Green synergistic non‐thermal processing of Xinhui Chenpi: targeted fraction screening and mechanistic elucidation of antioxidant enhancement in Caenorhabditis elegans

Abstract

BACKGROUND

Xinhui Chenpi (CP) is rich in polyphenols and flavonoids with excellent bioactivity, but its high‐value biomass utilization is restricted. Green non‐thermal processing can effectively maintain the structural integrity of bioactive small molecules, which is promising for the efficient valorization of CP resources. However, targeted preparation of high‐activity CP fractions and their in vivo antioxidant mechanisms, as well as potential targets against alcoholic fatty liver (AFL), remain poorly clarified.

RESULTS

A high‐activity CP fraction (CPSU‐H) was selectively prepared via pulsed electric field‐ultrasonic synergistic extraction, ethanol polarity grading fractionation and HPD100 macroporous resin adsorption purification. CPSU‐H significantly reduced malondialdehyde content to 0.32 ± 0.07 nmol mg protein⁻¹ in Caenorhabditis elegans, and increased total superoxide dismutase, glutathione peroxidase and catalase activities to 80.35 ± 2.15, 148.73 ± 9.05 and 9.74 ± 0.52 U mg protein⁻¹, respectively, showing strong in vivo antioxidant enhancement under non‐stressed conditions. CPSU‐H was dominated by neohesperidin and narirutin; nucleocytoplasmic transport pathway, longevity regulating pathway and nucleotide metabolism pathway were speculated to serve as the core signaling and metabolic pathways for its antioxidant enhancement. In addition, it could exert potential intervention effect on AFL through targeting proteins PTGS2, TNF, SRC, MMP9 and EGFR.

CONCLUSION

This study realizes the directional green preparation of CPSU‐H and clarifies its antioxidant mechanism under non‐stressed conditions and AFL‐related target proteins. The findings support the potential development of CPSU‐H as natural antioxidants and functional food components, and provide a theoretical basis for high‐value utilization of CP biomass and extended application of synergistic non‐thermal processing technologies. © 2026 Society of Chemical Industry.

INTRODUCTION

Xinhui Chenpi (CP) is a processed product obtained from the pericarp of Citrus reticulata ‘Chachiensis’ through harvesting, peeling and air‐drying. ¹ In the food industry, it is widely used as a culinary seasoning to impart a unique flavor. ² As a result of research on its physiological functions, CP has been found to regulate lipid metabolism, alleviate cough with excessive phlegm and other symptoms. ³ However, its long‐term natural drying and common hot‐water processing cause loss or degradation of thermosensitive bioactive components such as flavonoids, volatile oils and phenolic acids, reducing its nutritional and functional value. ⁴ , ⁵ In addition, nutritional studies on CP have focused on extractable macromolecular substances such as polysaccharides, pectin and dietary fiber, whereas insufficient research on the exploration, identification and functional verification of its high‐activity small molecules has resulted in the severe underutilization of resources and the lack of high‐value development in the food industry. ⁶ , ⁷ Therefore, it could provide technical support for comprehensively improving the nutritional evaluation system in the natural product chemistry of CP and enhancing the high‐value utilization rate of CP resources through exploring its key high‐activity characteristic small molecules components, analyzing the structure–activity relationship, and clarifying its functional targets and mechanisms.

Polyphenols and flavonoids are core high‐activity thermosensitive small molecules in CP with multiple phenolic hydroxyl groups in their molecular structures, which could be prone to oxidation and degradation under the influence of heat, oxygen, light, enzymes and other factors resulting in the loss of biological activity. ⁸ Traditional extraction technologies were based on organic solvent extraction with ethanol, methanol and other solvents, combined with auxiliary thermal processing methods, which had the disadvantages of low extraction efficiency and high solvent consumption. ⁹ , ¹⁰ Pulsed electric field (PEF) extraction technology could destroy the plant cell structure through the electroporation effect, accelerate the dissolution of small molecules from cells into the solvent, achieve efficient extraction at room temperature and typically shorten the extraction time to several minutes. ¹¹ , ¹² Ultrasonic‐assisted extraction (US) technology was found that it could utilize high‐frequency ultrasonic waves to generate cavitation and mechanical vibration effects in the solvent, which could produce local high pressure and micro‐jets in the reaction system to impact and rupture plant cell walls, realizing the efficient dissolution of polyphenol and flavonoid small molecules. ¹³ , ¹⁴ Applying non‐thermal technologies to CP extraction could mitigate thermal damage to target components, enable full‐value utilization of CP resources, reduce energy consumption and provide technical support for CP natural product chemistry research and food industry upgrading. ¹⁵

Nutritional studies on the functional components of CP have mostly focused on the evaluation of crude extracts and individual monomeric compounds. ¹⁶ , ¹⁷ However, it has not been established that the high‐efficiency technical system for the extraction, separation and purification adapted to the thermosensitive small molecules of CP, which made it difficult to fully elucidate the material basis for the functional properties of CP and restrict the high‐value development and application of CP in the field of functional foods, resulting in poor exploration, utilization and industrial transformation of its high‐activity small molecule resources. Therefore, it could fill the key gaps in the research on natural product nutrition of CP, improve the elucidation of the material basis for its functional properties, provide core technical support for the full‐component high‐value utilization of CP resources, and promote its innovative development and industrial application in the field of functional foods through conducting research on the directional screening and efficient extraction technologies for high‐activity thermosensitive small molecules from CP, systematically elucidating their structural characteristics, biological activities and mechanisms, and establishing a tailored separation and purification technical system.

This study prepared CP‐N through conventional pressing and CP‐PU through PEF‐US non‐thermal processing, determining their bioactive substances and effects on Caenorhabditis elegans to screen high‐activity extracts. The optimal extract was fractionated into alcohol‐soluble (CPPU‐SU) and insoluble (CPPU‐SE) components, purified via macroporous resins, and its small‐molecule composition was identified. Transcriptomic and metabolomic analyses explored its antioxidant mechanism in C. elegans, and major citrus flavonoids from the purified product (CPF) were screened, with their anti‐alcoholic fatty liver (AFL) targets predicted via network pharmacology and molecular docking. This study could establish a comprehensive development system for CP bioactive components, providing industrial references for PEF‐US technology, theoretical support for antioxidant functional foods and a green pathway for high‐value utilization of citrus biomass, advancing sustainable biomass processing.

MATERIALS AND METHODS

Preparation of CP‐N and CP‐PU

CP was purchased from the local agricultural market in Xinhui City, Guangdong Province (China).

Preparation of CP‐N

Fresh CP (50 g) was washed with clean water to remove surface dust and other foreign substances, placed into a homogenizer and mixed with water (300 mL), the supernatant was collected through homogenization and centrifugation, and CP‐N was obtained through freeze drying.

Preparation of CP‐PU

Fresh CP (50 g) was washed with clean water to remove surface dust and other foreign substances, placed in a homogenizer, mixed with water (300 mL), homogenized and crushed, and the mixed solution was transferred to a PEF material bin. PEF equipment equipped with a 700‐mL static treatment chamber (Guangzhou Paihu Technology Co., Ltd, Guangzhou, China). For power delivery, the input power parameters were: voltage of 3AC 380 V + PE, voltage fluctuation of ±10%, rated frequency of 50 Hz, input current of 20 A, and air cooling as the cooling method. The output power parameters included a pulse voltage ranging from 0 to 12 kV, a pulse current ranging from 0 to 300 A (with automatic system shutdown for protection when reaching 300 A), a pulse frequency ranging from 1 to 300 Hz (adjustable in 1 Hz steps), a pulse width ranging from 1 to 10 μs (adjustable in 1 μs steps) and a maximum average power of 10 kW. It was treated under the conditions of a pulse width of 8 μs, a pulse frequency of 5 Hz and an electric field strength of 1.2 kV cm⁻¹ for 5 min. The mixed solution was then transferred to the treatment chamber of SB25‐12DTD ultrasonic cleaner (Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China), and extracted at room temperature for 30 min under the condition of an ultrasonic power of 300 W. The supernatant was obtained by centrifugation, and AC‐PU was obtained after freeze‐drying. ¹⁵

Determination of active ingredients

The active ingredient contents of CP‐N and CP‐PU were determined, including polyphenol, carbohydrate, reducing sugar and protein contents. The polyphenol content was measured according to the method described by Lee et al., ¹⁸ the carbohydrate content was determined using the phenol‐sulfuric acid method, the reducing sugar content was measured using the dinitrosalicylic acid (i.e. DNS) method, and the protein content was determined using BCA quantitative assay kit (LABLEAD Inc., Beijing, China).

Study on antioxidant activity evaluation in vivo

The in vivo antioxidant activities of CP‐N and CP‐PU were determined by exploring their effects on the enhancement of antioxidant capacity markers under non‐stressed conditions in C. elegans, including malondialdehyde (MDA), total superoxide dismutase (T‐SOD), glutathione peroxidase (GSH‐Px) and catalase (CAT). It was completed with synchronization of C. elegans and amplification of OP50 (the food for C. elegans) in accordance with the method described by Lee et al. ¹⁸ After synchronization, C. elegans were randomly divided into three groups, including the non‐intervention group (NFD), the CP‐N intervention group (CP‐N mixed into OP50 to a final concentration of 0.5 mg mL⁻¹, CP‐N) and the CP‐PU intervention group (CP‐PU mixed into OP50 to a final concentration of 0.5 mg mL⁻¹, CP‐PU). The intervention concentration for the culture of C. elegans was determined based on our previous research experience, and 0.5 mg mL⁻¹ was identified as the safe and optimal evaluation concentration of Citrus reticulata cv. Chachiensis pulp extract for enhancing the antioxidant capacity of C. elegans. ¹⁸ Detailed grouping and intervention information is provided in Table 1. Caenorhabditis elegans were collected after being intervened with CP‐N and CP‐PU for 72 h, respectively. Lysis buffer was added to the C. elegans for homogenization on ice, and supernatant was obtained after centrifugation. The antioxidant‐related biochemical indicators MDA, T‐SOD, GSH‐Px and CAT in C. elegans of the NFD, CP‐N and CP‐PU groups were measured in accordance with the method provided in the reagent kit (Nanjing Jiancheng Biological Engineering Research Institute, Nanjing, China) and the protein concentration was quantified through the BCA reagent kit (Beijng LABLEAD Trading Co., Ltd, Beijng, China) as a quantitative evaluation indicator. The preparation scheme of CP extract with optimal in vivo enhancement of antioxidant capacity markers under non‐stressed conditions was selected for subsequent polar separation experiments.

Table 1.

Grouping and intervention conditions of Caenorhabditis elegans

Group	Intervention concentration	Intervention dose
NFD	OP50	0.1 mL
CP‐N	CP‐N was dissolved in OP50 to a final concentration of 0.5 mg mL−1	0.1 mL
CP‐PU	CP‐PU was dissolved in OP50 to a final concentration of 0.5 mg mL−1	0.1 mL
CPPU‐SU	CPPU‐SU was dissolved in OP50 to a final concentration of 0.5 mg mL−1	0.1 mL
CPPU‐SE	CPPU‐SE was dissolved in OP50 to a final concentration of 0.5 mg mL−1	0.1 mL
CPSU‐L	CPSU‐L was dissolved in OP50 to a final concentration of 0.5 mg mL−1	0.1 mL
CPSU‐A	CPSU‐A was dissolved in OP50 to a final concentration of 0.5 mg mL−1	0.1 mL
CPSU‐H	CPSU‐H was dissolved in OP50 to a final concentration of 0.5 mg mL−1	0.1 mL
CPSU‐N	CPSU‐N was dissolved in OP50 to a final concentration of 0.5 mg mL−1	0.1 mL
CPSU‐D	CPSU‐D was dissolved in OP50 to a final concentration of 0.5 mg mL−1	0.1 mL

Preparation, active component analysis and in vivo antioxidant activity evaluation of CP‐PU with different polarities

CP‐PU was separated based on polarity using an ethanol solution to explore specific components with high antioxidant activity. CP‐PU was mixed with ethanol (75%, 1:20 g mL⁻¹), stored at 4 °C for 24 h and centrifuged. The supernatant and sediment were collected separately and named CPPU‐SU and CPPU‐SE. ¹⁵ Their active components, including polyphenols, proteins, carbohydrates and reducing sugars, were determined according to the method as described above in the section ‘Determination of active ingredients’. The in vivo enhancement of antioxidant capacity markers under non‐stressed conditions of CPPU‐SU and CPPU‐SE were measured according to the method as described above in the section ‘Study on antioxidant activity evaluation in vivo’. The experimental design and the grouping and intervention information for intervention with C. elegans are shown in Table 1. The polar component of CP‐PU with the best in vivo antioxidant enhancement under non‐stressed conditions was selected for subsequent purification experiments.

Purification, in vivo antioxidant activity evaluation and screening of CPPU‐SU

CPPU‐SU was purified through macroporous resins LX‐8, AB‐8, HPD100, NKA‐9 and D101 to identify the purified component with the optimal antioxidant activity under non‐stressed conditions in vivo. LX‐8, AB‐8, HPD100, NKA‐9 and D101 macroporous resins were added to the CPPU‐SU (5 mg mL⁻¹) solution at a ratio of 0.1 g per 10 mL, and adsorbed in a constant temperature shaker at a speed of 120 rpm and a temperature of 25°C for 8 h. ¹⁵ The different types of macroporous resins adsorbed with CP‐PU small molecules were named CPSU‐L, CPSU‐A, CPSU‐H, CPSU‐N and CPSU‐D, respectively. These macroporous resins were transferred into ethanol solutions (75%) at a ratio of 0.1 g per 10 mL, and desorbed in a constant temperature shaker at 120 rpm and 25 °C for 8 h. After suction filtration, the desorption solution was collected, rotary evaporated and freeze‐dried to obtain different CPPU‐SU purified compounds. The antioxidant activity under non‐stressed conditions in vivo of different purified compounds was determined as described above in the section ‘Study on antioxidant activity evaluation in vivo’ and the optimal components were screened. The experimental design and the grouping and intervention information for intervention with C. elegans are shown in Table 1.

Identification of CPSU‐H components based on high‐performance‐liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) technology

The small molecule composition of CPSU‐H was identified using HPLC‐MS/MS technology with Waters HClass (Waters Corp., Milford, MA, USA) and Waters G2‐XS Qtof (Waters Corp.) equipment. The LC conditions and MS conditions were set based on the method described by Zheng et al. ¹⁹ The resulting data were compared and paired with the Waters Traditional Drug Library 2.0 (Waters Corp.) to identify the specific small molecule components of CPSU‐H. Small molecules with a response value greater than 10 000 and widely present in citrus fruits were selected as the key results of this study.

Transcriptomics analysis

After undergoing different CPSU‐H intervention treatments (0 or 0.5 mg mL⁻¹) for 3 days, C. elegans samples were collected and washed multiple times with M9 buffer to remove bacterial solution on their surface. Total RNA was extracted and its concentration and purity were detected to ensure that the samples met the requirements for constructing a library. mRNA was isolated, cDNA was obtained by reverse transcribing mRNA, ligating adaptors, purifying and classifying fragments, and performing PCR amplification. The amplified cDNA was sequenced on the NovaSeq X Plus platform (Illumina, San Diego, CA, USA). mRNA transcription information was mined by aligning the sequencing results with the reference genome, and differentially expressed mRNAs were screened according to the criteria of false discovery rate < 0.05 and |log₂FC| ≥ 1. The results were visually processed macroscopically through sample correlation analysis, principal component analysis (PCA) and volcano plot analysis. Goatools software (https://github.com/tanghaibao/goatools) was used to analyze the key Gene Ontology (GO) (http://geneontology.org) pathways enriched by characteristic mRNAs in C. elegans under CPSU‐H intervention, including biological processes (BP), cellular components (CC) and molecular functions (MF). Meanwhile, KEGG pathway enrichment analysis was performed on characteristic mRNAs to identify the key signaling pathways involved in the enhancement of antioxidant capacity markers under non‐stressed conditions in C. elegans induced by CPSU‐H intervention. ²⁰

Metabolomics analysis

After undergoing different CPSU‐H intervention treatments (0 or 0.5 mg mL⁻¹) for 3 days, C. elegans samples were collected and washed multiple times with M9 buffer to remove bacterial solution on their surface. Different C. elegans samples were homogenized and lysed on ice, and the supernatant containing metabolites was obtained through centrifugation and transferred to sample vials for instrument detection. The detection results were imported into Progenesis QI (Waters Corp.) software, and the metabolite information in C. elegans of the CPSU‐H group and NFD group was matched and qualitatively analyzed by comparing with the HMDB database (https://www.hmdb.ca) and Metlin database (https://metlin.scripps.edu). Characteristic metabolites in C. elegans of the CPSU‐H group and NFD group were obtained by calculating the variable importance in projection (VIP) and the P‐value of metabolites, with the screening criteria being VIP > 1 and P < 0.05. Meanwhile, characteristic metabolic pathways were analyzed by comparing and matching the characteristic metabolites with the Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg) database. ²¹

Network pharmacology analysis

The small molecule composition of CPSU‐H was obtained through HPLC‐MS/MS technology and small molecules with response values greater than 10 000 and widely present in citrus fruits were selected as the small molecule components (CPF) for network pharmacology analysis. The selected small molecule components were uploaded to the PubChem platform (https://pubchem.ncbi.nlm.nih.gov) to obtain structural information, the corresponding target proteins for each small molecule were retrieved from the SwissTarget Prediction database (https://www.swisstargetprediction.ch) and the target proteins with a screening score greater than 1 were considered as effective target proteins. ‘Alcoholic fatty liver’ (AFL) was used as a keyword to search in the GeneCards database, and target proteins with a screening score greater than 1 were identified as effective disease target proteins. The overlapping key target proteins of small molecules and AFL were preliminarily analyzed by uploading their respective effective target proteins to the JVENN platform (https://jvenn.toulouse.inrae.fr) and visualizing them through Cytoscape, version 3.8.0 (https://cytoscape.org). Meanwhile, the overlapping target proteins were uploaded to the David database (https://davidbioinformatics.nih.gov) for analysis, and the key GO enriched pathways and KEGG enriched pathways of CPF intervention in AFL diseases were obtained. In addition, the protein–protein interaction (PPI) network was obtained by uploading overlapping target proteins to the STRING platform (https://string-db.org) for analysis, and the PPI results were uploaded to Cytoscape, version 3.8.0 software, for visualization and analysis and screening of key target proteins for subsequent molecular docking studies. ²²

Molecular docking analysis

The 3D structure of the core target protein for screening was obtained by searching the PDB database (http://www.wwpdb.org) and importing it into AutoDock Tools, version 1.5.7 (https://autodock.scripps.edu) for dehydration and hydrogen bond addition treatment, serving as the protein receptor for molecular docking. The 2D structure of the CPF small molecule was obtained by searching the PubChem platform and uploaded into Avogadro software (http://two.avogadro.cc) for energy minimization and hydrogen bonding treatment, serving as the small molecule ligand for molecular docking. The binding energy information between small molecule ligands and macromolecular target proteins, as well as key docking sites, was explored through molecular docking operations using AutoDock Tools, version 1.5.7, and visualized in PyMOL (https://www.pymol.org) to obtain specific information on potential targets of CPF intervention in AFL disease. ²³

Statistical analysis

Data analysis and figure plotting were conducted using SPSS, version 26 (IBM Corp., Armonk, NY, USA) and Origin 2022 (OriginLab, Northampton, MA, USA), respectively. All experimental results were repeated at least three times and presented as the mean ± SD. One‐way analysis of variance (ANOVA) was employed to examine the significant inter‐group differences (P < 0.05) in the evaluation of the enhancement of antioxidant capacity markers under non‐stressed conditions of C. elegans by CP extracts prepared using different methods, polar separation products of CP‐PU and CPSU purified products. Significant differences (P < 0.05) in the active ingredient content between groups of CP extracts prepared by different methods and polar separation products of different CP‐PU were analyzed through Duncan's multiple comparison. The correlation between characteristic metabolites and the enhancement of antioxidant capacity markers under non‐stressed conditions of C. elegans under CPSU‐H intervention was investigated through Spearman correlation analysis, which was explored the potential relationship between the enhancement of antioxidant capacity markers under non‐stressed conditions of C. elegans under CPSU‐H intervention at the metabolic level. At the same time, the correlation between characteristic mRNAs and the enhancement of antioxidant capacity markers under non‐stressed conditions of C. elegans under CPSU‐H intervention was investigated through Spearman correlation analysis, aiming to explore the potential relationship between the enhancement of antioxidant capacity markers under non‐stressed conditions of C. elegans under CPSU‐H intervention at the gene expression level. In addition, the correlation between characteristic metabolites and characteristic mRNAs was explored through Spearman correlation analysis, providing a reference for elucidating the correlation between metabolites and mRNAs involved in the enhancement of antioxidant capacity under non‐stressed conditions of C. elegans treated by CPSU‐H.

RESULTS

Comparison of active component contents and in vivo antioxidant activities between CP‐N and CP‐PU

PEF and ultrasonic extraction, as representative non‐thermal processing technologies, have been shown to enable efficient extraction and effective activity retention of bioactive compounds from natural products, with particularly remarkable performance in the yield improvement and activity preservation of thermosensitive polyphenols. ¹⁸ Accordingly, it could provide technical support for the development of high‐efficiency extraction processes for high‐activity components from CP through comparing the bioactive component contents and in vivo antioxidant activities under non‐stressed conditions of CP extracts prepared by conventional pressing and the PEF‐ultrasonic combined non‐thermal extraction method. The contents of polyphenols, proteins, carbohydrates and reducing sugars in CP‐N were 20.86 ± 0.43 mg g⁻¹, 6.77 ± 0.44%, 37.40 ± 0.75% and 6.90 ± 0.03%, respectively. Compared with CP‐N, the contents of the above four components in CP‐PU were significantly increased by 20.94%, 35.43%, 18.72% and 42.69%, respectively (Fig. 1A–D). Meanwhile, the intervention with either CP‐N or CP‐PU could significantly enhance the in vivo antioxidant capacity of C. elegans under non‐stressed conditions (Fig. 1E–H). The levels of MDA, T‐SOD, GSH‐Px and CAT in the NFD group were 2.07 ± 0.44 nmol mg protein⁻¹, 40.30 ± 2.32 U mgrot⁻¹, 145.04 ± 9.10 U mgrot⁻¹ and 3.81 ± 0.30 U mgrot⁻¹, respectively (Fig. 1E–H). Compared with the NFD group, the MDA level in the CP‐N group was significantly decreased by 43.05%, whereas the levels of T‐SOD, GSH‐Px and CAT were significantly increased by 11.75%, 65.74% and 107.83%, respectively (Fig. 1E–H). In comparison with the CP‐N group, the levels of T‐SOD and GSH‐Px in the CP‐PU group were further significantly increased by 18.58% and 11.53%, respectively (Fig. 1F,G). These results demonstrated that the PEF‐ultrasonic combined non‐thermal extraction method outperformed conventional pressing in enhancing the contents of bioactive components (polyphenols, proteins, carbohydrates and reducing sugars) in CP extracts and further improving the in vivo enhancement of antioxidant capacity markers under non‐stressed conditions of C. elegans, providing critical technical support for the development of efficient extraction processes for high‐activity components from CP and expanding the application potential of non‐thermal processing technologies in natural product utilization.

Figure 1.

Effects of CP extracts (CP‐N and CP‐PU) prepared by different methods and fractions isolated through ethanol‐based polarity separation (CPPU‐SU and CPPU‐SE) on the bioactive substance contents and antioxidant capacity of C. elegans. Contents of polyphenol (A), protein (B), reducing sugar (C) and carbohydrate (D) in CP‐N and CP‐PU. Effects of CP‐N and CP‐PU on the activities of MDA (E), T‐SOD (F), GSH‐Px (G) and CAT (H) in C. elegans. Contents of polyphenol (I), protein (J), reducing sugar (K) and carbohydrate (L) in CPPU‐SU and CPPU‐SE. Effects of CPPU‐SU and CPPU‐SE on the activities of MDA (M), T‐SOD (N), GSH‐Px (O) and CAT (P) in C. elegans. Data are presented as the mean ± SD; n = 3 independent replicates. For (A) to (D) and (I) to (L), Student's t‐test was used to compare two groups; asterisks indicate significant differences between the two groups: P < 0.05, P < 0.01. For (E) to (H) and (M) to (P), one‐way analysis of variance (ANOVA) followed by Tukey's post‐hoc test was performed; bars with different lowercase letters indicate significant differences among groups at P < 0.05, whereas bars sharing the same letter are not significantly different (P ≥ 0.05).

Comparison of active component contents and in vivo antioxidant activities between CPPU‐SU and CPPU‐SE

CP‐PU was superior to CP‐N in bioactive compound content and in vivo antioxidant activity under non‐stressed conditions, and the key fractions responsible for its enhanced in vivo antioxidant activity under non‐stressed conditions were further identified through ethanol polarity fractionation technology for the separation of CP‐PU to investigate the bioactive compound contents and in vivo antioxidant activities under non‐stressed conditions of each separated fraction CPPU‐SU and CPPU‐SE (Fig. 1I–P). It was found that the contents of polyphenols, proteins, carbohydrates and reducing sugars in CPPU‐SU and CPPU‐SE were 48.90 ± 2.12 and 32.53 ± 1.18 mg g⁻¹, 37.98 ± 1.01 and 17.94 ± 0.34%, 52.01 ± 0.17 and 39.62 ± 0.87%, and 29.45 ± 0.11 and 12.48 ± 0.23%, respectively, and it was confirmed that CPPU‐SU was significantly superior to CPPU‐SE in bioactive compound content (Fig. 1I–L). Meanwhile, the levels of MDA, T‐SOD, GSH‐Px and CAT in C. elegans of the NFD group were 1.61 ± 0.11 nmol mg protein⁻¹, 48.74 ± 0.45 U mg protein⁻¹, 53.84 ± 3.71 U mg protein⁻¹ and 7.70 ± 0.17 U mg protein⁻¹, respectively (Fig. 1M–P). Compared with the NFD group, the MDA level in C. elegans of the CPPU‐SU group was significantly decreased by 72.72%, wheeras the levels of T‐SOD, GSH‐Px and CAT were significantly increased by 13.39%, 57.60% and 20.19%, respectively; the MDA level in C. elegans of the CPPU‐SE group was significantly decreased by 57.99%, and the levels of T‐SOD, GSH‐Px and CAT were significantly increased by 7.32%, 17.83% and 9.19%, respectively, and it was further verified that the in vivo antioxidant activity under non‐stressed conditions of CPPU‐SU was superior to that of CPPU‐SE (Fig. 1M–P). These results identified that CPPU‐SU, a fraction isolated from CP‐PU through ethanol gradient polarity fractionation, exhibited significantly higher contents of bioactive components and superior in vivo antioxidant activity of C. elegans compared to CPPU‐SE, pinpointing the key active fraction responsible for CP‐PU enhanced antioxidant efficacy under non‐stressed conditions and laying a foundation for the targeted development and utilization of high‐value bioactive components from CP.

Comparison of in vivo antioxidant activities of different purified products of CPPU‐SU

Small‐molecule extracts represented by polyphenols were the core functional components enabling non‐thermal processing technologies to efficiently retain thermosensitive bioactive substances in natural products, and it was confirmed that these components played a crucial role in enhancing the antioxidant capacity under non‐stressed conditions of C. elegans treated with CP‐PU and its separated fraction CPPU‐SU. ¹⁵ Therefore, it could provide important theoretical support and technical reference for the development of CP‐based functional products with high antioxidant activity under non‐stressed conditions through conducting macroporous resin purification, directional preparation and activity screening on CPPU‐SU (Fig. 2 A–D). The levels of MDA, T‐SOD, GSH‐Px and CAT in C. elegans of the NFD group were 0.93 ± 0.10 nmol mg protein⁻¹, 44.00 ± 0.93 U mg protein⁻¹, 77.32 ± 13.82 U mg protein⁻¹ and 4.35 ± 0.20 U mg protein⁻¹, respectively. Compared with the NFD group, the MDA level was significantly decreased, whereas the levels of T‐SOD, GSH‐Px and CAT were significantly increased in C. elegans treated with CPSU‐L, CPSU‐A, CPSU‐H, CPSU‐N and CPSU‐D, respectively. Among them, the MDA level in C. elegans of the CPSU‐H group was reduced to 0.32 ± 0.07 nmol mg protein⁻¹, which was significantly lower than that in the other CPSU‐purified product groups; the levels of T‐SOD and CAT were increased to 80.35 ± 2.15 U mg protein⁻¹ and 9.74 ± 0.52 U mg protein⁻¹, respectively, with the intervention effect significantly superior to that of the other CPSU‐purified product groups. In the investigation of the effects of different CPSU‐purified products on GSH‐Px, an antioxidant‐related biochemical index in C. elegans, the GSH‐Px levels in the CPSU‐H and CPSU‐N groups were 148.73 ± 9.05 U mg protein⁻¹ and 155.45 ± 10.01 U mg protein⁻¹, respectively, with no significant difference observed between the two groups. It was confirmed that CPSU‐H as the optimal purified product through macroporous resin purification and activity screening, providing potential theoretical support and technical reference for the development of high‐antioxidant‐activity functional products derived from CP.

Figure 2.

Effects of different CPSU purified products on MDA (A), T‐SOD (B), GSH‐Px (C) and CAT (D) in C. elegans and the key small‐molecule composition of CPSU‐H (E). (A–D) Data are presented as the mean ± SD; n = 3 independent biological replicates. Statistical analysis was performed using one‐way analysis of variance (ANOVA) followed by Tukey's post‐hoc test. Bars with different lowercase letters indicate significant differences among groups at P < 0.05, whereas bars sharing the same letter are not significantly different (P ≥ 0.05). NFD: normal food diet group; CPSU‐L, CPSU‐A, CPSU‐H, CPSU‐N and CPSU‐D represent different polarity fractions obtained from CPSU.

Identification of the small‐molecule component in CPSU‐H

Experimental support was provided clarifying the specific small molecular material basis responsible for its antioxidant activity through identification of the composition of key small molecular compounds in CPSU‐H, exploring high‐activity antioxidant functional components from CP sources and elucidating in vivo antioxidant mechanisms. The small molecular composition of CPSU‐H was identified through HPLC‐MS/MS technology, and the main fractions with a response value > 10 000 were screened out. It was found that neohesperidin, isorhamnetin‐3‐O‐neohesperidoside,3‐hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone, narirutin and kaempferol 3‐O‐beta‐d‐glucopyranosyl‐7‐O‐alpha‐l‐rhamnopyranoside were polyphenols and flavonoids widely present in citrus fruits (Fig. 2E and Table 2). Small‐molecule composition characterized by HPLC‐MS/MS could provide a critical material and experimental foundation for clarifying the specific small‐molecule basis of CP antioxidant activity and further exploring high‐activity antioxidant functional components from CP and in vivo mechanisms.

Table 2.

Identified small molecular compounds with response value >10 000 in CPSU‐H

Small molecule name	Chemical formula	Molecular weight (Da)	Observed retention time (min)	Observed m/z	Response intensity
Odoratin‐7‐O‐β‐d‐glucoside	C22H24O9	432.14203	13.31	433.1494	265 982
Neohesperidin	C28H34O15	610.18977	6.73	611.1966	236 154
Methyl ophiopogonanone A	C19H18O6	342.11034	11.86	343.1181	165 331
Cichoriin	C15H16O9	340.07943	2.56	385.0772	70 987
1,4‐Dicaffeoylquinic acid	C25H24O12	516.12678	5.5	561.1241	68 009
3‐Hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone	C20H20O8	388.11582	14.61	389.1228	44 381
Evodin	C26H30O8	470.19407	11.75	515.1914	42 515
Blumeatin	C16H14O6	302.07904	6.74	303.0865	38 558
Noririsflorentin	C19H16O8	372.08452	12.65	373.0919	36 142
2‐Methoxy‐4‐acetylphenol 1‐O‐α‐l‐rhamnopyranosyl‐(1′′→ 6′)‐β‐d‐glucopyranoside	C21H30O12	474.17373	0.52	473.1623	35 004
Strictosamide	C26H30N2O8	498.20022	0.53	533.1713	34 955
Isorhamnetin‐3‐O‐neohesperidoside	C28H32O16	624.16903	4.36	623.1604	34 955
2α,3β‐Dihydroxy nortropane	C7H13NO2	143.09463	0.52	144.1028	32 804
Kaempferol 3‐O‐beta‐d‐glucopyranosyl‐7‐O‐alpha‐l‐rhamnopyranoside	C27H30O15	594.15847	4.04	593.1499	29 902
Plantamajoside	C29H36O16	640.20034	8.67	639.1916	27 798
Rindoside	C35H42O21	798.22186	5.97	857.2334	26 523
Tribulusamide B	C36H34N2O9	638.22643	0.54	683.2243	24 956
Narirutin	C27H32O14	580.17921	6.17	579.1705	23 779
Methyl succinate	C6H10O4	146.05791	0.53	191.0561	23 565
1,8‐Dihydroxy‐9,10‐anthraquinone‐3‐methyl‐(2‐hydroxy) ropionate ester	C18H14O7	342.07395	13.86	343.0814	22 434
(2S)‐5,7‐Dihydroxy‐6‐methoxy‐flaanone‐7‐O‐β‐d glucopyranoside	C22H24O10	448.13695	6.74	449.1437	21 788
Terreside B	C39H62O14	754.41396	9.53	789.3771	19 665
Prunose II	C29H36O17	656.19525	6.75	655.1859	19 171
Thujaplicatin methyl ether	C21H24O8	404.14712	12.43	405.1542	18 898
Isopenniclavine	C16H18N2O2	270.13683	13.75	269.1309	17 777
Brazilide A	C16H14O7	318.07395	0.54	377.0855	15 589
Bufotalinin	C24H30O6	414.20424	15.55	415.2109	14 295
Moracin M‐3′‐O‐β‐d‐glucopyranoside	C20H20O9	404.11073	0.65	439.0766	14 036
Gentialutine	C9H11NO	149.08406	0.53	150.0917	12 908
Luteolin 7‐O‐β‐d‐(6′′‐acetyl)‐glucopyranoside	C23H22O12	490.11113	8.12	489.1022	11 079
20(R)‐Ginsenoside Rh2	C36H62O8	622.44447	31.16	621.435	10 769

Effects of CPSU‐H intervention on mRNA transcription in C. elegans

The enhancing effect of CPSU‐H on the antioxidant capacity of C. elegans under non‐stressed conditions has been clarified, and could provide theoretical support for elucidating the molecular regulatory mechanism underlying the in vivo antioxidant activity of CPSU‐H through investigating the effect of CPSU‐H on the mRNA transcription level of C. elegans by transcriptomics technology. In the correlation analysis of transcriptomic results between C. elegans in the NFD group and those treated with CPSU‐H intervention, it was found that the correlation coefficients among biological replicates within each group were higher than 0.995, indicating that the expression profiles of biological replicate samples in each group were highly consistent (Fig. 3A). By contrast, the correlation coefficient between the CPSUH group and the NFD group was 0.90–0.906, suggesting that there were significant differences in gene expression patterns between the two groups, which demonstrated that there were distinct differences in transcriptomic changes between C. elegans in the CPSUH group and those in the NFD group (Fig. 3A). Meanwhile, a macro‐level PCA analysis of the results confirmed significant differences in mRNA transcription between C. elegans in the CPSUH group and those in the NFD group, indicating that CPSU‐H could significantly regulate the mRNA transcription level of C. elegans (Fig. 3B). Compared with the NFD group, there were 2710 mRNAs significantly up‐regulated and 4781 mRNAs significantly down‐regulated in C. elegans of the CPSUH group (Fig. 3C). Potential signaling pathways were identified through GO pathway enrichment analysis of characteristic mRNAs in C. elegans of the CPSUH group, and it was found that the pathways involved in the CC of C. elegans treated with CPSU‐H included collagen trimer and extracellular region, those involved in MF included structural constituent of cuticle, and the pathways mainly involved in BP included neuropeptide signaling pathway and meiotic cell cycle process (Fig. 3D,E). This revealed that the differentially expressed genes were mainly enriched in the categories of metabolism (M), genetic information processing (GIP), environmental information processing (EIP) and organismal systems (OS) in KEGG enrichment analysis of differentially expressed mRNAs between C. elegans in the CPSUH group and NFD group (Fig. 3F,G). Among these, the M category included secondary pathways such as fatty acid degradation, propanoate metabolism and purine metabolism, whereas the OS, EIP, GIP and related pathways were significantly enriched in longevity regulating pathways, the FoxO signaling pathway and nucleotide excision repair pathways, which revealed that the molecular mechanism by which CPSU‐H affected the physiological state of C. elegans was through regulating material and energy metabolism, as well as antioxidant and aging‐related signaling pathways (Fig. 3F,G). In the longevity regulating pathway, the mRNA expression levels of daf‐9, fat‐7, ddl‐2, atl‐1, hsf‐1 and daf‐18 in C. elegans of the CPSUH group were significantly increased by 1.38, 8.90, 1.81, 1.24, 1.38 and 3.62 times compared to the NFD group, whereas those of lipl‐4 and daf‐12 were significantly decreased by 80.14% and 43.58% (Fig. 3H). Transcriptomic analysis revealed that CPSU‐H could significantly alter the mRNA transcription profile of C. elegans, with differentially expressed genes enriched in metabolism, antioxidant and aging‐related signaling pathways and key genes in the longevity pathway showing distinct expression changes, which clarified the molecular regulatory basis of CPSU‐H in vivo antioxidant activity under non‐stressed conditions and provided important theoretical support for further elucidating its underlying mechanisms.

Figure 3.

Effects of CPSU‐H intervention on mRNAs in C. elegans. (A) Correlation analysis. (B) PCA plot. (C) Volcano plot and statistical chart of characteristic mRNA screening. (D, E) GO pathway enrichment analysis of characteristic mRNAs in C. elegans treated with CPSU‐H. (F, G) KEGG pathway enrichment analysis of characteristic mRNAs in C. elegans treated with CPSU‐H. (H) Relative transcription levels of characteristic mRNAs in C. elegans treated with CPSU‐H. Data are presented as the mean ± SD; n = 3 independent biological replicates. Statistical analysis was performed using one‐way analysis of variance (ANOVA) followed by Tukey's post‐hoc test. Asterisks indicate significant differences compared to the NFD group: *P < 0.05, **P < 0.01.

Analysis of the potential mechanism underlying CPSU‐H intervention‐enhanced antioxidant capacity through characteristic mRNAs in C. elegans

CPSU‐H intervention could enhance the antioxidant capacity under non‐stressed conditions of C. elegans by regulating the synergistic effects of the nucleocytoplasmic transport pathway and longevity regulating pathway. In the nucleocytoplasmic transport pathway, the elevated transcriptional levels of ima‐2 and xpo‐2 mRNAs enhanced the nuclear‐cytoplasmic export efficiency of proteins and mRNAs, whereas the increased transcriptional levels of ddx‐19 and rae‐1 mRNAs could induce the nuclear entry of anti‐aging factors (Fig. 4A). In the longevity regulating pathway, the core factors of the INS pathway were activated, and the transcriptional levels of downstream antioxidant‐related mRNA gst‐4 was significantly increased, which enhanced the stress resistance of C. elegans (Fig. 4A). Meanwhile, the transcriptional level of pro‐aging‐related mRNA jnk‐1 was significantly decreased, which inhibited pro‐inflammatory signals (Fig. 4A). These findings confirmed that CPSU‐H exerted its effects by regulating nucleocytoplasmic transport efficiency, activating the insulin signaling pathway and the downstream antioxidant defense network, which could provide a reference for elucidating the mechanism underlying the enhancement of antioxidant capacity under non‐stressed conditions in C. elegans by CPSU‐H intervention at the molecular biological level (Fig. 4A).

Figure 4.

Mechanism analysis of CPSU‐H enhanced antioxidant capacity in C. elegans. (A): Molecular mechanism pathway for CPSU‐H enhancing antioxidant capacity in C. elegans. (B, C) Correlation analysis between antioxidant‐related biochemical indices and characteristic mRNAs in C. elegans treated with CPSU‐H.

The mechanism by which CPSU‐H intervention enhanced the antioxidant capacity of C. elegans under non‐stressed conditions through regulating characteristic mRNAs was further elucidated through analyzing the correlations between characteristic mRNAs and the enhancement of antioxidant capacity markers under non‐stressed conditions (Fig. 4B,C). It was found that MDA was significantly positively correlated with mRNAs spp‐11, C41G6.13, Y75B12B.8, scl‐13, Y71F9AM.8, scl‐12, F57F4.2 and Y71F9AM.11; GSH‐Px was significantly negatively correlated with mRNAs clec‐101 and col‐72; and CAT was significantly negatively correlated with mRNA F47B8.14 (Fig. 4B,C). These results clarified that CPSU‐H could enhance the antioxidant capacity under non‐stressed conditions of C. elegans by regulating the synergistic effects of the nucleocytoplasmic transport and longevity regulating pathways and revealed the specific correlation between characteristic mRNAs and the enhancement of antioxidant capacity markers under non‐stressed conditions, providing molecular biological basis for elucidating the antioxidant mechanism of CPSU‐H intervention in C. elegans.

Effects of CPSU‐H intervention on metabolite levels in C. elegans

The substances were fermented by gut microorganisms to produce metabolites after organisms ingested bioactive substances, which played an important role in improving organismal health. This could provide a basis for elucidating the mechanism by which CPSU‐H exerted antioxidant activity under non‐stressed conditions through metabolites by investigating the effect of CPSU‐H on metabolites in C. elegans, and offer a reference for the development of gut‐targeted foods derived from CP (Fig. 5). Macroscopic PCA and partial least squares discriminant analysis (PLS‐DA) of metabolites in C. elegans of the CPSUH group and NFD group revealed significant differences in metabolites between the two groups, and permutation test verified the reliability of this result, indicating that CPSU‐H intervention could significantly affect the metabolite levels in C. elegans (Fig. 5A–C).

Figure 5.

Effects of CPSU‐H intervention on metabolites in C. elegans. (A) PCA plot. (B) PLS‐DA plot. (C) Permutation testing. (D) Volcano plot and statistical chart of characteristic metabolite screening. (E) Characteristic metabolites screened by log₂FC values. (F) Characteristic metabolites screened by VIP values. (G) KEGG pathway enrichment analysis of characteristic metabolites in C. elegans treated with CPSU‐H. n = 3 independent biological replicates.

Compared with the NFD group, CPSU‐H intervention significantly increased the levels of 910 metabolites and significantly decreased the levels of 325 metabolites (Fig. 5D). It was found that the significantly up‐regulated characteristic metabolites mainly included leu ile asp glu leu, (2s,3r)‐2‐acetamido‐3‐hydroxy‐4‐methylpentanoate, liquiritigenin 4′‐[3‐acetylapiosyl‐(1‐>2)‐glucoside], beta‐funaltrexamine, aurantiumal, peiminine, cholyl‐l‐dopa, cloxacillin, desglucocheirotoxol, gluten exorphin C, trandolaprilat, 3‐deoxy‐d‐glycero‐d‐galacto‐2‐nonulosonic acid, bazedoxifene and chrysophanol‐9‐anthrone according to log₂FC value, whereas the significantly down‐regulated characteristic metabolites mainly included 25‐desacetyl rifapentine, smilagenin 3‐[2″‐glucosyl‐6″‐arabinosylglucoside] and malvidin 3‐caffeoyl‐glucoside (Fig. 5E). According to VIP scores, the significantly up‐regulated characteristic metabolites in C. elegans treated with CPSU‐H mainly included liquiritigenin 4′‐[3‐acetylapiosyl‐(1‐>2)‐glucoside], leu ile asp glu leu, (2s,3r)‐2‐acetamido‐3‐hydroxy‐4‐methylpentanoate, 3‐deoxy‐d‐glycero‐d‐galacto‐2‐nonulosonic acid, aurantiumal, kaempferol 3‐O‐alpha‐l‐(3‐trans‐p‐coumaroyl‐rhamnopyranoside), peiminine, 1,2‐dipentadecanoyl‐sn‐glycero‐3‐phosphocholine, cholyl‐l‐dopa, ethametsulfuron‐methyl, artemetin, 3′‐hydroxy‐4′,5,6,7,8‐pentamethoxyflavone, desglucocheirotoxol, bazedoxifene, 3‐hydroxy‐7,3′,4′,5′‐tetramethoxyflavone, gardenin B and altenusin, and the significantly down‐regulated characteristic metabolites mainly included malvidin 3‐caffeoyl‐glucoside, 25‐desacetyl rifapentine, physagulin D, (ho‐dimethyl‐3‐oxo‐hexadecahydro‐cyclopenta(a)phenanthren‐17‐yl)‐pentanoic acid, smilagenin 3‐[2″‐glucosyl‐6″‐arabinosylglucoside], afn911, glu ile phe lys, urolithin a 3,8‐O‐diglucuronide, lopinavir and leucomycin A5 (Fig. 5F). It was found that the top five potential metabolic pathways ranked by enrichment factor were one carbon pool by folate, axon regeneration, nucleotide metabolism, autophagy – animal and endocytosis through KEGG enrichment analysis of the characteristic metabolites in C. elegans after CPSU‐H intervention, whereas the top five potential metabolic pathways ranked by statistical significance were nucleotide metabolism, pyrimidine metabolism, purine metabolism, lysine degradation and tryptophan metabolism (Fig. 5G). CPSU‐H intervention could significantly alter the metabolite profile of C. elegans and enrich characteristic metabolites in key metabolic pathways related to nucleotide metabolism and autophagy, which may lay a potential metabolic basis for elucidating the antioxidant mechanism under non‐stressed conditions of CPSU‐H via metabolite regulation and provide a potential theoretical reference for the subsequent development of CP‐derived gut‐targeted functional foods.

Analysis of the potential mechanism underlying CPSU‐H intervention‐enhanced antioxidant capacity through characteristic metabolites in C. elegans

Nucleotide metabolism was the only core metabolic pathway that ranked among the top five in both enrichment factor ranking and statistical significance ranking through KEGG enrichment analysis of differentially expressed characteristic metabolites, indicating that this pathway played a key hub role in CPSU‐H regulating metabolism and exerting antioxidant activity under non‐stressed conditions (Fig. 6A). Compared with the NFD group, after CPSU‐H intervention, the relative contents of characteristic metabolites involved in purine nucleotide synthesis such as phosphoribosyl pyrophosphate, inosine monophosphate and other precursor substances and pyrimidine nucleotide salvage synthesis such as cytidine kinase substrates, uridine phosphorylation products and other related metabolites in the nucleotide metabolism pathway were significantly up‐regulated, whereas the relative contents of metabolites involved in nucleotide degradation such as xanthine, uracil and adenosine deaminase‐catalyzed products were significantly down‐regulated, which suggested that CPSU‐H could regulate the homeostasis of the nucleotide pool in C. elegans by activating nucleotide synthetic metabolism and inhibiting catabolic metabolism, providing a material basis for nucleic acid repair, synthesis of antioxidant enzymes, and maintenance of cellular homeostasis, supporting the enhancement of their antioxidant capacity under non‐stressed conditions (Fig. 6A).

Figure 6.

Mechanism analysis of CPSU‐H enhanced antioxidant capacity in C. elegans through mRNA transcription and metabolites. (A) Network diagram. (B) Correlation analysis between antioxidant‐related biochemical indices and characteristic metabolites in C. elegans treated with CPSU‐H. (C) Correlation analysis between characteristic mRNAs and characteristic metabolites in C. elegans treated with CPSU‐H.

Meanwhile, the nucleotide metabolism pathway exhibited close crosstalk and synergistic regulation with multiple core pathways (Fig. 6A). It was directly connected to purine metabolism and pyrimidine metabolism, and the up‐regulated purine/pyrimidine synthesis precursors induced by CPSU‐H could be further converted into functional nucleic acids and coenzymes through these two pathways, enhancing antioxidant under non‐stressed conditions (Fig. 6A). Meanwhile, metabolic flux communication with energy metabolism‐ATP and GTP produced during nucleotide synthesis could provide energy support for the maintenance of mitochondrial respiratory chain function and antioxidant stress responses, whereas the up‐regulation of intermediate metabolites such as pyruvate and citric acid in the energy metabolism pathway also reversely promoted the supply of substrates for nucleotide synthesis (Fig. 6A). In addition, the downstream products of the nucleotide metabolism pathway could regulate the synthesis of amino acids such as glycine and aspartic acid in amino acid metabolism, providing raw materials for the protein synthesis of antioxidant enzymes (Fig. 6A). These characteristic metabolites and metabolic pathway formed a synergistic regulatory network of nucleotide metabolism – energy supply – antioxidant defense, which jointly mediated the antioxidant activity under non‐stressed conditions of CPSU‐H.

In the correlation analysis between characteristic metabolites and the enhancement of antioxidant capacity markers under non‐stressed conditions of C. elegans treated with CPSU‐H, MDA was found to be significantly positively correlated with metabolites malvidin 3‐caffeoyl‐glucoside, (ho‐dimethyl‐3‐oxo‐hexadecahydro‐cyclopenta(a)phenanthren‐17‐yl)‐pentanoic acid and leucomycin A5, while exhibiting a significant negative correlation with metabolites gluten exorphin C, chrysophanol‐9‐anthrone, beta‐funaltrexamine, cholyl‐l‐dopa, desglucocheirotoxol, 3‐deoxy‐d‐glycero‐d‐galacto‐2‐nonulosonic acid and 2′‐deoxymugineic acid (Fig. 6B). Meanwhile, significant positive correlations were observed between T‐SOD and metabolite artemetin, whereas metabolite 3‐n‐ethyl‐1‐n‐[4‐(phenylcarbamoylamino)phenyl]piperidine‐1,3‐dicarboxamide showed a marked negative correlation with T‐SOD (Fig. 6B). For GSH‐Px, metabolite physagulin D was identified to have a significant negative correlation with this enzyme (Fig. 6B). Metabolite cloxacillin demonstrated a significant positive correlation with CAT (Fig. 6B). These results clarified the core intrinsic logic of multiple metabolic pathways synergistically mediating antioxidant activity through systematically elucidating the cross‐regulatory mechanisms and metabolic flux communication rules between the nucleotide metabolism pathway and purine/pyrimidine metabolism, energy metabolism, and amino acid metabolism, providing a theoretical reference for an in‐depth interpretation of the molecular mechanisms by which CPSU‐H enhanced the antioxidant capacity under non‐stressed conditions of C. elegans through multi‐target and multi‐pathway regulation.

Correlation analysis of characteristic mRNA transcription and characteristic metabolite level changes in C. elegans treated with CPSU‐H

The correlation analysis between the transcriptional changes of characteristic mRNAs and the level variations of characteristic metabolites in C. elegans under CPSU‐H treatment was of significance for revealing the potential transcription‐metabolism regulatory network mediating the antioxidant activity under non‐stressed conditions of CPSU‐H, providing a comprehensive molecular basis for elucidating its antioxidant mechanism (Fig. 6C). Characteristic mRNA col‐108 was found to be significantly positively correlated with metabolites afn911, 4‐(4‐fluorophenyl)‐3‐(4‐hydroxy‐3‐methoxyphenoxymethyl)piperidine, lopinavir, dihydrozeatin‐9‐n‐glucoside‐O‐glucoside and glu ile phe lys, at the same time as showing a significant negative correlation with metabolites leu ile asp glu leu, (2s,3r)‐2‐acetamido‐3‐hydroxy‐4‐methylpentanoate, liquiritigenin 4′‐[3‐acetylapiosyl‐(1‐>2)‐glucoside], aurantiumal, bazedoxifene and 1,2‐dipentadecanoyl‐sn‐glycero‐3‐phosphocholine (Fig. 6C). Meanwhile, a significant positive correlation was observed between characteristic mRNA D1086.9 and metabolites afn911, 4‐(4‐fluorophenyl)‐3‐(4‐hydroxy‐3‐methoxyphenoxymethyl)piperidine, lopinavir, dihydrozeatin‐9‐n‐glucoside‐O‐glucoside and glu ile phe lys, whereas metabolites leu ile asp glu leu, (2s,3r)‐2‐acetamido‐3‐hydroxy‐4‐methylpentanoate, liquiritigenin 4′‐[3‐acetylapiosyl‐(1‐>2)‐glucoside], beta‐funaltrexamine, aurantiumal, cholyl‐l‐dopa, desglucocheirotoxol, trandolaprilat, bazedoxifene, chrysophanol‐9‐anthrone, 1,2‐dipentadecanoyl‐sn‐glycero‐3‐phosphocholine and 2′‐deoxymugineic acid exhibited a marked negative correlation with mRNA Y71F9AM.11 (Fig. 6C). These results identified the specific correlation patterns between characteristic mRNAs and metabolites in C. elegans treated with CPSU‐H, which was crucial for revealing the potential transcription‐metabolism regulatory network mediating CPSU‐H antioxidant activity under non‐stressed conditions and provided a comprehensive molecular basis for fully elucidating its antioxidant mechanism.

Screening of target proteins and pathway analysis for CPF‐mediated regulation of AFL disease

CPSU‐H was identified as the key bioactive component responsible for CP‐mediated enhancement of antioxidant capacity under non‐stressed conditions in C. elegans, and the small‐molecule composition of CPSU‐H was characterized through HPLC‐MS/MS, which, when combined with the identification results, screened out five core bioactive components widely distributed in citrus natural products (CPF); namely, 3‐hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone (CPF1), isorhamnetin‐3‐O‐neohesperidoside (CPF2), kaempferol 3‐O‐beta‐d‐glucopyranosyl‐7‐O‐alpha‐l‐rhamnopyranoside (CPF3), narirutin (CPF4) and neohesperidin (CPF5). Furthermore, the potential regulatory mechanism of CPF in alcoholic fatty liver (AFL) in humans was investigated, aiming to provide theoretical references for the development of functional foods centered on antioxidant activity for AFL prevention. CPF had 128 potential target proteins, whereas AFL had 10 094 potential target proteins, with 112 overlapping target proteins between them, suggesting the potential application value of CPF in the prevention and treatment of AFL (Fig. 7A). The regulatory network of CPF against AFL consisted of 117 nodes and 169 edges through visualization analysis. Among them, CPF1 shared 88 target proteins with this network, whereas both CPF4 and CPF3 had 21 target proteins with the network, respectively (Fig. 7B and Table 3).

Figure 7.

Network pharmacology analysis of CPF in the intervention of AFL. (A) Venn diagram. (B) Network diagram. (C) PPI diagram. (D–F) Screening of key proteins. (G) GO pathway enrichment diagram. (H) KEGG pathway enrichment diagram.

Table 3.

Information on small molecule ligands in network pharmacology analysis of CPF and AFL disease

MOL ID	Compound	Degree
CPF1	3‐Hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone	88
CPF2	Isorhamnetin‐3‐O‐neohesperidoside	19
CPF3	Kaempferol 3‐O‐beta‐d‐glucopyranosyl‐7‐O‐alpha‐l‐Rhamnopyranoside	21
CPF4	Narirutin	21
CPF5	Neohesperidin	20

Eight key target proteins (EGFR, SRC, TNF, PTGS2, MCL1, AKT1, MMP9 and KDR) were screened out through performing PPI analysis on the above overlapping target proteins (Fig. 7C–F). Specifically, the betweenness centrality, closeness centrality and degree value of target proteins EGFR and AKT1 were 7.38, 1.0 and 36.0, respectively, and those of target protein SRC were 5.82, 0.95 and 34.0, respectively (Fig. 7F and Table 4). These results could support a foundation for further investigating the binding energy and binding site analysis of potential target proteins involved in CPF‐mediated intervention of AFL.

Table 4.

Core protein information in network pharmacology analysis of CPF and AFL disease

Name	Betweenness	Closeness	Degree
EGFR	7.377799977799976	1.0	36.0
SRC	5.818204018204017	0.9473684210526315	34.0
TNF	5.314307914307913	0.9473684210526315	34.0
PTGS2	3.227006327006327	0.9	32.0
MCL1	6.08097458097458	0.9473684210526315	34.0
AKT1	7.377799977799976	1.0	36.0
MMP9	5.314307914307913	0.9473684210526315	34.0
KDR	4.3809745809745815	0.9	32.0

GO functional enrichment analysis was performed on the overlapping target proteins of CPF in AFL intervention and the results demonstrated that, at the CC level, these target proteins were primarily involved in signaling pathways related to membrane, cytoplasm and plasma membrane (Fig. 7G). At the MF level, their core functions were concentrated in the protein binding, metal ion binding and nucleotide binding signaling pathways, whereas, at the BP level, the signal transduction, negative regulation of apoptotic process and lipid metabolic process signaling pathways represented the main enrichment directions (Fig. 7G). Additionally, KEGG pathway enrichment analysis revealed that the metabolic pathways, pathways in cancer and PI3K‐Akt signaling pathway pathways were potential key pathways through which CPF exerted its intervention effect on AFL (Fig. 7H). In‐depth exploration of the aforementioned enrichment analysis results could provide crucial reference for systematically clarifying the potential molecular mechanisms and core signaling pathway networks underlying CPF‐mediated regulation of AFL.

Mining of protein target information for CPF‐mediated regulation of AFL disease

The binding energies and binding sites between small molecule ligands from CPF and the screened macromolecular target proteins through molecular docking technology were investigated (Fig. 8 and Table 5). 3‐hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone was predicted to exhibit the strongest binding affinity to PTGS2, with a Vina score of −8.4 kcal mol⁻¹, and could achieve stable binding through conventional hydrogen bonds (binding site: ASNB375) and C—H hydrogen bonds (binding sites: ASNA375 and GLYB225) (Fig. 8 and Table 5). By contrast, the ligand was predicted to have relatively weak binding affinity to MMP9 and EGFR, with a Vina score of −6.9 kcal mol⁻¹ for both targets (Fig. 8 and Table 5). Additionally, significant differences were observed in binding affinity and interacting residues when different ligands acted on the same target TNF (Fig. 8 and Table 5). Kaempferol 3‐O‐beta‐d‐glucopyranosyl‐7‐O‐alpha‐l‐rhamnopyranoside was predicted to exhibit a higher binding affinity to TNF (−7.8 kcal mol⁻¹) than isorhamnetin‐3‐O‐neohesperidoside (−7.1 kcal mol⁻¹), and had more hydrogen bond‐interacting residues (binding sites: GLUC104, ALAA109, GLYA108, GLNB102, AGRB103, GLUA104 and GLUB104) compared to isorhamnetin‐3‐O‐neohesperidoside (binding sites: SERC9, LEUB157, LEUC157, LYSB11 and SERB52) (Fig. 8 and Table 5). These results could provide potential theoretical basis for the development of CP‐derived gene‐targeted foods with preventive and therapeutic effects against AFL disease.

Figure 8.

Binding energy and protein docking sites of CPF intervention in AFL through molecular docking analysis. (A) The binding energy and docking site information of the small molecule ligand 3‐hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone to the protein PTGS2. (B) The binding energy and docking site information of the small molecule ligand kaempferol 3‐O‐beta‐d‐glucopyranosyl‐7‐O‐alpha‐l‐rhamnopyranoside to the protein TNF. (C) The binding energy and docking site information of the small molecule ligand isorhamnetin‐3‐O‐neohesperidoside to the protein TNF. (D) The binding energy and docking site information of the small molecule ligand 3‐hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone to the protein SRC. (E) The binding energy and docking site information of the small molecule ligand 3‐hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone to the protein MMP9. (F) The binding energy and docking site information of the small molecule ligand 3‐hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone to the protein EGFR.

Table 5.

Key protein docking site information for CPF regulation of AFL disease

Number	Ligand name	Protein name	Vina score (kcal mol−1)	Conventional hydrogen bonds	C—H hydrogen bonds
A	3‐Hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone	PTGS2	−8.4	ASNB375	ASNA375, GLYB225
B	Kaempferol 3‐O‐beta‐d‐glucopyranosyl‐7‐O‐alpha‐l‐rhamnopyranoside	TNF	−7.8	GLUC104, ALAA109, GLYA108, GLNB102, AGRB103, GLUA104, GLUB104	ARGB103, PROC106
C	Isorhamnetin‐3‐O‐neohesperidoside	TNF	−7.1	SERC9, LEUB157, LEUC157, LYSB11, SERB52	/
D	3‐Hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone	SRC	−7.1	ARGA158	ARGA178
E	3‐Hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone	MMP9	−6.9	GLNA154, GLUA60	ALAA104, LYSA65
F	3‐Hydroxy‐3′,4′,5,7,8‐pentamethoxy‐flavone	EGFR	−6.9	THRA830, META769	LEUA694, PROA770, ASPA831

DISCUSSION

CP‐PU with favorable bioactive component content and in vivo antioxidant activity was prepared through PEF‐ultrasonic combined technology, and this result was consistent with the previous findings of Lee et al. ¹⁸ with respect to the preparation of polyphenol extracts from pineapple pulp using the PEF‐synergized ultrasonic technology. PEF could disrupt CP cell membranes to promote rapid diffusion of bioactive components, whereas ultrasonic treatment could rely on cavitation, mechanical vibration and turbulence effects to break down cellular barriers, facilitating intracellular component release. ²⁴ , ²⁵ , ²⁶ Meanwhile, the PEF and ultrasonic treatment conditions adopted in the present study ensured that the temperature of the extraction environment remained at a low level, which avoided the thermal degradation of thermosensitive bioactive components including polyphenols and flavonoids and preserved their nutritional and biological activities. ¹⁵ , ¹⁸ In addition, CPPU‐SU was found to have higher contents of bioactive components and superior in vivo antioxidant activity under non‐stressed conditions than CPPU‐SE. The core bioactive components responsible for the antioxidant activity in CP included small‐molecule substances such as polyphenols and flavonoids, as well as low‐molecular‐weight carbohydrates and proteins, and these components possessed solubility in alcoholic solutions, which resulted in a significant increase in the content of bioactive components in this fraction. ²⁷ By contrast, the alcohol precipitation precipitate CPPU‐SE was mainly composed of macromolecular substances with poor solubility in alcoholic solutions, such as macromolecular polysaccharides, proteins and dietary fiber. ²⁸ These substances had extremely weak antioxidant activity, and only a small number of bioactive components precipitate together with the macromolecules due to adsorption and complexation effects, leading to a low content of effective antioxidant components in CPPU‐SE. ²⁸

Component identification and analysis of the highly active CPSU‐H revealed that neohesperidin, isorhamnetin‐3‐o‐neohesperidoside, 3‐hydroxy‐3′,4′,5,7,8‐pentamethoxyflavone, narirutin and kaempferol 3‐O‐β‐d‐glucopyranosyl‐7‐O‐α‐l‐rhamnopyranoside were flavonoid small molecules widely distributed in citrus fruits, and the health‐promoting properties of these individual compounds have been proven. Neohesperidin could ameliorate intestinal pathology in colitis mice and accelerate wound healing; isorhamnetin‐3‐O‐neohesperidoside could inhibit angiogenesis/benign prostatic hyperplasia progression and exert anti‐inflammatory effects; and narirutin was shown to regulate gut microbiota and alleviates colitis‐related damage. ²⁹ , ³⁰ , ³¹ , ³² , ³³ These results could enrich the functional research of active flavonoids from citrus‐derived natural products, and further reveal the potential developmental prospect and application value of CPSU‐H in alleviating inflammation, repairing tissue damage and intervening in benign hyperplasia.

In the mechanistic investigation into the enhancement of antioxidant capacity under non‐stressed conditions of C. elegans by CPSU‐H intervention, multi‐omics analyses integrating transcriptomics and metabolomics identified the nucleocytoplasmic transport pathway and longevity‐regulating pathway as the potential key signaling pathways, alongside nucleotide metabolism as the potential core metabolic pathway. The nucleocytoplasmic transport pathway was identified to have pivotal for probiotic stress tolerance and linked to neurodegenerative disorders. ³⁴ , ³⁵ The longevity‐regulating pathway, a key mediator of bioactive compound‐induced antioxidant capacity in non‐stressed C. elegans, was found to play a crucial role in β‐sterol‐mediated healthspan improvement and regulate nematode behavioral traits and body length. ³⁶ , ³⁷ Nucleotide metabolism is a key pathway mediating CPSU‐H‐enhanced antioxidant capacity in non‐stressed C. elegans, which was aligned with the metabolic mechanism underlying the antioxidant capacity improvement of the nematode induced by pineapple pulp polyphenols in a previous study, and was identified as a critical metabolic pathway underlying the anti‐aging activity of polysaccharides. ¹⁵ , ³⁸

These results may provide multi‐omics evidence for deciphering the bioactive substance basis and regulatory network of CPSU‐H and fill the research gap regarding the mechanism by which citrus‐derived active components modulate the antioxidant activity under non‐stressed conditions of C. elegans. Meanwhile, these pathways are inferred to exert a wide range of extended physiological functions, including improving probiotic stress tolerance, intervening in neurodegenerative diseases, extending healthspan and regulating nematode phenotypes. These findings may deepen the current understanding of the action mechanisms of natural products, and offer a potential theoretical reference for the follow‐up exploration of CPSU‐H as probiotic preparations, neuroprotective candidates and functional foods.

AFL is a chronic progressive liver injury from long‐term excessive alcohol consumption and a major global cause of liver failure/transplantation because alcohol metabolism produces toxic substances (reactive oxygen species and acetaldehyde) that damage hepatocyte organelles (mitochondria and endoplasmic reticulum) and disrupt intracellular antioxidant systems. ³⁹ The present study provided a feasible potential basis for its application in preventing oxidative stress‐related liver injury in AFL by investigating the small‐molecule composition, in vivo antioxidant activity and underlying mechanism of CPSU‐H. Furthermore, the potential therapeutic targets for ameliorating AFL by screening CPF as the core small‐molecule component of CPSU‐H were explored, which offered a reference for the development of functional foods targeting the alleviation and treatment of AFL. CPF could exert potential preventive and therapeutic effects on AFL through targeted binding to PTGS2, TNF, SRC, MMP9 and EGFR proteins. Specifically, CPF might inhibit PTGS2‐mediated hepatic inflammatory responses and lipid peroxidation by targeting PTGS2, thereby alleviating alcohol‐induced inflammatory damage to hepatocytes. ⁴⁰ Targeting TNF exerted a suppressive effect on its pro‐inflammatory signal transduction, which in turn diminished the release of inflammatory factors and alleviated the impairment of hepatocytes caused by the hepatic inflammatory microenvironment. ⁴¹ The aberrant activation of downstream lipid synthesis‐associated pathways was inhibited by modulating SRC, which ameliorated hepatic lipid metabolic disorders and abrogated the abnormal lipid accumulation in hepatocytes. ⁴² MMP9 targeting abrogated its degradative action on the basement membrane of hepatic tissue, retarding the progression from alcoholic fatty liver to hepatic fibrosis. ⁴³ EGFR served as a therapeutic target for CPF, where its engagement modulated the EGFR‐mediated balance between hepatocellular proliferation and apoptosis, lessened excessive alcohol‐induced hepatocellular apoptosis and concomitantly suppressed hepatic stellate cell activation to abrogate the initiating phase of hepatic fibrosis. ⁴⁴

CP‐PU with a high content of bioactive components and potent in vivo antioxidant activity under non‐stressed conditions was prepared through PEF‐ultrasonic combined technology, and CPPU‐SU was identified as the core active fraction after alcohol precipitation. Citrus‐derived flavonoid small molecules including neohesperidin were confirmed as the core components of CPSU‐H, with the health‐regulating effects of each monomer validated by relevant evidence. The nucleocytoplasmic transport pathway, longevity‐regulating pathway and nucleotide metabolism were defined as the potential core pathways responsible for the enhancement of antioxidant capacity under non‐stressed conditions in C. elegans by CPSU‐H through integrated transcriptomic and metabolomic analyses, filling the research gap in the antioxidant regulatory mechanism of citrus‐derived active components. Additionally, CPF was screened as the core small molecule of CPSU‐H, and its potential to prevent and treat AFL through targeted binding to proteins including PTGS2 and TNF was discovered, providing novel targets for the development of functional foods against AFL. However, the research model for evaluating in vivo antioxidant activity was limited to C. elegans, lacking support from mammalian models and clinical data. The conservation of core pathways in higher organisms and the efficacy of CPSU‐H was remained unvalidated. Moreover, the synergistic mechanism of flavonoid components in CPSU‐H and the in vivo metabolic absorption process of CPSU‐H have not been investigated, making it impossible to determine the targeted action sites of active components and the activity of their metabolites. Therefore, future studies should construct mammalian models to verify the antioxidant, hepatoprotective and other effects of CPSU‐H and its core pathways in higher organisms, clarify the in vivo metabolic regularity of CPSU‐H and the targeted action sites of its active components, and develop functional foods for AFL based on the target proteins of CPF, aiming to promote the high‐value transformation of CP deep processing industry.

CONCLUSIONS

In the present study, CPSU‐H was directionally prepared through PEF‐ultrasonic synergistic extraction, ethanol‐based polarity fractionation, supernatant collection and HPD100 macroporous resin adsorption. It was found that CPSU‐H significantly reduced the MDA level in C. elegans to 0.32 ± 0.07 nmol mg protein⁻¹, at the same time as elevating the activities of T‐SOD, GSH‐Px and CAT to 80.35 ± 2.15, 148.73 ± 9.05 and 9.74 ± 0.52 U mg protein⁻¹, respectively, demonstrating potent in vivo antioxidant activity under non‐stressed conditions. A component of CPSU‐H was identified through HPLC‐MS/MS and found to be predominantly composed of citrus‐derived flavonoid small molecules, including neohesperidin, isorhamnetin‐3‐O‐neohesperidoside, 3‐hydroxy‐3′,4′,5,7,8‐pentamethoxyflavone, narirutin and kaempferol 3‐O‐β‐d‐glucopyranosyl‐7‐O‐α‐l‐rhamnopyranoside. Transcriptomic and metabolomic integrative analyses further predicted the potential underlying mechanism of CPSU‐H in regulating the antioxidant capacity under non‐stressed conditions of C. elegans, suggesting that the nucleocytoplasmic transport pathway and longevity‐regulating pathway acted as the key signaling pathways, with nucleotide metabolism putatively serving as the core metabolic pathway. Additionally, network pharmacology and molecular docking studies were conducted on CPF, which was the core small‐molecule fraction of CPSU‐H, and it was revealed that CPF exerted potential AFL‐intervening activity through targeted binding to PTGS2, TNF, SRC, MMP9 and EGFR proteins. These results collectively provided multi‐dimensional evidence for the bioactive substance basis and regulatory mechanisms of CPSU‐H, laying a foundation for the development of CPSU‐H as potential natural antioxidants and functional food ingredients targeting AFL, and also provided novel insights into the directional preparation of high‐activity components from CP and their high‐value utilization in the food industry.

AUTHOR CONTRIBUTIONS

SZ, PC and QL were responsible for conceptualization. SZ was responsible for reviewing and editing, supervision and project administration. SZ, LC and ZW were responsible for funding acquisition. PC and QL were responsible for writing the original draft, formal analysis, methodology and visualization. YW was responsible for supervision.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

DATA AVAILABILITY

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