Metabolic Profiling of Alpinetin in Rat Plasma, Urine, Bile and Feces after Intragastric Administration

Alpinetin, a bioactive flavonoid, has been known to have a diverse therapeutic effect, with namely anti-inflammatory, anticancer and antioxidant effects with low systemic toxicity. This study aimed to obtain metabolic profiles of alpinetin in orally administrated rats. The metabolites of alpinetin were systematically analyzed and identified by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The chromatographic separation was achieved on a High Strength Silica (HSS) T3 (1.8 μm, 2.1 × 100 mm) column with the mobile phase consisting of water containing 0.1% formic acid and acetonitrile with 0.1% formic acid via gradient elution. An extracted ion chromatogram strategy based on multiple prototype/metabolite intermediate templates and 71 typical metabolic reactions was proposed to comprehensively profile the metabolites of alpinetin. With the metabolite profiling strategy, altogether 15 compounds were recognized from urine, plasma, bile and feces of rats after intragastric administration of alpinetin for the first time. The prototype, glucuronide conjugates and phenolic acids metabolites were the probable predominant form of alpinetin in rats. This work showed a comprehensive study of the probable metabolic pathways of alpinetin in vivo, which could provide meaningful information for future pharmacological studies.


Introduction
Alpinia katsumadai Hayata seeds, known as CaoDouKou in China, have been used in traditional Chinese medicine for thousands of years to cure digestive and inflammatory diseases [1]. Alpinetin is known as a natural flavonoid, and was firstly extracted from Amomum subulatum Roxb's seed. It is considered as the primary active ingredient of Zingiberaceae (Alpinia katsumadai Hayata) [2]. Modern pharmacological research shows a series of pharmacological activities that are involved in alpinetin, including anti-inflammatory [3][4][5][6], anti-cancer [7][8][9][10], anti-oxidant effects [11]. Alpinetin was found to improve the sensitivity of drug-resistant lung cancer cells to cis-diammined dichloridoplatium [10], which may be used as a potential drug for combination therapy. Recently, it has been reported that alpinetin may be deemed as a good candidate for some possible treatment of liver injury and brain diseases [12,13]. Because of its multiple therapeutic activities, alpinetin is considered as a potential candidate drug for further clinical studies, and has recently attracted more and more attention from medical and pharmaceutical fields [14][15][16]. Therefore, a better understanding of the efficacy of alpinetin needs to elucidate its biological fate in the body.
For an active component of natural medicine, the substance basis of pharmacological action may include not only the prototype drug component but also the metabolite formed by the biotransformation of the prototype compound [17]. Due to profound conjugative metabolism, alpinetin, which is known as a flavonoid with one hydroxyl group, tends to be featured with poorer oral bioavailability [18,19]. Previous metabolism studies found that the glucuronide conjugates are the major metabolites of alpinetin, but the biological and pharmacological activity of the glucuronide conjugated metabolites may be completely different from that of the prototype [20]. This suggests that not only alpinetin itself, but also its metabolites may be responsible for its numerous pharmacological effects. Therefore, the exploring of the metabolic characteristics of alpinetin is of great significance. To our knowledge, there are no relevant reports about the metabolic characteristics of alpinetin. In this study, the biological samples (plasma, urine, bile and feces) after intragastric administration of alpinetin were analyzed by ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The structures of metabolites were further characterized by high resolution mass (HRMS) and MS/MS fragment (MS n ) data. We hope that some meaningful information for the enlightenment of future pharmacological researches of alpinetin will be provided by the results.

Materials
Alpinetin (99.0% purity) was purchased from Chengdu Munster Biotechnology Co., Ltd. (SChuan, China). A Milli-Q system (Millipore, Bedford, MA, USA) prepared the ultra-pure water. Methanol, HPLC-grade formic acid, and acetonitrile were purchased in Sigma-Aldrich Co (St. Louis, MO, USA). Further, the other reagents and chemicals were of analytical grade.

Drug Administration and Animals
Male Sprague-Dawley (SD) rats (200 ± 20 g) were purchased from the Experimental Animal Center of Wenzhou Medical University (Wenzhou, China). All experimental procedures were approved by the Animal Ethics Committee in Wenzhou Medical University (No. WMU2018-0983). The animals were maintained in an environmentally controlled breeding room before experiments at a temperature of 23-25 • C and 45-55% humidity and then fasted for 12 h prior to the experiment.
Twelve rats were divided into four groups (three rats per group): Plasma, urine, bile and feces groups. The rats were given alpinetin by intragastric administration at a dose of 40 mg/kg. Bile samples were collected from bile intubated rats prior to dose and 0-4 h, 4-8 h, 8-12 h, 12-24 h, 24-36 h after administration. The samples of urine and feces were collected 4 h pre-dose and 0-12 h, 12-24 h, 24-36 h post-dose. Approximately 150 µL blood samples were collected from the heparinized tail vein at 0, 1, 5, 15, 30, 45 min, and 1, 3, 5, 8, 12, 24, 36 h after administration. Furthermore, the samples of plasma were acquired by samples of centrifuging blood for 10 min at 4500 rpm. All the plasma samples were mixed and pooled for the time points of 1-15 min, 30-60 min, 3-8 h, and 12-36 h across rats in equal volumes. Bile, urine, and feces samples were pooled at each time period in equal volumes or weights. All biological samples were frozen at −20 • C before analysis.
The MS experiments were performed by the positive mode via an electrospray ionization (ESI) interface. The following sets show the optimal MS parameters: Source temperature, 100 • C; capillary voltage, 2.5 KV; cone gas flow, 50 L/h; cone voltage, 40 V; desolvation gas flow, 800 L/h. Compounds were detected by precursor ions and fragment ions using elevated collision energies (MS E ) centroid analysis from m/z 100-1200 Da at a resolving power of 30,000 with a scan time of 0.2 s. Nitrogen was used as cone gas, and argon as collision gas. Collision-induced dissociation was performed with low-energy and high-energy functions. In the low-energy function, collision energy was off to acquire HRMS data; in the high-energy function, a collision energy ramp 20-40 V was used to obtain MS n data.

Sample Preparation
The protein precipitation method was performed to pre-treated samples. Pooled plasma (2 mL), urine (1 mL), and bile samples (1 mL) were extracted with methanol (10 mL) in test tubes. Freeze-dried pooled fece samples were milled and weighed (100 mg). Each fece sample was ultrasonically extracted with methanol (1 mL) for 60 min. After extraction, all biological samples were vortexed for 5 min and centrifuged at 4500 rpm for 10 min. The supernatants were evaporated in a nitrogen stream at 30 • C to dryness. The residues were dissolved in methanol of 100 µL and vortexed for 5 min. The sample was then centrifuged at 13,000 rpm for 10 min, and 10 µL of the supernatant was injected into the UPLC-Q-TOF-MS system for analysis.

Data Analysis
The data of UPLC-Q-TOF-MS were obtained and processed with MassLynx Version 4.1 software (Waters Co.). The process of metabolite profiling was divided into two steps. Firstly, an in-house developed software was designed to predict expected alpinetin metabolites automatically. This method used alpinetin and its metabolic intermediate as templates, inputted their chemical formula, calculated the accurate high resolution mass spectrometric data with the ionization set as [M + H] + and [M − H] − , and predicted expected alpinetin metabolites based on 71 typical template metabolic reactions. Secondly, screening and validation of potential metabolites by extracted ion chromatogram was performed with mass tolerance set to 5 ppm, and MS 2 confirmation manually.

Mass Fragmentation of Alpinetin
Drug metabolism is a process of structural modification of drugs through enzymatic systems in vivo and in vitro. Since the prototype compounds and their metabolites are similar in chemical structure, it is necessary to study the mass spectrometric decomposition of prototype compounds before studying the metabolites of alpinetin.
The alpinetin was detected in plasma, urine, bile and feces, which was eluted at 16.52 min. It exhibited an accurate [M + H] + ion at m/z 271.0943 (C 16 H 15 O 4 ). The fragment ion at m/z 257.1538 resulted from a fragment ion m/z 271.0943 by the loss of -CH 2 . Fragmentation ions of m/z 104.0645, and 167.0298 were produced by the Retro Diels-Alder reaction, which is a typical cleavage method for flavonoids. The MS 2 spectra showed a characterized fragmentation pathway of m/z: 167.0297→151.0581→123.1169 (with a loss of -O and -CO, respectively). Comparison of the retention time between P and the alpinetin standard further indicated that P was alpinetin.

Identification of Metabolites
The biological samples (plasma, urine, bile and feces) of alpinetin were analyzed by way of the UPLC-Q-TOF-MS method. The biological samples of alpinetin and blank samples were obtained and processed with MassLynx Version 4.1 software. Firstly, 71 typical metabolic reactions were incorporated in an in-house developed software. Multiple templates were selected based on an extensive literature review and experience-rich structural analysis to generate a list of potential metabolites. Following that, potential metabolites would further be validated with multiple-dimension data, including the extracted ion chromatogram (EIC) strategy (HRMS data), mass difference match (HRMS data), and fragment confirmation (MS n data). The maximum mass errors between the measured and calculated values were <5 ppm. With the metabolite profiling strategy, a total of 14 metabolites were identified from biological samples after intragastric administration of alpinetin (Table 1).   Figure 2, the prototype, glucuronide conjugates and phenolic acids metabolites were the predominant form of alpinetin in rats. The structure and the MS 2 spectrum of the alpinetin and its probable metabolites are shown in Figure 3. This work showed a comprehensive study of the probable metabolic pathways of alpinetin in vivo. Future work will focus on the synthesis of newly identified major metabolites and assess their biological activity and toxicity.   Figure 2, the prototype, glucuronide conjugates and phenolic acids metabolites were the predominant form of alpinetin in rats. The structure and the MS 2 spectrum of the alpinetin and its probable metabolites are shown in Figure 3. This work showed a comprehensive study of the probable metabolic pathways of alpinetin in vivo. Future work will focus on the synthesis of newly identified major metabolites and assess their biological activity and toxicity.

Discussion
Alpinetin, a bioactive flavonoid, has been known to have diverse therapeutic effects, namely anti-inflammatory, anticancer and antioxidant effects. The main objective of our study was to analyze the metabolites of alpinetin in rats. Alpinetin, which is known as a flavonoid with one hydroxyl group, tends to be characterized with poorer bioavailability due to a profound conjugative metabolism [21]. We demonstrated in this study that significant glucuronidation was undergone by alpinetin in rats. It was noteworthy that the glucuronide conjugates could be detected in rat plasma, urine and bile. This was supported by the fact that intestine-specific enzyme uridine diphospho-glucuronosyltransferase1A10 (UGT1A10) is characterized with the poorest ability to catalyze alpinetin glucuronidation [18]. It has been reported that the UGT1A1, 1A3, 1A9 and 2B15 were determined to participate in the alpinetin glucuronidation in human liver microsomes [20]. Therefore, due to the prevalence of genetic polymorphism in UGT enzymes, individuals with different polymorphisms may exhibit different metabolic activities of alpinetin.
In our study, the metabolites of alpinetin can be explained by eleven proposed pathways: Deoxidation, reduction, oxidation, desaturation, acetylation, glycine conjugation, sulfation, glucuronidation, demethylation, hydration and cleavage. In recent years, more and more attention has been paid to the metabolic process of flavonoids [22]. The metabolic pathways of flavonoids mainly include conjugation, cleavage and oxidation in vivo [23,24], which is consistent with the conclusions of the metabolic characteristics of alpinetin in our study. However, to date, there are no systematic and detailed data on alpinetin metabolites. This study enriched the known metabolic types of alpinetin and made the study of alpinetin metabolism more comprehensive.
From our current studies, major metabolites in the biosamples were different from each other. It was found that the prototype, glucuronide conjugates and phenolic acids were the predominant compounds of alpinetin in rat plasma. The predominant metabolites of alpinetin in rat urine and bile were the prototype, glucuronide conjugates, deoxidated and phenolic acids metabolites. The prototype and sulfate conjugates were the main metabolites of alpinetin in rat feces. It can be preliminarily speculated that alpinetin was absorbed into the blood in the small intestine of rats as prototype and phenolic acids. The absorbed prototype was glucuronidated in the blood and liver to form glucuronide conjugates and converted into bile or blood circulation. Finally, the prototype,  Figure 3. Structure and the MS 2 spectrum of the alpinein and its probable metabolites.

Discussion
Alpinetin, a bioactive flavonoid, has been known to have diverse therapeutic effects, namely anti-inflammatory, anticancer and antioxidant effects. The main objective of our study was to analyze the metabolites of alpinetin in rats. Alpinetin, which is known as a flavonoid with one hydroxyl group, tends to be characterized with poorer bioavailability due to a profound conjugative metabolism [21]. We demonstrated in this study that significant glucuronidation was undergone by alpinetin in rats. It was noteworthy that the glucuronide conjugates could be detected in rat plasma, urine and bile. This was supported by the fact that intestine-specific enzyme uridine diphospho-glucuronosyltransferase1A10 (UGT1A10) is characterized with the poorest ability to catalyze alpinetin glucuronidation [18]. It has been reported that the UGT1A1, 1A3, 1A9 and 2B15 were determined to participate in the alpinetin glucuronidation in human liver microsomes [20]. Therefore, due to the prevalence of genetic polymorphism in UGT enzymes, individuals with different polymorphisms may exhibit different metabolic activities of alpinetin.
In our study, the metabolites of alpinetin can be explained by eleven proposed pathways: Deoxidation, reduction, oxidation, desaturation, acetylation, glycine conjugation, sulfation, glucuronidation, demethylation, hydration and cleavage. In recent years, more and more attention has been paid to the metabolic process of flavonoids [22]. The metabolic pathways of flavonoids mainly include conjugation, cleavage and oxidation in vivo [23,24], which is consistent with the conclusions of the metabolic characteristics of alpinetin in our study. However, to date, there are no systematic and detailed data on alpinetin metabolites. This study enriched the known metabolic types of alpinetin and made the study of alpinetin metabolism more comprehensive.
From our current studies, major metabolites in the biosamples were different from each other. It was found that the prototype, glucuronide conjugates and phenolic acids were the predominant compounds of alpinetin in rat plasma. The predominant metabolites of alpinetin in rat urine and bile were the prototype, glucuronide conjugates, deoxidated and phenolic acids metabolites. The prototype and sulfate conjugates were the main metabolites of alpinetin in rat feces. It can be preliminarily speculated that alpinetin was absorbed into the blood in the small intestine of rats as prototype and phenolic acids. The absorbed prototype was glucuronidated in the blood and liver to form glucuronide conjugates and converted into bile or blood circulation. Finally, the prototype, deoxidated metabolites, glucuronide conjugates and sulfate conjugates were excreted through feces and urine.

Conclusions
This study aimed to obtain metabolic profiles of alpinetin in plasma, urine, bile and feces of the rats after intragastric administration. With the metabolite profiling strategy, altogether 15 metabolites were detected, including 12 metabolites in urine, 7 metabolites in bile, 9 metabolites in feces and 4 metabolites in plasma. The prototype, glucuronide conjugates and phenolic acids metabolites were the predominant form of alpinetin in rats. This research provides meaningful information for future pharmacological studies on alpinetin, and leads to a better understanding of the bio-transformations and the pharmaceutical applications of alpinetin.