Acetonitrile (HPLC-grade), methanol (HPLC-grade), and formic acid (HPLC-grade) were purchased from Aladdin Industrial Corporation. Ultrapure water was prepared by Milli-Q water purification system (Madrid, Spain). Sodium carboxymethyl cellulose (CMC-Na) was purchased from Aladdin Industrial Corporation. Standards of procyanidin B2 and (−)-epicatechin were purchased from Tauto Biotech Corporation. (Shanghai, China). The purities of all standards were over 99% based on HPLC analysis.

Eighteen male 10-week C57BL/6 mice (23 ± 2 g) were obtained from SLAC Laboratory Animal center (Shanghai, China). All animals were housed in an environmentally controlled breeding room (24 ± 2°C, 60 ± 10% humidity) for 7 days with unrestricted chow and water. Then, the mice were randomly divided into 2 groups with nine mice per group (Group A: the procyanidin B2 group and Group B: control group) before the beginning of experiments. The procyanidin B2 was dissolved in 0.5% CMC-Na water solution and orally administered to group A at a dose of 800 mg/kg body weight, and the mice in group B were administered 0.5% CMC-Na water solution in the same way. The mice were administered for 3 days at 9:00 am. All experiments were performed in compliance with the Chinese legislation on the use and care of laboratory animals and were approved by the Shanghai Institute of Technology Committee on Animal Care and Use.

Urine sample was collected within 2 days after the first administration. Finally, all urine samples from one group were merged into one sample. Whereafter, the urine samples were dried by Boyikang FD-80 freezer dryer (Beijing, China). Next, 1.00 g of dried sample was redissolved by 2 mL methanol. Then, the solution was centrifuged at 2,795 g for 15 min (Sigma 3k-18, Sigma, Germany) after ultrasonic bath for 30 min. Afterward, the supernatant was stored in −80°C(MDF-1156, SANYO, Japan) before the next step of analysis (Liang et al., 2013).

The blood samples were collected in heparinized tubes at 1, 1.5, and 2 h by extracting eye bowl after the first administration of the last day under anesthesia state, and then centrifuged for 15 min at 1,789 g for obtaining the plasma samples. All plasma samples from one group were merged into one sample. Finally, each plasma sample (3 ml) was deproteinized with ice-cold acetonitrile (9 ml) in an ultrasonic bath for 20 min, and then was centrifuged at 2,795 g for 15 min to remove precipitated protein, and the solid residue was washed for three times using ice-cold acetonitrile (1 ml). Then, the merged supernatant was dried with a gentle flow of nitrogen in ice bath. The residue was redissolved in 3 ml methanol and was centrifuged at 2,795 g for 15 min after vortex oscillation. The supernatant was reconstituted to dryness, and was quickly redissolved in 200 μL methanol with vortex oscillation. The treated samples were centrifuged at 11,180 g for 10 min before injecting the system of high-performance liquid chromatography.

The pretreatment of heart, liver, brain, spleen, lungs, kidneys, or small intestine was homogenized by a homogenizer (PRO 200, Oxford, USA), which was suspended in methanol (1/10, g/ml). Then, the homogenates were in the ice-cold ultrasonic bath for 30 min and centrifuged at 2,795 g for 15 min. Afterward, the supernatant was evaporated to dryness in vacuum at 45°C, and then quickly redissolved in 2 mL methanol. The pretreated samples were stored at −80°C before analysis. All the samples were filtered through 0.45 μm nylon membrane before injecting the system (Liang et al., 2013).

The UPLC-DAD-ESI-IT-TOF analysis of the metabolites was performed using Shimadzu LC-30AD instrument equipped with two LC-30AD pumps, an SIL-30AC autosampler, SPD-M20A, CTO-20AC column oven, and CBM-20A system controller, and coupled to an IT-TOF mass spectrometer with an ESI interface.

The chromatography separations were performed on a Shim-pack HR-ODS column (150 mm × 2.1 mm, 3 μm). The column was eluted with a gradient mobile phase A of water-formic acid (100: 0.1, v/v) and B of acetonitrile, The elution gradient was 0–2 min, 5–10% B, 2–4 min, 10–12% B, 4–7 min, 12–18% B, 7–13 min, 18–27% B, 13–32 min, 27–56% B, 32–37 min, 56–100% B, and 37–47 min 100% B. The flow rate was 0.2 mL/min; the injection volume was 5 μL and the column oven temperature was kept at 30°C.

The tandem mass spectrometry analyses were carried out on an IT-TOF (Shimadzu, Japan) with the full scan over m/z 100–900 (MS¹) and m/z 50–900 (MS² and MS³) in the ion model of negative(NI) and positive(PI). The parameters were as follows: heat block and curved desolvation line temperature, 200°C; nebulizing nitrogen gas flow, 1.5 L/min; interface voltage: (+), 4.5 kv; (−), 3.5 kv; detector voltage, 1.56 kv; relative collision-induced dissociation energy, 50%.

To find more information of metabolites, in this study, we adopted a specially pattern for metabolites screening and identification. The procedures included three steps, (1) collecting the information of metabolites of procyanidin B2 by searching the literature databases and bioinformatic databases on line (Chemspider: http://www.chemspider.com, http://metlin.scripps.edu/); (2) searching the possible mass-to-charge ratio of metabolites by extracted ion chromatograms (EICs) in the total ion chromatograms (TICs) comparing with the TICs of control group using Profiling Solution version 3.60 and metabolites with the aid of MetlID Solution 1.2 software; (3) the known metabolites were confirmed by the databases described above. The unknown metabolites were tentatively identified in several steps: firstly, the type of substitution was identified based on the characteristic fragment ions and neutral losses in MSⁿ spectrum; second, the skeleton structure was speculated in comparison with the mass fragmentation patterns of reference standard procyanidin B2; third, the proposed isomeric metabolites were further confirmed by characteristic fragment ions in MSⁿ spectrum, n-octanol/water partition coefficient (ClogP value) and predominant conjugation site reported by other literature (Liang et al., 2013, 2014). All the data was processed by Shimadzu software of Formula Predictor version 1.20.

Since it has been well proven that the mass fragmentation patterns of metabolites are always similar to the parent compounds, the fragmentation patterns analysis of parent compound is very helpful for the metabolite characterizations. Therefore, this study conducted a detailed fragmentation of procyanidin B2 and (−)-epicatechin authentic standard in UPLC-DAD-ESI-IT-TOF (shown in Figure 2), which provided guidance in the subsequent structure of metabolites. Procyanidin B2 showed the [M-H]⁻ at m/z 577.1260 (C₃₀H₂₅O₁₂), which was broken into characteristic fragment ions at m/z 425.0845 (C₂₂H₁₇O₉), 407.0764 (C₂₂H₁₅O₈), 289.0715 (C₁₅H₁₄O₆), and m/z 245.0796 (C₁₄H₁₄O₄) in the MS² spectrum. The (−)-epicatechin showed the characteristic fragment ions at m/z 245.0799 (C₁₄H₁₄O₄), 205.0476 (C₁₁H₁₀O₄), 137.0258 (C₇H₆O₃), and m/z 125.0258 (C₆H₆O₃).

The MS full-scan might reveal all the ion chromatograms of the expected or unexpected metabolites. The peaks detected in the ion chromatograms of sample group correspond to the mass-to-charge ratio (m/z) of the possible metabolites compared with the ion chromatograms of control group (shown in Figures 3 and 4). According to predicted gains and neutral losses in molecular masses of the metabolites compared with the molecular mass of the parent compound, the multiple metabolites from biological matrix could be effectively revealed based on the advantage of LC-IT-TOF technology in absence of authentic standards for calibration compounds.

A total of 53 compounds were detected as metabolites of procyanidin B2 (45 of them were identified). As for 45 preliminarily identified metabolites, 27 metabolites (M_r < 577.13) were degraded from procyanidin B2 by cleavage of the flavanol interflavanic bond C4–C8. Additionally, 14 conjugates were formed, including 9 methylation metabolites, 8 sulfation conjugates, 5 hydration metabolites, 2 hydroxylation metabolites, 1 hydrogenation metabolite, and 1 glucuronidation metabolite. Interestingly, 24 compounds as metabolites of procyanidin B2 were newly found, including 8 methylation metabolites, 9 sulfation conjugates, 2 hydroxylation metabolites, 7 hydration metabolites, 1 hydrogenation metabolite, 1 glucuronidation metabolite, and 8 unidentified metabolites. The data was summarized in Tables 1, 2.

M1 was calculated to be C₁₅H₁₄O₆ on the basis of high-resolution mass spectrometry (HRMS) data. Since the typical fragment ions at m/z 245.07, 205.04, and 137.02 were noticed, which were in high accordance with the reference (−)-epicatechin, as generated by Retro-Diels-Alder (RDA) cleavage in accordingly with the previous reports (Liang et al., 2014), the M1 was confirmed as (−)-epicatechin formed by cleavage of interflavanic bond of procyanidin B2.

In the MSⁿ spectra of M2-M6, the same fragmentation ions at m/z 289.06, 245.07, and 137.02 were found, which were similar with the fragment behaviors of (−)-epicatechin. Therefore, M2-M6 were the metabolic compounds of (−)-epicatechin. The molecular formula of M2 and M3 showed the same [M-H]⁻ ion at m/z 369.03, which could fragment into [M-SO₃-H]⁻ at m/z 289.07 by neutral loss of 79.95 Da. Since the typical A-ring fragment ions at m/z 216.98 and 137.02 of M2 and M3 were noticed, which were produced by RDA cleavage, we could deduce that the sulfonic group should be conjugated at A-ring. Hence, it was concluded that M2 and M3 were (−)-epicatechin-5/7-O-sulfate. M4 and M5 manifested the same [M-H]⁻ at m/z 383.04 in their MSⁿ spectra, and the same [M-CH₂-H]⁻ at m/z 369.03 was detected by neutral loss of 14.01 Da (elemental composition: CH₂). Accordingly, M4 and M5 were presumed to be methylation M2/M3 with the preferable site of C-3′-OH of 3′-O-methyl-5/7-O-sulfate (−)-epicatechin (Selma et al., 2009; Liang et al., 2014). Because the molecular formula of M6 was determined to be C₂₁H₂₂O₁₅S with the [M-C₆H₈O₆-H]⁻ at m/z 369.03 by a neutral loss of 176.03 Da (elemental composition: C₆H₈O₆) in the NI MS² spectra, M6 was supposed to be one metabolite of (−)-epicatechin, which was formed through glucuronidation and sulfation. But we could not determine its exact binding site, so it was tentatively inferred to be 5/7-O-sulfate-epicatechin-O-glucuronide (Liang et al., 2014).

The molecular formulas of M7-M9 were calculated to be C₁₅H₁₆O₆, C₂₁H₂₄O₁₂, and C₁₅H₁₈O₇, respectively, with the [M-H]⁻ at m/z 291.08, 467.12, and 309.09 based on the HRMS data. In the NI MSⁿ spectra, M7, M8, and M9 had the same characteristic fragment ions at m/z 247.09 (elemental composition: C₁₄H₁₅O₄), 167.03 (elemental composition: C₈H₇O₄), and 123.04 (elemental composition: C₇H₇O₂), showing the similar fragmentation pathways of 1-(3′,4′-dihydroxyphenyl)-3-(2′,4′, 6′-trihydroxyphenyl)-propan-2-ol (3, 4-diHPP-2-ol) (Liang et al., 2014; Margalef et al., 2014). As a result, M7 was assigned as 3,4-diHPP-2-ol. M8 showed the [M-C₆H₈O₆-H]⁻ at m/z 291.08 by neutral loss 176.03 Da (elemental composition: C₆H₈O₆), so it was preliminarily identified as glucuronic acid conjugates of 3,4-diHPP-2-ol. According to the glucuronidation of (−)-epicatechin preferably occurred at the C-5-OH of A-ring (Shali et al., 1991), M8 was tentatively identified as 5-O-glucuronide-3,4-diHPP-2-ol. As for M9, the neutral loss of 18.01 Da (elemental composition: H₂O) was observed. Therefore, M9 was identified as hydration products of 3,4-diHPP-2-ol.

The molecular formula of M10-M11 were calculated to be C₁₁H₁₂O₇S with the same [M-SO₃-H]⁻ at m/z 207.06 by the neutral loss of 79.95 Da (elemental composition: SO₃), and the precursor ion [M-SO₃-H]⁻ at m/z 207.06 can break into characteristic fragment ions at m/z 163.07, and m/z 122.04. Moreover, the fragmentation pathways of the aglucon were in good accordance with that of 5-(3,4-dihydroxyphenyl)-γ-valerolactone (Appeldoorn et al., 2009a; Urpi-Sarda et al., 2009). Thus, they were confirmed to be 5-(3,4-dihydroxyphenyl)-γ-valerolactone sulfate.

Summarily, 16 aromatic acid metabolites are tentatively identified, including 2 phenylvalenic acid metabolites, 7 phenylpropionic acid metabolites, 3 phenylacetic acids metabolites, and 4 benzoic acid metabolites. Their structures are shown in Figure 5.

A total of 17 conjugated metabolites were identified, which had the same characteristic fragmentation behaviors at m/z 425.08 and 407.07, as generated from RDA cleavage (Shown in Figure 1) of procyanidin B2.

M45 had the molecular formula C₃₁H₃₀O₁₂ with the [M-CH₂-H]⁻ at m/z 579.15 and m/z 289.07 in the NI MSⁿ spectra, which was confirmed as methyl C-ring cleavage of procyanidin B2 based on previous reports. Although there should be two potential sites for reductive cleavage of the C-ring (upper unit and lower unit), the site of upper (−)-epicatechin unit was more accessible to reaction (Stoupi et al., 2010b). As results, M45 could be defined as upper C-ring cleavage of M29.

In NI MSⁿ spectra of all compounds, the [M-H]⁻ at m/z 702.1426, 676.1156 691.1194, 685.1241, 705.1387, 627.1335, 714.1452, and 325.0501 were detected, respectively, which were identified as the metabolites of procyanidin B2 in comparison with the fragmentation behaviors (shown in Table 2). In addition, m/z 591.14 was observed in MSⁿ spectra of M50 and M51, respectively. Hence, M50 and M51 should be the methyl procyanidin B2, but the structure of all compounds consisting of complicated aglucon and misty conjugated sites were not fully identified with pure high-resolution mass spectrometry data in the absence of applicable standard and guidance from previous literature.

The MSⁿ data with high mass accuracy provided much information to tentatively analyze the structure of metabolites under negative ion mode. The primary objective was to characterize the relatively comprehensive metabolites of the procyanidin B2 in mice in the present study. Therefore, we analyzed and compared the metabolites in urine, plasma, and organ tissue samples following oral routes of administration. The base peak chromatograms of the mice urine and plasma samples detected in negative ion mode are shown in Figures 3 and 4, respectively. 53 metabolites of procyanidin B2 (24 new metabolites) showed that the procyanidin B2 was metabolized diversely (shown in Tables 1, 2). Although the previous study reported that glucuronide or sulfate metabolites of dimeric procyanidins had not been detected in biological fluids, we identified the intact procyanidin B2 conjugates of 10 sulfate metabolites and 1 glucuronide metabolites in this work (Appeldoorn et al., 2009a,b). Meanwhile, aromatic acid metabolites were very common in the metabolic pathways of procyanidins after cleavage of the interflavan bond.

Furthermore, a possible metabolic pathway was proposed in Figure 5. The metabolites of procyanidin B2 could be categorized into two major groups: (a) cleavage of interflavanic bond (C4–C8) of procyanidin B2 (M_r < 577) and formation of small molecule material, including (−)-epicatechin, C-ring cleavage of epicatechin metabolites, phenylvalerolactone metabolites, and aromatic acid metabolites; (b) metabolites with intact interflavanic bond C4–C8, including procyanidin B2 conjugates, partial heterocyclic ring cleavage products, and their conjugates.

In general, the conjugation process (glucuronidation, sulfation, and methylation) affected the bioactive properties of the parent compound. Sulfation and glucuronidation had been reported to improve a considerable attenuation of biological activity. Additionally, the case of methylation seemed to be more complex because the incorporation of methyl groups reduced the number of available OH group, but increased the lipophilic nature of the compound, which could be advantageous for cellular uptake by passive diffusion (Donovan et al., 2007; Monagas et al., 2010). In particular, we found 32 sulfation metabolites, 3 glucuronidation metabolites, and 14 methylation metabolites, respectively. Besides, M4 and M5 exhibited the potential inhibition of lipid oxidation and inhibition of apoptosis induced by oxidized LDL. M1 was identified as beneficial effects in vascular disaster (Schroeter et al., 2001; Su et al., 2004). In the case of M10, M11, M21, and M22, they have been reported to be a slight increase in antioxidant activity (Grimm et al., 2004). In addition, the biological activities of metabolites of procyanidin B2 were still largely unknown, thus the bioactivities of metabolites were also worthy to further research.

In this work, the metabolic profile of procyanidin B2 in mice urine, plasma and organs were qualitatively described by UPLC-DAD-ESI-IT-TOF method. The numbers of all detected metabolites in plasma, urine, small intestine, liver, heart, kidney, lung, brain and spleen were 37, 34, 23, 13, 12, 7, 3, 1, and 3, respectively.

According to previous reports, liver had more importance in the metabolization process of polyphenols showing a higher metabolite concentration (Appeldoorn et al., 2009a). In this work, all metabolites found in the liver was M_r > 577, which indicated that cleavage of interflavanic bond could not occur in the liver, and conjugation reaction (glucuronidated, sulfated, and methylated) was the major reaction formation with intact procyanidin B2. 34 metabolites in urine and 5 in the kidney showed that the urinary path was the main procyanidin B2 excretion pathway, which was consistent with the conclusion of previous literature (Serra et al., 2011). There were 12 metabolites observed in the heart, which would enhance their described beneficial effects. The presence of products in such tissues as the heart may be related to the potential health benefits of procyanidin B2, especially in the context of cardiovascular health effect (Fraga et al., 2011). 23 metabolites were detected in small intestine, including M1, M4–M5, M10–M11, M16, M19, M22–M23, M29–M32, M34–M36, M44–M45, M46–M49, and M53. Furthermore, 14 metabolites were the conjugation metabolites with intact procyanidin B2, suggesting that the procyanidin B2 could pass intact through the gastrointestinal tract, reaching the small intestine where it was transformed by the intestinal microbiota before absorption, and these metabolites could reach the systemic circulation to be transported to other tissue where they could be further biotransformed according to the previous reports (Aura, 2008; Crozier et al., 2009; Monagas et al., 2010; Margalef et al., 2015).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.