Metabolites of Medicarpin and Their Distributions in Rats

Medicarpin is a bioactive pterocarpan that has been attracting increasing attention in recent years. However, its metabolic fate in vivo is still unknown. To clarify its metabolism and the distribution of its metabolites in rats after oral administration, the HPLC-ESI-IT-TOF-MSn technique was used. A total of 165 new metabolites (13 phase I and 152 phase II metabolites) were tentatively identified, and 104, 29, 38, 41, 74, 28, 24, 15, 42, 8, 10, 3, and 17 metabolites were identified in urine, feces, plasma, the colon, intestine, stomach, liver, spleen, kidney, lung, heart, brain, and thymus, respectively. Metabolic reactions included demethylation, hydrogenation, hydroxylation, glucuronidation, sulfation, methylation, glycosylation, and vitamin C conjugation. M1 (medicarpin glucuronide), M5 (vestitol-1’-O-glucuronide) were distributed to 10 organs, and M1 was the most abundant metabolite in seven organs. Moreover, we found that isomerization of medicarpin must occur in vivo. At least 93 metabolites were regarded as potential new compounds by retrieving information from the Scifinder database. This is the first detailed report on the metabolism of ptercarpans in animals, which will help to deepen the understanding of the metabolism characteristics of medicarpin in vivo and provide a solid basis for further studies on the metabolism of other pterocarpans in animals.


MS Fragmentation Characteristics of Medicarpin in ESI− Mode and Identification of Medicarpin in Rats
In order to facilitate the description of the ESI− MS characteristics of medicarpin and its metabolites, we proposed a nomenclature for the fragmentation pathways and fragment ions. The four rings were named A, B, C and D, respectively. The cleavable C-C bonds in the skeleton were designated by numbers 1-8, as shown in

MS Fragmentation Characteristics of Medicarpin in ESI− Mode and Identification of Medicarpin in Rats
In order to facilitate the description of the ESI− MS characteristics of medicarpin and its metabolites, we proposed a nomenclature for the fragmentation pathways and fragment ions. The four rings were named A, B, C and D, respectively. The cleavable C-C bonds in the skeleton were designated by numbers 1-8, as shown in Figure 2.

Profiling 165 Metabolites of Medicarpin in Rats
The metabolites of medicarpin were screened by comparing the HPLC chromatograms and MS base peak chromatograms (BPCs) of drug and blank group samples obtained through HPLC-ESI-IT-TOF-MS n analysis, and then confirmed by comparison of extracted ion chromatograms (EICs) between the drug and blank groups. The metabolic reactions were judged by the accurate mass (elemental composition) differences between medicarpin and its metabolites. The accurate mass (elemental composition) differences of −14.01 Da (CH2), +14.01 Da (CH2), +2.01 Da (H2), and +15.99 Da (O) indicated demethylation, methylation, hydrogenation and hydroxylation. The loss of 176.03 Da (C6H8O6) from precursor ion and/or an anion at m/z 175.02 in MS 2 spectra indicated that the metabolite was a glucuronide; the loss of 79.95 Da (SO3) from precursor ion indicated that the metabolite was a sulfate; the loss of 162.05 Da (C6H10O5) indicated a hexoside (more likely to be a glucoside); the loss of 158.02 Da (C6H6O5) indicated a vitamin C conjugate [35].
A total of 165 metabolites of medicarpin were confirmed and tentatively identified by the abovementioned method, and the proposed metabolic pathways of medicarpin are shown in Figure 3.

Profiling 165 Metabolites of Medicarpin in Rats
The metabolites of medicarpin were screened by comparing the HPLC chromatograms and MS base peak chromatograms (BPCs) of drug and blank group samples obtained through HPLC-ESI-IT-TOF-MS n analysis, and then confirmed by comparison of extracted ion chromatograms (EICs) between the drug and blank groups. The metabolic reactions were judged by the accurate mass (elemental composition) differences between medicarpin and its metabolites. The accurate mass (elemental composition) differences of −14.01 Da (CH 2 ), +14.01 Da (CH 2 ), +2.01 Da (H 2 ), and +15.99 Da (O) indicated demethylation, methylation, hydrogenation and hydroxylation. The loss of 176.03 Da (C 6 H 8 O 6 ) from precursor ion and/or an anion at m/z 175.02 in MS 2 spectra indicated that the metabolite was a glucuronide; the loss of 79.95 Da (SO 3 ) from precursor ion indicated that the metabolite was a sulfate; the loss of 162.05 Da (C 6 H 10 O 5 ) indicated a hexoside (more likely to be a glucoside); the loss of 158.02 Da (C 6 H 6 O 5 ) indicated a vitamin C conjugate [35].
A total of 165 metabolites of medicarpin were confirmed and tentatively identified by the above-mentioned method, and the proposed metabolic pathways of medicarpin are shown in Figure 3.
M3 ( Figure 5) 11 and their molecular formulae were predicted to be C22H22O10, so they were isomers. The fragment ions at m/z 269.08 and m/z 175.02 can be detected in both of their MS 2 spectra. Therefore, M1 and M2 were determined to be glucuronides of medicarpin. However, medicarpin only has one hydroxyl group, i.e., one glucuronidation site ( Figure 4a). Hence, we deduced that isomerization of medicarpin (configuration change or shift of substituent) must occur.
M3 ( Figure 5)    , and its molecular formula was predicted to be C16H16O4, which had two more hydrogen atoms than that of medicarpin. Therefore, one ring of medicarpin must be cleaved. In its MS 2 spectra, the fragment ions at m/z 147.0469 (C9H7O2, 6 3,5 A − or 2,4 A − ) and m/z 109.0321 (C6H5O2, 2,5 A − + 2H) were detected. According to the characteristic fragment ion at m/z 135.0466 (C8H7O2, 3,4 A − or 3,4 B − ), we could deduce that medicarpin had undergone ring cleavage at the bond of C11-C11a as shown in Figure 6a. Hence, M4 was determined to be vestitol. The characteristic fragment ion at m/z 135.05 (C8H7O2) was very useful, because its existence indicated that the C11-C11a bond was cleaved and the hydrogenated medicarpin had the skeleton of a vestitol. The MS 2 spectrum of M4 is shown in Figure S45. and m/z 121.0354 (C7H5O2) were detected in MS 3 spectra. This indicated that M10 was a hydrogenated medicarpin glucuronide sulfate. Besides, based on m/z 135.0498 (C8H7O2), we could deduce that the bond of C11-C11a in medicarpin was cleaved. Moreover, the fragment ion at m/z 254.9829 (C10H7O6S,), which was derived from precursor ion m/z 527.0901, indicated that the sulfonic group (-SO3H) was linked to the fragment of C10H7O3 ( 1,5 B − − 4H), and the fragment ion at m/z 229.0156 (C9H9O5S), which was obtained from precursor ion m/z 351.0505, indicated that -SO3H was linked to the fragment of C9H9O2 ( 3,5 B − ). Hence, we could deduce that the sulfonic group was linked to the hydroxyl group of B ring, while the glucuronyl group was linked to the hydroxyl group of A ring. Therefore, M10 was unambiguously identified as vestitol-7-O-glucuronide-1'-O-sulfate. The MS spectra of M10 are shown in Figure S46.  Figure 6a. Hence, M4 was determined to be vestitol. The characteristic fragment ion at m/z 135.05 (C 8 H 7 O 2 ) was very useful, because its existence indicated that the C11-C11a bond was cleaved and the hydrogenated medicarpin had the skeleton of a vestitol. The MS 2 spectrum of M4 is shown in Figure S45. , we could deduce that the bond of C11-C11a in medicarpin was cleaved. Moreover, the fragment ion at m/z 254.9829 (C 10 H 7 O 6 S,), which was derived from precursor ion m/z 527.0901, indicated that the sulfonic group (-SO 3 H) was linked to the fragment of C 10 H 7 O 3 ( 1,5 B − − 4H), and the fragment ion at m/z 229.0156 (C 9 H 9 O 5 S), which was obtained from precursor ion m/z 351.0505, indicated that -SO 3 H was linked to the fragment of C 9 H 9 O 2 ( 3,5 B − ). Hence, we could deduce that the sulfonic group was linked to the hydroxyl group of B ring, while the glucuronyl group was linked to the hydroxyl group of A ring. Therefore, M10 was unambiguously identified as vestitol-7-O-glucuronide-1'-O-sulfate. The MS spectra of M10 are shown in Figure S46. Demethylated medicarpin only has two metabolism sites (two free hydroxyl groups), but it has three glucuronidation metabolites (M11-M13), seven sulfation metabolites (M14-M20) and three glucuronidation and sulfation metabolites (M21-M23). Therefore, we can confirm that isomerization of medicarpin (configuration change or shift of substituent) must occur. Demethylated medicarpin only has two metabolism sites (two free hydroxyl groups), but it has three glucuronidation metabolites (M11-M13), seven sulfation metabolites (M14-M20) and three glucuronidation and sulfation metabolites (M21-M23). Therefore, we can confirm that isomerization of medicarpin (configuration change or shift of substituent) must occur.  Figure 7. The MS spectrum of M37 is shown in Figure S47.    at m/z 287.0943 and the molecular formula was calculated to be C16H16O5, which was two hydrogen atoms and one oxygen atom more than that of medicarpin. Therefore, we speculated that M112 was hydrogenated and hydroxylated medicarpin. In its MS 2 spectra, a characteristic fragment ion at m/z 125.0181 (C6H5O3, 2,5 A − + 2H) was observed, indicating that the newly added hydroxyl group was linked to the A ring, so it has three possibilities O, P or Q. Besides, based on the fragment ion at m/z 151.0451 (C8H7O3, 3,4 A − ), we could determine that the bond of C11-C11a was cleaved. Finally, the structure of M112 was determined to be O. Furthermore, the fragment ions of M112 including m/z 163.0388 (C9H7O3, 6 Figure S48. at m/z 287.0943 and the molecular formula was calculated to be C 16 H 16 O 5 , which was two hydrogen atoms and one oxygen atom more than that of medicarpin. Therefore, we speculated that M112 was hydrogenated and hydroxylated medicarpin. In its MS 2 spectra, a characteristic fragment ion at m/z 125.0181 (C 6 H 5 O 3 , 2,5 A − + 2H) was observed, indicating that the newly added hydroxyl group was linked to the A ring, so it has three possibilities O, P or Q. Besides, based on the fragment ion at m/z 151.0451 (C 8 H 7 O 3 , 3,4 A − ), we could determine that the bond of C11-C11a was cleaved. Finally, the structure of M112 was determined to be O. Furthermore, the fragment ions of M112 including m/z 163.0388 (C 9 H 7 O 3 , 6 Figure S48. , we could deduce that the aglycon of M114 was O2 or P. However, because the same fragment ions could be produced from O2 or P, it was impossible to judge the position of hydroxylation based on the current mass spectrometry data. In addition, the elemental composition of m/z 229.0108 (C9H9O5S) suggested that the sulfonic group was connected to the fragment ion of m/z 149.0653 (C9H9O2). Hence, we could determine the sulfate was linked to the hydroxyl group of B ring. The MS 2 spectrum of M114 is shown in Figure S49. , we could deduce that the aglycon of M114 was O2 or P. However, because the same fragment ions could be produced from O2 or P, it was impossible to judge the position of hydroxylation based on the current mass spectrometry data. In addition, the elemental composition of m/z 229.0108 (C 9 H 9 O 5 S) suggested that the sulfonic group was connected to the fragment ion of m/z 149.0653 (C 9 H 9 O 2 ). Hence, we could determine the sulfate was linked to the hydroxyl group of B ring. The MS 2 spectrum of M114 is shown in Figure S49.  , we could identify that the newly added hydroxyl group was linked to the D ring of medicarpin and the C11-C11a bond of medicarpin was cleaved. Moreover, m/z 230.9941 was predicted to be C 8 H 7 O 6 S, which indicated that the sulfonic group was connected to the fragment ion of m/z 151.0440 (C 8 H 7 O 3 , 3,4 B − ). We therefore confirmed that the sulfonic group was linked to the hydroxyl group of B ring. The probable structure and characteristic fragment ions of M115 are shown in Figure 11. The MS 2 spectrum of M115 is shown in Figure S50. was cleaved. Moreover, m/z 230.9941 was predicted to be C8H7O6S, which indicated that the sulfonic group was connected to the fragment ion of m/z 151.0440 (C8H7O3, 3,4 B − ). We therefore confirmed that the sulfonic group was linked to the hydroxyl group of B ring. The probable structure and characteristic fragment ions of M115 are shown in Figure 11. The MS 2 spectrum of M115 is shown in Figure S50. According to the fragment ion at m/z 147.0451 (C9H7O2), we could exclude P, R and T. Besides, based on the fragment ion at m/z 137.0308 (C7H5O3), we could exclude E. Therefore, we could determine that the aglycon of M144 was O or Q. Furthermore, based on the fragment ion at m/z 147.0451 (C9H7O2), we finally deduced that the aglycon of M144 was O1, O2 or Q (Figure 12). The MS 2 spectrum of M144 is shown in Figure S51. , we could deduce that the aglycon of M144 might be O, P, Q, R, S or T. According to the fragment ion at m/z 147.0451 (C 9 H 7 O 2 ), we could exclude P, R and T. Besides, based on the fragment ion at m/z 137.0308 (C 7 H 5 O 3 ), we could exclude E. Therefore, we could determine that the aglycon of M144 was O or Q. Furthermore, based on the fragment ion at m/z 147.0451 (C 9 H 7 O 2 ), we finally deduced that the aglycon of M144 was O1, O2 or Q (Figure 12). The MS 2 spectrum of M144 is shown in Figure S51.

Analysis of Metabolites
The molecular formulae of M147-M149 were predicted as C 21   Based on the fragment ion at m/z 123.0137 (C 6 H 3 O 3 , 2,5 A − ), we assured that one hydroxyl group must be linked to the A ring. Besides, the fragment ion at m/z 124.0254 (C 6 H 4 O 3 , 6,7 D − + 2H − CH 3 •) suggested that one hydroxyl group must be linked to the D ring. Furthermore, according to the fragment ion at m/z 203.9750 (C 6 H 4 O 6 S), we could determine that the sulfonic group was linked to the hydroxyl group of D ring. The fragmentation pathways of M159 are shown in Figure 13. The MS spectrum of M159 is shown in Figure S52. Based on the fragment ion at m/z 123.0137 (C6H3O3, 2,5 A − ), we assured that one hydroxyl group must be linked to the A ring. Besides, the fragment ion at m/z 124.0254 (C6H4O3, 6,7 D − + 2H − CH3•) suggested that one hydroxyl group must be linked to the D ring. Furthermore, according to the fragment ion at m/z 203.9750 (C6H4O6S), we could determine that the sulfonic group was linked to the hydroxyl group of D ring. The fragmentation pathways of M159 are shown in Figure 13. The MS spectrum of M159 is shown in Figure S52.

Analysis of Metabolites M162 Having the Skeleton of Hydrogenated and Dihydroxylated Medicarpin
Its molecular formula was calculated to be C16H16O6 based on [M − H] − at m/z 303.0861. Compared with the C16H14O4 of medicarpin, it had two more hydrogen atoms and two more oxygen atoms. Accordingly, M162 was determined as hydrogenated and dihydroxylated medicarpin.

Analysis of Metabolites M162 Having the Skeleton of Hydrogenated and Dihydroxylated Medicarpin
Its molecular formula was calculated to be C 16 2 and one more oxygen atom. Therefore, M165 was determined as a demethylated, hydrogenated and dihydroxylated medicarpin sulfate.

Distribution of 165 Metabolites in Rats
The distribution of 165 metabolites (including 104 metabolites in urine, 29 metabolites in feces, 38 metabolites in plasma, 41 metabolites in the colon, 74 metabolites in the intestine, 28 metabolites in the stomach, 24 metabolites in the liver, 15 metabolites in the spleen, 42 metabolites in the kidney, 8 metabolites in the lung, 10 metabolites in the heart, 3 metabolites in the brain and 17 metabolites in the thymus) of medicarpin and their relative contents in each biological sample are shown in Figure 14.
The relative content of a metabolite in each biosample was calculated by (peak area of a metabolite in the sample/total peak area of all metabolites detected in the sample) × 100%. The peak area of a metabolite was calculated from its extracted ion chromatogram.
The metabolic reactions of medicarpin included demethylation, hydrogenation, hydroxylation, glucuronidation, sulfation, glycosylation, methylation, and conjunction of vitamin C. The relative contents of metabolic reactions and phase I metabolites in each biosample are shown in Table 1, which were calculated by summing the relative contents of all metabolites generated through it and all phase I metabolites, respectively. In urine, the contents of sulfation metabolites, glucuronidation metabolites and vitamin C conjugates were 74.60%, 25.83% and 1.08%, respectively. In feces, the contents of sulfation metabolites, glucuronidation metabolites and vitamin C conjugates were 73.53%, 1.62% and 15.54%, respectively. This indicated that medicarpin was mainly excreted in the form of sulfates. In plasma, the major metabolic reaction was glucuronidation (relative content: 67.87%). The glycosylation metabolite (M50) was only found in urine. Furthermore, the contents of phase I metabolites of medicarpin in most biosamples were very low (<10%), except in the colon (17.57%), which suggested that phase II metabolites are major metabolites.  ------------104  29  38  41  74  28  24  15  42  8  10  3 17 Total detected metabolites in the matrix Figure 14. The distribution of 165 metabolites of medicarpin and their relative contents in 13 biosamples ( a , new compounds). The relative content of a metabolite in each biosample was calculated by (peak area of a metabolite/total peak area of all detected metabolites) × 100%. Figure 14. The distribution of 165 metabolites of medicarpin and their relative contents in 13 biosamples ( a , new compounds). The relative content of a metabolite in each biosample was calculated by (peak area of a metabolite/total peak area of all detected metabolites) × 100%.