Microbial Metabolism of Naringin and the Impact on Antioxidant Capacity

Naringin is a dietary flavonoid glycoside with broad bioactivities, and it has been found to undergo extensive microbial metabolism in human gut. Microbial metabolites are believed to play an important role in the overall bioactivity of naringin. However, knowledge is scarce about its microbial metabolism in laboratory rats, which are the most commonly used animal model for naringin-related biomedical studies. Herein, we profiled the microbial metabolism of naringin in rat by an in vitro anaerobic fermentation combined with LC-MS/MS methods. A total of 35 microbial metabolites were identified, and corresponding metabolic pathways were proposed. Naringin and its metabolites were further quantified in fermentation samples. Rhoifolin, neoeriocitrin, neohesperidin, naringenin, methylated naringin, and hydroxylated naringin were detected as the primary microbial metabolites. Moreover, antioxidant capacity assays suggested that fermentation-associated microbial metabolites exhibited higher antioxidant activity than original naringin. Obtained results contribute to a more comprehensive understanding of the microbial metabolism and antioxidant capacity of naringin.

Gut-microbiota-related gastrointestinal digestion is believed to contribute significantly to the bioavailability of dietary polyphenols [11], as well as their physiological function (antioxidant [12], antimicrobial [13], and anti-inflammatory activities [14]). To date, a number of studies have reported the metabolic profiles of naringin in vitro and in vivo [9,15]. In the gastrointestinal tract, microbial enzymes could eliminate the glycoside of naringin to yield its aglycone naringenin, metabolize it into other flavonoid metabolites, and further degrade it into a variety of ring-fission products [16][17][18]. Recent studies have revealed the important role of these microbial metabolites in the overall bioactivity of naringin [19][20][21]. In fact, the physiological effects of naringin cannot be fully achieved in the absence of microbial metabolites due to the extensive biotransformation of parent compound [22]. Therefore, it is important to profile the gut microbial metabolism of naringin.
In vitro anaerobic fermentation is a practical method to investigate gut microbiotamediated metabolism of natural products, such as flavonoids [23], ginsenosides [24], and theaflavins [25]. To date, several studies have reported the human intestinal microbial metabolism of naringin [26][27][28]. However, information concerning the microbial

In Vitro Fermentation of Naringin with Rat Gut Microbiota
The in vitro fecal fermentation of naringin was conducted in the light of the methodology reported by Ma et al. [30], with some optimizations. Fecal materials were collected from the small intestinal and caecal lumen, processed separately, and used for subsequent fermentation assays to investigate whether there were differences in the metabolic capacity of intestinal microbes at different locations. After collection, fecal material (1.0 g) was accurately weighed and homogenized with cold saline in a ratio of 1:4 (w/v) to afford fecal slurry. The fecal slurry was centrifuged at 5000× g for 30 min at 4 • C to obtain corresponding rat fecal suspension. Subsequently, an aliquot of 190 µL rat fecal suspen-sion was spiked with 1710 µL fluid medium (obtained by dissolving the agar medium base in water and then autoclaving at 121 • C) and 100 µL naringin solution (prepared by dissolving the solid naringin in methanol at a concentration of 10 µmol/mL). The final concentrations of naringin were 500 nmol/mL. The mixture was then incubated at 37 • C for 12 h in an anaerobic condition. To identify and quantify the metabolites derived from naringin, aliquots of 100 µL samples were taken after 12 h fermentation and immediately vortex-mixed with 200 µL cold acetonitrile (containing internal standard at a concentration of 1 µg/mL) for 3 min. After centrifugation (15,000× g, 30 min, 4 • C), the supernatants were collected and stored at −70 • C until LC-MS/MS analysis.

Identification of the Microbial Metabolites of Naringin by UHPLC-Q-TOF-MS/MS
To identify the microbial metabolites of naringin, obtained supernatants were analyzed with a UHPLC system (Nexera X2; Shimadzu, Tokyo, Japan) tandem hybrid triple quadrupole time-of-flight mass spectrometer (Triple TOF 5600 plus; Sciex, Foster City, FL, USA) equipped with an electrospray ionization (ESI) source. Detailed parameters of chromatographic separation and MS/MS detection were the same as our preliminary studies [18,31]. Instrument operation and data acquisition were performed with Analyst software (version TF 1.6; Sciex, Foster City, FL, USA), while the data processing was conducted with PeakView software (version 1.2; Sciex, Foster City, FL, USA).

Quantification of the Microbial Metabolites of Naringin by UHPLC-Q-Trap-MS/MS
To quantitatively profile the microbial metabolism of naringin in rat fecal fermentation samples, a UHPLC-Q-Trap-MS/MS was developed and validated for the simultaneous determination of naringin and its primary metabolites, including rhoifolin, neoeriocitrin, neohesperidin, naringenin, apigenin, eriodictyol, hesperetin, p-coumaric acid, and caffeic acid. Chromatographic separation was conducted with a UHPLC system (XR; Shimadzu, Tokyo, Japan) equipped with a communications bus module, a degassing unit, a binary pump, an autosampler, and a column oven. The separation of metabolites was carried out on an ACQUITY UPLC ® BEH C 18 column (50 mm × 2.1 mm, 1.7 µm; Waters, Milford, DE, USA) with the column temperature operated at 40 • C. Mobile phases consisting of water (A) and methanol (B), both containing 0.1% formic acid (v/v), were employed at a flow rate of 0.4 mL/min. Mobile phases were eluted as the following linear gradient elution program: 5% to 35% B at 0-7 min, 35-100% B at 7-11 min, and a 4 min post-run time for equilibration. The temperature of autosampler was maintained at 15 • C, and the injection volume was set as 10 µL.
Following the chromatographic separation, UHPLC effluent was directly introduced to a hybrid triple quadrupole linear ion trap mass spectrometer (Q-Trap 6500 + ; Sciex, Foster City, CA, USA) equipped with an ESI source. The mass spectrometer was operated in negative ion mode and multiple reactions monitoring (MRM) scan mode. Nitrogen was engaged as auxiliary gas, curtain gas, and collision gas. The common parameters of the mass spectrometer were set as follows: ion spray voltage, −4500 V; atomization temperature, 550 • C; ion source gas 1, 55 psi; ion source gas 2, 55 psi; curtain gas, 35 psi; collision gas, medium level. The MRM transitions, declustering potentials (DP), and collision energies (CE) were optimized as Table 1. Raw MS/MS data were acquired with Analyst software (version TF 1.7 with HotFix 3; Sciex, Foster City, CA, USA) and then processed with OS software (version 1.4; Sciex, Foster City, CA, USA).
The developed method was further validated for specificity, carry-over effect, calibration curve, lower limit of quantification (LLOQ), precision, accuracy, matrix effect, dilution integrity (20 times), and stability (long-term cryopreserved at −70 • C for 1 month, incubation at 37 • C for 12 h, placement in autosampler at 15 • C for 24 h) in light of the Guidelines for Validation of Quantitative Analytical Method of Biological Samples documented in the Pharmacopoeia of the People's Republic of China (ChP) of 2020 edition [32]. The detailed experimental processes of methodology validation referred to our reported studies [33,34]. In this work, the quantitative results in the cecal contents group were chosen to evaluate the impact of microbial metabolism on the antioxidant capacity of naringin. The pre-fermented working solution of naringin was prepared at the concentration of 363.4 nmol/mL. The post-fermented working solution was prepared by spiking the stock solution of naringin and its metabolites rhoifolin, neoeriocitrin, neohesperidin, naringenin, apigenin, eriodictyol, hesperetin, p-coumaric acid, and caffeic acid in the determined proportion, so as to obtain a total molarity the same as the pre-fermented working solution of naringin. The total antioxidant capacity (TAOC) of the above two working solutions were evaluated with ABTS (2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonate) radical scavenging assay, ferric reducing antioxidant power (FRAP) assay, DPPH (α,α-diphenylβ-picrylhydrazyl) radical scavenging assay, and hydroxyl radical (·OH) scavenging assay, following the protocols of corresponding kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Data Analysis
Raw LC-MS/MS data were acquired with SCIEX Analyst software (Version 1.7) and then processed with SCIEX OS software (Version 1.4). Data were expressed as mean ± standard deviation (SD) and evaluated by Student's t-test. p-values less than 0.05 or 0.01 were considered to be statistically significant. Quantitative results were illustrated with GraphPad Prism software (Version 7.00).

Identification of the Microbial Metabolites of Naringin after In Vitro Fecal Fermentation
Metabolites identification was performed in light of the following two points: (I) full understanding of the data obtained with available reference standards, including retention time (RT) and MS/MS fragmentation patterns; (II) reported results associated with the metabolism of naringin and other flavonoids. Herein, a total of 35 metabolites were identified or tentatively characterized in the fermentation samples. Their detailed information, including tentative identifications, molecular formulas, RT, detected mass-to-charge ratios (m/z) of deprotonated quasi-molecular ion, and corresponding mass errors, as well as the characteristic MS/MS fragment ions, are listed in Table 2.     On the basis of their chemical structures, these metabolites could be categorized into flavonoid metabolites and phenolic catabolites. Compounds in the class of "flavonoid metabolites" contained the characteristic carbon skeleton C6-C3-C6. A total of 29 flavonoid metabolites were characterized after the fecal fermentation. In light of the corresponding reference standards, M1, M14, M16, M18, M19, M27, and M29 were identified as rhoifolin, neoeriocitrin, neohesperidin, naringenin, apigenin, eriodictyol, and hesperetin, respectively. For most other metabolites, due to the unavailability of corresponding commercial standards, they were identified on the basis of data obtained with mentioned reference compounds and the published literature.
The MS/MS fragmentation of detected flavonoid metabolites showed typical retro-Diels-Alder (RDA) reactions, aligned with reported studies [35,36]. Taking deprotonated naringenin as an example, it was fragmented into product ions at m/z 151 and 119 by RDA reaction, which could be considered as diagnostic product ions for naringenin and its derivates. In particular, the MS/MS fragmentation of apigenin, eriodictyol, hesperetin, and corresponding conjugates also gave the ion signal at m/z 151, which was yielded by RDA reaction similar to naringenin. Meanwhile, a loss of 120 Da (C 4 H 8 O 4 ) was observed in the MS/MS fragmentation of flavonoid-O-glucosides, such as naringin, rhoifolin, naringin monoglucoside, and so on. The characteristic loss was proposed as a fragment of the glucose moiety.
M2 and M3 both gave [M-H] − at m/z 621, 42 Da (C 2 H 2 O) more than that of naringin, were presumed to be acetylated naringin. Considering that naringin has two binding sites, 4 -OH and 5-OH, for acetyl, these two metabolites could be 4 -O-acetyl-naringin or 5-O-acetyl-naringin. As shown in Table 2, most of the fragment ion signals of M2 and M3 were coincident, except that M3 has an exclusive fragment ion at m/z 408. The product ion at m/z 408 could be inferred to be formed by M25 and M26 exhibited the same [M-H] − with M27 (eriodictyol, i.e., 3 -hydroxynaringenin) and were proposed to be hydroxyl naringenin. Through the same RDA reaction occurring on the bonds 1 and 3 in C ring, M25 and M26 gave the product ions at m/z 167 and m/z 119, while eriodictyol formed the fragment ions at m/z 151 and m/z 135. Based on the difference in MS/MS fragmentation pattern, M25 and M26 were inferred to be 6/8-hydroxyl-naringenin. Combined with reported relative retention time data [37], M25 and M26 were tentatively characterized as carthamidin (6-hydroxy-naringenin) and isocarthamidin (8-hydroxy-naringenin), respectively. In another similar case, M13 were presumed to be 6/8-hydroxy-naringin, an isomer of neoeriocitrin. Based on similar correlation between chemical structure and MS/MS fragmentation pattern, M11, M12, M15, M17, M23, M24, and M28 were tentatively identified as the flavonoid metabolites described in Table 2. M5, M6, M7, and M8 had the same [M-H] − at m/z 741, 162 Da (C 6 H 10 O 5 ) more than that of naringin, and showed same product ion signals at m/z 271 and m/z 151. Several studies [38,39] have reported the synthesis of naringin glycosides through Bacilus speciesmediated transglucosylation. Herein, these four compounds were proposed as the glucose conjugates of naringin. Given the chemical structure of naringin, the new glucose moiety could bind to either the hydroxyl of flavonoid skeleton (4 -OH, 5-OH) or the hydroxyl of 7-O-neohesperidose moiety. Further methodologies are required to determine the exact binding sites for glucose moiety in these four compounds. Similarly, M20, M21, and M22 were inferred to be glucosyl naringenin. In the structure of naringenin, 4 -OH, 5-OH, and 7-OH are the three possible conjunct sites for the glucose group. On the basis of relative retention time reported in previous studies [40,41], M20, M21, and M22 were tentatively identified as naringenin-5-O-glucoside, naringenin-7-O-glucoside, and naringenin-4 -Oglucoside, respectively.
Phenolic catabolites were the downstream metabolites that flowed from the ring fission of the flavonoid skeleton. As shown in Table 2, a total of six phenolic catabolites were detected in the fermentation samples. Compared with authentic standards, M30, M31, and M33 were unambiguously identified as p-coumaric acid, caffeic acid, and 3-(4hydroxyphenyl)propionic acid, respectively. M32, M34, and M35 were presumed to be cinnamic acid, 3-(3 , 4 -dihydroxyphenyl)propionic acid, and 3-phenylpropionic acid based on their MS/MS fragmentation patterns and reported results [23,42]. Due to carrying the carboxyl group, these phenolic catabolites all gave characteristic product ions with the neutral loss of CO 2 (44 Da).

Biotransformation of Naringin during In Vitro Fecal Fermentation
In this work, microbial-mediated degradation of naringin gave rise to a number of metabolites. On the basis of identified metabolites listed in Table 2, naringin was considered to be engaged in a series of microbial-mediated biotransformations, including dehydrogenation, acetylation, sulfation, glucosylation, methylation, dehydroxylation, hydroxylation, and hydrolysis. With the successive loss of a rhamnose and a glucose moiety, naringin was hydrolyzed to the aglycone naringenin. Generated naringenin was subsequently involved in dehydrogenation, glucosylation, dehydroxylation, hydroxylation, and methylation. Meanwhile, naringenin undergo ring fission and yield p-coumaric acid, which was further employed in hydrogenation, hydroxylation, and dehydroxylation, giving rise to several phenolic catabolites. The proposed biotransformation pathways of naringin during in vitro fecal fermentation are illustrated in Figure 1.
To date, human-gut-microbiota-mediated metabolism of naringin has been investigated in several studies. Using the HPLC method, Xiong et al. [26] determined the degradation profile of naringin co-incubated with feces of healthy people, and found that naringin was extensively metabolized into naringenin. With the extension of time, the concentration of naringenin in the incubation system also gradually decreased, suggesting that naringenin could be further degraded by fecal microorganisms, but the corresponding downstream metabolites were not identified. Zou et al. [27] investigated the human intestinal microbial metabolism of naringin with LC-MS/MS technology. Naringin was found to be metabolized into naringenin and further degraded into 3-(4 -hydroxyphenyl)propionic acid. The ability of human intestinal microorganisms to mediate naringenin metabolism to produce 3-(4 -hydroxyphenyl)propionic acid showed significant individual differences. In another study, Chen et al. [28] detected the human-intestinal-microbes-associated metabolic profile of naringin with stable isotope-labeling strategy coupled with high-resolution LC-MS/MS. A total of 13 microbial metabolites were identified, and naringin was found to undergo extensive phase I metabolism mediated by human intestinal microbes. In this study, rat intestinal microbial metabolism of naringin was first investigated. Thanks to more available reference standards and high-resolution UFLC-Q-TOF-MS/MS system, a total of 35 metabolites were detected in the fermentation samples, far more than in previous studies. Based on these metabolites, more comprehensive microbial-mediated naringin metabolic pathways were proposed, as shown in Figure 1.  s 2022, 14, x FOR PEER REVIEW 11 of 17 Figure 1. Potential pathways for the metabolism of naringin by rat gut microbiota. The numbers of metabolites in brackets corresponds to those in Table 1. Figure 1. Potential pathways for the metabolism of naringin by rat gut microbiota. The numbers of metabolites in brackets corresponds to those in Table 1.

Quantification of the Microbial Metabolites of Naringin by UHPLC-Q-Trap-MS/MS
Based on the results of metabolite profiling and the availability of corresponding reference standards, naringin and its primary metabolites (including rhoifolin, neoeriocitrin, neohesperidin, naringenin, apigenin, eriodictyol, hesperetin, p-coumaric acid, and caffeic acid) were assigned as the target analytes in quantitative analysis. Isoquercitrin was selected as the internal standard (IS). A UHPLC-Q-Trap-MS/MS method was developed and validated for simultaneous determination of these compounds.
Fine peaks were obtained for each target analyte and IS, and no significant interference peaks at their respective RT range were observed. The residual interferences of ten target analytes after the injection of upper limit of quantification sample were all less than 20% of that in LLOQ sample, while that for IS was within 5%. All calibration curves of target analytes achieved acceptable linearity with correction coefficients greater than 0.99 (detailed in Table S1 in the Supplementary Material). The LLOQ of naringin, rhoifolin, neoeriocitrin, neohesperidin, naringenin, apigenin, eriodictyol, hesperetin, p-coumaric acid, and caffeic acid were 4.783, 5.185, 5.000, 5.100, 5.350, 0.4715, 0.4464, 0.5510, 0.4804, and 0.5000 ng/mL, respectively. The intra-batch and inter-batch precision (expressed by relative standard deviation, RSD) of LLOQ samples were <12.4% with accuracy (expressed by relative error, RE) in the range of −10.8% to 6.2% for all analytes (Table S2). The intra-batch and interbatch assay of QC samples (LQC, MQC, HQC) obtained acceptable precision of <11.4%, and accuracy varied from −8.1% to 8.9% for target analytes (Table S2). The IS-normalized matrix factors (IS-MF) of ten target analytes were tested at LQC and HQC concentration levels, and achieved the precision of <5.5% (Table S2). The precision of analytes diluted 20 times was less than 12.4%, with the accuracy varying from −8.4% to 8.7%. In long-term stability, incubation stability, and placement stability tests, the accuracy of LQC and HQC samples were all in the range of −8.3% to 12.7%, and the precision within 13.0% (Table S3). The incubation stability tests showed that naringin was stable when incubated at 37 • C for 12 h, that is to say, naringin would not undergo metabolism in the absence of intestinal microorganisms. All methodology validation assay results were within the acceptable criteria, suggesting that the present method was stable and reliable for simultaneous determination of the abovementioned target analytes in rat fecal fermentation samples.
The validated method was successfully applied to the quantitative determination of naringin and its primary microbial metabolites in rat fecal fermentation samples. Measured concentrations of target analytes are plotted in Figure 2 (detailed in Table S4 in the Supplementary Material). As shown in Figure 2, whether incubated with intestinal or caecal contents, the abundances of rhoifolin, neoeriocitrin, neohesperidin, and naringenin in fermentation samples were much higher than those of other microbial metabolites. It should be noted that the concentrations of some metabolites yielded by incubation with intestinal or caecal contents were significantly different. For example, compared with that in the intestinal contents group, the concentrations of naringenin and apigenin in caecal contents group were significantly higher, while the concentration of p-coumaric acid was significantly lower. These results suggest that there was a difference in the capacity of degrading naringin between intestinal or caecal microbiota, probably associated with the community diversity at different locations [43,44]. Li et al. [45] investigated the regional differences of microbiota along the gastrointestinal tract in rat, and found that the community diversity was closely related to biogeographic location. Lactate-producing bacteria (including Lactobacillus, Turicibacter, and Streptococcus) dominated the bulk of small-intestinal microbiota, while a greater proportion of anaerobic Lachnospiraceae and Ruminococcaceae were found to be the core microbiota in the large intestine. After the fermentation, the total molarity of naringin and its nine microbial m lites in intestinal contents group and caecal contents group were 334.3 and 363.4 nmo which were equivalent to 66.9% and 72.7% of the original naringin input. These r suggest that a considerable amount of naringin was metabolized into other metab As shown in Table 2, in addition to the nine compounds quantified, a series of oth crobial metabolites were also detected in the fermentation samples. To quantify thes tabolites, fermentation samples were further detected in full scan mode and the optim chromatographic separation conditions of the above UHPLC-Q-Trap-MS/MS metho quired peak area data revealed that acetylated, methylated, dehydroxylated, and hy ylated metabolites of naringin were also abundant in the fermentation samples. S quently, the MRM parameters for these abundant metabolites were optimized as fo However, due to the una bility of corresponding authentic standards, these metabolites were quantified usin calibration curves of corresponding structural analogues. Specifically, naringin was to quantify its acetylated, methylated, and dehydroxylated metabolites, while riocitrin was used to quantify hydroxylated naringin. Measured concentrations of metabolites are listed in Table S4 in the Supplementary Material. Taking these metab into account, the overall recovery of naringin in the intestinal contents group and contents group after incubation reached 86.5% and 92.1%, respectively.
To date, two studies [27,46] have quantified a microbial metabolite of narin human fecal fermentation samples, i.e., 3-(4′-hydroxyphenyl)propionic acid, which detected in this study. Herein, a rapid UHPLC-Q-Trap-MS/MS method was firstly lished for simultaneous determination of nine microbial metabolites derived from ingin. Moreover, six other metabolites with high abundance in fermentation samples After the fermentation, the total molarity of naringin and its nine microbial metabolites in intestinal contents group and caecal contents group were 334.3 and 363.4 nmol/mL, which were equivalent to 66.9% and 72.7% of the original naringin input. These results suggest that a considerable amount of naringin was metabolized into other metabolites. As shown in Table 2, in addition to the nine compounds quantified, a series of other microbial metabolites were also detected in the fermentation samples. To quantify these metabolites, fermentation samples were further detected in full scan mode and the optimized chromatographic separation conditions of the above UHPLC-Q-Trap-MS/MS method. Acquired peak area data revealed that acetylated, methylated, dehydroxylated, and hydroxylated metabolites of naringin were also abundant in the fermentation samples. Subsequently, the MRM parameters for these abundant metabolites were optimized as follows: However, due to the unavailability of corresponding authentic standards, these metabolites were quantified using the calibration curves of corresponding structural analogues. Specifically, naringin was used to quantify its acetylated, methylated, and dehydroxylated metabolites, while neoeriocitrin was used to quantify hydroxylated naringin. Measured concentrations of these metabolites are listed in Table S4 in the Supplementary Material. Taking these metabolites into account, the overall recovery of naringin in the intestinal contents group and caecal contents group after incubation reached 86.5% and 92.1%, respectively.
To date, two studies [27,46] have quantified a microbial metabolite of naringin in human fecal fermentation samples, i.e., 3-(4 -hydroxyphenyl)propionic acid, which is also detected in this study. Herein, a rapid UHPLC-Q-Trap-MS/MS method was firstly established for simultaneous determination of nine microbial metabolites derived from naringin. Moreover, six other metabolites with high abundance in fermentation samples were also quantified with constructed calibration curves of authentic standards with similar structures. This work provides a feasible quantitative method for other studies concerning the microbial metabolism of flavonoids.
In this study, the metabolic profiles of naringin mediated by rat gut microbiota were investigated qualitatively and quantitatively; however, information concerning the association between microbial species and specific metabolic pathways is still limited. Characterization of the compositional profile of microbiota is essential to confirm mentioned association. The integration of in vitro fermentation and sequencing methods (e.g., 16S rDNA) should be a feasible strategy to clarify the correlation between certain microbial species and specific metabolic pathways.

Effect of Fecal Fermentation on the Antioxidant Capacity
In this work, ABTS, FRAP, DPPH, and ·OH assays were applied to evaluate the TAOC changes before and after fecal fermentation. Obtained results are plotted in Figure 3. Compared with the pre-fermented working solution, the post-fermented working solution showed a significant increase in the TAOC values. In other words, fermentation-associated microbial metabolites exhibited higher antioxidant activity than original naringin. On the one hand, naringin was metabolized into some flavonoid compounds with polyhydroxyl groups and higher antioxidant activity [9]. On the other hand, gut-microbiotarelated gastrointestinal digestion led to better overall bioavailability and bioaccessibility of naringin [14,17]. A number of studies [47][48][49] have indicated that gastrointestinal tract is constantly being challenged by free radicals, including diet-derived oxidants and endogenous reactive oxygen species. Therefore, the presence of fermentation-associated metabolites could help to relieve oxidative stress in the gastrointestinal tract.
utrients 2022, 14, x FOR PEER REVIEW 14 In this study, the metabolic profiles of naringin mediated by rat gut microbiota investigated qualitatively and quantitatively; however, information concerning the ciation between microbial species and specific metabolic pathways is still limited. C acterization of the compositional profile of microbiota is essential to confirm menti association. The integration of in vitro fermentation and sequencing methods (e.g. rDNA) should be a feasible strategy to clarify the correlation between certain micr species and specific metabolic pathways.

Effect of Fecal Fermentation on the Antioxidant Capacity
In this work, ABTS, FRAP, DPPH, and •OH assays were applied to evaluat TAOC changes before and after fecal fermentation. Obtained results are plotted in F 3. Compared with the pre-fermented working solution, the post-fermented working tion showed a significant increase in the TAOC values. In other words, fermentatio sociated microbial metabolites exhibited higher antioxidant activity than original ingin. On the one hand, naringin was metabolized into some flavonoid compounds polyhydroxyl groups and higher antioxidant activity [9]. On the other hand, gut-mic ota-related gastrointestinal digestion led to better overall bioavailability and bioacc bility of naringin [14,17]. A number of studies [47][48][49] have indicated that gastrointes tract is constantly being challenged by free radicals, including diet-derived oxidants endogenous reactive oxygen species. Therefore, the presence of fermentation-assoc metabolites could help to relieve oxidative stress in the gastrointestinal tract.

Conclusions
In summary, rat-fecal-microbiota-mediated metabolism of naringin and its effe the antioxidant capacity was investigated in this work. UFLC-Q-TOF-MS/MS-based tabolite identification results showed that rat fecal microbiota could metabolize nar into a number of metabolites, including 29 flavonoid metabolites and 6 phenolic ca

Conclusions
In summary, rat-fecal-microbiota-mediated metabolism of naringin and its effect on the antioxidant capacity was investigated in this work. UFLC-Q-TOF-MS/MS-based metabolite identification results showed that rat fecal microbiota could metabolize naringin into a number of metabolites, including 29 flavonoid metabolites and 6 phenolic catabolites. Naringin was found to undergo a series of microbial-mediated biotransformations, and corresponding metabolic pathways were proposed. Moreover, a UHPLC-Q-Trap-MS/MS method was developed for simultaneous determination of naringin and its metabolites in fecal fermentation samples. Rhoifolin, neoeriocitrin, neohesperidin, and naringenin, as well as the methylated and hydroxylated metabolites of naringin, were detected as the primary microbial metabolites. Further antioxidant capacity assays indicated that fermentation-associated microbial metabolites showed higher antioxidant activity than original naringin. The present results provide new insights in understanding the microbial metabolism and in vivo antioxidant capacity of naringin.
Author Contributions: Conceptualization, X.Z. and W.S.; methodology, X.Z., Y.Z., Y.H. and J.Z.; writing-original draft preparation, X.Z.; writing-review and editing, X.Z. and W.S.; project administration, W.P.; funding acquisition, X.Z. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement: The animal study protocol was approved by the Institutional Animal Care and Use Committee in Sun Yat-Sen University (protocol code SYSU-IACUC-221-B1305, approved on 12 July 2021).