Flavonoids contribute most to discriminating aged Guang Chenpi (Citrus reticulata ‘Chachi’) by spectrum‐effect relationship analysis between LC‐Q‐Orbitrap/MS fingerprint and ameliorating spleen deficiency activity

Abstract To further explore the mechanism of “the longer storage time, the better bioactivity” of aged Guang Chenpi, the dry pericarp of Citrus reticulata ‘Chachi’ (CRC), a series of activity assessments were performed on spleen deficiency mice. The constituents in CRC with different storage years were analyzed by LC‐Q‐Orbitrap/MS. A total of 53 compounds were identified, and CRC stored for more than 5 years showed higher flavonoid content, especially that of polymethoxyflavones. Anti‐spleen deficiency bioactivity analysis among various CRC with different storage years showed aged CRC (stored for more than 3 years) could significantly alleviate fatigue and depression behaviors much better, increase D‐xylose and gastrin secretion, and upregulate the expression of the linking protein occludin in the colon walls. Results from 16S rDNA sequencing showed that aged CRC could downregulate the abundance of Enterococcus, Gemmata, Citrobacter, Escherichia_Shigella, and Klebsiella, which were significantly overrepresented in the model group. Bacteroides, Muribaculum, Alloprevotella, Paraprevotella, Alistipes, Eisenbergiella, and Colidextribacter were downregulated in the model group but enriched in the CRC groups. At last, the spectrum‐effect relationship analysis indicated that flavonoids such as citrusin III, homoeriodictyol, hesperidin, nobiletin, and isosinensetin in aged CRC showed the highest correlation with better activity in ameliorating spleen deficiency by regulating gut microbiota. Flavonoids contribute most to discriminating aged CRC and could disclose the basis of “the longer storage time, the better bioactivity” of aged Guang Chenpi.


| INTRODUC TI ON
"The longer storage time, the better bioactivity" is known as "Chen Jiu Zhe Liang" in Chinese, which means that herbal medicine should be stored for many years before being used in the clinic (Nijat et al., 2022).This is one of the important methods of preprocessing traditional Chinese medicine that has been recorded for many Chinese medicines (Luo et al., 2019).Guang Chenpi is the dry pericarp of the fruits of Citrus reticulata "Chachi" (CRC) that is mainly planted in Guangdong province.It is recorded in the Chinese Pharmacopeia (2020 edition) to exert bioactivities such as regulating qi and strengthening the spleen, drying dampness, and eliminating phlegm (Yu et al., 2018).Guang Chenpi is also widely used as a medicinal food in South China and other Asian countries (Xu et al., 2014).Currently, the market price of Guang Chenpi is related to its storage time.However, there is not enough evidence to confirm the relationship between its bioactivity and extended storage time.Therefore, studying the correlation between bioactivity and extended storage time may explain the mechanism of the traditional uses of "Chen Jiu Zhe Liang." The chemical constituents of Guang Chenpi mainly include flavonoids, volatile oils, alkaloids, and polysaccharides (Yu et al., 2018).
Evidence indicates that flavonoids and volatile oils can exert liver protection, hyperglycemia, pancreatic protective, and anticancer effects owing to their anti-inflammatory, antioxidant, antibacterial, and glycosidase-inhibiting effects (Bhandari et al., 2021;Elhelaly et al., 2019;Manthey et al., 2001;Zeng et al., 2018).The flavonoid glycoside and polymethoxyflavonoid content of Guang Chenpi obtained from different areas of cultivation and after varying harvest times differ considerably (Zheng et al., 2021).Furthermore, some studies show that its polymethoxylated flavone and antioxidant content increase with an increase in storage years (Fu et al., 2017;Zeng et al., 2017).Headspace-solid phase microextraction combined with GC-MS has been used not only to analyze flavonoids but also the volatile oil components in Citri Reticulatae Pericarpium (CRP) after different harvest times (Zheng et al., 2018).Furthermore, "activity fingerprints" have been established for Chenpi samples from different cultivars (Zheng, Chao, et al., 2020), and near-infrared spectroscopy has been used to establish a qualitative analysis model of CRP to distinguish between samples from different origins (Li et al., 2020).The DNA barcoding method has also been used to effectively distinguish Guang Chenpi and CRP from other related species.It provides a molecular basis for the identification of medicinal materials (Liu et al., 2021).The above evidence shows that chemical analysis can reveal the components that are present; however, studies on their correlation with the bioactivity of Guang Chenpi are lacking.Therefore, it is meaningful to develop an accurate method to explore the changes in bioactivity resulting from different years of storage.
Long-term and extensive clinical applications of Guang Chenpi reveal that it ameliorates spleen deficiency-related diseases, including indigestion, lack of appetite, abdominal fullness, and distention (Yu et al., 2018).Its mechanism of alleviating spleen deficiency has been reported to be related to the modulation of the gut microbiota.
16S rDNA gene sequencing shows that CRP can restore the abundance of gut microbiota in rats with spleen deficiency not only by upregulating some of the short-chain fatty acid-producing and antiinflammatory bacteria but also by downregulating certain spleen deficiency-aggravating bacteria (Zheng, Zeng, et al., 2020).CRP also modulates the immune system (Zhou et al., 2021).CRP flavonoids can modulate the mRNA expression of gastric hormones CD3~+ and TFF3 in rats with spleen deficiency.
Although a few studies on active differences between aged and within-year Guang Chenpi have been reported, no data are available on whether the aged samples have better activities.Hene, the gut microbiota composition, gastric hormone levels, and normal behaviors of rats were determined in this study.The chemical spectra and component differentiations of Guang Chenpi samples stored for 1-36 years were analyzed.The model of spectrum-effect relationship established in this study will pave the way to exploring the theoretical mechanism of "Chen Jiu Zhe Liang" in the preprocessing of Guang Chenpi.

| Guang Chenpi sample collection and preparation
Guang Chenpi (Citrus reticulata 'Chachi') was collected from the Xinhui area in the Guangdong Province and identified by Professor Yang Quan at Guangdong Pharmaceutical University, Guangzhou, China.
The storage period ranged from 1 to 36 years.
Smashed CRC samples (passed through a 40-mesh sieve) were boiled with water and filtered twice.The filtrates were combined and concentrated to 1 g/mL.The positive control drug, Shen Ling Bai Zhu powder, was dissolved in water to yield a concentration of 1.5 g/mL (Luo et al., 2022).The aqueous extract of senna leaf was boiled in water for 5 min in a 1:5 ratio of material: liquid, filtered, and concentrated to 1.5 g/mL (Ma et al., 2019).

| LC-MS analysis
The chemical constituents of Guang Chenpi water extract were analyzed using high-performance liquid chromatography (HPLC) and a Waters Symmetry C18 column (4.6 mm × 250 mm, 5 μm).The mobile phases were aqueous 0.1% formic acid (A) and acetonitrile (B).The gradient elution was as follows: 0-10 min, 10%-25% B; 10-20 min, 25%-60% B; 20-30 min, 60%-95% B. The flow rate was 1 mL/min, the column temperature was 30°C, and the injection volume was 3 μL.Mass spectrometry (MS) was performed using a Q Exactive Focus mass spectrometer (Thermo Fisher Scientific Co., Waltham, USA) combined with an electrospray ion source and positive and negative ion switching scanning modes.The scanning range was 150-2000 m/z, the full MS primary resolution was 35,000, and the secondary resolution was 17,500.The ion-spray voltage was 3.5 kV, the ion-source temperature was 320°C, the sheath gas flow rate was 35 arb, the auxiliary gas flow rate was 15 arb, and the ion-transport tube temperature was 320°C.The collision energy gradient was 20, 40, and 60 eV.Identification of compounds was done using Compound Discoverer software (version 3.0), and the original data were matched with mzCloud and the Chinese Medicine Compounds (OTCML) database.

| Establishment of the mouse model of spleen deficiency
An equal number of 6-7-week-old male and female SPF-grade Swiss mice weighting 20-25 g were purchased from Guangdong Medical Laboratory Animal Center (SCXK2018-0002).All mice were housed at a constant temperature (26 ± 2°C) and standard humidity and subjected to appropriate light/dark conditions.Mice were allowed free access to water.All procedures were approved by the Institutional Animal Use Committee (Ethics Committee of GDPU on Laboratory Animal Care, No. gdpulacspf2017448).
Except for those in the control group, all other mice were administered aqueous senna extract intragastrically (i.g.0.2 mL/20 g).Mice in the control group received a similar volume of normal saline by gavage for 14 days.Mice in each group were forced to swim once daily in a water tank for 14 consecutive days.The water temperature was 25°C, and the tank depth was 30 cm.Moreover, mice in each group were fasted every other day for 14 days (Zhang et al., 2021).Mice in the control and model groups were administered normal saline (i.g.0.2 mL/20 g), whereas those in the CRC groups received a corresponding dose of CRC extract (i.g. 2 g/kg) and Shen Ling Bai Zhu powder for 14 days.The administered dose is calculated as 6-9 g for one person per day, according to the Chinese Pharmacopeia (2020 edition).

| Sugar preference test
After adaptive sugar-water training, two identical water bottles of equal volumes were placed in each cage.For the first 24 h, 1% sucrose water was filled into both water bottles.Then, the solution in one of the bottles was replaced with pure water, whereas the contents of the other bottle were not changed.The sugar-water preference test was conducted on mice in each group on the 7th day after administration.Mice in each group were fasted for 24 h before the formal experiment.The consumption volume of sugar and pure water for 1 h was recorded (Zhang, Niu, Zhang, et al., 2022).The sugar water preference rate (W%) = sugar water intake/(sugar water intake + pure water intake) × 100%.

| Tail-suspension test
The tail-suspension test was performed after 8 days of drug administration.Briefly, the tail of each mouse was fixed on top of an iron platform such that the mouse was inverted and suspended with its head approximately 15 cm from the ground.Mice were observed for 6 min, and the activity in the last 4 min was recorded.The immobility time of each mouse was calculated (Yang et al., 2022).

| Forced-swimming test
After drug administration for 9 days, mice were forced to swim in a 15-cm-deep glass beaker for 6 min.The 6 min for which they floated were recorded, and the duration of immobility for the last 4 min was calculated (Zhu et al., 2022).

| Open-field test
The open-field test (OFT) was performed using a KW-OPF openfield activity box (Guangzhou Bite Biotechnology Co., Ltd., Guangzhou, China) after 10 days of drug administration.Mice were gently placed in an open box with black walls and a bottom.The test was conducted in absolute silence and without any direct light source.
Behavioral changes within 5 min, including the total distance and the time spent in the central area (25 × 25 cm), were recorded using a synchronous camera system (Hu et al., 2022).After each experiment, 75% ethanol was sprayed on the bottom and surrounding surfaces of the open-field activity box and dried with a paper towel to avoid the influence of the residual odor of rats on subsequent tests involving a different batch of rats.

| Determination of D-xylose and gastrin levels
For the determination of serum D-xylose levels, mice were fasted for 12 h before the i.g.administration (5%, 0.5 mL) of D-xylose solution.Serum from mice was collected after 30 min.Mice were anesthetized using 1.25% avertin, and their whole blood was collected after removing their eyeballs.The blood was allowed to stand for 4 h at 4°C.The upper layer of serum was collected for analysis.Serum D-xylose (20210823, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and gastrin (202104, Wuhan Aidi Biotechnology Co., Ltd., Wuhan, China) levels were determined using the phloroglucinol method according to the manufacturer's instructions.

| Determination of intestinal propulsive ratio and gastric residual rate
Mice were administered a mixture of activated carbon and 10% sodium carboxymethyl cellulose.After 1 h, they were anesthetized using 1.25% avertin.Blood was collected, the pylorus was ligated, and the mesentery was stripped.The intestine from the pylorus to the ileocecal junction was cut and gently placed on the tray in a straight line.The total length of the small intestine was measured, and the distance from the pylorus to the furthest point to which the carbon had advanced in the intestine was recorded (Deng et al., 2018).The intestinal propulsive ratio was calculated as the distance traveled by carbon in the intestine/ the total length of the small intestine × 100%.The gastric residual rate was calculated as follows: (total weight of the stomach−net weight of the stomach)/weight of the carbon paste × 100%.

| 16S rDNA gene sequencing of fecal samples
The DNA from the samples was extracted using an E.Z.N.A.® Stool DNA kit according to the manufacturer's instructions.Total DNA samples were diluted with 50 μL elution buffer and stored at −80°C until a polymerase chain reaction was performed (LC-BioTechnology Co., Ltd.).The V4 regions of 16S rDNA were amplified using 341F (5′-CCTAC GGG NGG CWG CAG-3′), 805R (5′-GACTA CHV GGG TAT CTA ATCC-3′).Samples were sequenced on the NovaSeq PE250 platform according to the standard protocols.Sequencing data were processed using the Quantitative Insights Into Microbial Ecology (QIIME, v1.8.0) software.

| Histopathology analysis
Colon tissues were immersed in 4% paraformaldehyde to fix for dehydration and then paraffin-embedded and sectioned for hematoxylin and eosin (H&E) staining.Pathological changes were observed using the OLYMPUS/BX fluorescence microscope (Guangzhou Zhongchuang Biotechnology Co., Ltd., Guangzhou, China).

| Western blotting
The total protein of colon tissue was extracted by adding a RIPA lysis solution containing a phosphatase inhibitor and protease inhibitor (1:100).The total protein concentration was determined using a bicinchoninic acid assay kit (BCA) (P0012S, Beyotime Biotechnology, Shanghai, China).After quantification, 40 μg protein was used for the electrophoretic separation on a 10% separation gel.Next, the protein was transferred to a polyvinylidene fluoride membrane.
After blocking, they were incubated at 4°C with the primary antibody occludin (1:10,000, ab167161, Abcam, English) for 12 h.After washing three times with TBST solution (10 min/wash), the secondary antibody (1:10,000, NO. 7074S, Cell Signaling Technology, USA) was added and further incubated at 26°C for 1 h.An enhanced chemiluminescence chromogenic solution was added for imaging, and the gray value of the bands was measured using ImageJ software.

| Gray relational analysis (GRA)
GRA is a method to study the degree of similarity or dissimilarity of development trends among factors.It is used to measure the degree of association between different factors and to evaluate the degree of correlation between the components and pharmacological effects of traditional Chinese medicine (Xiao et al., 2022;Yang et al., 2022;Zhang, Yu, Yang, et al., 2022).In this study, pharmaceutical factors correlating with spleen deficiency from CRC samples with different storage years were used as the mother sequence, and each characteristic peak quantification index was used as the sub-sequence.
The original data were dimensionless and processed using the mean value method.The resolution coefficient (ξ) was 0.5, and the gray correlation coefficients and correlation magnitudes were calculated (Qi et al., 2022).At the same time, the mean value was also used to calculate a composite score for spectral efficiency correlation, and the scores were ranked.By analyzing the magnitude of correlation between compounds and pharmacological effects, the contribution of each characteristic peak of CRC to ameliorating spleen deficiency symptoms was determined.

| Statistical analysis
Data were analyzed using GraphPad Prism, and the results are expressed as the mean ± standard error of the mean.Statistically significant differences were analyzed using one-way analysis of variance (ANOVA) with Tukey's post hoc test.p < .05 was considered significant.Compound Discoverer 3.0 software was used to accurately fit the molecular formula and primary mass spectrum of compounds.
The results were matched with those in the mzCloud network database and the OTCML database, and the chromatographic peaks were determined.To further speculate on and identify the structure of compounds, the ion information of the primary and secondary segments of the filtered target compounds was compared with the corresponding reference materials.Results from GRA were imported into Origin software 2021 to generate a heat map, which was used to visualize changes in the spectrum efficiency correlation coefficient.

| Guang Chenpi could rescue spleen deficiency-induced fatigue
Guang Chenpi is the dry pericarp from the fruit of CRC obtained from the Guangdong province in China."Chen Jiu Zhe Liang" means "longer storage years and better activities."An extended storage period is a representative approach used in traditional Chinese medicine (Fu et al., 2017).To explore the scientific mechanism of this method, we first performed a pharmacological evaluation with respect to the amelioration of spleen deficiency using Guang Chenpi samples stored for different years.Spleen deficiency is associated with Qi deficiency, digestive dysfunction, and other symptoms of fatigue in Chinese medicine theory (Wang et al., 2019).To mimic these symptoms, mice were subjected to the starvation and overeating method in combination with the i.g.administration of aqueous senna leaf extract and to physical exhaustion by forced swimming (Zhang et al., 2020).In agreement with previous studies, mice with spleen deficiency exhibited symptoms of fatigue and digestive dysfunction, indicating the successful establishment of the model (Zhang, Yu, Zhang, et al., 2022).The complete experimental protocols are listed in Figure 1a.After the treatment on the 7th day, the model mice had loose stools, appeared tired, had unpolished fur, did not move freely (Figure S1a), and lost weight (Figure 1b) compared with control mice, suggesting the successful establishment of the mouse model of spleen deficiency.Mice with spleen deficiency traveled a shorter total distance and exhibited lesser in-zone duration in the OFT (p < .01,p < .001),suggesting that the spontaneous movement of model mice had decreased (Figure 1c,d).In the tail-suspension and forced swimming tests, model mice exhibited more immobility time than control mice (p < .01,p < .01),which was indicative of fatigue (Figure 1e,f).In the sucrose preference test, the preference rate of model mice was lower than that of control mice (p < .01)(Figure 1g).Treatment with SLBZ or CRC could rescue behavioral dysfunction in mice with spleen deficiency.The immobility time of mice in the CRC groups with long storage years (7-36 years) was shorter compared with that of mice in the CRC groups with short storage years (1-5 years) (p < .01).This finding indicated that Guang Chenpi extracts with different storage years could rescue spleen deficiencyinduced fatigue and that the fatigue-ameliorating effect increased with an increase in storage years.

| Guang Chenpi could ameliorate spleen deficiency-induced digestive dysfunction
We further evaluated the amelioration of digestive dysfunction, gastric residual rate, and gastric emptying rate after administration of different Guang Chenpi samples.As shown in Figure 2a,b, the gastric residual rate in spleen deficiency mice was significantly higher (p < .01).The intestinal propulsive rate was lower than that in mice with spleen deficiency, but the difference was not significant.After the administration of Guang Chenpi extracts, intestinal propulsive rates were nearly identical to those of the control mice, indicating that Guang Chenpi could promote the digestive ability of mice with spleen deficiency.D-xylose is a pentose that is not detected in the blood.D-xylose levels in the blood can be used to indirectly evaluate the absorption function of intestinal mucosa after administering a certain dose of D-xylose solution (You et al., 2020).secretion.Spleen deficiency can also influence the immune system.Thus, we determined the organ indices of the spleen and thymus.As shown in Figure 2e,f, these indices were significantly decreased in model mice (p < .01)and rescued after the administration of Guang Chenpi samples.However, there were no significant differences in outcomes after the use of within-year and aged samples.
The ameliorative effect increased with an increase in storage years compared with that of the within-year sample.In addition, there were no significant differences among all indices, except Dxylose and GAS levels, when Guang Chenpi extracts from different storage years were used.Additionally, aged Guang Chenpi samples could influence the digestive system by ameliorating gastric and intestinal function much more than samples aged for only 1-5 years.

| Guang Chenpi with different storage years could modulate the abundance and composition of the gut microbiota
Intestinal integrity is altered by dysfunction resulting from certain diseases (Shi et al., 2020).To evaluate the intestinal permeability and ameliorative effects of Guang Chenpi in mice with spleen deficiency, levels of the linking protein occludin were determined and its distribution in the colon was monitored (Figure S1b).Occludin expression in colon tissues in the model group was significantly lower than that in the control group (p < .05).Occludin expression was significantly higher in the CRC-36Y group than in the model group (p < .05);however, there were no significant differences in expression between the CRC-1Y and model groups.In addition, H&E staining of colon tissues from the model group showed a thinner intestinal wall, which could be rescued by treatment with Guang Chenpi (Figure S1c).
These findings showed that CRC-1Y, CRC-5Y, and CRC-36Y could repair the intestinal barrier in mice with spleen deficiency.The gut microbiota plays an important role in good health, and its dysfunction has been associated with many diseases (Qian et al., 2021;Tung et al., 2018).To further explore the correlation between gut microbiota and Guang Chenpi samples (different storage years) in mice with spleen deficiency, 16S rRNA gene sequencing was used to analyze the fecal samples of mice after the oral administration of Guang Chenpi stored for 5 and 36 years.Therefore, feces samples from mice treated with CRC-5Y and CRC-36Y were chosen for gut microbiota analysis.
A key metric for analyzing microbial diversity was diversity, which was used to determine Chao1, Shannon, Simpson, and goods-coverage diversity measures.According to this information, the model group showed a lower level of alpha diversity compared with the control and CRC groups (Figure 3a Furthermore, linear discriminant analysis (LDA) effect size (LEfSe) was used to determine the differential microbial composition among groups.According to a cladogram that records the hierarchical taxonomic structure of the gut microbiota, there are notable differences in the phylogenetic distributions among the phyla and genera among the groups (Figure 4a).By using a logarithmic cutoff point of 3.0, the LDA score has been calculated to indicate that the genera Enterococcus, Gemmata, Citrobacter, Esch-erichia_Shigella, and Klebsiella were significantly overrepresented in the model group.Administration of aged Guang Chenpi extracts downregulated these levels.Bacteroides, Muribaculaceae_unclassified, Alloprevotella, Paraprevotella, Alistipes, Eisenbergiella, and Colidextribacter were downregulated in the model group but enriched in the CRC groups.The heat map further demonstrated the differences in gut microbiota among the three groups (Figure 4b and Figure S2).
The relative abundance of the genera Enterobacter, Enterococcus, Citrobacter, and Escherichia-Shigella was significantly lower in the model group compared with that in the CRC group, whereas that of Alloprevotella and Tannerellaceae_unclassified increased after CRC treatment in mice with spleen deficiency (Figure 5).Some of these results overlap with those from LEfSe, suggesting that these microbes may be the target of CRC.To further compare the microbiota abundance between the CRC-5Y and CRC-36Y groups, the biomarker in LEfSe showed that Eubacterium_coprostanoligenes_ group_unclassified was significantly higher in the CRC-36Y than in the CRC-5Y group (Figure S2).There were significant differences in abundance between the CRC-5Y and CRC-36Y groups (p < .05).
These data showed that aged Guang Chenpi extracts could restore intestinal wall integrity as well as enteric microbial richness and diversity in mice with spleen deficiency.

| Characteristics of Guang Chenpi with different storage years determined using LC-Q-Exactive Orbitrap/MS
To compare the chemical constituents among Guang Chenpi of different storage years and their association with bioactivities, LC-Q-Exactive Orbitrap/MS was used to analyze the chemical constituents  1. Analysis of the chemical composition of Guang Chenpi revealed a change in the chemical contents with storage years.Flavonoids account for 89% of the total compounds, and 42 common peaks were identified in all samples, including alkaloids (1, 25), organic acids (21), limonin (22, 24, 39), and flavonoids (2-20, 23, 26-28, 40-53).These peak numbers were listed in Table 1.Flavanone aglycone, flavone-c-glycosides, flavone-O-glycosides, and polymethoxyflavones (PMFs) were the flavonoids in the samples.Changes in the contents of chemical constituents, including 3′,4′,5 ,5′,7-pentamethoxyflavone, 3′,4′,3,5,6,7-hexameth oxyflavone, nobiletin isomer, and orientin, were observed with an increase in storage years.Furthermore, the PMF content of samples from different storage years was compared; the levels were high during the first 5 years and decreased beyond 7 years of storage (Figure 6e).Overall, the sample stored for 5 years showed a higher flavonoid content, especially that of PMFs, compared with those stored for other time periods.
The flavone and PMFs in Guang Chenpi exert several effects such as antioxidant, digestion-promoting, cough-and asthmarelieving, and anticancer (Bai et al., 2017;Benavente-Garcia & Castillo, 2008;Keshari et al., 2016;Roohbakhsh et al., 2015).Especially, citrus PMFs can attenuate the metabolic syndrome by regulating the gut microbiome and amino acid metabolism (Zeng et al., 2020).Thirty-one metabolites have been identified to distinguish Citri Reticulatae Chachi Pericarpium in samples from different storage years.The metabolite levels increase when stored for 3-10 years and decrease after 15-30 years of storage (Luo et al., 2019).Polymethoxyflavones tangeretin and 3,5,6,7,8,3′,4′ -heptamethoxyflavone have been reported as the most important PMFs in distinguishing various Guang Chenpi according to storage years, which contributed most to anticancer effects (Tao et al., 2022).The existing studies also focus on the differences in harvest time, such as Guang Chenpi collected in the early months F I G U R E 5 Comparison of the gut microbiota among the Model, CRC5Y, and CRC36Y groups.Differences in the relative abundance of gut microbiota at the genus level (Mann-Whitney U test).The box presents the 95% Cls, and the line inside denotes the median.containing higher concentrations of bioactive flavonoids and exhibiting potent anti-lipase activity (Zeng et al., 2018).Nonvolatile components are considered the characteristic components for identifying Guang Chenpi harvest at different stages (Zheng et al., 2021).Targeted metabolomics technology combined with total antioxidant capacity analysis showed that flavones in CRP with different harvest times were endowed with different efficacy and usage (Liang et al., 2022).As for the anti-spleen deficiency bioactivity identified in this research, some PMFs were also identified as the most important compounds in distinguishing storage years, especially the 5-year ones.As a result, flavone compound content was mostly different among Guang Chenpi samples stored for various years.

| Spectrum-effect relationship between chemical fingerprints and amelioration of spleen deficiency
A total of 53 peaks and ten activity indices were used for gray relational analysis (GRA).The mean coefficient of the 29 peaks was >0.8, indicating a strong correlation, and the mean correlation coefficient of the other 13 peaks was between 0.7 and 0.8, indicating a moderate correlation (Table 2).These findings indicate that Guang Chenpi contains multiple compounds that exert synergistic effects.Peaks 18,15,14,30,and 35 were the top five ranked peaks that correlated with the activity of ameliorating spleen deficiency and, hence, may be the major bioactive components of Guang Chenpi

TA B L E 2
The correlation coefficient between indexes of spleen deficiency and the content of common peaks. NO.

F
I G U R E 1 Guang Chenpi with different storage years could alleviate fatigue induced by spleen deficiency.(a) Experimental procedure.(b) Body weight loss was reversed by Guang Chenpi.(c, d) Total distance and inzone duration of each group in the open-field test.(e) Immobility time of each group in the tail-suspension test.(f) Immobility time in the forced swimming test.(g) Sucrose preference test for each group.(#p < .05,##p < .01,###p < .001,compared with control group; *p < .05,**p < .01,***p < .001,compared with model group; △p < .05,△△p < .01,△△△p < .001,compared with CRC-1Y group).All data are presented as mean ± SEM.
Serum D-xylose levels reflect the absorption ability of the small intestine.As shown in Figure 2c, D-xylose levels in model mice were significantly lower than those in control mice (p < .01),suggesting absorption dysfunction in the intestine.Guang Chenpi samples with different storage years could increase D-xylose levels, especially when samples stored for longer than 7 years were used.GAS secreted by the G cells in the gastric antrum is an important index that indicates the physiological function of the gastrointestinal tract (Niu et al., 2020).Gastrin is another important index for measuring the physiological function of the gastrointestinal tract.Guang Chenpi samples could restore lowered gastrin levels induced by spleen deficiency (Figure 2d).Guang Chenpi samples stored for more than 7 years could promote gastrin F I G U R E 2 Guang Chenpi with different storage years could ameliorate gastric and immune organ dysfunction.(a, b) The effect of Guang Chenpi on gastric remnants and emptying.(c) The effect of Guang Chenpi on serum D-xylose levels.(d) The effect of Guang Chenpi on serum gastrin levels.(e, f) Spleen and thymus indices of each group.(#p < .05,##p < .01,###p < .001,compared with control group; *p < 0.05, **p < 0.01, ***p < .001,compared with model group; △p < .05,△△p < .01,△△△p < .001,compared with CRC-1Y group).All data are presented as mean ± SEM.
-e), suggesting that Guang Chenpi treatment might increase the abundance of gut microbes.Principal coordinate analysis (PCoA) of weighted UniFrac distance matrices was used to analyze the bacterial communities of these groups.According to PCoA's first principle, the model group is separated from the control and CRC groups.It was revealed that bacteria belong to the Bacteroidetes, Firmicutes, and Proteobacteria phyla, which were the most dominant in mice's colons (62.93%, 17.74%, and 14.73%) (Figure 3f).As shown in Figure 3g, different groups had different dominant phyla.A model group was dominated by proteobacteria, firmicutes, and bacteroidetes, whereas CRCs and controls were dominated by bacteroidetes and firmicutes.Using the unweighted pair group method with the arithmetic mean tree, almost all mice in the model CRC groups were found to be clustered together, distinctly separated from the model group, and clustered together with the control group (Figure 3g).These findings showed that similar alpha and beta diversity was observed in the intestinal microbiota in the control and CRC groups.The abundance and composition of the gut microbiota in the model group changed markedly.

F
Gut microbial abundance at the phylum level.(a-e) Alpha diversity based on observing OTUs, the Chao1 index, the Shannon index, Simpson index and goods_coverage index.(f) PCoA plots were constructed using the weighted UniFrac PCoA method.(g) Composition and relationship of intestinal microbiota in the Control, Model, CRC5Y, and CRC36Y groups.(*p < .05,**p < .01,***p < .001).All data are presented as mean ± SEM.F I G U R E 4 Differences in the composition of the gut microbiota among different groups.(a) Taxonomic cladogram obtained from LEfSe analysis, representing the abundance of microbiota.(b) Heatmap plot depicting the normalized abundance of each microbiota genus in the Control, Model, CRC5Y, and CRC36Y groups.(*p < .05,**p < .01,***p < .001).All data are presented as mean ± SEM. of Guang Chenpi water extract.The procedure is shown in Figure 6a.The total ion current from the positive and negative ion modes reveals different chemical constituents (Figure 6b,c).The similarities and differences can also be observed in the representative chromatograms of different storage years (Figure 6d).A total of 53 compounds were identified from Guang Chenpi samples.Quasi-molecular ions exhibited equivalence to [M+H] + and [M−H] − and were unambiguously or tentatively identified through alignment with accurate molecular weights within the mass accuracy of <10 ppm.Detailed information on the chemical compounds and their distribution in Guang Chenpi samples with different storage years is shown in Table

F
I G U R E 6 Analysis of the chemical components of Guang Chenpi water extracts using LC-Q-Orbitrap/MS.(a) Procedure of the spectrum-effect analysis.(b, c) Typical total ion chromatograms of samples by LC-QOrbitrap/MS in positive and negative ion modes.(d) The fingerprints of different samples.(e) Contents of different flavones in different samples.TA B L E 1 Identification based on UPLC-Q-Exactive Orbitrap/MS of Citri Reticulatae Pericarpium with different storage years.

(
Figure 7a).The structures of these five compounds (alkaloid, flavonoid, and PMFs) are identified in Figure 7b.There was a change in the levels of chemical contents after different storage years.As shown in Figure 7c, with an increase in storage years, peak areas of 18, 30, and 35 tended to increase, whereas peak areas of 15 and 14 did not change significantly.Spectrum-effect analysis revealed that besides the uncommon peaks, the common peaks from different samples, especially F I G U R E 7 Spectrum-effect correlation between the relative content of the common peaks and bioactivity indices using gray relational grade.(a) Heatmap of spectrum-effect correlation analysis created using gray relational grade.(b) Structures of the most correlated compounds based on gray relational grade.(c) Relative contents of the five compounds in different samples.