Systematic Identification, Fragmentation Pattern, And Metabolic Pathways of Hyperoside in Rat Plasma, Urine, And Feces by UPLC-Q-Exactive Orbitrap MS

Hyperoside is a natural flavonol glycoside, which has antioxidation, antitumor, and anticancer activities together with other healthy effects like improving cardiovascular function, protecting the liver, and regulating the immune system. It is a popular compound used in the traditional Chinese medicine and different studies on hyperoside are present in the literature. However, studies on the metabolism of hyperoside in vivo were not comprehensive. In this study, UPLC-Q-Exactive Orbitrap MS technology was used to establish a rapid and comprehensive analysis strategy to explore the metabolites and metabolic process of hyperoside in rats. The metabolites of hyperoside were systematically identified in rat plasma, urine, and feces. According to the hyperoside standard substance and relevant works of literature, a total of 33 metabolites were identified, including 16 in plasma, 31 in urine, and 14 in feces. Among them, the metabolites quercetin and dihydroquercetin were unambiguously confirmed by comparison with standard substances. In addition, 13 metabolites had not been reported in hyperoside metabolism-related articles at present. The metabolic reactions of hyperoside in vivo were further explored, including phase I metabolism (hydroxylation, dehydroxylation, glycoside hydrolysis, hydrogenation, and hydration) and phase II metabolism (methylation, acetylation, sulfation, and glucuronide conjugation). The fragment ions of hyperoside and its metabolites were usually produced by glucoside bond hydrolysis, the neutral loss of (CO + OH), COH, CO, O, and Retro-Diels Alder (RDA) cleavage. In conclusion, this study comprehensively characterized the metabolism of hyperoside in rats, providing a basis for exploring its various biological activities.

In view of many biological activities and pharmacological effects, hyperoside has been widely studied [13]. As the basis for research, studies on its metabolites in vivo have been reported. For example, Guo et al. [14] used UPLC-Q-TOF-MS to characterize 12 different types of hyperoside metabolites (including the prototype) in rat plasma, urine, and bile. Yang et al. [15] studied the interaction between hyperoside and human intestinal bacteria and detected the prototype compound and six other metabolites from the isolated bacterial samples. However, the metabolites analysis and metabolic process of hyperoside in vivo are not comprehensive at present. erefore, it is necessary to study the metabolites of hyperoside systematically in rat plasma, urine, and feces, so as to deeply make a further comprehensive characterization of hyperoside in rats and explore its pharmacological mechanism.
At present, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) are commonly used in metabolism analysis in vivo [16]. UPLC-Q-Exactive Orbitrap MS has the advantages of high resolution, high-quality accuracy, high sensitivity, and wide scanning range compared with other detection instruments [17]. It can realize efficient and rapid identification and analysis of compounds. For example, San Miao Wan (SMW) is a prescription that consists of Phellodendri Chinensis Cortex, Atractylodis Lanceae Rhizoma, and Achyranthis Bidentatae Radix and has a complex composition. Ma et al. [18] used UPLC-Q-Exactive Orbitrap MS to characterize 76 constituents of SMW and to detect 113 metabolites in rat plasma. erefore, in this study, UPLC-Q-Exactive Orbitrap MS was used to systematically analyze hyperoside and its metabolites in rats after an oral administration, which would help to find potential active metabolites of hyperoside.
is study aimed to use UPLC-Q-Exactive Orbitrap MS to comprehensively and accurately analyze hyperoside and its metabolites in rats after an oral administration. e metabolites of hyperoside in rat plasma, urine, and feces were identified systematically, the fragmentation patterns were summarized, and the metabolic pathways of hyperoside were further explored, which would help to find the target of active components, promote the efficient development and utilization of hyperoside.

Animal
Experiments. Ten healthy Sprague-Dawley rats, (male, SPF grade), weighing 200 ± 20 g, were purchased from Beijing huafukang Biotechnology Co., Ltd. (Beijing), license number: SCXK (jing) 2019-0008. All rats were kept in an animal room with darkness and light alternated for 12 hours. e room temperature was 23 ± 2°C and the relative humidity was 60 ± 5%. Free access to food and water. After keeping the adaptation period for 3 days, the rats were randomly divided into two groups: group A, the plasma group; group B, the urine and fecal group. Before the experiment, they fasted for 12 hours but drank freely. All experiments were carried out in accordance with the approved Regulations on the Management of Experimental Animals and the Detailed Rules on the Management of Chinese Medical Experimental Animals. All animal experiments were approved by the Medical Ethics Review Committee of Animal Experiments of Beijing University of Chinese Medicine (NO: BUCM-4-2022040602-2003).
Hyperoside was dissolved in 0.1% carboxymethyl cellulose sodium salt (CMC-Na) buffer solution up to 50 mg/ mL and given to rats at a dose of 350 mg/kg. In group A, the plasma samples were collected from the orbital venous plexus of rats at 10 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, and 6 h after administration, respectively. e obtained plasma samples were centrifuged at 3500 rpm, 4°C for 10 min, and the supernatant was taken. Rats in group B were placed in metabolic cages after administration, and the urine and feces were collected in time periods: 0-4 h, 4-8 h, 8-12 h, and 12-24 h. e collected urine samples were centrifuged at 12000 rpm in a 4°C high-speed freezing centrifuge for 15 min, and then the supernatant was taken. e fecal samples were dried in the fume hood, then crushed and sealed. e plasma of group A and the urine and feces of group B collected before administration were as blank samples. All the above-given samples were stored at − 80°C for later use.

Preparation of Plasma
Samples. 50 μL of plasma was taken from different blood collection points of each rat from group A and combined. 5.25 mL of acetonitrile was added to precipitate the proteins, followed by 5 min of a vortex and a centrifugation step at 12000 rpm for 15 min at 4°C. e supernatants were collected and dried under a nitrogen stream.

Preparation of Urine
Samples. 20 μL urine of each rat from group B was collected at 4 time periods: 0-4 h, 4-8 h, 8-12 h, and 12-24 h and mixed evenly. 1.2 mL of acetonitrile was added to precipitate the proteins, followed by 5 min of a vortex and a centrifugation step at 12000 rpm for 15 min at 4°C. e supernatants were collected and dried under a nitrogen stream. Before analysis, all pretreated samples were dried under nitrogen, and the residue was redissolved with 200 μL 10% methanol, followed by 5 min of a vortex, centrifugation at 12000 rpm for 15 min at 4°C, and then the supernatant was taken. Finally, the supernatant of each sample was injected into UPLC-Q-Exactive Orbitrap MS for analysis.
For mass spectrum acquisition, a Q-Exactive Orbitrap MS ( ermo Fisher, USA) was used with electrospray ionization source (ESI) in negative mode and MS conditions were optimized as follows: sheath gas flow rate was 40 arb and aux gas flow rate was 10 arb; spray voltage was at 3.0 kV; capillary temperature was at 350°C; high-resolution MS Journal of Analytical Methods in Chemistry scanning was performed under full scan/dd-MS 2 covered the range from m/z 100 to 1200 with a full scan at a resolution of 70000 and dd-MS 2 resolution of 17500.

Data Processing and
Analysis. e chromatograms were imported into Xcalibur 3.0 software to calculate the mass to charge ratio of possible excimer ions of the compound. Based on this, data processing such as peak extraction and peak matching were performed on the collected spectrograms. Based on the retention time and characteristic fragment ions of standards, the compounds were quickly annotated. e error was less than 10 ppm.

Results and Discussion
Hyperoside is a flavonol glycoside and has multiple phenolic hydroxyl groups. So it tends to lose H and has a negative charge when ionized. e fragment ion spectra and proposed fragmentation pathways of hyperoside in negative mode are shown in Figure 2. e [M-H] − ion at m/z 463.0887 lost galactoside and galactoside radical, respectively, to generate aglycone ion at m/z 301.0356 and aglycone radical ion at m/z 300.0274. e ion at m/z 300.0274 further lost (CO + OH) to generate the product ion at m/z 255.0297. Other product ions at m/z 271.0247, 243.0299, and 227.0348 were also generated after a consecutive loss of COH, CO, and O from the ion at m/z 300.0274. In addition, the aglycone ion was prone to Retro-Diels Alder (RDA) cleavage, resulting in characteristic ions at m/z 151.0038 and 149.0249. e results showed that a total of 33 metabolites were identified, including 16 in plasma, 31 in urine, and 14 in feces. As shown in Figure Table 1. Among them, M6, M8, M10, M14-15, M17-M23, and M27 were newly discovered metabolites in this study, which had not been reported in the identification literature up to now.

Metabolite Analysis in Plasma.
A total of 16 metabolites including the prototype were identified in plasma compared with the blank control group. e total ion chromatogram (TIC) of blank plasma is shown in Figure 4(a) and hyperoside-contained plasma is shown in Figure 4(b). e extract ion chromatogram (EIC) of hyperoside and its metabolites in rat plasma is shown in Figure 5

Metabolite Analysis in
Urine. 31 compounds were detected in rat urine, of which 16 were also identified in plasma samples. Figures 4(c) and 4(d) show the TIC of blank urine and drug-contained urine respectively. Figure 5(b) shows the EIC of hyperoside and its metabolites in rat urine.    erefore, M7 was probably acetylhyperoside.
In the results of this experiment, M6, M8, M10, M14-15, M17-M23, and M27 were hyperoside metabolites that had not been reported previously, and these metabolites might also have other biological activities [20]. For example, hyperoside was hydrolyzed to form its aglycone quercetin, as an intermediate metabolite, quercetin continued to undergo hydro reduction reaction to produce dihydroquercetin (M19).
Dihydroquercetin, also known as taxifolin, has anticancer, antibacterial, scavenging free radicals, and other effects [21] but has a low content in plants [22]. And, this metabolic pathway had not been reported in the literature on hyperoside or quercetin metabolism in vivo. erefore, new research contents were added to this study. In addition, dihydroquercetin is a flavonoid compound with high antioxidant, capillary-protective, and anti-inflammatory activity [23]. Some studies have shown that dihydroquercetin was more effective than quercetin in inhibiting the superoxide produced by xanthine oxidase and has a stronger antioxidant effect [24,25].
ere were also studies that showed dihydroquercetin pretreatment markedly alleviated cardiac dysfunction, scavenged free radicals, reduced lipid peroxidation, and increased the activity of antioxidant enzymes ex vivo and in vitro [26]. So it was proposed to be useful in the prevention and treatment of cardiovascular disease.

Proposed Metabolic Pathways of Hyperoside in Rats.
In this experiment, UPLC-Q-Exactive Orbitrap MS was used to systematically identify hyperoside and its metabolites in rat plasma, urine, and feces, so as to clarify the metabolic pathways of hyperoside in vivo. A total of 33 metabolites were identified, including 10 phase I metabolites and 23 phase II metabolites. e metabolic pathways of hyperoside in rats included phase I metabolic reactions such as glycoside hydrolysis, hydroxylation, dehydroxylation, hydrogenation, and hydration, and phase II metabolic reactions such as methylation, acetylation, glucuronidation, and sulfation. e analysis results showed that the type of phase II metabolites 8  Journal of Analytical Methods in Chemistry detected in urine was the most abundant, indicating that hyperoside was easy to combine with some endogenous substances such as glucuronide and sulfate after entering the body, resulting in the increasing of polarity and water solubility of the metabolites, which were more likely to be excreted through the urine [27]. e nine metabolic reactions of hyperoside involved and the number of metabolites in rat plasma, urine, and feces are shown in Table 2 (duplicate metabolites in diverse samples were only counted at once). e proposed metabolic pathways of hyperoside in rats are shown in Figure 8. In total, 10 metabolites were produced by phase I metabolism. Hydroxylation, dehydroxylation, glucoside hydrolysis, hydrogenation, and hydration were the main first-stage metabolic pathways. Hyperoside lost the hydroxyl group to produce M4 and reacted with the hydroxyl group to      glucuronidation. Compared with hyperoside, the polarity and water solubility of these metabolites increased, which was conducive to excretion.

Conclusion
In this study, UPLC-Q-Exactive Orbitrap MS was used to establish an accurate and systematic analysis method to study the biotransformation of hyperoside in rats. e fragmentation patterns of hyperoside in rats were summarized. Hyperoside was prone to glycoside hydrolysis in vivo, resulting in producing aglycone quercetin. Quercetin, as an intermediate product, further went through other metabolic transformations in vivo.
e neutral loss of (CO + OH), COH, CO, O, and RDA cleavage were also used as the specific fragmentation rules of this compound, which provided the basis for rapid screening of hyperoside metabolites. us, a total of 33 metabolites were found in rats, including 16 in plasma, 31 in urine, and 14 in feces, of which 10 were phase I metabolites and 23 were phase II metabolites. Besides, the metabolites quercetin and dihydroquercetin were unambiguously confirmed by comparison with standard substances. e metabolic pathways of hyperoside in rats included hydrolysis, hydroxylation, dehydroxylation, hydro reduction, hydration, methylation, acetylation, glucuronidation, and sulfation. In this study, 13 metabolites had not been reported in the articles related to hyperoside or quercetin metabolism in vivo, and these metabolites might also have many potential drug activities.

Data Availability
e data used to support the findings of this study are included within the manuscript and are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.