In Vivo Metabolite Profiling of DMU-212 in ApcMin/+ Mice Using UHPLC-Q/Orbitrap/LTQ MS

3,4,5,4’-Trans-tetramethoxystilbene (Synonyms: DMU-212) is a resveratrol analogue with stronger antiproliferative activity and more bioavailability. However, the metabolite characterization of this component remains insufficient. An efficient strategy was proposed for the comprehensive in vivo metabolite profiling of DMU-212 after oral administration in ApcMin/+ mice based on the effectiveness of the medicine. Ultra-high performance liquid chromatography-quadrupole/orbitrap/linear ion trap mass spectrometry (UHPLC-Q/Orbitrap/LTQ MS) in the AcquireXTM intelligent data acquisition mode, combining the exact mass and structural information, was established for the profiling and identification of the metabolites of DMU-212 in vivo, and the possible metabolic pathways were subsequently proposed after the oral dose of 240mg/kg for 3 weeks in the colorectal adenoma (CRA) spontaneous model ApcMin/+ mice. A total of 63 metabolites of DMU-212 were tentatively identified, including 48, 48, 34 and 28 metabolites in the ApcMin/+ mice’s intestinal contents, liver, serum, and colorectal tissues, respectively. The metabolic pathways, including demethylation, oxidation, desaturation, methylation, acetylation, glucuronide and cysteine conjugation were involved in the metabolism. Additionally, further verification of the representative active metabolites was employed using molecular docking analysis. This study provides important information for the further investigation of the active constituents of DMU-212 and its action mechanisms for CRA prevention.


Introduction
Colorectal cancer (CRC) is a malignant tumor of the gastrointestinal tract that is highly likely to metastasize and recur, and it is associated with an extremely high mortality rate [1]. Colorectal adenoma (CRA) is a frequent intestinal mucosal disease, which can progress into CRC in the adenoma-carcinoma sequence [2]. Early chemopreventive interventions of precancerous CRA are the most effective measure for the reduction in mortality and morbidity associated with CRC [3]. Currently, nonsteroidal anti-inflammatory drugs, COX-2 inhibitors, and calcium are potential candidates as chemopreventive agents for CRC. However, the limitation of a lower effectiveness and side effects hindered further application [4].
3,4,5,4'-Trans-tetramethoxystilbene (Synonyms: DMU-212, Figure 1) is a methylated analogue of resveratrol possessing greater bioavailability and stronger anti-cancer activity [5]. It has been examined as a potential chemopreventive agent that can prevent CRC progression through reducing the number of adenomas and suppressing tumor formation [5]. It has been examined as a potential chemopreventive agent that can progression through reducing the number of adenomas and suppressing tu in the colon of the Apc Min/+ mice; the inhibitory effect of DMU-212 on CRA g related to the interference with prostaglandin E-2 generation in cells [6]. study of ours, we demonstrated that the mechanisms of CRA prevention u were related to gut microbiota and its metabolites (unpublished data) an 212 has the potential for use as a chemopreventive agent to prevent CR outstanding efficacy of DMU-212 for anti-CRA, the active constituent metabolism study of DMU-212 related to the pharmacological effect are un According to one study, DMU-212 undergoes metabolic oxidation, and O-demethylation reactions in liver extracts of mice and an incubate microsomes, as a result of which five metabolites are generated: DMU-2 DMU-291, DMU-295 and DMU-807. DMU-212, administrated to mice, pr levels of drug in the small intestine, colon mucosae, and brain tha Furthermore, DMU-212 is more easily able to cross the blood-brain resveratrol due to its higher lipophilicity [7]. It is of utmost importance metabolites and metabolic pathways of a drug during drug discovery and However, taking into highly complex biological matrices, discove metabolites, and identifying the significant features remains a big challen few corresponding metabolites of DMU-212 have been identified using th Q tandem quadrupole mass spectrometer upgraded to the Quattro MK II sp healthy mice [7]. This was not sufficient for the research on the active co action mechanisms of DMU-212 for anti-CRA. Consequently, it is necess efficient strategies that could identify and targeted detect the numerous prototypes and metabolites from the complex biological matrix to systema and characterize the absorbed components of DMU-212 in CRA spont Apc Min/+ mice.
The Orbitrap ID-X Tribrid mass spectrometer with a quadrupole, orb and linear ion trap has an enhanced mass resolution power, scan speed, an perform MS n for better structural characterization. The AcquireX TM da workflow includes an automatic background subtraction, fast polarity-sw isotope in-tensity-filtering, and other real-time decision-making feature Discoverer 3.3 software carried out the metabolite analysis and compou Thus, the chemical constituents in complex matrix samples can be detected in a sensitive and high-resolution platform. In the present study, a c strategy was proposed for the rapid identification of the metabolites of DM oral dose of 240mg/kg for 3 weeks in CRA spontaneous model Apc Min/+ According to one study, DMU-212 undergoes metabolic oxidation, hydroxylation and O-demethylation reactions in liver extracts of mice and an incubate of mouse liver microsomes, as a result of which five metabolites are generated: DMU-214, DMU-281, DMU-291, DMU-295 and DMU-807. DMU-212, administrated to mice, produced higher levels of drug in the small intestine, colon mucosae, and brain than resveratrol. Furthermore, DMU-212 is more easily able to cross the blood-brain barrier than resveratrol due to its higher lipophilicity [7]. It is of utmost importance to identify the metabolites and metabolic pathways of a drug during drug discovery and development. However, taking into highly complex biological matrices, discovering possible metabolites, and identifying the significant features remains a big challenge [8]. Only a few corresponding metabolites of DMU-212 have been identified using the Quattro Bio-Q tandem quadrupole mass spectrometer upgraded to the Quattro MK II specifications in healthy mice [7]. This was not sufficient for the research on the active constituents and action mechanisms of DMU-212 for anti-CRA. Consequently, it is necessary to develop efficient strategies that could identify and targeted detect the numerous trace-absorbed prototypes and metabolites from the complex biological matrix to systematically identify and characterize the absorbed components of DMU-212 in CRA spontaneous model Apc Min/+ mice.
The Orbitrap ID-X Tribrid mass spectrometer with a quadrupole, orbitrap analyzer, and linear ion trap has an enhanced mass resolution power, scan speed, and the ability to perform MS n for better structural characterization. The AcquireX TM data acquisition workflow includes an automatic background subtraction, fast polarity-switching mode, isotope in-tensity-filtering, and other real-time decision-making features. Compound Discoverer 3.3 software carried out the metabolite analysis and compound annotation. Thus, the chemical constituents in complex matrix samples can be detected at trace levels in a sensitive and high-resolution platform. In the present study, a comprehensive strategy was proposed for the rapid identification of the metabolites of DMU-212 after the oral dose of 240 mg/kg for 3 weeks in CRA spontaneous model Apc Min/+ mice based on ultra-high performance liquid chromatography-quadrupole/orbitrap/linear ion trap mass spectrometry (UHPLC-Q/Orbitrap/LTQ MS) combined with the data processing software "Compound Discoverer 3.3" and the AcquireX TM data acquisition workflow. The representative active metabolites were supported by data from molecular docking assays. This study provides valuable information regarding DMU-212's active constituents and its mechanism of action for preventing CRA.

Establishment of the Analytical Strategy
In this paper, an effective strategy was established for metabolite identification of DMU-212 in the Apc Min/+ mice's blood, liver, colorectal tissues and intestinal contents using UHPLC-Q/Orbitrap/LTQ MS coupled with multiple data-processing methods. Firstly, data extraction was used to obtain and interpret the data from the normal and CRA samples using qualitative Acquire X TM iterative data dependent MS 2 (dd-MS 2 ) and quantitative full-MS analyses. The inclusion and exclusion lists are automatically generated in Deep Scan mode upon the injection of the blank and sample, respectively. In the iterative injections of the drug-containing sample, both lists (inclusion/exclusion) are automatically updated, allowing a comprehensive acquisition of the relevant features, excluding those present in the blanks. Then, the raw control and sample files are imported into the Compound Discovery software, and the DMU-212 structure was analyzed to acquire the cleavage pathways and diagnostic product ions for metabolite identification. The workflow parameters include the type of transformation steps and list of possible adduct ions, and it also performs a retention time alignment, unknown compound detection, and compound grouping. An expected ion list was generated based on the above. Thirdly, the metabolite templates were summarized and established using the reported metabolic transformations of the ingredients in the literature. Compared with the blank biosamples, including blood, liver, colorectal tissues and intestinal contents, the DMU-212 metabolites in the biosamples were identified using multiple metabolite templates, the exact mass, retention time, and structural information. The summary analytical strategy diagram is shown in Figure 2.

Comprehensive Characterization of Metabolites of DMU-212 In Vivo
After intragastric gavage (i.g.) administration to the Apc Min/+ mice, DMU-212 (M0) was detected in the blood, liver, colorectal tissues and intestinal contents samples, according to the retention time (t R = 7.454 min), the accurate quasimolecular ion, as well as the characteristic product ions. DMU-212 was detected in all samples in vivo. A total of 63 DMU-212 metabolites were detected and characterized from the Apc Min/+ mice's blood, liver, colorectal tissues and intestinal contents samples according to the suggested transformation pathways, molecular formula, and mass spectrometry fragment ions by means of the UHPLC-Q/Orbitrap/LTQ MS method coupled with the established strategy. All the information on the 63 metabolites of DMU-212 detected using UHPLC-Q/Orbitrap/LTQ MS are listed in Table S2. In addition, Supplementary Figure S4 shows the MS/MS spectrum of DMU-212 and its metabolite.

Comprehensive Characterization of Metabolites of DMU-212 In Vivo
After intragastric gavage (i.g.) administration to the Apc Min/+ mice, DMU-212 (M was detected in the blood, liver, colorectal tissues and intestinal contents sampl according to the retention time (t R = 7.454 min), the accurate quasimolecular ion, as w as the characteristic product ions. DMU-212 was detected in all samples in vivo. A total 63 DMU-212 metabolites were detected and characterized from the Apc Min/+ mice's bloo liver, colorectal tissues and intestinal contents samples according to the suggest transformation pathways, molecular formula, and mass spectrometry fragment ions is 30 Da (O) more than that of DMU-212. The difference in mass corresponds to the binding of two oxygen atoms and the loss of two hydrogen atoms. In the MS 2 spectra, these metabolites had common fragments with the parent drug, indicating they are the oxidized and desaturated derivatives of DMU-212.
Demethylated and dehydrated (and reduced/desaturated) metabolites: Metabolites M23 and M24 were detected at the retention time of 6.331 and 6.144 min, respectively. They both showed the [M + H] + ions at around m/z 255 (C 16 O 4 , suggesting that it lost two or three methyl moieties and added two hydrogen atoms. All these metabolites had common fragments with the parent drug.
Other Phase I metabolites: The fitted chemical formula of M11 was C 17 H 16 O 5 , with [M + H] + ions at around m/z 301, a difference of (-CH 4 +O) with DMU-212, which indicated the loss of the alkyl chain moiety, oxidation in the benzene ring and desaturation, and the characteristic ions were in accordance with the speculative structure. Metabolite M12, with [M + H] + ions at around m/z 305, was eluted at 5.554 min. The metabolite was 4 Da more than DMU-212 with the formula C 17 H 20 O 5 , which was yielded via demethylation of the alkyl chain and hydration, and the characteristic fragment ions at the MS 2 spectra, such as m/z 241, 121 were able to be detected. Metabolite M59 was found at 5.846 min with [M + H] + ions at around m/z 319 (with a mass shift of 18 Da, exactly bound to a water molecule), so this metabolite showed the characteristic fragments and could be a hydrated metabolite of DMU-212.

Phase II Metabolites Identification
Glucuronide-conjugated and/or methylated metabolites: Metabolite M56 was eluted at 6.112 min and exhibited a molecular formula of C 23

Metabolic Profiles of DMU-212
A total of 63 metabolites with different structures were observed and identified in the biosamples in vivo ( Figure 4A). Among them, 34 metabolites were detected in the Apc Min/+ mice's serum and all of them can be observed in the Apc Min/+ mice's liver (48 metabolites) except for two unique metabolites. Another three unique metabolites were also identified in the Apc Min/+ mice's colorectal tissues and the rest of the 25 metabolites in colorectal tissues can be found in the serum, liver or intestinal contents, of which 2 metabolites can only be detected in the intestinal contents (50 metabolites) and 1 metabolite can only be obtained in the livers of the Apc Min/+ mice. Apc Min/+ mice's serum and all of them can be observed in the Apc Min/+ mice's liver metabolites) except for two unique metabolites. Another three unique metabolites w also identified in the Apc Min/+ mice's colorectal tissues and the rest of the 25 metabolites colorectal tissues can be found in the serum, liver or intestinal contents, of which metabolites can only be detected in the intestinal contents (50 metabolites) and metabolite can only be obtained in the livers of the Apc Min/+ mice. Summarizing the metabolic pathways ( Figure 5) of the 63 DMU-212 metaboli extracted and identified in the blood, liver, colorectal tissues and intestinal contents of Apc Min/+ mice administrated with DMU-212. A total of 43 Phase I metabolites of DMUwere identified, most of which were produced through demethylation, oxidati desaturation, dehydration, reduction and hydration. A total of 20 Phase II metaboli were identified. Among them, most were methylation, acetylation, glucuronide, cystei glycine and glutamine conjugation. As shown in Figure 4B, the methylated a demethylated metabolic reaction in the Apc Min/+ mice's intestinal contents, demethylated, dehydrated and demethylated reaction in the liver, and the demethylat oxidized and demethylated reaction in the serum and colorectal tissue, were the prim metabolic steps according to the relative percentage of metabolite type. Additionally, prototype DMU-212 was found in all serum, liver, colorectal tissues and intesti contents. The parent drug that was most abundant in the liver, colorectal tissues a Summarizing the metabolic pathways ( Figure 5) of the 63 DMU-212 metabolites extracted and identified in the blood, liver, colorectal tissues and intestinal contents of the Apc Min/+ mice administrated with DMU-212. A total of 43 Phase I metabolites of DMU-212 were identified, most of which were produced through demethylation, oxidation, desaturation, dehydration, reduction and hydration. A total of 20 Phase II metabolites were identified. Among them, most were methylation, acetylation, glucuronide, cysteine, glycine and glutamine conjugation. As shown in Figure 4B, the methylated and demethylated metabolic reaction in the Apc Min/+ mice's intestinal contents, the demethylated, dehydrated and demethylated reaction in the liver, and the demethylated, oxidized and demethylated reaction in the serum and colorectal tissue, were the primary metabolic steps according to the relative percentage of metabolite type. Additionally, the prototype DMU-212 was found in all serum, liver, colorectal tissues and intestinal contents. The parent drug that was most abundant in the liver, colorectal tissues and intestinal contents was not also the most abundant in the serum, and demethylated metabolite M1 was detected as the most abundant metabolite found in the serum. Except for the parent drug, metabolites M1, M51 and M44 were more abundant in the intestinal contents of the Apc Min/+ mice, and metabolites M1, M24 and M19 were most abundant in the liver; metabolites M3, M54 and M26 were most abundant in the colorectal tissues. The data are presented in Figure 6.
intestinal contents was not also the most abundant in the serum, and demethyla metabolite M1 was detected as the most abundant metabolite found in the serum. Exc for the parent drug, metabolites M1, M51 and M44 were more abundant in the intesti contents of the Apc Min/+ mice, and metabolites M1, M24 and M19 were most abundan the liver; metabolites M3, M54 and M26 were most abundant in the colorectal tissues. T data are presented in Figure 6.

Molecular Docking
Microbial bile salt hydrolases contribute to the pathogenesis of colon cancer throug the bile acid signaling pathway [9]. BSHs may be a potential target for colorectal canc therapy. Based on the results described above, combined with the literature, prototyp demethylated metabolites, oxidized metabolites, demethylated and oxidized metabolite which have higher relative percentages in colorectal tissue, were molecularly docke using AutoDock Vina, as shown in Table S3. The absolute value of the binding energ indicates the affinity of the ligands with the target and the conformational stability. Th absolute value greater than 4.25 kcal/mol, 5.0 kcal/mol, and 7.0 kcal/mol indicates certain, good and strong binding activity, respectively [10]. The molecular docking resul showed that the binding ability of all DMU-212 metabolites to BSHs was stronger, and th absolute values of the binding energy of each active component were stronger than th of DMU-212 (7.0 kcal/mol). Clusters having a relatively higher absolute value of bindin energy were selected and the specific binding patterns were processed and optimize (Figure 7).

Molecular Docking
Microbial bile salt hydrolases contribute to the pathogenesis of colon cancer through the bile acid signaling pathway [9]. BSHs may be a potential target for colorectal cancer therapy. Based on the results described above, combined with the literature, prototype, demethylated metabolites, oxidized metabolites, demethylated and oxidized metabolites, which have higher relative percentages in colorectal tissue, were molecularly docked using AutoDock Vina, as shown in Table S3. The absolute value of the binding energy indicates the affinity of the ligands with the target and the conformational stability. The absolute value greater than 4.25 kcal/mol, 5.0 kcal/mol, and 7.0 kcal/mol indicates a certain, good and strong binding activity, respectively [10]. The molecular docking results showed that the binding ability of all DMU-212 metabolites to BSHs was stronger, and the absolute values of the binding energy of each active component were stronger than that of DMU-212 (7.0 kcal/mol). Clusters having a relatively higher absolute value of binding energy were selected and the specific binding patterns were processed and optimized (Figure 7).

Discussion
Identifying drug metabolites can be crucial to finding potential drug targets, developing safe drug treatments in clinics, and even rationally modifying drugs [11]. On one hand, for the present drug metabolite identification studies, the components in biological samples and their metabolites are obtained with healthy or disease-model animal-based approaches [12,13]. We believe that a core approach to obtaining the active ingredients that serve a therapeutic role can be based on the effectiveness of the medicine [14]. Only in this case, where a specific medication proves to be efficacious, are the components in biological samples analyzed to clarify the material basis and mechanism of action for the drug's efficacy. On the other hand, in order to identify metabolites and to better understand the structure of drugs, high-quality tandem mass spectra are essential [15]. Without previous experience and prediction of metabolic pathways, identifying low

Discussion
Identifying drug metabolites can be crucial to finding potential drug targets, developing safe drug treatments in clinics, and even rationally modifying drugs [11]. On one hand, for the present drug metabolite identification studies, the components in biological samples and their metabolites are obtained with healthy or disease-model animal-based approaches [12,13]. We believe that a core approach to obtaining the active ingredients that serve a therapeutic role can be based on the effectiveness of the medicine [14]. Only in this case, where a specific medication proves to be efficacious, are the components in biological samples analyzed to clarify the material basis and mechanism of action for the drug's efficacy. On the other hand, in order to identify metabolites and to better understand the structure of drugs, high-quality tandem mass spectra are essential [15]. Without previous experience and prediction of metabolic pathways, identifying low abundant metabolite ions in in vivo samples with a complicated biological matrix presents a substantial challenge [8]. In this study, UHPLC-Q/Orbitrap/LTQ MS combining AcquireX TM , a novel data-dependent acquisition workflow, was used to analyze DMU-212 metabolic profiles in vivo based on the effectiveness of the medicine. Through the AcquireX TM data acquisition workflow, background subtraction and method updating were performed in real-time. Hence, only the MS 2 of potential metabolites were triggered using the updated method without any user intervention. In total, 63 DMU-212 metabolites were identified after the oral dose of 240 mg/kg for 3 weeks in CRA spontaneous model Apc Min/+ mice, including 48 metabolites in the intestinal contents, 48 metabolites in the liver, 34 metabolites in the serum, and 28 metabolites in the colorectal tissues, centering on the further reaction of the prototype, demethylated DMU-212 and oxidized DMU-212.
As we know, before oral drugs can be used to enter the blood circulation in the body, they must first be digested in the gastrointestinal tract in order to be absorbed in a suitable form [16]. As a result, the potential metabolic role of gut microbiota on drug metabolism is gaining increasing attention [17][18][19][20]. Our research found that the gut microbiota may be an important site of DMU-212 metabolism in vivo, and DMU-212 mainly undergoes rapid Phase I, such as demethylation, desaturation, dehydration and oxidation, and Phase II, including methylation metabolism, in the gut microbiota. There is increasing evidence that the gut microbiota affects drug metabolism by altering the structural and functional properties of drugs, which is primarily mediated by unique enzymes encoded within the microbiome [21][22][23]. That DMU-212 is being metabolized by gut microbiota may also have something to do with the fact that the gut microbiota contain a wide range of enzymes. Firmicutes and Bacteroidetes were the dominating taxa in the gut microbiota, which have many enzymes, such as oxidase, reductase, and esterase, allowing for many catalytic processes such as oxidation (hydration, hydrogenation, hydroxylation, methylation, oxygenation), and reduction (dehydration, dehydroxylation, demethylation) [24,25]. After being processed by gut microbes, most drugs, including DMU-212, are reabsorbed by the intestinal epithelium and then enter the enterohepatic circulation. In addition to being the main metabolic organ, the liver is also the main site of drug metabolism. As we expected, 48 metabolites were observed in the Apc Min/+ mice's liver, which inferred that the liver possessed high metabolic activity for DMU-212. In the liver, CYP450 plays an important role in drug metabolism [26]. A previous study demonstrated that via CYP1A1, DMU-212 may be demethylated to DMU-218 and oxidized to DMU-214, and the biological activity of the parent compound may be dependent on its conversion to DMU-214 and the level of this enzyme [27]. In contrast, our results showed that DMU-212 not only included demethylation and oxidation in the Apc Min/+ mice's liver but was also involved in many other reactions including Phase I, such as desaturation and dehydration, and even Phase II reactions, such as acetylation, glucuronide and cysteine conjugation, which were responsible for the identification of abundant DMU-212 metabolites. In general, the drug is metabolized in the liver and then distributed into the blood. During previous research, DMU-212 was rapidly cleared from the blood within an hour after administration, and a small amount of the prototype was detected [7]. In this paper, in the serum of the Apc Min/+ mice orally administered with DMU-212, the parent drug was also low, which indicated that DMU-212 was metabolized by intestinal bacteria or was absorbed into the blood and underwent hepatic first-pass elimination, followed by Phase I and II metabolism to be quickly transformed into the other metabolites, especially the demethylated, dehydrated and oxidized metabolites involved in Phase I, and the acetylated, glycine/glucuronide-bound metabolites involved in Phase II observed in this paper.
Medicines enter the bloodstream and are delivered to the target tissue where they exert their therapeutic impact. Colorectal tissue is the target tissue in which DMU-212 might prevent malignancy or delay its onset. In our study, in addition to the most abundant parent drug detected in the Apc Min/+ mice's colorectal tissue, demethylated, desaturated and oxidized metabolites were also found. In addition, the oxidized and cysteine-bound metabolite M54 was also a major Phase II metabolite and found at a relatively high level in colorectal tissue. Phase II reactions convert compounds to more water-soluble and often less active or toxic derivatives to increase excretion [28]; therefore, metabolite M54 may not be the pharmacologically active constituent. Bile acid metabolism associated with gut microbiota may contribute to the pathogenesis of colon cancer. In addition, our recent findings underline the regulatory roles of DMU-212 in dysregulated gut microbiota and BA metabolism to prevent CRA in Apc Min/+ mice (our unpublished data). Microbial BSHs, which initiate bile acid metabolism, are highly related to CRC, these enzymes have been considered a promising target in the manipulation of gut microbiota to benefit human health [9]. Therefore, we further explored the possible molecular interactions of the metabolites obtained in the colorectal tissue with BSHs using molecular docking experiments. Molecular docking showed that the target BSHs had a strong binding activity with the main metabolites. In addition to the reported five in vivo metabolites [7], several novel demethylated and/or oxidized metabolites have been characterized from the Apc Min/+ mice's colorectal tissue, which may affect bile acid metabolism to prevent CRA by acting on the BSHs' target. These provided a hint that DMU-212 metabolic products may be the pharmacodynamic material basis of DMU-212 to prevent CRA. However, it is still necessary to conduct further experiments to confirm this speculation.

Chemicals and Reagents
The resveratrol analogue DMU-212 was synthesized according to the reference method [29]. Its purity (>98%) (Figures S1 and S2) and structure were determined using nuclear magnetic resonance spectroscopy ( Figure S3, Table S1). Methanol, formic acid and acetonitrile (HPLC grade) were purchased from Fisher Scientific (Fisher Scientific, Waltham, MA, USA).

Animal Experiments
All of the experiments were approved by the Animal Care Welfare Committee of the Heilongjiang University of Chinese Medicine. In total, 20 Apc Min/+ mice (males aged 4 w) were obtained from GemPharmatech Co. Ltd. (Nanjing, China) and housed under a standard 12-h light-dark cycle at 25 ± 2 • C and 60 ± 5% humidity with free access to water and a high-fat diet (Research Diets, D12492; 60% fat by calories). At 7 weeks of age, the Apc Min/+ mice were randomly assigned to the vehicle-treated Apc Min/+ (MOD) and DMU-212-treated Apc Min/+ groups (DMU) (10 mice per group). The MOD group was orally administrated 0.5 % sodium carboxymethyl cellulose (CMC-Na) per day. The DMU group was orally administrated 240 mg/kg of DMU-212 per day. Drug treatment was performed once daily for 28 successive days. The efficacy of DMU-212 prevention for CRA was evaluated using blood feces, colon length, spleen index, adenoma number, histopathology and inflammatory cytokines, and CRA-related protein expression in colonic tissues. The data is being published in detail in a different publication.

Collection and Preparation of Biological Samples
Blood, liver, colorectal tissues and intestinal contents samples were collected at 1 h after the last administration. The serum was obtained through the centrifugation of blood in a refrigerated centrifuge at 3000 rpm (4 • C). A 300 µL volume of the serum sample was added to 900 µL of methanol and vortexed for 1.0 min to precipitate the protein and the sample was centrifuged at 12,000 rpm and 4 • C for 10 min. The supernatants were then concentrated using a speed vacuum concentrator and the residue was redissolved using 100 µL of methanol and centrifuged at 12,000 rpm for 10 min. In total, 0.1 g intestinal contents were collected and resuspended in 1 mL of methanol followed by centrifugation (12,000× g rpm, 4 • C, 10 min). The supernatants were dried in vacuo and resuspended in 200 µL of methanol and centrifuged at 12,000 rpm for 10 min. A total of 0.2 g of the mice's liver tissue and colorectal tissues were weighed, respectively, and homogenized in 1 mL of methanol, and then centrifuged at 12,000 g for 10 min at 4 • C. The supernatant was concentrated in vacuo and redissolved in 200 µL of methanol, and centrifuged at 4 • C at 12,000× g rpm for 10 min. Finally, the supernatants collected from the four kinds of samples were passed through a 0.22 µm membrane filter and a 2 µL sample was injected into the UHPLC-Q/Orbitrap/LTQ MS system for analysis.
A heated electrospray ionization interface, working in positive mode, was used in the mass spectrometer. The parameters were the following: spray voltage of 3.5 kV; sheath gas of 40 arb; and auxiliary gas of 15 arb. The ion transfer tube and the vaporizer temperature were at 350 • C. The scanning mode was full MS/DD-MS 2 based on the AcquireX TM intelligent data acquisition technology (Thermo Fisher Scientific), with an Orbitrap resolution of 120,000 and a mass range of m/z 50-1200.

Data Processing Software
The Xcalibur 4.0 workstation software (Thermo Fisher Scientific) was used for raw data recording and processing. Using Compound Discoverer 3.3 (Thermo Fisher Scientific), post-processing of the data was performed to extract the metabolite-related datasets based on the structural correlation between the drug and its metabolites. A maximum tolerance of 5 ppm was set for the mass error. Using blank samples, the workflow subtracted the chemical backgrounds, aligned the retention times, and found the expected compounds and metabolites. Fragment ion search (FISH) scoring was used, and each annotation was then manually evaluated based on the HCD, DDA spectra, molecular formula, and FISH coverage.

Molecular Docking
The 3D structure of bile salt hydrolases (BSHs) was downloaded from the RCSB PDB (https://www.rcsb.org/, accessed on 18 April 2023). The PubChem database (https: //pubchem.ncbi.nlm.nih.gov/, accessed on 18 April 2023) was used to download the SDF format files of the 2D structure of DMU-212. The structures of another core active component were made with ChemDraw (http://www.perkinelmer.com/category/chemdraw, accessed on 18 April 2023). Molecular docking with the main active components of the main active ingredients and BSHs was performed using PyMoL 2.3.0 and AutoDock Vina 18. The binding activity was assessed using binding energy.

Conclusions
In this paper, the metabolic profiles of DMU-212 in Apc Min/+ mice's serum, liver, colorectal tissues and intestinal contents were systematically and comprehensively investigated based on the effectiveness of the medicine. A total of 63 DMU-212 metabolites were determined and summarized through quick, sensitive and accurate UHPLC-Q/Orbitrap/LTQ MS combined with the data processing software "Compound Discoverer 3.3" and the AcquireX TM data acquisition workflow. In addition, further verification of the representative active metabolites was employed using molecular docking analysis. The Phase I metabolites of DMU-212 were mainly produced via demethylation, oxidation, desaturation, dehydration, reduction and hydration, while the major Phase II metabolites were methylation, acetylation, glucuronide, cysteine, glycine and glutamine conjugation products. As a result of this study, a simple method for studying drugs' metabolism in vivo is provided, as is scientific and reliable support for a complete understanding of DMU-212's metabolism and transformation. In addition, as a reference for the further development of new drugs, this is also important for a deeper understanding of the active constituents of DMU-212 and its action mechanisms for CRA prevention.
Author Contributions: H.S. and X.W. designed the experiment; J.L., X.L., X.Z., L.Y. and L.K. performed the experiments; J.L., G.Y., and Y.H. analyzed and calculated the data; J.L. reviewed the data and wrote the paper; H.S. and X.W. reviewed and revised the paper. All authors have read and agreed to the published version of the manuscript.