 −0.17 151.0038, 255.0301, 271.0244, 300.0272 Flavonoids
55 4.70 Myricetin 3-O-rutinoside C27H30O17 [M − H]− 625.1410 625.1408 −0.36 92.5929, 151.0039, 161.0237, 178.9989, 255.0290, 271.0243, 287.0197, 299.0197, 316.0216 Flavonoids
56 4.70 Rutin C27H30O16 [M + H]+ 611.1607 611.1596 −1.81 303.0495, 465.1023 Flavonoids
57 4.72 4,12-dimethyl-14,19-dioxa-17-azaheptacyclo[10.7.2.22,5.02,7.08,18.08,21.013,17]tricosane-4,20-diol C22H33NO4 [M + H − H2O]+ 358.2376 358.2370 −1.74 357.2251 Alkaloids
58 4.77 Butyl (S)-3-hydroxybutyrate [arabinosyl-(1-> 6)-glucoside] C19H34O12 [M − H]− 453.1978 453.1977 −0.02 103.0400, 112.9857, 113.0246, 119.0346, 143.0356, 163.0608, 205.0713, 248.9588, 407.1904 Glycosides
59 4.81 Quercetin-3-O-glucuronide C21H18O13 [M − H]− 477.0675 477.0673 −0.34 71.0138, 89.0245, 92.5887, 119.0503, 149.0608, 151.0039, 163.0401, 178.9987, 301.0352 Flavonoids
60 4.81 Nuciferoside C22H38O11 [M + FA − H]− 523.2396 523.2394 −0.40 152.9955, 161.0455, 163.0608, 165.0557, 179.0722, 205.0730, 331.1747, 361.1655, 477.2361 Glycosides
61 4.83 Hyperoside C21H20O12 [M + H]+ 465.1028 465.1021 −1.46 85.029, 303.0497 Flavonoids
62 4.83 Myricitrin C21H20O12 [M + Na]+ 487.0847 487.0838 −1.87 325.0312 Flavonoids
63 4.85 Apigenin 5-O-glucoside C21H20O10 [M − H]− 431.0984 431.0988 0.91 113.0236, 119.0348, 181.0330, 205.1233, 269.0452, 283.0605, 311.0549, 341.0659, 430.1841 Flavonoids
64 4.85 Quercetin 3-o-(6″-galloyl)-beta-d-glucopyranoside C28H24O16 [M − H]− 615.0992 615.0992 0.10 151.0037, 178.999, 300.0285, 301.035 Flavonoids
65 4.85 (16-Hydroxy-5,5,9-trimethyl-14-methylidene-15-oxo-2-tetracyclo[11.2.1.01,10.04,9]hexadecanyl) acetate C22H32O4 [M + NH4]+ 378.2639 378.2633 −1.70 360.2528, 377.2516 Terpenes
66 4.85 Suberic acid C8H14O4 [M − H]− 173.0819 173.0821 0.79 93.0346, 104.9539, 111.0815, 129.0921, 130.0875, 146.0362, 172.0976 Fatty acid and derivatives
67 4.85 Ellagic acid C14H6O8 [M − H]− 300.9990 300.9990 0.06 92.5915 Phenols
68 4.88 m-Anisaldehyde C8H8O2 [M + H]+ 137.0597 137.0595 −1.37 95.0861, 96.0451, 109.0650, 110.0604, 120.0447, 136.0218, 136.0617, 136.0748 Carboxylic acid and derivatives
69 4.90 Kaempferol-3-O-rutinoside C27H30O15 [M + H]+ 595.1657 595.1646 −1.90 71.0498, 85.0289, 287.0546, 449.1073 Flavonoids
70 4.90 Kaempferol 3-O-robinobioside C27H30O15 [M − H]− 593.1512 593.1511 −0.10 227.0353, 255.0265, 284.0323 Flavonoids
71 4.96 L-3-Phenyllactic acid C9H10O3 [M − H]− 165.0557 165.0558 0.68 97.0407, 98.0248, 119.0504, 120.0456, 121.0295, 121.0661, 122.0614, 147.0453 Phenylpropanoids
72 5.03 Kaempferol-3-O-glucuronoside C21H18O12 [M − H]− 461.0725 461.0726 0.04 71.0138, 85.0296, 113.0246, 229.0504, 257.0465, 285.0409 Flavonoids
73 5.05 Trifolin C21H20O11 [M + Na]+ 471.0898 471.0890 −1.76 309.0364 Flavonoids
74 5.05 Songoramine C22H29NO3 [M + H]+ 356.2220 356.2213 −2.03 338.2112 Alkaloids
75 5.06 Quercitrin C21H20O11 [M + H]+ 449.1078 449.1070 −1.85 85.0289, 287.0547, 303.049 Flavonoids
76 5.13 Vitexin C21H20O10 [M − H]− 431.0984 431.0986 0.58 59.0139, 89.0244, 268.0375, 269.0444 Flavonoids
77 5.13 Sophoricoside C21H20O10 [M + H]+ 433.1129 433.1123 −1.49 271.0599 Flavonoids
78 5.28 2-(4-methoxyphenyl)-7-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one C22H22O9 [M + FA − H]− 475.1246 475.1245 −0.26 134.0374, 149.0609, 163.0401, 236.0484, 253.0499, 295.0613, 312.0633, 313.1073 Flavonoids
79 5.30 Aloeresin D C29H32O11 [M − H2O − H]− 537.1766 537.1749 −3.07 190.0270, 205.0494, 225.1749, 298.7839, 341.1386, 342.3734, 355.4550, 367.1013, 474.4010 Others
80 5.52 Okanin C15H12O6 [M − H]− 287.0561 287.0562 0.24 92.9275, 96.9604, 125.0245, 149.061, 243.0663, 259.0609 Flavonoids
81 5.99 Steppogenin C15H12O6 [M − H]− 287.0561 287.0560 −0.32 107.014, 135.0452, 139.0403, 151.0036 Flavonoids
82 6.01 Chrysophanein C21H20O9 [M + FA − H]− 461.1089 461.1089 −0.13 253.0503 Others
83 6.03 Sebacic acid C10H18O4 [M − H]− 201.1132 201.1133 0.51 74.0249, 89.0243, 116.9285, 139.1129, 183.1023 Fatty acid and derivatives
84 6.07 Luteolin C15H10O6 [M − H]− 285.0405 285.0405 0.05 133.0295 Flavonoids
85 6.08 (5S,10S,13R,14R,15S,17R)-15-hydroxy-17-[(Z,2R)-7-hydroxy-6-methylhept-5-en-2-yl]-4,4,10,13,14-pentamethyl-1,2,5,6,12,15,16,17-octahydrocyclopenta[a]phenanthren-3-one C30H46O3 [M + NH4]+ 472.3786 472.3776 −2.03 119.0859,454.3658 Terpenes
86 6.10 Quercetin C15H10O7 [M + H]+ 303.0499 303.0493 −2.04 229.0494 Flavonoids
87 6.10 Morin C15H10O7 [M − H]− 301.0354 301.0352 −0.49 107.0139, 121.0296, 151.0037, 178.9988 Flavonoids
88 6.25 Syringaresinol C22H26O8 [M + H − H2O]+ 401.1594 401.1587 −1.90 291.0995, 315.0858, 330.1095, 331.1151, 339.1240, 343.1169, 351.1234, 369.1332, 383.1486 Phenylpropanoids
89 6.37 3-O-Methylquercetin C16H12O7 [M − H]− 315.0510 315.0510 −0.01 255.029, 271.025, 300.0269 Flavonoids
90 6.74 Naringenin chalcone C15H12O5 [M − H]− 271.0612 271.0611 −0.38 93.0347, 107.0139, 119.0504, 151.0037, 227.072 Flavonoids
91 6.79 Apigenin C15H10O5 [M − H]− 269.0455 269.0455 −0.11 117.0347, 149.0242, 151.0035 Flavonoids
92 6.92 Kaempferol C15H10O6 [M − H]− 285.0405 285.0404 −0.32 165.991,270.0542 Flavonoids
93 7.09 Alpinetin C16H14O4 [M + H]+ 271.0965 271.0958 −2.47 131.0496, 135.0028, 167.0337 Flavonoids
94 7.28 2-Methoxycinnamaldehyde C10H10O2 [M + H]+ 163.0754 163.0750 −2.27 117.0701, 133.0284, 135.0441, 145.0647 Phenylpropanoids
95 7.62 Dodecanedioic acid C12H22O4 [M − H]− 229.1445 229.1444 −0.61 116.9286, 167.1441, 211.1337 Fatty acid and derivatives
96 8.07 3-Hydroxycapric acid C10H20O3 [M − H]− 187.1340 187.1340 0.42 59.014, 61.9882, 125.0972 Carboxylic acid and derivatives
97 8.23 p-Hydroxyphenethyl trans-ferulate C18H18O5 [M + H]+ 315.1227 315.1220 −2.33 131.0488, 196.0363, 211.0597 Phenylpropanoids
98 8.65 4′,5-Dihydroxyflavone C15H10O4 [M − H]− 253.0506 253.0506 −0.31 116.9284, 209.1545 Flavonoids
99 8.65 Dihydroartemisinin C15H24O5 [M − H2O − H]− 265.1445 265.1442 −1.16 61.9885,129.9759,221.1548 Terpenes
100 8.93 Acacetin C16H12O5 [M − H]− 283.0612 283.0611 −0.42 268.0372 Flavonoids
101 9.08 Desmethoxyyangonin C14H12O3 [M + H]+ 229.0859 229.0855 −1.86 131.0491, 141.0697 Phenylpropanoids
102 9.11 L-Borneol C10H18O [M + NH4]+ 172.1696 172.1692 −2.59 126.0914, 130.0498, 130.9665, 145.0984, 148.9768, 149.9401, 171.1491 Terpenes
103 9.46 15-Hydroxydehydroabietic acid C20H28O3 [M + H − H2O]+ 299.2005 299.1999 −2.01 145.101, 280.2628 Terpenes
104 9.85 Cardamonin C16H14O4 [M − H]− 269.0819 269.0819 −0.23 89.0244, 165.0193, 226.0626, 254.0583 Flavonoids
105 9.89 12,13-Dihydroxyoctadec-9-enoate C18H34O4 [M − H]− 313.2384 313.2384 −0.20 99.082, 129.0923, 183.1388, 201.1134, 295.2281 Fatty acid and derivatives
106 9.89 3-Hydroxydodecanoic acid C12H24O3 [M − H]− 215.1653 215.1652 −0.28 59.0139 Carboxylic acid and derivatives
107 10.30 Hexadecanedioic acid C16H30O4 [M − H]− 285.2071 285.2070 −0.55 223.2065, 267.1961 Fatty acid and derivatives
108 10.41 Pelargonic acid C9H18O2 2[M − H]− 315.2541 315.2538 −0.85 297.2423, 313.2381 Fatty acid and derivatives
109 10.43 Pinostrobin C16H14O4 [M + H]+ 271.0965 271.0958 −2.47 131.0492, 167.0338 Flavonoids
110 10.46 Pellitorine C14H25NO [M + NH4]+ 241.2275 241.2268 −2.81 88.0762, 200.2006 Alkaloids
111 10.96 Ricinoleic acid C18H34O3 [M + H − H2O]+ 281.2475 281.2468 −2.50 175.1479, 179.1429, 179.1785, 189.1632, 193.1585, 207.1742, 221.2258, 245.2260, 263.2365 Fatty acid and derivatives
112 11.04 Lupenone C30H48O [M + H − H2O]+ 407.3672 407.3663 −2.12 109.1015, 121.1015, 123.1170, 135.1169, 147.1167, 149.1317, 189.1642, 203.1792, 335.1665 Terpenes
113 11.04 Octadecanedioic acid C18H34O4 [M − H]− 313.2384 313.2383 −0.38 251.2377, 295.2271, 312.1719 Fatty acid and derivatives
114 11.19 2-Hydroxytetradecanoic acid C14H28O3 [M − H]− 243.1966 243.1964 −0.66 197.1912 Fatty acid and derivatives
115 12.74 Oleamide C18H35NO 2[M + H]+ 563.5510 563.5502 −1.51 69.0707, 83.0862, 97.1017, 247.242, 265.2523, 282.2792 Fatty acid and derivatives
Open in a new tab

3.1.1. Identification of Flavonoids

Thirty seven flavonoids were identified in the FLCWK capsule, including kaempferol 3-sophoroside-7-glucosid (33), catechin (37), afzelechi (41), epicatechin (43), manghaslin (44), butin-7-O-β-D-glucopyranoside (50), mauritianin (51), quercetin 3-O-neohesperidoside (54), myricetin 3-O-rutinoside (55), rutin (56), and so on. For example, compound 56 had the precursor ion [M + H]+ at m/z 611.1596, indicating the formula of C27H30O16. Its MS/MS spectrum shows the ions resulting from the loss of rhamnose at m/z 465.1023 [M + H-C6H10O4]+ and of glucose-rhamnose at m/z 303.0495 [M + H-C6H10O4-C6H10O5]+ (Figure 2(a)). By comparison with reference compounds, compound 56 was assigned as rutin.

Figure 2.

Figure 2

Open in a new tab

MS/MS spectra and proposed cleavage pathways of rutin (Compound 56) (a), asperulosidic acid (Compound 31) (b), 6-methylnicotinamide (Compound 19) (c), roseoside (Compound 42) (d), desmethoxyyangonin (Compound 101) (e), and protocatechuic acid (Compound 25) (f).

3.1.2. Identification of Terpenes

Fifteen terpenes were identified in the FLCWK capsule, including geniposidic acid (26), mussaenosidic acid (27), 8-epi-loganic acid-6′-O-beta-D-glucoside (28), asperulosidic acid (31), and so on. Compound 31 showed the deprotonated molecule [M − H]− at m/z 431.1192, indicating the formula of C18H24O12. The fragment ion m/z 269.0666 ([M − H-C6H10O5]−) was formed by [M-H]− removing a molecule of glucose. The fragment ion m/z 165.0563 ([M − H-C6H10O5-H2O-CO2-C2H2O]−) was generated by eliminating H2O, CO2, and C2H2O from m/z 269.0666 in succession (Figure 2(b)). The proposed fragmentation pathway of compound 31 was in accordance with the reference compound of asperulosidic acid.

3.1.3. Identification of Alkaloids

Five alkaloids were identified in the FLCWK capsule, including stachydrine (12), 6-methylnicotinamide (19), 4,12-dimethyl-14,19-dioxa-17-azaheptacyclo[10.7.2.22, 5.02, 7.08, 18.08, 21.013, 17]tricosane-4,20-diol (57), songoramine (74), and pellitorine (110). Compound 19 showed the precursor ion [M + H]+ at m/z 137.0708, indicating the formula of C7H8N2O. The fragment ion m/z 94.0655 ([M + H-CONH]+) was generated by eliminating CONH from the precursor ion (Figure 2(c)). By comparison with the fragmentation pathway of the reference compound, compound 19 was assigned as 6-methylnicotinamide.

3.1.4. Identification of Glycosides

Nine glycosides were identified in the FLCWK capsule, including 4-O-beta-glucopyranosyl-cis-coumaric acid (29), trans-ferulic acid-4-beta-glucoside (34), syringin (36), 2-[4,5-dihydroxy-2-(hydroxymethyl)-6-[(5-methyl-2-propan-2-yl-2H-furan-5-yl)oxy]oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol (39), roseoside (42), and so on. Compound 42 showed the precursor ion [M + FA-H]− at m/z 431.1922 and [M − H]− at m/z 385.1848, indicating the molecular formula of C19H30O8. The fragment ion m/z 223.1345 ([M − H-C6H10O5]−) was formed by [M-H]− removing a molecule of glucose (Figure 2(d)). By comparison with the fragmentation pathway of the reference compound, compound 42 was assigned as roseoside.

3.1.5. Identification of Phenylpropanoids

Seven phenylpropanoids were identified in the FLCWK capsule, including oxyresveratrol 2-O-beta-D-glucopyranoside (40), lyoniresinol 9′-O-glucoside (53), L-3-phenyllactic acid (71), syringaresinol (88), 2-methoxycinnamaldehyde (94), p-hydroxyphenethyl trans-ferulate (97), and desmethoxyyangonin (101). Compound 101 showed the precursor ion [M + H]+ at m/z 229.0855, indicating the molecular formula of C14H12O3. As shown in Figure 2(e), the fragment ion m/z 141.0697 ([M + H-CH3O-C2HO2]+) was generated by eliminating CH3O and C2HO2 from the precursor ion. The fragment ion m/z 131.0491 ([M + H-C5H6O2]+) was formed by [M + H]+ removing C5H6O2 (Figure 2(e)). By comparison with the fragmentation pathway of the reference compound, compound 101 was assigned as desmethoxyyangonin.

3.1.6. Identification of Phenols

Four phenols were identified in the FLCWK capsule, including gallic acid (23), protocatechuic acid (25), isovanillic acid (46), and ellagic acid (67). Compound 25 showed a deprotonated molecule [M − H]− peak at m/z 153.0195, indicating the molecular formula of C7H6O4. The deprotonated molecule lost a CO2 moiety to form a fragment ion [M − H-CO2]− at m/z 109.0296. Then, it was dehydrated to form the [M − H-CO2-H2O]− fragment ion of m/z 91.0301. The fragmentation pathways of compound 25 are shown in Figure 2(f). Compound 25 was identified as protocatechuic acid by comparing its MS/MS fragmentation pattern and retention time of the reference standard.

3.2. Network Pharmacology

3.2.1. Screening of Active Ingredients and Potential Targets of the FLCWK Capsule

The active constituents of the FLCWK capsule, characterized by favorable drug-like and pharmacokinetic properties, were identified utilizing the SwissADME platform. Compounds that did not fully meet the predefined criteria were also included if their pharmacological activity was substantiated by literature evidence. After removing compounds without targets and duplicate potential targets, 46 active ingredients were successfully screened, and their 551 potential targets were predicted by the SwissTargetPrediction platform. More than half of these ingredients are flavonoids, which aligns with the previous literature indicating that the active ingredients of FLCWK contain no less than 12% flavonoids by weight [22]. Experimental pharmacological studies have confirmed that these flavonoids, such as apigenin, quercetin, and quercitrin, exhibit anticolitis effects by suppressing inflammatory mediators [23]. Morin has been shown to alleviate DSS-induced ulcerative colitis in mice through the inhibition of inflammation and modulation of intestinal microbiota [24]. Alpinetin is associated with a dose-dependent reduction in intestinal inflammation and oxidative stress, and it also regulates the expression of tight junctions between cells in ulcerative colitis mice [25]. In addition to flavonoids, the remaining active ingredients include terpenes, phenylpropanoids, phenols, and alkaloids. These compounds are also crucial components of FLCWK, and experimental pharmacological studies have demonstrated that they exhibit a wide range of anti-inflammatory biological activities [26–28]. The information on the active ingredients of the FLCWK capsule is shown in Table 2.

Table 2.

The information on the active ingredients of the FLCWK capsule.

No. Compound GI absorption Lipinski Ghose Veber Egan Muegge Number of yes
FL1 6-Methylnicotinamide High Yes No Yes Yes No 3
FL2 Gallic acid High Yes No Yes Yes No 3
FL3 Protocatechuic acid High Yes No Yes Yes No 3
FL4 Geniposidic acid Low Yes No No No No 1
FL5 3,17-dihydroxy-4,4,8,10,14-pentamethyl-2,3,5,6,7,9-hexahydro-1H-cyclopenta[a]phenanthrene-15,16-dione High Yes Yes Yes Yes Yes 5
FL6 Kaempferol 3-sophoroside-7-glucoside Low No No No No No 0
FL7 Roseoside Low Yes No Yes No Yes 3
FL8 Manghaslin Low No No No No No 0
FL9 Isovanillic acid High Yes Yes Yes Yes No 4
FL10 Neridienone A High Yes Yes Yes Yes Yes 5
FL11 Rutin Low No No No No No 0
FL12 Quercetin-3-O-glucuronide Low No No No No No 0
FL13 Hyperoside Low No No No No No 0
FL14 Myricitrin Low No Yes No No No 1
FL15 (16-Hydroxy-5,5,9-trimethyl-14-methylidene-15-oxo-2-tetracyclo[11.2.1.01,10.04,9]hexadecanyl) acetate High Yes Yes Yes Yes Yes 5
FL16 Ellagic acid High Yes Yes No No Yes 3
FL17 Kaempferol-3-O-rutinoside Low No No No No No 0
FL18 Kaempferol 3-O-robinobioside Low No No No No No 0
FL19 L-3-Phenyllactic acid High Yes Yes Yes Yes No 4
FL20 Songoramine High Yes Yes Yes Yes Yes 5
FL21 Quercitrin Low No Yes No No No 1
FL22 Vitexin Low Yes Yes No No No 2
FL23 Sophoricoside Low Yes Yes No No No 2
FL24 2-(4-methoxyphenyl)-7-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one High Yes Yes Yes No Yes 4
FL25 Okanin High Yes Yes Yes Yes Yes 5
FL26 Steppogenin High Yes Yes Yes Yes Yes 5
FL27 Luteolin High Yes Yes Yes Yes Yes 5
FL28 (5S,10S,13R,14R,15S,17R)-15-hydroxy-17-[(Z,2R)-7-hydroxy-6-methylhept-5-en-2-yl]-4,4,10,13,14-pentamethyl-1,2,5,6,12,15,16,17-octahydrocyclopenta[a]phenanthren-3-one High Yes No Yes No No 2
FL29 Quercetin High Yes Yes Yes Yes Yes 5
FL30 Morin High Yes Yes Yes Yes Yes 5
FL31 Syringaresinol High Yes Yes Yes Yes Yes 5
FL32 3-O-Methylquercetin High Yes Yes Yes Yes Yes 5
FL33 Naringenin chalcone High Yes Yes Yes Yes Yes 5
FL34 Apigenin High Yes Yes Yes Yes Yes 5
FL35 Kaempferol High Yes Yes Yes Yes Yes 5
FL36 Alpinetin High Yes Yes Yes Yes Yes 5
FL37 2-Methoxycinnamaldehyde High Yes Yes Yes Yes No 4
FL38 p-Hydroxyphenethyl trans-ferulate High Yes Yes Yes Yes Yes 5
FL39 4′,5-Dihydroxyflavone High Yes Yes Yes Yes Yes 5
FL40 Dihydroartemisinin High Yes Yes Yes Yes Yes 5
FL41 Acacetin High Yes Yes Yes Yes Yes 5
FL42 L-Borneol High Yes No Yes Yes No 3
FL43 15-Hydroxydehydroabietic acid High Yes Yes Yes Yes Yes 5
FL44 Cardamonin High Yes Yes Yes Yes Yes 5
FL45 Pinostrobin High Yes Yes Yes Yes Yes 5
FL46 Pellitorine High Yes Yes Yes Yes Yes 5
Open in a new tab

3.2.2. Colitis Target Prediction and Intersection With Targets of the FLCWK Capsule

1135 and 6086 colitis-related targets were collected from the DisGeNET and GeneCards databases, respectively. After combining the results and removing duplicates, 6381 target genes were obtained. 352 potential targets for FLCWK against colitis were obtained after intersecting the targets of the FLCWK active ingredients and the colitis-related targets by the Venny software (Figure 3).

Figure 3.

Figure 3

Open in a new tab

Venn diagram of targets of the FLCWK capsule and colitis.

3.2.3. PPI Network Construction

Three hundred and fifty-two potential targets for FLCWK against colitis were imported into the STRING database to get PPI information. After removing disconnected targets, a PPI network with 259 nodes and 1066 edges was constructed by the Cytoscape software (Figure 4). In the PPI network, each target was represented by a node, and the interactions between the targets were represented by the edges linking the nodes. A node degree value indicates the number of connections of each node, and the larger a node's degree is, the more it interacts with others. The top 10 targets according to their degree value were TP53 (degree = 49), SRC (degree = 44), PIK3R1 (degree = 41), PIK3CA (degree = 40), HSP90AA1 (degree = 39), STAT3 (degree = 38), PIK3CB (degree = 38), PIK3CD (degree = 37), AKT1 (degree = 36), and EGFR (degree = 32). These may be the key targets when the FLCWK capsule treats colitis.

Figure 4.

Figure 4

Open in a new tab

PPI network of FLCWK against colitis. The node became larger, and its color changed from yellow to red with the increased degree of the targets. Network nodes represent proteins. Edges represent protein-protein associations.

3.2.4. GO and KEGG Enrichment Analysis

GO and KEGG pathways (p < 0.05) were considered significantly enriched. A total of 1042 significantly enriched GO entries were obtained from the DAVID database, including 763 BP, 86 CC, and 193 MF. For visual analysis, the results of GO and KEGG were drawn into bar and bubble charts using a bioinformatics analysis platform (https://www.bioinformatics.com.cn/). As shown in Figure 5(a), 5(b), 5(c), 5(d) and Tables S2-S3, the top 20 GO entries and KEGG pathways were chosen according to the p value and counts of hit genes. BP mainly involves protein phosphorylation, response to xenobiotic stimulus, regulation of the apoptotic process, inflammatory response, and so on. CC mainly involves the plasma membrane, receptor complex, cytosol, membrane raft, cytoplasm, and so on. MF mainly involves ATP binding, protein kinase activity, RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding, and so on. Pathogenesis of colitis is always regulated through protein phosphorylation [29]. Furthermore, when the intestinal mucosa is exposed to xenobiotic stimuli, it may produce aberrant responses, resulting in significant inflammation and intestinal damage, such as colitis [30]. Excessive apoptosis of intestinal epithelial cells can lead to epithelial dysfunction and gut microbiology imbalance, which also play an important role in the pathogenesis and progression of colitis [31]. The results of GO suggested that FLCWK may play a role in anticolitis treatment by regulating the abovementioned biological processes via effecting ATP binding, protein kinase, RNA polymerase, and DNA binding. In the KEGG analysis, a total of 164 pathways were enriched, including pathways in cancer, EGFR tyrosine kinase inhibitor resistance, AGE-RAGE, PI3K-Akt, MAPK signaling pathway, and so on. The top 20 pathways were taken to construct the compound-target-pathway-disease network for further analysis of the anticolitis mechanism of the FLCWK capsule.

Figure 5.

Figure 5

Open in a new tab

GO and KEGG pathway enrichment analyses of the potential targets for FLCWK against colitis. The bar chart of top 20 BP (a), CC (b), and MF (c) and the bubble chart of top 20 KEGG signaling pathways (d). The redder the color, the more significant the value.

3.2.5. Network Construction

The compound-target-pathway-disease network was constructed and analyzed by Cytoscape. As shown in Figure 6, active ingredients and their corresponding targets were represented by nodes, and each ingredient was linked to its target genes with edges. Using the Network Analyzer in the Cytoscape software, the topological parameters of the network were calculated. Among the topological parameters, the degree, which refers to the number of edges associated with a node, was selected as a measure of node importance. According to the results of the topological analysis, 4′,5-dihydroxyflavone, pinostrobin, naringenin chalcone, apigenin, morin, and alpinetin were among the top 10 important compounds, suggesting significant anticolitis effects (Table 3). The top 10 core protein targets (Table 4), including EGFR, AKT1, PIK3R1, PIK3CB, MAPK1, IGF1R, and MET, were partially consistent with the results of PPI analysis. Pathways in cancer, MAPK, and PI3K-Akt signaling pathways were among the 10 important pathways in this network (Table 5). These compounds may primarily bind to these core targets to regulate relevant pathways, thereby inhibiting the development of colitis.

Figure 6.

Figure 6

Open in a new tab

Compound-target-pathway-disease network. Diamonds, ellipses, and rectangles represent active ingredients in the FLCWK capsule, pathways, and target genes, respectively. Each ingredient was linked with its potential pathways and targets. The node became larger with the increased degree of the ingredients, pathways, and target genes.

Table 3.

Top 10 important compounds of the compound-target-pathway-disease network.

No. Compound Degree
FL39 4′,5-Dihydroxyflavone 79
FL45 Pinostrobin 76
FL33 Naringenin chalcone 75
FL34 Apigenin 75
FL30 Morin 74
FL36 Alpinetin 74
FL28 (5S,10S,13R,14R,15S,17R)-15-hydroxy-17-[(Z,2R)-7-hydroxy-6-methylhept-5-en-2-yl]-4,4,10,13,14-pentamethyl-1,2,5,6,12,15,16,17-octahydrocyclopenta[a]phenanthren-3-one 72
FL29 Quercetin 72
FL32 3-O-Methylquercetin 72
FL35 Kaempferol 72
Open in a new tab
Table 4.

Top 10 targets of the compound-target-pathway-disease network.

No. Target Degree
1 EGFR 30
2 CA2 29
3 CA4 29
4 AKT1 28
5 AKR1B1 27
6 PIK3R1 26
7 MAPK1 23
8 PIK3CB 22
9 IGF1R 22
10 MET 22
Open in a new tab
Table 5.

Top 10 pathways of the compound-target-pathway-disease network.

No. Pathway Degree
hsa05200 Pathways in cancer 91
hsa04151 PI3K-Akt signaling pathway 58
hsa04010 MAPK signaling pathway 52
hsa05417 Lipid and atherosclerosis 49
hsa05205 Proteoglycans in cancer 43
hsa05208 Chemical carcinogenesis—reactive oxygen species 43
hsa04014 Ras signaling pathway 43
hsa05167 Kaposi sarcoma-associated herpesvirus infection 41
hsa05207 Chemical carcinogenesis—receptor activation 41
hsa05161 Hepatitis B 38
Open in a new tab

Colitis is characterized by chronic relapsing inflammation with intestinal epithelial injury and immune homeostasis disruption [32]. The MAPK signaling pathway is a classical inflammatory signaling pathway, while the PI3K-Akt signaling pathway can activate NF-κB and increase proinflammatory cytokine production (e.g., IL-6, IL-1β, and TNF-α), playing a critical role in colitis pathogenesis [33–35]. Network pharmacology results showed that the compounds of the FLCWK capsule interact with key targets in the MAPK and PI3K-Akt signaling pathways. For example, pinostrobin binds to critical MAPK pathway genes (SRC, FGFR1, and MAPKAPK2) and interacts with key targets in the PI3K-AKT signaling cascade (PIK3CA, PIK3CB, PIK3CD, PIK3CG, MTOR, and AKT1). The previous literature reported that pinostrobin attenuates azoxymethane-induced bowel inflammation in rats [36]. Thus, pinostrobin may alleviate bowel inflammation by inhibiting the MAPK and PI3K-AKT signaling pathways. Apigenin interacts with key MAPK signaling cascade targets (EGFR, SRC, and PIK3R1) and binds to critical PI3K-AKT pathway genes (PIK3R1, AKT1, and GSK3B). These findings are consistent with the previous literature demonstrating that apigenin downregulates inflammatory cytokine expression by modulating the MAPK pathway and inhibits the PI3K-AKT pathway, supporting its anti-inflammatory potential [37]. Morin interacts with crucial MAPK pathway genes (SRC and MAPK) and pivotal PI3K-AKT signaling cascade targets (PIK3R1, PIK3CG, and GSK3B). This is consistent with previous research showing that morin intervention mitigates ulcerative colitis severity in mice by suppressing the MAPK pathways [24]. Alpinetin interacts with a crucial MAPK pathway gene (MAPKAPK2) and key PI3K-AKT pathway targets (PIK3CD, PIK3CB, PIK3CG, PIK3CA, and MTOR). Previous studies indicated that alpinetin improves the disease activity index, colonic shortening, histological scores, and myeloperoxidase activity in mice with ulcerative colitis [25]. Therefore, alpinetin may inhibit colitis via the PI3K-AKT and MAPK signaling pathways. In conclusion, interaction with key targets in the MAPK and PI3K-Akt signaling pathways may be one of the mechanisms by which the FLCWK capsule attenuates the inflammatory response in colitis.

4. Conclusions

In this study, an integrated approach combining UHPLC-Q-Exactive Orbitrap MS and network pharmacology analysis was adopted to explore the potential active ingredients and anticolitis mechanisms of the FLCWK capsule. 115 compounds in the FLCWK capsule were identified. According to the results of the compound-target-pathway-disease network, the anticolitis effect of the FLCWK capsule is mainly attributed to 46 active ingredients such as 4′,5-dihydroxyflavone, pinostrobin, naringenin chalcone, apigenin, and morin, which act on 352 core protein targets, such as EGFR, AKT1, PIK3R1, PIK3CB, and MAPK1, thereby modulating relevant pathways, such as MAPK and PI3K-Akt signaling pathways. In conclusion, the integrated approach provided valuable insights into the potential active ingredients and anticolitis mechanisms of the FLCWK capsule. Based on the current findings, further confirmation through in vitro and in vivo experiments in subsequent studies is required to establish a reliable foundation for its clinical application.

Acknowledgments

This work was supported by the Hainan Provincial Natural Science Foundation of China (grant number 825QN314, 2025 and grant number 822MS072, 2022), the National Natural Science Foundation of China (grant number 82460823, 2025), Undergraduate Training Programs for Innovation and Entrepreneurship of the Hainan Medical University (grant number S202411810051), and the Education Department of Hainan Province (grant number Hnky2023-33).

Contributor Information

Xizhe Sun, Email: hy0308028@hainmc.edu.cn.

Yanfei Chen, Email: hy0308024@hainmc.edu.cn.

Data Availability Statement

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Tingting Liu, Zhijiang He, and Witiao Lv contributed equally to this work and should be considered cofirst authors.

Funding

This work was supported by the Hainan Provincial Natural Science Foundation of China (grant number 825QN314, 2025 and grant number 822MS072, 2022), the National Natural Science Foundation of China (grant number 82460823, 2025), Undergraduate Training Programs for Innovation and Entrepreneurship of the Hainan Medical University (grant number S202411810051), and the Education Department of Hainan Province (grant number Hnky2023-33).

Supporting Information

Supporting Information

Additional supporting information can be found online in the Supporting Information section.

2948965.f1.docx (42.1KB, docx)

The name, molecular formula, batch number, and company of reference compounds are shown in Table S1. The top 20 GO entries and KEGG pathways obtained from GO and KEGG enrichment analysis are shown in Tables S2-S3.

References

  • 1.Wei Y., Fan Y., Ga Y., Zhang Y., Han J., Hao Z. Shaoyao Decoction Attenuates DSS-Induced Ulcerative Colitis, Macrophage and NLRP3 Inflammasome Activation Through the MKP1/NF-Κb Pathway. Phytomedicine . 2021;92:p. 153743. doi: 10.1016/j.phymed.2021.153743. [DOI] [PubMed] [Google Scholar]
  • 2.Yang Y., Zhou X., Jia G., et al. Network Pharmacology Based Research Into the Effect and Potential Mechanism of Portulaca oleracea L. Polysaccharide Against Ulcerative Colitis. Computers in Biology and Medicine . 2023;161:p. 106999. doi: 10.1016/j.compbiomed.2023.106999. [DOI] [PubMed] [Google Scholar]
  • 3.Yang Y., Hua Y., Chen W., et al. Therapeutic Targets and Pharmacological Mechanisms of Coptidis Rhizoma Against Ulcerative Colitis: Findings of System Pharmacology and Bioinformatics Analysis. Frontiers in Pharmacology . 2022;13:p. 1037856. doi: 10.3389/fphar.2022.1037856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Zhai L., Yang W., Li D., Zhou W., Cui M., Yao P. Network Pharmacology and Molecular Docking Reveal the Immunomodulatory Mechanism of Rhubarb Peony Decoction for the Treatment of Ulcerative Colitis and Irritable Bowel Syndrome. Journal of Pharmacy & Pharmaceutical Sciences . 2023;26:p. 11225. doi: 10.3389/jpps.2023.11225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Wei M., Li H., Li Q., et al. Based on Network Pharmacology to Explore the Molecular Targets and Mechanisms of Gegen Qinlian Decoction for the Treatment of Ulcerative Colitis. BioMed Research International . 2020;2020(1):p. 5217405. doi: 10.1155/2020/5217405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Niu K., Lin D., Wang X., Huang L., Yang F. Clinical Efficacy on the Treatment of Chronic Colitis With Feng-Liao-Chang-Wei-Kang Granules Combined With Mesalazine. Medicinal Plants . 2022;13(03):53–55. [Google Scholar]
  • 7.Huang L., Ni P., Tian S., et al. Feng-Liao-Chang-Wei-Kang Is Synergistic With 5-Fluorouracil in Inhibiting Proliferation of Colorectal Cancer. Asian Pacific Journal of Tropical Medicine . 2019;12(14):p. 41. doi: 10.4103/1995-7645.271979. [DOI] [Google Scholar]
  • 8.Zhong L., Yang F., Gan L., et al. Feng-Liao-Chang-Wei-Kang Combined With 5-Fluorouracil Synergistically Suppresses Colitis-Associated Colorectal Cancer via the IL-6/STAT3 Signalling Pathway. Evidence-based Complementary and Alternative Medicine: eCAM . 2020;2020(1):p. 1395954. doi: 10.1155/2020/1395954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Hou Y., Gan A. Efficacy and Safety of Fengliao Stomach Granules Combined with Mesalazine in the Treatment of Recurrent Ulcerative Colitis. Practical Pharmacy and Clinical Remedies . 2014;17(2):225–227. [Google Scholar]
  • 10.Fu S., Cheng R., Deng Z., Liu T. Qualitative Analysis of Chemical Components in Lianhua Qingwen Capsule by HPLC-Q Exactive-Orbitrap-MS Coupled With GC-MS. Journal of Pharmaceutical Analysis . 2021;11(6):709–716. doi: 10.1016/j.jpha.2021.01.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Zhang H., Aisa H. A., Liu Y., Tohtahon Z., Xin X., Abdulla R. Characterisation and Identification of Chemical Constituents in Aqueous Extract of Fomes officinalis Ames Based on Ultrahigh‐Performance Liquid Chromatography Tandem quadrupole‐Orbitrap High‐Resolution Mass Spectrometry. Phytochemical Analysis . 2023;35(2):369–379. doi: 10.1002/pca.3295. [DOI] [PubMed] [Google Scholar]
  • 12.Qin S., Liu M., Tang S., et al. Rapid Characterization and Action Mechanism of the Antidiabetic Effect of Diospyros lotus L Using UHPLC-Q-Exactive Orbitrap MS and Network Pharmacology. Journal of Analytical Methods in Chemistry . 2022;2022:1–19. doi: 10.1155/2022/8000126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhang X., Chu Y., Wang M., et al. Rapid and Comprehensive Identification of Chemical Constituents in Mai-Luo-Shu-Tong Pill by UHPLC-Q-Orbitrap HRMS Combined With a Data Mining Strategy. Analytical Methods . 2022;14(47):4990–5000. doi: 10.1039/d2ay01453j. [DOI] [PubMed] [Google Scholar]
  • 14.Li Z., Zuo L., Shi Y., et al. Combination of High-Resolution Accurate Mass Spectrometry and Network Pharmacology Provides a New Method for the Chemical Constituents’ Study and Target Prediction In Vivo of Garcinia Multiflora. Journal of Separation Science . 2020;43(5):978–986. doi: 10.1002/jssc.201900755. [DOI] [PubMed] [Google Scholar]
  • 15.Zhang X., Zhang J., Zhou Z., et al. Integrated Network Pharmacology, Metabolomics, and Transcriptomics of Huanglian-Hongqu Herb Pair in Non-Alcoholic Fatty Liver Disease. Journal of Ethnopharmacology . 2024;325:p. 117828. doi: 10.1016/j.jep.2024.117828. [DOI] [PubMed] [Google Scholar]
  • 16.Jiang H., Yang L., Sun Q., et al. Glyoxalase I Inhibitors Targeting Breast Cancer-Associated Endothelial Cells: An Integrated Network Pharmacology and Experimental Investigation. Journal of Molecular Structure . 2025;1334:p. 141868. doi: 10.1016/j.molstruc.2025.141868. [DOI] [Google Scholar]
  • 17.Duan Z. L., Wang Y. J., Lu Z. H., et al. Wumei Wan Attenuates Angiogenesis and Inflammation by Modulating RAGE Signaling Pathway in IBD: Network Pharmacology Analysis and Experimental Evidence. Phytomedicine . 2023;111:p. 154658. doi: 10.1016/j.phymed.2023.154658. [DOI] [PubMed] [Google Scholar]
  • 18.Liu J., Liu J., Tong X., et al. Network Pharmacology Prediction and Molecular Docking-Based Strategy to Discover the Potential Pharmacological Mechanism of Huai Hua San Against Ulcerative Colitis. Drug Design, Development and Therapy . 2021;15:3255–3276. doi: 10.2147/dddt.s319786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Wu Y., Liu X., Li G. Integrated Bioinformatics and Network Pharmacology to Identify the Therapeutic Target and Molecular Mechanisms of Huangqin Decoction on Ulcerative Colitis. Scientific Reports . 2022;12(1):p. 159. doi: 10.1038/s41598-021-03980-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Daina A., Michielin O., Zoete V. SwissADME: a Free Web Tool to Evaluate Pharmacokinetics, Drug-Likeness and Medicinal Chemistry Friendliness of Small Molecules. Scientific Reports . 2017;7(1):p. 42717. doi: 10.1038/srep42717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Wang W., Zhang Y., Yang Y., Gu L., Singla R. K. Network Pharmacology and Molecular Docking to Explore the Mechanism of Kangxian Decoction for Epilepsy. Evid-Based Compl Alt . 2022;1-12 doi: 10.1155/2022/3333878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Zhang J., Liu X., Fu N., Liu M., Tan Y. Systemic Exposure of Quercetin After Administration of Feng-Liao-Chang-Wei-Kang Granules to Rats. Journal of Ethnopharmacology . 2011;133(2):911–913. doi: 10.1016/j.jep.2010.10.014. [DOI] [PubMed] [Google Scholar]
  • 23.Wang J., Dempsey E., Corr S. C., Kukula-Koch W., Sasse A., Sheridan H. The Traditional Chinese Medicine Houttuynia Cordata Thunb Decoction Alters Intestinal Barrier Function via an EGFR Dependent MAPK (ERK1/2) Signalling Pathway. Phytomedicine . 2022;105:p. 154353. doi: 10.1016/j.phymed.2022.154353. [DOI] [PubMed] [Google Scholar]
  • 24.Qiu L., Yan C., Yang Y., et al. Morin Alleviates DSS-Induced Ulcerative Colitis in Mice via Inhibition of Inflammation and Modulation of Intestinal Microbiota. International Immunopharmacology . 2024;140:p. 112846. doi: 10.1016/j.intimp.2024.112846. [DOI] [PubMed] [Google Scholar]
  • 25.Tan Y., Zheng C. Effects of Alpinetin on Intestinal Barrier Function, Inflammation and Oxidative Stress in Dextran Sulfate Sodium-Induced Ulcerative Colitis Mice. The American Journal of the Medical Sciences . 2018;355(4):377–386. doi: 10.1016/j.amjms.2018.01.002. [DOI] [PubMed] [Google Scholar]
  • 26.Bairagi J., Saikia P. J., Boro F., Hazarika A. A Review on the Ethnopharmacology, Phytochemistry and Pharmacology of Polygonum Hydropiper Linn. Journal of Pharmacy and Pharmacology . 2022;74(5):619–645. doi: 10.1093/jpp/rgab175. [DOI] [PubMed] [Google Scholar]
  • 27.Zhong J., Wang H., Zhang Q., Gao S. The Chemistry of Daphniphyllum Alkaloids. The Alkaloids-Chemistry and Biology . 2021;85:113–176. doi: 10.1016/bs.alkal.2021.01.001. [DOI] [PubMed] [Google Scholar]
  • 28.Liu M., Liu C., Zhang X., Sheng L., Zhang J., Kang S. Two New Drimane Sesquiterpenoids From Compound Changweikang and Their Inhibitory Activity Against Nitric Oxide Production. Chemical and Pharmaceutical Bulletin . 2010;58(9):1224–1226. doi: 10.1248/cpb.58.1224. [DOI] [PubMed] [Google Scholar]
  • 29.Tao S., Long X., Gong P., Yu X., Tian L. Phosphoproteomics Reveals Novel Insights Into the Pathogenesis and Identifies New Therapeutic Kinase Targets of Ulcerative Colitis. Inflammatory Bowel Diseases . 2023;30(8):1367–1378. doi: 10.1093/ibd/izad291. [DOI] [PubMed] [Google Scholar]
  • 30.Nieves K. M., Hirota S. A., Flannigan K. L. Xenobiotic Receptors and the Regulation of Intestinal Homeostasis: Harnessing the Chemical Output of the Intestinal Microbiota. American Journal of Physiology-Gastrointestinal and Liver Physiology . 2022;322(2):G268–G281. doi: 10.1152/ajpgi.00160.2021. [DOI] [PubMed] [Google Scholar]
  • 31.Wan Y., Yang L., Jiang S., Qian D., Duan J. Excessive Apoptosis in Ulcerative Colitis: Crosstalk Between Apoptosis, ROS, ER Stress, and Intestinal Homeostasis. Inflammatory Bowel Diseases . 2022;28(4):639–648. doi: 10.1093/ibd/izab277. [DOI] [PubMed] [Google Scholar]
  • 32.Yang S., Li F., Lu S., et al. Ginseng Root Extract Attenuates Inflammation by Inhibiting the MAPK/NF-κB Signaling Pathway and Activating Autophagy and P62-Nrf2-Keap1 Signaling In Vitro and In Vivo. Journal of Ethnopharmacology . 2022;283:p. 114739. doi: 10.1016/j.jep.2021.114739. [DOI] [PubMed] [Google Scholar]
  • 33.Wang X., Cheng K., Liu Z., et al. Bioactive Constituents of Mosla Chinensis-Cv. Jiangxiangru Ameliorate Inflammation through MAPK Signaling Pathways and Modify Intestinal Microbiota in DSS-Induced Colitis Mice. Phytomedicine . 2021;93:p. 153804. doi: 10.1016/j.phymed.2021.153804. [DOI] [PubMed] [Google Scholar]
  • 34.Ye Z., Li Y., She Y., et al. Renshen Baidu Powder Protects Ulcerative Colitis via Inhibiting the PI3K/Akt/NF-Κb Signaling Pathway. Frontiers in Pharmacology . 2022;13:p. 880589. doi: 10.3389/fphar.2022.880589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Dong L., Du H., Zhang M., et al. Anti‐Inflammatory Effect of Rhein on Ulcerative Colitis via Inhibiting PI3K/Akt/mTOR Signaling Pathway and Regulating Gut Microbiota. Phytotherapy Research . 2022;36(5):2081–2094. doi: 10.1002/ptr.7429. [DOI] [PubMed] [Google Scholar]
  • 36.Ali Abed Wahab B., Ain Salehen N., Abdulla M. A., et al. Pinostrobin Attenuates Azoxymethane-Induced Colorectal Cytotoxicity in Rats Through Augmentation of Apoptotic Bax/Bcl-2 Proteins and Antioxidants. SAGE Open Medicine . 2023;11:p. 20503121231216585. doi: 10.1177/20503121231216585. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 37.Abid R., Ghazanfar S., Farid A., et al. Pharmacological Properties of 4′, 5, 7-Trihydroxyflavone (Apigenin) and Its Impact on Cell Signaling Pathways. Molecules . 2022;27(13):p. 4304. doi: 10.3390/molecules27134304. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information

Additional supporting information can be found online in the Supporting Information section.

2948965.f1.docx (42.1KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Articles from Journal of Analytical Methods in Chemistry are provided here courtesy of Wiley

RESOURCES