Identification of Xanthine Oxidase Inhibitors from Celery Seeds Using Affinity Ultrafiltration–Liquid Chromatography–Mass Spectrometry

Celery seeds have been used as an effective dietary supplement to manage hyperuricemia and diminish gout recurrence. Xanthine oxidase (XOD), the critical enzyme responsible for uric acid production, represents the most promising target for anti-hyperuricemia in clinical practice. In this study, we aimed to establish a method based on affinity ultrafiltration–liquid chromatography–mass spectrometry (UF–LC–MS) to directly and rapidly identify the bioactive compounds contributing to the XOD-inhibitory effects of celery seed crude extracts. Chemical profiling of celery seed extracts was performed using UPLC-TOF/MS. The structure was elucidated by matching the multistage fragment ion data to the database and publications of high-resolution natural product mass spectrometry. Thirty-two compounds, including fourteen flavonoids and six phenylpeptides, were identified from celery seed extracts. UF–LC–MS showed that luteolin-7-O-apinosyl glucoside, luteolin-7-O-glucoside, luteolin-7-O-malonyl apinoside, luteolin-7-O-6′-malonyl glucoside, luteolin, apigenin, and chrysoeriol were potential binding compounds of XOD. A further enzyme activity assay demonstrated that celery seed extract (IC50 = 1.98 mg/mL), luteolin-7-O-apinosyl glucoside (IC50 = 3140.51 μmol/L), luteolin-7-O-glucoside (IC50 = 975.83 μmol/L), luteolin-7-O-6′-malonyl glucoside (IC50 = 2018.37 μmol/L), luteolin (IC50 = 69.23 μmol/L), apigenin (IC50 = 92.56 μmol/L), and chrysoeriol (IC50 = 40.52 μmol/L) could dose-dependently inhibit XOD activities. This study highlighted UF–LC–MS as a useful platform for screening novel XOD inhibitors and revealed the chemical basis of celery seed as an anti-gout dietary supplement.


Introduction
Gout is a common type of arthritis with increasing prevalence in China. Gout stones formed by monosodium urate (MSU) deposit in the joints, especially the toes, causing inflammatory arthritis and severe pain in the affected joints [1]. The cumulative incidence of gout attacks is positively correlated with serum uric acid (sUA) levels in patients. Beyond the saturation point of MSU (68 mg/L), new MSU crystals can form in joints. In contrast, a low sUA (<60 mg/L) leads to the dissolution of existing crystals, reducing the risk of gout recurrence [2]. In this context, medicines that control sUA have been widely used in clinical practice to prevent gout recurrence [3]. Xanthine oxidase (XOD) is the ratelimiting enzyme in uric acid production from purine metabolism [4]. XOD inhibitors such as allopurinol and febuxostat efficiently reduce sUA and are therefore often used to treat gout. Currently, XOD remains the most promising therapeutic target for the development of novel anti-gout drugs.
Natural products are recognized as important sources of bioactive compounds. Celery seed (Apium graveolens L.) is rich in various phytochemicals such as flavones [5], coumarin [6], volatile oils [7], and fatty acids [8], making it a popular dietary supplement with antioxidant, anti-inflammatory, hypoglycemic, hypolipidemic, and antihypertensive effects [8,9]. Recently, the anti-gout effects of celery seed have received increasing attention due to its role in XOD inhibition and sUA lowering [10,11]. Meanwhile, the chemical profiles of celery seed extracts were predicted to contain potential XOD inhibitors in a neural network drug-target interaction model, supporting the anti-gout effects of celery seed [12]. The conventional methods for isolating XOD inhibitors from celery seed extracts, which require multistep procedures including compound separation and biological activity assays, are costly and time-consuming. On the other hand, affinity ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS), simultaneously exploiting protein-ligand interaction and chemical analysis methods, could be a straightforward approach to isolate novel enzyme modulators from chemistry libraries or plant extracts [13]. UF-LC-MS is now widely used to screen drugs for diabetics [14,15], hyperlipidemia [16], and inflammatory disorders [17]. In this study, we applied UPLC-TOF/MS to profile celery seed chemical compositions and developed a UF-LC-MS-based method to screen the compounds contributing to celery seed-mediated XOD inhibition.

Chemical Profiles of Celery Seed Extracts
Celery seed extracts were analyzed using UPLC-Q-TOF/MS. The multi-level structural characterization of reconstituted extracts was matched to the database and publications on high-resolution natural product mass spectrometry. A total of 32 compounds were identified, including 14 flavonoids, 6 phenylpeptides, and 3 quinic acids. The total ion current chromatogram (TICC) of the extracts in positive and negative models was shown in Figure 1. The 32 peaks were elucidated and are reported in Table 1. As examples, the MS analyses of luteolin 7-O-malonyl-apiosylglucoside and sedenenolide are demonstrated in Figure 2.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 13 gout. Currently, XOD remains the most promising therapeutic target for the development of novel anti-gout drugs. Natural products are recognized as important sources of bioactive compounds. Celery seed (Apium graveolens L.) is rich in various phytochemicals such as flavones [5], coumarin [6], volatile oils [7], and fatty acids [8], making it a popular dietary supplement with antioxidant, anti-inflammatory, hypoglycemic, hypolipidemic, and antihypertensive effects [8,9]. Recently, the anti-gout effects of celery seed have received increasing attention due to its role in XOD inhibition and sUA lowering [10,11]. Meanwhile, the chemical profiles of celery seed extracts were predicted to contain potential XOD inhibitors in a neural network drug-target interaction model, supporting the anti-gout effects of celery seed [12]. The conventional methods for isolating XOD inhibitors from celery seed extracts, which require multistep procedures including compound separation and biological activity assays, are costly and time-consuming. On the other hand, affinity ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS), simultaneously exploiting protein-ligand interaction and chemical analysis methods, could be a straightforward approach to isolate novel enzyme modulators from chemistry libraries or plant extracts [13]. UF-LC-MS is now widely used to screen drugs for diabetics [14,15], hyperlipidemia [16], and inflammatory disorders [17]. In this study, we applied UPLC-TOF/MS to profile celery seed chemical compositions and developed a UF-LC-MS-based method to screen the compounds contributing to celery seed-mediated XOD inhibition.

Chemical Profiles of Celery Seed Extracts
Celery seed extracts were analyzed using UPLC-Q-TOF/MS. The multi-level structural characterization of reconstituted extracts was matched to the database and publications on high-resolution natural product mass spectrometry. A total of 32 compounds were identified, including 14 flavonoids, 6 phenylpeptides, and 3 quinic acids. The total ion current chromatogram (TICC) of the extracts in positive and negative models was shown in Figure 1. The 32 peaks were elucidated and are reported in Table 1. As examples, the MS analyses of luteolin 7-O-malonyl-apiosylglucoside and sedenenolide are demonstrated in Figure 2.

Validation of Inhibitory Activities
The inhibitory effects of crude extracts and six binding compounds on XOD enzymatic activities were assessed as described in the methods. As shown in Figure 5  Extracted ion current chromatogram of XOD ligands from celery seeds. Red and blue peaks denote the febuxostat-and DMSO-treated groups, respectively. Numbers refer to peak numbers in Table 1.  . Extracted ion current chromatogram of XOD ligands from celery seeds. Red and blue peaks denote the febuxostat-and DMSO-treated groups, respectively. Numbers refer to peak numbers in Table 1.

Validation of Inhibitory Activities
The inhibitory effects of crude extracts and six binding compounds on XOD enzymatic activities were assessed as described in the methods. As shown in Figure 5

Validation of Inhibitory Activities
The inhibitory effects of crude extracts and six binding compounds on XOD enzymatic activities were assessed as described in the methods. As shown in Figure 5
In flavonoid glycoside (10) and aglycone (25) with the best activity were selected to reveal the interaction mechanism between compounds and xanthine oxidase. In Figure 6, 3D and 2D diagrams of the binding mode of luteolin-7-O-glucoside (10)/xanthine dehydrogenase(A-B) and chrysoeriol (25)/xanthine dehydrogenase(C-D) complex are shown. These complexes maintain mutual attraction mainly through hydrogen bonds, pi-pi stacking (presumptive noncovalent pi interactions between the pi bonds of aromatic rings), and hydrophobic interaction.

Identification of Chemical Constituents in Celery Seed Extracts
The chemical composition analysis results of celery seeds show that flavonoids, including luteolin, apigenin, and chrysoeriol and its derivatives, are the main constituents in celery seed ethanol extract. This result is consistent with the results obtained through traditional separation and purification in previous reports [30]. Flavonoid glycoside defragmentation is generally cleaved through glycoside. broken down to m/z 535.1115 fragment ion with the loss of apiosyl, followed by production of m/z 287.0558 fragment ion after malonyl glucoside breakdown. The phenylpeptides in celery seeds, which contain ester groups and often carry hydroxyl or butyl groups, are prone to generating fragmented ion peaks that remove C 4

Identification of XOD-Binding Compounds from Celery Seed Extracts
Screening with the UF-LC-MS method, luteolin-7-O-apinosyl glucoside (9), luteolin-7-O-glucoside (10), luteolin-7-O-malonyl apinoside (14), luteolin-7-O-6 -malonyl glucoside (15), luteolin (21), apigenin (24), and chrysoeriol (25) are identified as potential ligands of febuxostat binding domain in the XOD, which indicated that flavone aglycones and flavonoids derived from luteolin were potential XOD inhibitors in celery seeds. As another common chemical class in celery seeds [11], phenylpeptides have rarely been reported in previous studies as having xanthine oxidase-inhibitory activity. In the present UF-LC-MS study, we did not identify any significant difference in the peak area corresponding to phenylpeptides between XOD with and without febuxostat. This result suggests that phenylpeptides in celery seeds exert weak or no competitive blocking activity on XOD.

Validation of Inhibitory Activities
Flavonoids are common polyphenols in plants and have a wide range of pharmacological effects [31]. Studies have shown that the activity of flavonoids is closely related to their chemical structure [32,33]. In this study, the structure-activity analysis of the hitting compounds showed that the inhibitory effects of compounds on XOD appear to be positively associated with the number of hydroxyl groups on ring B. On the other hand, higher glycosylation in flavonoids represents weaker XOD-inhibitory activities. For example, luteolin-7-O-apinosyl glucoside (9), luteolin-7-O-glucoside, and luteolin-7-O-6malonyl glucoside (15) are derivatives of luteolin (21), differing only in the number of glycosides. The enzymatic assay demonstrated that luteolin (21) has lower IC 50 values than its derivatives.

The Study of Molecular Recognition
By predicting the binding strength and affinity between compounds and target proteins, molecular docking can quickly identify active ingredients in plants, and has unique advantages in the virtual screening of active ingredients based on targets. The lower docking affinity reflects the stronger binding ability between the compound and its targets.

UF-LC-MS Assay for Screening Novel XOD Inhibitors
The traditional method for screening xanthine oxidase inhibitors from plant extracts involves multiple laborious steps, including extraction, purification, and determination of the compound library and activity detection [30,34]. Compared to traditional procedures, UF-LC-MS combines affinity capture of active molecules against protein targets of interest with ultrafiltration, liquid chromatography, and high-resolution mass spectrometry, which can simultaneously screen and identify bioactive components from complex mixtures, greatly improving efficiency, and reducing sample and reagent consumption [35,36].
Many previous affinity UF-LC-MS studies used mass spectrometry to identify the compounds in the extracts and determined the candidates by comparing the peak area of each compound in the liquid chromatogram [37,38]. However, co-elution and peak overlapping often occur in the liquid chromatogram [39], and some compounds, such as saponins, alkaloids, and amines, have poor UV absorption, resulting in missed screening. The extracted ion current chromatogram used in this study selects specific ion fragments for peak area comparison, effectively avoiding the pitfalls of conventional methods and increasing the hitting rate.
Febuxostat is one of the most commonly prescribed medications to treat gout and hyperuricemia due to its potent inhibitory effect on XOD. Febuxostat is considered superior to allopurinol at standard doses, particularly in patients who are intolerant to allopurinol [40]. Mechanistically, febuxostat non-competitively binds to the molybdenum pterin site of XOD, thereby inhibiting the oxidase activity and preventing the production of uric acid. In this regard, compounds that can competitively displace febuxostat binding in the XOD are candidates for anti-hyperuricemia drugs. The present affinity UF-LC-MS assay compares the binding profiles of XOD with and without febuxostat. The differential compounds between two profiles are therefore considered as candidates sharing a similar binding domain of XOD with febuxostat. A subsequent enzymatic assay confirmed that the potential molybdenum-pterin binding compounds inhibited XOD activity. Taken together, we concluded that UF-LC-MS assay is suitable for the rapid identification of novel XOD inhibitors from crude plant extracts.

Preparation of Celery Seed Extract
Dried celery seeds were extracted twice at 85 • C in a water bath, the first time with 8 times the amount of 70% (v/v) ethanol for 1.5 h, and the second time with 6 times the amount of 70% (v/v) ethanol for 1 h. The ethanol extracts were combined and then concentrated using rotary vaporization at 60 • C under reduced pressure. The extract powder was obtained by drying the concentrated solution using a freeze dryer and stored in a refrigerator at −20 • C until use. For analysis in LC-MS, the extract powder was ultrasonically dissolved in 70% (v/v) methanol to make a 2 mg/mL solution, followed by filtration before use.

XOD Activity Assay
The inhibitory effects of compounds were determined in the XOD activity assay according to a previous report [41] with minor modifications. Celery seed extracts or the "hit" compounds from the UF-LC-MS screening assay were dissolved in PBS and prepared for a series of test solutions using 2-fold serial dilutions. Test solutions (50 µL) were mixed with XOD (50 µL at 0.02 U/mL) and incubated at 37 • C for 5 min. This was followed by the addition of XOD substrate xanthine (150 µL at 0.48 mM) and further incubation at 37 • C for 30 min. The optical density (OD) of each tube was then measured at 290 nm. Percent XOD inhibition was calculated using the following formula: (%) = 1 − A1−A2 A3−A4 × 100 where A1 was the absorbance of the production with the sample and XOD, A2 was the absorbance of the production with the sample, A3 was the absorbance of the production with PBS and XOD, and A4 was the absorbance of the production with PBS.

Screening for XOD Inhibitors Using UF-LC-MS
Identification of potential XOD inhibitors from celery seed extract was performed using UF-LC-MS according to a previous study with modification [42]. To prepare the XOD interaction solution, 250 µL of XOD (0.5 U/mL) was incubated with 50 µL of febuxostat (the XOD inhibitor, 500 µg/mL in 10% DMSO-PBS, masked group) to mask the druggable binding domain of XOD or 10% DMSO-PBS (vehicle control, unmasked group) at 37 • C for 45 min. The extract (50 µL at 2 mg/mL in methanol) was then added to the interaction solution for a further 45 min. After incubation, the mixtures were transferred to a 10 kDa molecule weight cut-off ultrafiltration filter and centrifuged at 14,500 rpm for 20 min. The membrane was washed 3 times with 450 µL ddH 2 O to remove the unbound molecules. The binding molecules, including febuxostat, selectively and unselectively binding compounds, were dissociated by treatment with 200 µL of 70% methanol for 10 min under ultrasonication. The elution solution was then ultrafiltered in the new filter units as described above. The final products in the bottom of the filter units were analyzed using a HPLC-MS system.

Molecular Docking
AutoDock Vina 1.1.2 program [43] was used to model the interaction between the active compounds and the xanthine dehydrogenase protein. The crystal structure of xanthine dehydrogenase was downloaded from the Protein Data Bank database (PDB ID: 1N5X) and pre-processed with PyMol 2.5.2 [44] to remove water molecules and ligands and add hydrogen atoms. The Mo-pt, FE/SI, and FAD structures in the crystal structure were retained. Then, for them, a grid box size of 25 × 25 × 25 Å3 containing the entire binding site was set. Before molecular docking, all proteins and compounds were converted to PDBQT format using AutoDock Tools 1.5.6 program [45], and the charge information of Mo ions was manually set. Docking was performed with default parameters, and the pose with the lowest score was extracted and visualized using Maestro (Schrödinger, New York, NY, USA).

Conclusions
Celery seed is a common spice rich in volatile oil and flavonoids. In this study, a comprehensive LC-MS analysis of the chemical profile confirmed the presence of various flavonoids, phenylpeptides, and quinic acids in celery seed. Using UF-LC-MS techniques, we successfully established a screening assay to identify XOD inhibitors from celery seed extracts. We found that flavonoids represent the major class of compounds in celery seed with potential inhibitory effects on XOD and identified Luteolin-7-O-6'-malonyl glucoside as a new XOD inhibitor for the first time. Furthermore, the XOD activity assay confirmed that these binding compounds inhibited XOD activities in a dose-dependent manner. Our study revealed the chemical basis of celery seed-mediated anti-gout and anti-hyperuricemia effects and provided potential XOD inhibitors for the prevention of recurrent gout.