Study on Antipyretic Properties of Phenolics in Lonicerae Japonicae Flos Based on Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry Combined with Network Pharmacology

Xiong, Lewen; Huang, Wenjing; Liu, Yan; Zhao, Hongwei; Wang, Yang; Jin, Ying; Zhang, Longfei; Zhang, Yongqing

doi:https://doi.org/10.1155/2023/8883860

Journal of Food Biochemistry

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Abstract Introduction Materials and Methods Results Discussion Conclusion Data Availability Additional Points Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 8883860 | https://doi.org/10.1155/2023/8883860

Study on Antipyretic Properties of Phenolics in Lonicerae Japonicae Flos Based on Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry Combined with Network Pharmacology

Lewen Xiong,¹Wenjing Huang,²Yan Liu,¹Hongwei Zhao,¹Yang Wang,¹Ying Jin,¹Longfei Zhang,¹and Yongqing Zhang¹

Academic Editor: Muhammad Babar Khawar

Received05 Jul 2023

Revised12 Oct 2023

Accepted24 Nov 2023

Published08 Dec 2023

Abstract

Objective. To identify and quantify the active phenolic components in Lonicerae japonicae flos (LJF) for fever treatment and their mechanism of action using network pharmacology and molecular docking. Methods. Based on qualitative analysis of LJF, 194 phenolics were obtained, including 81 phenolic acids and 113 flavonoids. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to identify potential targets for these components to interact with fever. Molecular docking with microsomal PGE2 synthase-1, EP1, EP2, EP3, and EP4 targets was used to determine antipyretic components. The antipyretic efficacy of the main components was verified by in vivo experiments. Finally, high-performance liquid chromatography-tandem mass spectrometry was used to quantify the main antipyretic components of LJF. Results. Phenolics in LJF may prevent and treat fever by participating in calcium signaling, regulating TRP channels, and cAMP signaling. Luteolin-7-O-glucoside, apigenin-7-O-glucoside, 3,5-O-dicaffeoylquinic acid, luteolin, and other components have a good docking effect with PGE2 synthase-1 and its four subtypes. 3,5-O-dicaffeoylquinic acid, luteolin-7-O-glucoside, and apigenin-7-O-glucoside have good antipyretic effects in a yeast-induced pyrexia model. The content of these antipyretic components varies with the developmental period of LJF. Phenolic acids are the main components that distinguish the different developmental periods of LJF. Conclusion. The potential antipyretic components and molecular mechanisms of phenolics provide a basis for the traditional medicinal effects and future development and utilization of LJF.

1. Introduction

Lonicerae japonicae flos (LJF), the flower buds of Lonicera japonica Thunb. (LJT), is a traditional Chinese medicine used for medicine and food, with potential antipyretic and anti-inflammatory activities [1]. It was used to treat various diseases, including exopathogenic wind-heat, epidemic febrile diseases, sores, carbuncles, and infectious diseases [2]. The LJF has a complex chemical component with phenolic acids, flavonoids, iridoids, volatile oils, and triterpenoid saponins [3]. Phenolic acids have antiviral [4, 5], antibacterial [6, 7], anti-inflammatory [8, 9], antitumor [10], and medicinal activities. These activities are related to the traditional efficacy of LJF, as shown by modern pharmacological studies. Phenolic acids are the primary medicinal substances of LJF. The 2000 edition of the “Chinese Pharmacopoeia” established the content of chlorogenic acid as the quality control index of LJT, with a minimum requirement of 1.5% [11]. The 2020 edition of the “Chinese Pharmacopoeia” increased the quality standards of LJT by increasing the content index of phenolic acid components, including chlorogenic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, which should not be less than 3.8% in total [1, 12]. Flavonoids in LJT have various antibacterial [13, 14], anti-inflammatory [15, 16], antioxidant [17], antitumor [18, 19], and neuroprotective [20, 21] effects. According to Chinese Pharmacopeia, luteolin-7-O-glucoside in flavonoids is used as an evaluation and control index for the quality of LJF [1]. Phenolic acids and flavonoids are both phenolics. Therefore, phenolics in LJF deserve extensive attention.

Fever is a common symptom of infectious and inflammatory disease. Prostaglandin E2 is the final mediator of fever. Inflammatory stimulation may induce fever by activating cyclooxygenase-2 and microsomal PGE synthase in brain endothelial cells [22–24] or perivascular cells [25]. Simultaneously, brain-bound cells release PGE2, which causes fever by affecting neurons expressing the EP receptor [26, 27]. Microsomal PGE2 synthase-1 (mPGES-1) is considered a key target for developing new anti-inflammatory drugs due to its association with various human diseases and pathological states, including pain, fever, and rheumatoid arthritis [28]. Hence, mPGES-1 presents a crucial target protein in the pursuit of antipyretic drugs. Additionally, the PGE receptors exhibit four distinct subtypes, namely, EP1, EP2, EP3, and EP4, each performing a unique function in the acute phase response of fever [29–32]. For instance, activation of EP4 receptors escalates cAMP levels, whereas activation of the EP1 receptor boosts intracellular Ca²⁺ levels, and activation of the EP3 receptor either diminishes or augments cAMP levels based on its isoform [33, 34]. Consequently, EP receptors are intimately associated with fever and could potentially offer an avenue to regulate it.

This study explored the antipyretic mechanism of phenolics in LJF using network pharmacology. Molecular docking was conducted between LJF components and fever-related targets, including mPGES-1, EP1, EP2, EP3, and EP4. In vivo experiments were used to verify the main antipyretic components and quantify those using ESI-QqQ-MS/MS. These findings provide a scientific explanation for LJF’s traditional heat-clearing and detoxifying effects and can be used as a reference for quality control. The research process is shown in Figure 1.

2. Materials and Methods

2.1. Network Pharmacology of Phenolic Components of LJF [35]

2.1.1. Analysis of the Phenolic Components of LJF

The phenolics of LJF were identified by preliminary experiments and used as the component database of LJF.

2.1.2. Potential Target Mining

Targets of active components were predicted by the SwissTargetPrediction database (https://www.swisstargetprediction.ch/). With “fever,” “febrile,” and “heat” as keywords, the DrugBank database (https://go.drugbank.com/), GeneCards database (https://www.genecards.org/), and TTD database (https://db.idrblab.net/ttd/) were used to retrieve disease targets.

The potential targets of LJF for the treatment of fever were obtained by repeatedly screening the disease targets and the predicted targets of potential active components. The potential targets and their related components imported Cytoscape 3.7.1 software and built a multivariate network.

2.1.3. Gene Ontology (GO) Analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Analysis

The Metascape website (https://metascape.org) [36] and the KEGG database (https://www.kegg.jp/) [37] were used for GO analysis and KEGG pathway analysis of potential targets.

2.1.4. Molecular Docking

Data for MPGES1 (PDB ID: 4AL0), EP1 (Uniprot ID: P34995), EP2 (PDB ID: 7CX2), EP3 (PDB ID: 6M9T), and EP4 (PDB ID: 5YWY) were downloaded from the RSCB database (https://www.rcsb.org/) and the UniProt database (https://www.uniprot.org/). PyMOL 1.7.2.1 software was used to extract the 3D structure of the target protein. The structure was optimized using AutoDock Tools 1.5.6 software and then stored in PDBQT format. The molecular docking grid was set as surrounding residues centered on the primary ligand of the target protein. AutoDock Vina 1.1.2 and Python scripts were used to conduct molecular docking and to record and sort the binding energies.

2.2. In Vivo Experiment of Antipyretic Effects

2.2.1. Chemicals and Materials

Yeast (Saccharomyces cerevisiae) was purchased from Angel Yeast Co., Ltd. (Hubei, China). Paracetamol injection was obtained from Chenxin Pharmaceutical Co., Ltd. (Jining, China). HH801B high-accuracy thermocouple thermometers were purchased from Omega Engineering (Shanghai, China). PGE2, IL-6, TNF-α, and IL-1β ELISA kits were provided by Shanghai Enzyme Linked Biotechnology Co., Ltd. (Shanghai, China).

2.2.2. Animals and Ethics Statement

C57BL/6N mice (male, 8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice were maintained in the SPF Laboratory Animal Center of the Shandong University of Traditional Chinese Medicine (SDUTCM). The animals were maintained at constant room temperature (25 ± 2°C) on a 12 h light/dark cycle with free access to food and water for 24 h or more before experiments. All experiments were performed in accordance with international guidelines and approved by the animal ethics committee of SDUTCM (No. YYLW2023000011).

2.2.3. Drugs Preparation

Low dose (10 mg/kg) and high dose (50 mg/kg) of 3,5-O-dicaffeoylquinic acid were prepared. Low dose (20 mg/kg) and high dose (80 mg/kg) of luteolin-7-O-glucoside and apigenin-7-O-glucoside were prepared, respectively. The positive drug (paracetamol injection) was 2 mL (0.25 g), and the dose was 100 mg/kg. Yeast powder was dissolved in normal saline to prepare 20% yeast suspension and incubated at 37°C for 30 min.

2.2.4. Antipyretic Activity Test

The base rectal temperature of each animal was measured by a thermometer in the rectum before the yeast injection. Mice with a base rectal temperature of 36.5–37.5°C were selected for antipyretic assays. 20% aqueous suspension of yeast was injected subcutaneously behind the neck of the animal (10 mL/kg) [38, 39]. The body temperature of the mice increased by >0.5°C at 5 h after injection, which was recognized as successfully prepared, and the temperature at this time was recorded as T0 (°C). Mice groups (9 groups of 5 mice each) were administered with their respective drugs. The intraperitoneal injection was performed in all groups. The temperatures (Ti, °C) were recorded after the administration of the drugs. After 8 h of mice body temperature determination, ΔT (Ti-T0, °C), mouse serum was collected.

2.3. Quantitative Study on Antipyretic Components Instruments

2.3.1. Materials and Reagents

Chlorogenic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, protocatechuic acid, chlorogenic acid methyl ester, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, caffeic acid, ferulic acid, quercetin-3-O-rutinoside, isoquercitrin, luteolin-7-O-glucoside, luteolin-7-O-neohesperidoside, kaempferol-3-O-rutinoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside, apigenin-7-O-glucoside, tricin-7-O-glucoside, luteolin, quercetin, diosmetin, apigenin, and tricin were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). 3,5-O-dicaffeoylquinic acid methyl ester and caffeic acid ethyl ester were purchased from Yunnan Xili Biotechnology Co., Ltd. (Yunnan, China). Quercetin-3-O-galactoside was purchased from ChemFaces Biochemical Co., Ltd. (Hubei, China). The purities of all reference standards is above 98%. Methanol, acetonitrile, and phosphoric acid were HPLC grade and obtained from Fisher Corporation (Waltham, MA, USA).

The LJF samples were collected from the medicinal botanical garden of Shandong University of Traditional Chinese Medicine (the germplasm is “Siji Hua”). The samples were divided into 6 periods (S1–S6) [40]. All dried material was ground to 65 mesh by liquid nitrogen.

2.3.2. Apparatus

The KQ-500DE digital ultrasonic cleaning instrument was purchased from Henan Brother Instruments Co., Ltd. (Zhengzhou, China). A ME204/02 Precision electronic balance was purchased from Mettler-Toledo Instruments Co., Ltd. (Shanghai, China).

Chromatographic analysis was performed on an Agilent 1290 series UPLC system (Agilent Technologies, Santa Clara, CA, USA). Mass spectrometry detection was performed using an Agilent 6460 series triple-quadrupole tandem mass spectrometer (QqQ-MS/MS) (Agilent Technologies, Santa Clara, CA, USA).

2.3.3. Quantitative Experimental Method

The preparation of samples, preparation of solutions, analysis methods, and method validation were referenced in previous studies [40, 41]. The information and HPLC-MS/MS parameters for MRM on phenolic acids and flavonoids are shown in Supplementary Table 1.

2.4. Statistical Analysis

All values were presented as mean ± S.D. Statistical analysis was performed using a one-way analysis of variance (ANOVA) model by SPSS 21.0. PCA and OPLS-DA were carried out by SIMCA software 14.1 version.

3. Results

3.1. The Phenolic Components of LJF

In total, 194 phenolics of LJF were identified, including 81 phenolic acids and 113 flavonoids. These active components are summarized in Supplementary Table 2. The MS chromatogram is shown in Supplementary Figure 1.

3.2. Target Mining

From the DrugBank database, GeneCards database, and TDD database, 11103 targets associated with fever were retrieved. 853 predicted targets for phenolic components of LJF were obtained. After the repeated screening, 586 potential targets were obtained. By taking the phenolic components directly related to potential targets as nodes, a multicomponent network was constructed (Figure 2). The figure contains 78 phenolic acids and 108 flavonoids, which are closely related to fever.

3.3. GO Analysis

586 potential targets were subjected to GO cluster analysis. It mainly included biological process (BP), molecular function (MF), and cell composition (CC). According to the value of GO enrichment analysis to filter results (Figure 3), BP is mainly involved in protein phosphorylation. CC mainly involves membrane rafts, and MF mainly involves protein serine/threonine/tyrosine kinase activity.

3.4. KEGG Pathway Analysis

Overall, 586 potential targets received 252 pathways. The top 15 pathways were selected for visual analysis using gene number, value, and gene ratio as study parameters (Figure 4).

Among them, lipid and atherosclerosis, chemical carcinogenesis-receptor activation, calcium signaling pathway, inflammatory mediator regulation of TRP channels, cAMP signaling pathway, and NF-kappa B signaling pathway were closely related to fever and inflammation.

3.5. Molecular Docking

Molecular docking was performed between the 194 potential antipyretic components of LJF and the target proteins. Molecular docking results showed that the binding energies of the 194 potential antipyretic components with five kinds of protein were all less than 0.

The docking binding energies of each component and the five proteins were summed and sorted (Figure 5). 3,5-O-dicaffeoylquinic acid had the best docking and binding energy with mPGES-1, EP1, and EP4, with the value of −7.0, −9.8, and −10.0, respectively. The binding energy of luteolin-7-O-glucoside and EP2 was the best, which was −10.3, whereas, for EP3 binding, apigenin-7-O-glucoside and EP3 presented the best binding energy, with a value of −10.8. The potential antipyretic component and the amino acid residues of the target protein in the binding site mainly undergo hydrophobic binding via pi-cation, pi-alkyl, pi-donor hydrogen bond, pi-sigma, pi-sulfur, pi-pi-T-shaped, van der Waals, carbon-hydrogen bond, and conventional hydrogen bond interactions. These interactions increased the binding between the target protein and the active component.

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The components with good binding energy and suitable for quantitative determination are summarized in Table 1. The results showed that luteolin-7-O-glucoside, apigenin-7-O-glucoside, 3,5-O-dicaffeoylquinic acid, and luteolin had the best binding energy.

3.6. Three Components Exert Antipyretic Effect during Yeast-Induced Fever

To observe the impact of three components on thermoregulatory responses during yeast-induced fever, recordings of ΔT (°C) were conducted for 8 hours after administration of the components (Figure 6). Results indicated no significant difference between the control and the PAP groups or the control groups and API-H groups. However, there were significant differences between the control and other groups (), demonstrating that PAP and API-H groups have superior antipyretic effects. Significant differences were observed between the model group and other groups at the partial time node, indicating that both high and low doses of the three components possess antipyretic effects.

3.7. Effect of Three Components on the Levels of Endogenous Pyrogen and Febrile Mediator

PGE2, IL-6, TNF-α, and IL-1β are endogenous pyrogen or febrile mediators that play key roles in the fever response [42, 43]. The present study evaluated the effect of three components on the levels of PGE2, IL-6, TNF-α, and IL-1β in the yeast-induced fever mice with paracetamol as a positive control. As shown in Figure 7, compared with the control without yeast injection, the plasma levels of PGE2, IL-6, TNF-α, and IL-1β of yeast-induced fever mice were markedly increased. The differences were statistically significant (). However, these three components inhibited the yeast-induced increase in PGE2, IL-6, TNF-α, and IL-1β in the plasma.

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3.8. Validation of Methodology

The investigation found that all components had repeatability RSD of ≤3.02%, intraday precision RSD of ≤3.17%, interday precision RSD of ≤3.44%, stability within 48 h of ≤3.47%, and recovery RSD of ≤3.05%. The sample recovery ranged from 92.80% to 107.40%. The validation results are presented in Table 2.

3.9. Changes of Component Contents in Different Developmental Periods of LJF

3.9.1. Change of Antipyretic Components in LJF

The phenolic acids and flavonoids in LJF at different developmental periods are presented in Supplementary Table 3. The content changes of the four components with the best binding energy are shown in Figure 8. F4 (luteolin-7-O-glucoside) and F9 (apigenin-7-O-glucoside) had increased and decreased content, reaching a peak in the S4 period; P2 (3,5-O-dicaffeoylquinic acid) reached its peak in S3 period, and F11 (quercetin) showed a gradual decline. Interestingly, 3,5-O-dicaffeoylquinic acid and apigenin-7-O-glucoside were quality control components of LJF in Chinese Pharmacopoeia. These two components partly reflect the antipyretic effect of LJF, which has clinical significance.

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The study found that chlorogenic acid comprised 68% of the total component content. To avoid high-content components affecting trace components, the content ratio of different components in different developmental periods was analyzed (Figure 9). The figure showed that the total proportion of each component increased and then decreased with the developmental period. This change can reflect the distribution of each component’s content and indirectly reflect the antipyretic effect of LJF in different periods. A higher total proportion of components resulted in a better antipyretic effect.

Although the overall phenolics demonstrated effective antipyretic properties during the S3 and S4 periods, the number of individual components varied across different periods, as evidenced in Figure 10. Notably, the content of P5 (methyl caffeate), P6 (chlorogenic acid methyl ester), P8 (4-O-caffeoylquinic acid), and P10 (caffeic acid) was at its lowest during the S3 period, while the content of P4 (3,4-dihydroxybenzoic acid) and P8 (4-O-caffeoylquinic acid) peaked during the S6 period. Chlorogenic acid is a phenylpropanoid substance formed by the shikimic acid pathway through the activity of phenylalanine ammonia-lyase, whereas caffeic acid serves as an important precursor substance in the synthesis process. Interestingly, the variation in caffeic acid content depicted in Figure 10 exhibits an opposing trend to that of chlorogenic acid, indicating a potential connection between this outcome and chlorogenic acid synthesis. Compared to phenolic acids, the distribution of flavonoids was more consistent, with most flavonoids demonstrating higher content during the S3 period.

3.9.2. Comparative Analysis of Phenolic Acid Quality Control Components in LJF

In addition to 3,5-O-dicaffeoylquinic acid, chlorogenic acid and 4,5-O-dicaffeoylquinic acid were also quality control components of LJF. We plot the contents of the above three acids in Figure 11. The chlorogenic acid content of the sample in this study reached the content standard of 1.5%, and five periods of the sample reached the standard of 3.8% phenolic acid component stipulated by pharmacopeia. Therefore, there was a certain contradiction between the high chlorogenic acid content and the quality standard of phenolic acid stipulated by the pharmacopeia of LJF. If the chlorogenic acid content of LJF was up to the standard (≥1.5%), the content of the other two phenolic acids was of little significance. Even if the two phenolic acids were not contained in the medicine, this batch met LJF pharmacopeia standards. In different developmental periods of LJF, 3,5-O-dicaffeoylquinic acid content accounted for 0.408∼0.505% of the chlorogenic acid content and 4,5-O-dicaffeoylquinic acid content accounted for 0.023∼0.038% of the chlorogenic acid content. When the chlorogenic acid reached the standard of 1.5%, the content of 3,5-O-dicaffeoylquinic acid was 0.612∼0.758% and 4,5-O-dicaffeoylquinic acid content was 0.035∼0.057%. Therefore, the total acid content was 1.931∼2.043%, far from the standard of 3.8% stipulated by the pharmacopeia. As the previous study pointed out, 3,5-O-dicaffeoylquinic acid was crucial to the antipyretic effect of LJF, so its content standard should be strictly controlled. However, the current LJF quality standard has defects in the control of 3,5-O-dicaffeoylquinic acid content. The total acid content of LJF includes chlorogenic acid, which accounted for 0.648∼0.695%. However, when the total acid content reached 3.8%, the content of chlorogenic acid in LJF was about 2.462∼2.641%, much higher than the standard of 1.5%.

3.9.3. Multivariate Statistical Analysis

Principal component analysis (PCA) is an unsupervised pattern recognition method. PCA can visually display the data differences in each group through dimensionality reduction of data. Since the antipyretic components of LJF were not consistent in different periods, SIMCA 14.1 software was used to reduce the dimensionality of the data (Figure 12). Figure 12 visually represents the variations between samples. LJF samples at different developmental periods formed a single cluster, while different periods showed a notable separation trend.

PCA is an unsupervised analysis method that has limitations. In-group errors and random errors cannot be ignored or eliminated during analysis. Therefore, supervised orthogonal partial least-squares discriminant analysis (OPLS-DA) is needed to determine the different components of different developmental periods. First, the permutation model was used to verify the fitting degree of the OPLS-DA model. In the positive ion model, the parameters of 200 permutation models were R² = 0.999 and Q² = 0.999, indicating that the model was effective and that the differential chemical components could be further identified. The study employed OPLS-DA analysis to detect distinct components in varying developmental periods. Differential components were screened based on VIP >1, and potential differential components of LJF were examined across different periods. The analysis identified 10 components, comprising 8 phenolic acids and 2 flavonoids (Figure 13).

Based on the analysis, phenolic acids exert a more pronounced effect on the differentiation of samples in distinct periods than flavonoids. Specifically, the content difference of phenolic acids in different periods is evident. Therefore, the selection of picking time bears a greater impact on phenolic acids, ultimately resulting in an indirect effect on the antipyretic effect of LJF.

4. Discussion

Using network pharmacology, LJF’s phenolic acid and flavonoid active components were analyzed to construct a network of key potential components and potential targets. The study found 186 potential components that act directly on fever targets and may be the key antipyretic components in LJF’s efficacy.

In the GO biological process analysis results, the protein phosphorylation showed the greatest enrichment. The process of signal transduction involves reversible phosphorylation of a variety of proteins. Proteins can amplify and transmit signals through reversible phosphorylation and different cascade reactions. In conclusion, protein phosphorylation plays an essential role in cell signaling [44, 45]. Protein serine/threonine/tyrosine kinase activity significantly enriched GO molecular function analysis. It has implications in regulating cell morphology, motility, and cell transformation [46]. Serine/threonine/tyrosine kinase 1 (STYK1) was upregulated in nonsmall cell lung cancer (NSCLC) and correlated with poor clinical outcomes. Moreover, STYK1 hindered forkhead box O1 actions, prompting metastasis and epithelial-mesenchymal transition in NSCLC [47]. The cellular components of GO were mainly enriched in membrane rafts, which are fundamental for maintaining cellular functions such as signal transduction [48], receptor activation [49], and intracellular lipid and protein trafficking [50]. Interfering with this process leads to abnormal growth, differentiation, metabolism, and biological traits, resulting in various illnesses.

The lipid and atherosclerosis signaling pathway was closely related to the calcium signaling pathway, NF-kappa B signaling pathway, toll-like receptor signaling, apoptosis, and TNF signaling pathway. As the responder and transmitter of calcium signals, calcium-ion-binding protein plays an essential role in the signaling pathway [51]. Proper regulation of calcium concentration is necessary for systemic metabolism, and disruption of calcium homeostasis is associated with various metabolic diseases in vivo [52]. Inflammation mainly refers to the complex reaction caused by tissue damage or pathogen infection, leading to various inflammatory diseases. TRP channels were a class of ion channels that responded to various chemical and physical stimuli derived from harmful agents and contributed to increased intracellular cation concentrations [53]. Inflammatory diseases cause TRP ion channels to malfunction [54], increasing calcium influx into the cell [55, 56]. This increase in calcium ion concentration can trigger reactions, including the production and release of arachidonic acid [57] and respiratory burst [58].

The anti-inflammatory responses have been induced by cAMP, and its pathways have been extensively used for treating inflammatory diseases. cAMP is one of the important inflammatory mediators of the central nervous system, a crucial information-conveying molecule in response to external stimuli, and the main positive heating medium in the central nervous system. Increased cAMP content is a common intermediate link in various pyrogen-induced fevers, which can participate in thermoregulation through the endogenous pyrogen-hypothalamic Na⁺/Ca²⁺-cAMP pathway [59–62]. The cAMP-dependent protein kinase signal transduction pathway may be involved in the central pyrogenic mechanisms in rats [63]. Therefore, LJF can exert antipyretic effects via the cAMP signaling pathway. In conclusion, the KGEE analysis indicated that the antipyretic mechanism of phenolic acids and flavonoids in LJF is modulated mainly through regulating these pathways.

The binding energy of phenolic acids and flavonoids in LJF with mPGES-1, EP1, EP2, EP3, and EP4 targets is satisfactory. The four most important components were luteolin-7-O-glucoside, apigenin-7-O-glucoside, 3,5-O-dicaffeoylquinic acid, and luteolin. Luteolin-7-O-glucoside and apigenin-7-O-glucoside have pharmacological activities to reduce oxidative stress and inflammation. Luteolin-7-O-glucoside could inhibit the activation of NF-kappa B and phosphorylation of Akt [64] and significantly reduce the content of cholesterol hydroxylated species such as 7-α-hydroxicholesterol and 7-β-hydroxicholesterol [65]. Apigenin-7-O-glucoside could significantly inhibit H₂O₂-induced ROS production in RAW 264.7 cells, free radical-induced oxidative damage on erythrocytes, and LPS-induced NF-kappa B/NLRP3/caspase-1 signaling in RAW 246.7 cells [66]. 3,5-O-dicaffeoylquinic acid has shown a protective effect against inflammation-related diseases and attenuated inflammation-mediated pain hypersensitivity by enhancing autophagy by inhibiting MCP3-induced JAK2/STAT3 signaling [67]. Additionally, 3,5-O-dicaffeoylquinic had a protective effect against acute lung injury caused by LPS in mice [68]. Luteolin reduced the expression of proinflammatory molecules (TNF-α, ICAM-1) in vivo [69] and inhibited LPS-induced IL-6 production in the brain by inhibiting the JNK signaling pathway and activation of AP-1 in microglia [70]. In addition, luteolin exhibited anti-inflammatory activity at micromolar concentrations [71], indicating its potential as a promising component for further development. Binding to an EP receptor on the cell membrane is essential for PGE2 to exert its biological work. The EP1 receptor activates protein kinase C by upregulating intracellular Ca²⁺ while partially coupled to G protein Gαs. The EP2 receptor activates the PKA pathway by increasing intracellular cAMP levels and partially coupling to G protein Gαs [72]. The docking results showed that LJF components were well connected to EP1 and EP2 receptors. The results of the KEGG analysis also indicated that the calcium signaling and cAMP signaling pathways were the main antipyretic pathways of LJF. Therefore, it was hypothesized that LJF’s antipyretic mechanism involves interaction between its main antipyretic components and PGE2 receptors. This interaction regulates calcium signaling, cAMP signaling, and other pathways, ultimately resulting in antipyretic effects.

Yeast-induced pyrexia by increasing prostaglandin synthesis is a useful model for screening drugs for their antipyretic effect [73]. Combined with previous experiments, we found that LPS-induced body temperature changes were susceptible to ambient temperature and manipulation stimuli. In contrast, yeast-induced fever was more stable and persistent. Therefore, the yeast heating model was selected for the experiment. In the present study, intraperitoneal administration of three components (high-low dose), especially API-H, significantly attenuated the rectal temperature of yeast-induced febrile mice. In addition, we tested the effect of three components on PGE2, IL-6, TNF-α, and IL-1β secretion by yeast-induced pyretic animals. IL-6, TNF-α, and IL-1β are important endogenous pyrogen, which can directly or indirectly cause the release of PGE2 and increase body temperature [42, 43]. The data showed that three high- and low-dose components reduced the endogenous pyrogen or febrile mediator levels in febrile mice. Therefore, it is reasonable to choose 3,5-O-dicaffeoylquinic acid and luteolin-7-O-glucoside as the quality control components of LJF in the Chinese pharmacopeia.

Using molecular docking, LJF’s antipyretic properties were analyzed and several related phenolic acids and flavonoids were identified. Consequently, changes in the content of these components can serve as an indirect indicator of LJF’s antipyretic effect. According to the proportion of each component content in different developmental periods of LJF, the antipyretic effect of LJF in S3 and S4 was better than that in other periods, suggesting that the harvesting period of the LJF should not be too early. By discussing the content of three phenolic acids in LJF, defects in quality control of LJF in Chinese Pharmacopoeia were detected. The “Siji Hua” germplasm of LJF had an individual problem, while other germplasms had a general problem. The main antipyretic component, 3,5-O-dicaffeoylquinic acid, has a relatively loose restriction condition, which affects the efficacy of LJF. The LJF total acid content standard ≥3.8% specified in the 2020 Chinese Pharmacopoeia needs to be discussed. Quality control of phenolic acid components should be increased to ensure the quality of LJF, as the picking time significantly influences these components.

5. Conclusion

This paper investigated the antipyretic mechanism of LJF’s phenolic acids and flavonoids using network pharmacology and molecular docking technology, providing a modern scientific explanation of the curative effect of traditional heat-clearing and detoxification of LJF. 3,5-O-dicaffeoylquinic acid, luteolin-7-O-glucoside, and apigenin-7-O-glucoside were found to have significant antipyretic activity in a yeast-induced pyrexia model. The ESI-QqQ-MS/MS method analyzed antipyretic components to guide LJF’s quality control. However, more research is necessary to explore the molecular mechanism and develop new drugs.

Data Availability

The data supporting the findings of the study included within the article or supplementary materials.

Additional Points

Sample Availability: samples are not available from the authors.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Lewen Xiong, Wenjing Huang, and Yan Liu developed the methodology; Lewen Xiong and Yang Wang performed the software analysis; Yan Liu, Ying Jin, and Hongwei Zhao validated the manuscript; Lewen Xiong prepared the original draft of the manuscript; Lewen Xiong, Longfei Zhang, and Yongqing Zhang reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript. Lewen Xiong and Wenjing Huang contributed equally to this work.

Acknowledgments

This research was funded by the Major Scientific and Technological Innovation Projects in Shandong Province (2019JZZY011020), National Key Research and Development Program of China (2017YFC1701503), and Shandong Natural Science Foundation Project (ZR2021QH238).

Supplementary Materials

Supplementary Table 1: information and HPLC-MS/MS parameters for MRM on phenolic acids and flavonoids in LJF. Supplementary Table 2: phenolic acids and flavonoids in LJF. Supplementary Table 3: contents of phenolic acids and flavonoids in LJF at different developmental periods. Supplementary Figure 1: MS chromatogram of the LJF extract. (Supplementary Materials)

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