Systematically Exploring the Chemical Ingredients and Absorbed Constituents of Polygonum capitatum in Hyperuricemia Rat Plasma Using UHPLC-Q-Orbitrap HRMS

Polygonum capitatum as an ethnic medicine has been used to treat urinary tract infections, pyelonephritis and urinary calculi. In our previous study, P. capitatum was found to have anti-hyperuricemia effects. Nevertheless, the active constituents of P. capitatum for treating hyperuricemia were still unclear. In this study, an ultra-high-performance liquid chromatography coupled to quadrupole/orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to comprehensively detect the chemical ingredients of P. capitatum and its absorbed constituents in the plasma of hyperuricemia rats for the first time. Xcalibur 3.0 and Compound Discoverer 2.0 software coupled to mzCloud and ChemSpider databases were utilized for qualitative analysis. A total of 114 chemical components including phenolics, flavonoids, tannins, phenylpropanoids, amino acids, amides and others were identified or tentatively characterized based on the exact mass, retention time and structural information. Compared to the previous P. capitatum study, an additional 66 different components were detected. Moreover, 68 related xenobiotics including 16 prototype components and 52 metabolites were found in the plasma of hyperuricemia rats. The metabolic pathways included ring fission, hydrolysis, decarboxylation, dehydroxylation, methylation, glucuronidation and sulfation. This work may provide important information for further investigation on the active constituents of P. capitatum and their action mechanisms for anti-hyperuricemia effects.


Introduction
Hyperuricemia (HUA) is one of the most common metabolic conditions characterized by abnormally increased serum urate levels. Long-term HUA is a main etiologic factor for the deposition of monosodium urate crystals (MSU) in joints and soft tissues resulting in gout [1]. Moreover, HUA is associated with incidences of hypertension, diabetes, obesity and chronic kidney disease [2]. Allopurinol, febuxostat and benzbromarone were selected as anti-hyperuricemic agents, although these agents exhibited some adverse effects including hypersensitivity, cardiovascular mortality risk and hepatic toxicity [3][4][5].
Traditional Chinese medicine (TCM) and ethnic medicine have been applied to treat hyperuricemia and gout for over thousands of years with their own unique advantages. TCM and ethnic medicine were considered important resources for discovering multitarget drugs for the treatment of hyperuricemia and gout. Polygonum capitatum Buch.-Ham. ex 2 of 22 D. Don, named Touhualiao in Chinese, was utilized as Miao ethnic medicine in China to treat urinary tract infections, pyelonephritis and urinary calculi [6]. In our previous study, P. capitatum was found to reduce serum urate levels to treat hyperuricemia and gouty arthritis without renal toxicities. The underlying action mechanism of P. capitatum involved inhibiting the expression and function of xanthine oxidase and decreasing the expressions of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) [7]. Despite the remarkable efficacy of P. capitatum for anti-hyperuricemia and anti-gouty arthritis, the active constituents of P. capitatum related to the pharmacological effect are still not clear.
A range of the active constituents of TCM and ethnic medicine is an essential prerequisite for executing pharmacological effects. Profiling the chemical ingredients, the absorbed constituents and metabolites is beneficial for elucidating the pharmacological materials of TCM and ethnic medicine. Traditional separation technologies have been used to isolate and obtain pure components from P. capitatum including triterpenes [8], phenolics [8][9][10], flavonoids [8][9][10], lignans [8,9], alkaloids [10] and tannins [11], although the technology was time-consuming and it was often difficult to acquire the substances at low concentrations. Only partial phenolics of P. capitatum were identified by ultra-high-performance liquid chromatography-photodiode array detection coupled with triple quadrupole mass spectrometry (UHPLC-PDA-QqQ-MS) [12] and UHPLC with time-of-flight mass spectrometry (UHPLC-TOF-MS) [13]. These were not sufficient for the research on the active constituents and action mechanisms of P. capitatum for anti-hyperuricemia and anti-gouty arthritis. Therefore, it is necessary to comprehensively identify and characterize the chemical constituents of P. capitatum and its absorbed components in hyperuricemia rats.
UHPLC coupled with quadrupole/orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) technology provided a sensitive and high-resolution platform for the analysis of chemical constituents at µg/kg concentration levels in complex matrix samples. The data-dependent acquisition mode of Q-Orbitrap HRMS provides MS/MS spectra with accurate mass data [14,15] which are beneficial for identifying and characterizing unknown compounds in TCM and ethnic medicine. Q-Orbitrap HRMS was also a powerful analytical technology for elucidating the metabolism of TCM and ethnic medicine in vivo due to its high sensitivity, high resolution and fast scanning capability.
In this study, a UHPLC-Q-Orbitrap HRMS method was employed to systematically clarify the chemical constituents of P. capitatum for the first time. Additionally, the prototypes and metabolites of P. capitatum in hyperuricemia rat plasma were also analyzed by the UHPLC-Q-Orbitrap HRMS technology based on neutral loss and metabolism types of representative components. Ultimately, 114 chemical constituents were tentatively identified or characterized from P. capitatum. Compared to the previous P. capitatum study using LC-MS, additional 66 different components were detected in this study. Among these, two new compounds were found, and 7 compounds were discovered in P. capitatum for the first time. A total of 68 related xenobiotics including 16 prototypes and 52 metabolites were detected in the hyperuricemia rats. Among them, 14 prototypes and 50 metabolites were reported for the first time. This study helped illustrate the active components and action mechanisms of P. capitatum for anti-hyperuricemia and anti-gouty arthritis.

UHPLC-Q-Orbitrap HRMS Analysis of P. capitatum Extract
UHPLC-Q-Orbitrap HRMS method was employed to profile the chemical constituents in P. capitatum extract. Under the optimized UHPLC-Q-Orbitrap HRMS conditions, the total ion current (TIC) chromatograms of P. capitatum extract in negative and positive ion modes are shown in Figure 1. The elemental compositions for the compound and the fragment ion were predicted within a mass tolerance of ±5 ppm. The chemical structures of the components in P. capitatum were elucidated by comparing their retention time, exact mass and structural information with those of authentic standards or available literature data. As a result, a total of 114 components from P. capitatum were unambiguously identified or tentatively characterized (Table S1 in Supplementary Materials), including 30 phenolic  acids, 38 flavonoids, 16 phenylpropanoids, 10 tannins, 10 phenolics, 3 amino acids, 3 amides  and 4 others. The chemical structures of the detected constituents were shown in Figure S1 in Supplementary Materials. Among these compounds, compounds 59, 77, 84, 96, 97, 110 and 112 were found in P. capitatum for the first time. Although positive and negative ion modes were employed, more peak signals and higher sensitivities were obtained in the negative ion mode. The fragment ions in the negative and positive ion modes are listed in Table S1.
ion modes are shown in Figure 1. The elemental compositions for the compound an fragment ion were predicted within a mass tolerance of ±5 ppm. The chemical struc of the components in P. capitatum were elucidated by comparing their retention time, mass and structural information with those of authentic standards or available liter data. As a result, a total of 114 components from P. capitatum were unambiguously tified or tentatively characterized (Table S1 in Supplementary Materials), includi  phenolic acids, 38 flavonoids, 16 phenylpropanoids, 10 tannins, 10 phenolics, 3 amin  ids, 3 amides and 4 others. The chemical structures of the detected constituents shown in Figure S1 in Supplementary Materials. Among these compounds, compo 59, 77, 84, 96, 97, 110 and 112 were found in P. capitatum for the first time. Although tive and negative ion modes were employed, more peak signals and higher sensiti were obtained in the negative ion mode. The fragment ions in the negative and po ion modes are listed in Table S1.

Phenolic Acids
The mass signals for phenolic acids in negative ion mode were observed. The q molecular ions of phenolic acids preferred to produce the corresponding product io neutral loss of H2O, CO and CO2. Compounds 4 and 14 were unambiguously iden as gallic acid and protocatechuic acid, respectively, by comparing their retention tim mass data with those of reference standards. The [M − H] − of gallic acid at m/z 169

Phenolic Acids
The mass signals for phenolic acids in negative ion mode were observed. The quasimolecular ions of phenolic acids preferred to produce the corresponding product ions by neutral loss of H 2 O, CO and CO 2 . Compounds 4 and 14 were unambiguously identified as gallic acid and protocatechuic acid, respectively, by comparing their retention time and mass data with those of reference standards. ) was also observed in the tandem mass spectrometry (MS 2 ) of compound 50. Therefore, compound 50 was tentatively identified as the reported ethyl gallate [16]. Different from the fragmentation pattern of compound 50, compound 52 generated the product ions at m/z 182.0212 ([M − H − CH 3 ·] − ) and 166.9976 ([M − H − 2 × CH 3 ·] − ) suggesting the presence of two methyl groups in compound 52.

Tannins
Tannins identified in P. capitatum in this study were classified into proanthocyanidins and ellagitannins. Proanthocyanidins were condensed tannins composed of oligomers and polymers of flavan-3-ol moieties linked mainly through 4-8 bonds. RDA reaction, heterocyclic ring fission (HRF) and quinone methide (QM) cleavage were the main mass fragmentation patterns of proanthocyanidins [24]. Ellagitannins belonging to hydrolyzable tannins consisted of hexahydroxydiphenoyl (HHDP) groups and related acyl groups. The characteristic fragment ion at m/z 300.9991 (C 14

Tannins
Tannins identified in P. capitatum in this study were classified into proanthocyanidins and ellagitannins. Proanthocyanidins were condensed tannins composed of oligomers and polymers of flavan-3-ol moieties linked mainly through 4-8′ bonds. RDA reaction, heterocyclic ring fission (HRF) and quinone methide (QM) cleavage were the main mass fragmentation patterns of proanthocyanidins [24]. Ellagitannins belonging to hydrolyzable tannins consisted of hexahydroxydiphenoyl (HHDP) groups and related acyl groups. The characteristic fragment ion at m/z 300.9991 (C14H5O8 − ) in the MS 2 spectrum of ellagitannin was corresponding to an ellagic acid moiety.
A The characteristic fragment ions at 615.0638 and 309.0245 were generated through losing a C13H9O9 moiety, which implies the existence of the residue region composed of D-and E-rings. Compound 33 was tentatively attributed to phyllanthusiin C. The MS 2 fragmentation pathway ( Figure 4C) of phyllanthusiin C was proposed for the first time.

Other Phenolics
Ten phenolics with a small molecular mass (<350 Da) and less than 15 carbons were tentatively identified in P. capitatum. The common fragment characteristic of phenolics is a successive or simultaneous loss of H2O and CO groups in their MS 2 spectra. Compound The characteristic fragment ions at 615.0638 and 309.0245 were generated through losing a C 13 H 9 O 9 moiety, which implies the existence of the residue region composed of D-and Erings. Compound 33 was tentatively attributed to phyllanthusiin C. The MS 2 fragmentation pathway ( Figure 4C) of phyllanthusiin C was proposed for the first time.

Other Phenolics
Ten phenolics with a small molecular mass (<350 Da) and less than 15 carbons were tentatively identified in P. capitatum. The common fragment characteristic of phenolics is a successive or simultaneous loss of H 2 O and CO groups in their MS 2 spectra. Compound 29 at t R 5.58 min gave a deprotonated ion at m/z 247.0246 (C 12 H 7 O 6 − ). The MS 2 spectrum of compound 29 showed the fragment ions at m/z 219.0293 and 203.0343 resulting from the neutral loss of CO and CO 2 unit from quasi-molecular ion, respectively. Furthermore, the ion at m/z 219.0293 went on losing a CO and a CO 2 unit in succession to form the ions at 191.0342 and 147.0440. Since brevifolin has previously been found in P. capitatum in the reported literature [20], compound 29 was tentatively identified as brevifolin. A lactone moiety or a carboxyl group was implied to be present in compound 46. In addition, the ion at m/z 191.0341 was generated through an RDA fragmentation reaction from the ion at m/z 231.0291. Compound 46 was tentatively identified as urolithin M5. Urolithin M5 was an intestinal bacterial metabolite of ellagitannin davidiin from P. capitatum [26] and was also found in natural higher plants from diverse families [27,28]. The compound might be biosynthesized through the polyketide pathway [29,30] by endophytic fungi residing in raw P. capitatum.

UHPLC-Q-Orbitrap HRMS Analysis of the Prototype Compounds in Hyperuricemia Rat Plasma
The TIC chromatograms and mass data of rat plasma from hyperuricemia and drugtreated groups were compared to analyze P. capitatum-related exogenous components. The peaks that appeared at the same positions in the TIC chromatograms of both the dosed rat plasma and the herb extract but not in the chromatogram of the model rat plasma were regarded as prototype constituents. As a result, 16 prototype components of P. capitatum were found in hyperuricemia rat plasma. The detailed mass information is shown in Table S1. Among these, ellagic acid, 5,7-dihydroxychromone, quercetin-3-O-glucuronide, quercitrin, 3,3 -di-O-methylellagic acid, flazin, salidroside, 3,4,5-trimethoxyphenol-1-O-β-D-glucopyranoside, fructose-phenylalanine, nudiposide, quercetin-3-O-β-D-galactoside, quercetin-3-O-β-D-glucopyranoside, kaempferol-4-O -rutinoside, N-feruloyltyramine and afzelin were found in rat plasma after oral administration of P. capitatum extracts for the first time.

UHPLC-Q-Orbitrap HRMS Analysis of P. capitatum Metabolites in Hyperuricemia Rat Plasma
The procedures for identification of metabolites included speculating probable metabolites according to the biotransformation rules of original compounds, extracting the [M − H] − or [M + H] + ions of probable metabolites from dosed plasma in full-scan mass mode and analyzing the MS 2 information of the detected peak. The detected metabolic mechanism of P. capitatum in hyperuricemia rats involved ring fission, hydrolysis, decarboxylation, dehydroxylation, methylation, glucuronidation and sulfation. In this study, a total of 52 metabolites of P. capitatum in rat plasma were tentatively identified. Among them, 50 metabolites were revealed for the first time. The details of the characterized metabolites are listed in Table 1.   − ) ascribed to the continuous losses of an SO 3 unit and a glucuronic acid residue. The ion at m/z 315.0511 lost a methyl group to yield the product ion at m/z 300.0275 and furtherly was subjected to an RDA fragmentation reaction to form the ion at m/z 148.0155. The cracking path of the ion at m/z 315.0511 was similar to 3-O-methylquercetin [35]. M41 was speculated as glucuronidation and sulfation of 3-O-methylquercetin.

Characterization of Phenolic-Related Metabolites
Ring cleavage was a common metabolic pathway of (epi)catechin in vivo [36,37]. These metabolites generated through ring cleavage were further bio-transformed through sulfation or glucuronidation. were consistent with the fragment pathway of m-coumaric acid or p-coumaric acid [39]. M16 was tentatively assigned as m-coumaric acid sulfate or p-coumaric acid sulfate. Based on the above data, the possible metabolic pathways of (epi)catechin in hyperuricemia rats administered orally with P. capitatum extract are shown in Figure 6A

Characterization of Tannis-Related Metabolites
According to the reported literature, although ellagitannins were not absorbed in vivo, ellagitannins located at the distal segment of the gastrointestinal tract could be bio-transformed by the intestinal bacteria into dibenzo-α-pyrones derivatives [40]. Compared to the base peak chromatography (BPC) of the plasma from the hyperuricemia group, a peak with high intensity at 10.13 min (M38) was detected in the BPC of dosed rat plasma.

Material and Reagents
The herb of P. capitatum was collected from Qianxi county, Guizhou province, China and was identified by Qingde Long (Guizhou Medical University) as the whole plant of Polygonum capitatum Buch.-Ham. ex D. Don. The voucher specimen of P. capitatum (No.: PC20201103) was deposited in the Herbarium of Guizhou Medical University.
HPLC-grade methanol and acetonitrile were acquired from Honeywell Burdick & Jackson Company (Morristown, NJ, USA). Formic acid (MS grade) was obtained from Fisher Scientific (Madrid, Spain). Deionized water for HPLC analysis was prepared using a Milli-Q water purification system (Millipore, Milford, MA, USA). All other reagents were of analytical grade.

Preparation of Mixed Standard Solutions
The stock solutions of standards were prepared by weighting appropriate amounts of 13 reference substances individually and dissolving them in methanol at a concentration of 1.0 mg/mL. The final mixed standard solution (200 ng/mL) was obtained by mixing the appropriate volumes of the stock solutions and diluting with 60% methanol before qualitative analysis.

Preparation of P. capitatum Samples
The dried raw herb of P. capitatum (1453 g) was weighed and crushed into power. The obtained powder was immersed in a ten-fold volume of distilled water for 30 min and decocted three times by boiling for 1 h. The decoctions were filtered to remove the herbal residue. The supernatants were merged together and concentrated to yield the extract residue (291.1 g, the extraction rate 20.03%).
10 mg of the obtained extract residue was dissolved in 1 mL of 60% (v/v) methanol and ultrasonicated for 30 min at 100 kHz. After centrifuging at 12,000 rpm for 10 min, 10 µL of the supernatant was used for UHPLC-Q-Orbitrap HRMS analysis.

Animal Treatment and Drug Administration
A total of 18 male Sprague-Dawley (SD) rats (weighing 200 ± 20 g) were obtained from Changsha Tianqin Biotechnology Company (Hunan, China). The rats were housed under a standard 12-h light-dark cycle at 25 ± 2 • C and 60 ± 5% humidity with free access to water and a normal diet for 7 days. All of the experiments were approved by the Animal Care Welfare Committee of Guizhou Medical University (approval number 2100138) and performed according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health).
Rats were randomly divided into three groups with six animals per group (control, hyperuricemia and drug-treated groups). Hyperuricemia was induced in the rats according to the described method previously [42]. Briefly, except for the control group, intragastric hypoxanthine (500 mg/kg/day) and intraperitoneal injection of potassium oxonate (100 mg/kg/day) were given to the rats for 7 days. The animals in the control group received physiological saline in a similar fashion. On the 4th day of hyperuricemia induction, the P. capitatum extract (5 g/kg/day) was administered orally to the rats in the drug-treated group at 1 h after dosing of the modeling agents for 3 days. The serum urate levels in the control and hyperuricemia rat groups were detected by urate assay kits during the experimental period. The serum urate levels in the hyperuricemia rat group significantly increased compared to those of the control rats (p < 0.05), indicating the successful establishment of the hyperuricemia model.

Collection and Preparation of Plasma Sample
Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h after the last administration from the retro-orbital plexus into heparinized tubes. Plasma was obtained through centrifugation at 3000 rpm for 10 min. All plasma samples at different time points from each group of rats were combined to produce the pooled sample for eliminating the individual variability. A 500 µL volume of the pooled plasma sample was added with 1.5 mL of acetonitrile and vortexed for 1.0 min to precipitate protein. The sample was centrifuged at 12,000 rpm and 4 • C for 10 min. The supernatant was evaporated to dryness under a gentle flow of nitrogen at room temperature. The residue was redissolved with 200 µL of 60% methanol in water and centrifuged at 12,000 rpm for 10 min. The supernatant was injected into the UHPLC-Q-Orbitrap HRMS system for analysis.

UHPLC-Q-Orbitrap HRMS Conditions
A Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) consisted of a quaternary solvent delivery system, a column compartment and a refrigerated auto-sampler. The sample separation was performed on an ACQUITY UPLC ® BEH C18 column (2.1 mm × 100 mm, 1.7 µm) eluted with acetonitrile (A) and 0.1% aqueous formic acid (B). The flow rate was set at 0.3 mL/min with an initial mobile phase of 5% (A). The chromatographic elution program was set: 5-5% A at 0-1. A Q-Exactive™ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) equipped with a heated electrospray ionization source (HESI) was used for qualitative analysis. The analysis was carried out both in positive and negative ion modes. The collision and nebulizing gases were ultra-high purity helium (He) and high purity nitrogen (N 2 ). The parameters were set as follows: ion spray voltage: +3.0 kV and −2.5 kV, capillary temperature: 320 • C, S-lens RF level: 60%. The flow rates of sheath gas and auxiliary gas were set to 35 and 10 arbitrary units, respectively. A full MS/dd-MS 2 acquisition program was executed with resolutions of 70,000 and 17,500 FWHM. For the full MS experiments, the scan range was from 80 to 1200 m/z, the automatic gain control (AGC) target was defined as 1e 6 and the maximum injection time (IT) was set as auto. For the dd-MS 2 experiments, AGC target: 2e 5 , maximum IT: auto, loop count: 1, the isolation window was 3.0 m/z. The stepped normalized collision energies (NCE) were 20, 40, and 60 eV.

Data Analysis
The information of chemical constituents from P. capitatum, including CAS number, molecular formula and molecular weight, were obtained by retrieving SciFinder Scholar and Dictionary of Natural Product databases. An in-house library containing potential compounds from P. capitatum extract was established. Data analysis was performed through Xcalibur 3.0 software (Thermo Fisher Scientific, Waltham, MA, USA) and Compound Discoverer 2.0 software coupled to mzCloud© and ChemSpider© databases. The data processing workflow for the identification of chemical ingredients from P. capitatum and its metabolites was shown in Figure 7.

Conclusions
In this study, a sensitive and accurate UHPLC-Q-Orbitrap HRMS method was utilized to systematically analyze the chemical constituents of P. capitatum and its absorbed components in hyperuricemia rats. A total of 114 compounds including phenolic acids, Figure 7. The data processing workflow for identification of chemical ingredients from P. capitatum and its absorbed constituents in hyperuricemia rat plasma.

Conclusions
In this study, a sensitive and accurate UHPLC-Q-Orbitrap HRMS method was utilized to systematically analyze the chemical constituents of P. capitatum and its absorbed components in hyperuricemia rats. A total of 114 compounds including phenolic acids, flavonoids, phenylpropanoids, tannins, phenolics, amino acids, amides and others were identified or characterized. At the same time, 68 P. capitatum-related xenobiotics were found in the hyperuricemia rats' plasma. These exogenous components in hyperuricemia rats might be the potential active constituents of P. capitatum for anti-hyperuricemia and anti-gouty arthritis. The detected metabolic pathway of P. capitatum in hyperuricemia rats included ring fission, hydrolysis, decarboxylation, dehydroxylation, methylation, glucuronidation and sulfation. This study not only supplied a basis for the further investigation of the active components and pharmacokinetics of P. capitatum, but also provided insight into the anti-hyperuricemia mechanism and quality control of P. capitatum.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27113521/s1, Table S1: Chemical constituents identified and characterized in P. capitatum by UHPLC-Q-Orbitrap HRMS in negative and positive ion modes. Figure S1: The chemical structures of the constituents from P. capitatum analyzed by UHPLC-Q-Orbitrap HRMS.