==== Front Molecules Molecules molecules Molecules 1420-3049 MDPI 33256251 10.3390/molecules25235560 molecules-25-05560 Article Exploring the In Vivo Existence Forms (23 Original Constituents and 147 Metabolites) of Astragali Radix Total Flavonoids and Their Distributions in Rats Using HPLC-DAD-ESI-IT-TOF-MSn Liu Li-Jia Li Hong-Fu https://orcid.org/0000-0002-9642-6406Xu Feng * Wang Hong-Yan Zhang Yi-Fan Liu Guang-Xue Shang Ming-Ying Wang Xuan Cai Shao-Qing * State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191, China; lijialiu@bjmu.edu.cn (L.-J.L.); 1716383012@bjmu.edu.cn (H.-F.L.); wanghy@pku.org.cn (H.-Y.W.); zhangyf0911@pku.org.cn (Y.-F.Z.); guangxl@bjmu.edu.cn (G.-X.L.); myshang@bjmu.edu.cn (M.-Y. S.); xuanwang6818@bjmu.edu.cn (X.W.) * Correspondence: xufeng76@bjmu.edu.cn (F.X.); sqcai@bjmu.edu.cn (S.-Q.C.); Tel.: +86-10-8280-2534 (F.X.); +86-10-8280-1693 (S.-Q.C.) 26 11 2020 12 2020 25 23 556001 11 2020 23 11 2020 © 2020 by the authors.2020Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Astragali Radix total flavonoids (ARTF) is one of the main bioactive components of Astragali Radix (AR), and has many pharmacological effects. However, its metabolism and effective forms remains unclear. The HPLC-DAD-ESI-IT-TOF-MSn technique was used to screen and tentatively identify the in vivo original constituents and metabolites of ARTF and to clarify their distribution in rats after oral administration. In addition, modern chromatographic methods were used to isolate the main metabolites from rat urine and NMR spectroscopy was used to elucidate their structures. As a result, 170 compounds (23 original constituents and 147 metabolites) were tentatively identified as forms existing in vivo, 13 of which have the same pharmacological effect with ARTF. Among 170 compounds, three were newly detected original constituents in vivo and 89 were new metabolites of ARTF, from which 12 metabolites were regarded as new compounds. Nineteen original constituents and 65 metabolites were detected in 10 organs. Four metabolites were isolated and identified from rat urine, including a new compound (calycoisn-3’-O-glucuronide methyl ester), a firstly-isolated metabolite (astraisoflavan-7-O-glucoside-2’-O-glucuronide), and two known metabolites (daidzein-7-O-sulfate and calycosin-3’-O-glucuronide). The original constituents and metabolites existing in vivo may be material basis for ARTF efficacy, and these findings are helpful for further clarifying the effective forms of ARTF. Astragali RadixflavonoidsmetabolismLC-MSeffective forms ==== Body 1. Introduction Astragali Radix total flavonoids (ARTF) is one of the main bioactive components of Astragali Radix (AR, Huangqi in Chinese) [1]. Many studies have proven its cardiovascular protective effect, owing that it exhibited a protective effect on an ischemia-reperfusion model by effectively inhibiting the free radical spectrum [2,3], and exhibited vasorelaxant and endothelial-protective effect via the Akt/eNOS signaling pathway [4]. ARTF has an obvious protective effect on the inflammatory response in brain tissue of a natural aging rat by reducing the expression level of the downstream inflammatory factors [5]. ARTF also has immunostimulatory and anti-inflammatory effects via regulating MAPK (Mitogen-Activated Protein Kinase) and NF-κB signaling pathways [6,7]. ARTF has a protective effect against hepatic damage induced by paracetamol [8] or reperfusion [9] as well. In a word, ARTF has a wide range of pharmacological actions. Up to now, besides studies on the pharmacological actions of AR, many investigations have been conducted in the field of phytochemistry. Over 70 flavonoid compounds have been isolated and identified from AR by modern chromatographic and spectroscopic methods [10,11,12], and 421 flavonoids have been detected and characterized from AR (the roots of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao) by HPLC-MS (High Performance Liquid Chromatography-Mass Spectrometry) technology [13]. All these findings indicate that many kinds of constituents exist in ARTF. The metabolism of some high content flavonoid compounds in ARTF have been reported by our research group. Forty-one and 21 metabolites have been identified from the urine of rats after administration of calycosin-7-O-glucoside [14] and ononin [15], respectively. Twenty-six and 14 metabolites have been identified when calycosin [14] and formononetin [15] were incubated with rat liver S9. Relatively, the metabolic studies of isoflavan and pterocarpan are pretty few, and only our research group has done several before. Twenty-one and 20 metabolites of astrapterocarpan-3-O-glucoside, astraisoflavan-7-O-glucoside have been identified in rats, respectively [15]. Forty and 19 metabolites of astrapterocarpan [16] and astraisoflavan [15] have been detected when incubated with rat liver S9, respectively. And many other research groups also have identified some metabolites of these four main isoflavones (calycosin [17], calycosin-7-O-glucoside [18], formononetin [19,20], ononin [21]) in different test systems, such as human gut microbiota [18], zebrafish larvae [17], sheep [19], human liver microsome [20,21], and so on. However, the forms existing in vivo (i.e., absorbed constituents and metabolites) of ARTF remains unclear. Our research group had done a study on ARTF metabolism before, and identified 127, 43, and 22 compounds in the urine, plasma, and feces, respectively [22]. But in that study, no glucuronides of ARTF were found, which should be main metabolites of flavonoid compounds; the distribution of ARTF was not investigated either. And the dose schedule of ARTF was once per day for seven days. As is well known, it usually needs a long dose period for traditional Chinese medicines to treat diseases in clinical practice. As for AR, it can be used for a long time [23]. Therefore, to better simulate the long dose period of AR and understand the existence forms in vivo of ARTF under long-term administration situation, this study was performed. The rats were orally administrated with ARTF twice a day for 31 days, and the samples of urine, plasma, feces, and organs were collected to identify original constituents and metabolites that existed in vivo, and to study the distribution of the existence forms in vivo. After that, the ARTF-containing urine was used to isolate some major metabolites. 2. Results and Discussion To know which compounds exist in vivo, which is one of the prerequisites for the compounds to be effective forms, the compounds in the bio-samples including urine, plasma, feces, and organs of the rats after oral administration of ARTF were analyzed. In total, one hundred and seventy compounds (23 original constituents and 147 metabolites) were identified, among which 12 were regarded as new compounds (they are all metabolites) by retrieving information from the SciFinder database, three were newly detected original constituents, and 89 were new metabolites of ARTF. In 147 metabolites, nine were phase I and 138 were phase II metabolites. Among 138 phase II metabolites, ninety-two sulfates, twenty-six glucuronides, thirteen both sulfuric acid, and glucuronic acid conjugates and seven methyl conjugates were included, which indicated that sulfates were the most important existence forms of ARTF. To better understand the distribution of compounds, ten different organs of the rat were analyzed as well. Nineteen original constituents including six ones detected only in the organs (F18–F23) and 65 metabolites containing three ones detected only in the organs (M145–M147) were identified. Three, 5, 4, 3, 5, 17, 7, 7, 6 original constituents together with 3, 18, 5, 6, 22, 30, 46, 21, 6 metabolites were identified in the heart, liver, spleen, lungs, kidneys, stomach, small intestine, colon intestine, and thymus, respectively. However, no compounds were found in the brain. Four metabolites were isolated from the ARTF-containing urine and identified by NMR. To better understand potential effective forms among those compounds in vivo, we retrieved over 40 compounds which have specific structures from SciFinder, and 13 compounds including six original constituents, and seven metabolites were found to possess the same pharmacological effects as ARTF. Most of the phase Ⅱ metabolites were not found to possess bioactivity, perhaps owing that this kind of material was difficult to gain and to study. 2.1. Identification of Original Constituents and Metabolites of ARTF in Rats 2.1.1. Identification of Original Constituents The peaks which appeared at the same position in LC-MS chromatograms of both the administrated bio-samples and the ARTF but did not exist in the blank bio-samples were regarded as original constituents. By comparison of their extracted ion chromatograms (EICs) and base peak chromatograms (BPCs), twenty-three original constituents were identified (Table 1, F1–F23, and F18–F23 only detected in the organs). They were composed of 15 flavones or isoflavones, namely calycosin and its isomers (F1–F3), calycosin-7-O-glucoside (F4), ononin (F5), formononetin (F6), isomers of pratensein (F7, F8), daidzein (F11), trihydroxyisoflavone/flavone (F13), isomer of odoratin (F14), dihydroxy dimethoxyisoflavone/flavone (F16), naringin (F18), pratensein glucoside and its isomer (F22, F23); four pterocarpans and isoflavanes, namely, astraisoflavane isomer (F15), astraptercarpan (F17), astrapterocarpan pentose glucoside (F20), 3,10-dihydroxy-9-methoxypterocarpan (F21); four dihydroisoflavones/flavones and chalcones, namely trihydroxy-dihydroisoflavone/flavone (F9), trihydroxy-tetrahydroisoflavone/flavone (F10), trihydroxychalcone (F12), dihydrocalycosin pentose glucoside (F19). Among the 23 original constituents, three were newly found original constituents of ARTF, namely F3, F7, and F8. Seventeen original constituents (F1–F17) were found in the urine (Figure 1); F4–F6 (Figure S1) were detected in the plasma; F1, F6, F12, and F17 (Figure S2) existed in the feces. The remaining six original constituents (F18–F23; Table 1) were detected only in the organs. 2.1.2. Identification of Metabolites The peaks only appearing in LC-MS chromatograms of ARTF-treated rat bio-samples, but not existing in either blank bio-samples or ARTF were regarded as metabolites. By comparing the EICs and BPCs of them, 147 peaks were assigned as metabolites (M1–M147; Table 2). M145–M147 were only found in the organs. One hundred and six, 64, and 17 metabolites were identified in the urine (Figure 2), plasma (Figure S3), and feces (Figure S4), respectively. Among the 147 metabolites, 89 were new metabolites of ARTF, from which 12 were regarded as new compounds by searching information from SciFinder database (their MS information was shown in Table S1 and Figures S5–S16). Eighty-nine new metabolites included the sulfates of the ring cleavage products of flavone, sulfates of oxidized, reduced, methylated astraisoflavan, and all the glucuronides as well as disulfates. And 12 potential new compounds were sulfates, disulfates, glucuronides, diglucuronides of tetrahydrocalycosin. By analyzing the structures of 147 metabolites, we speculated they were mainly derived from calycosin and its glycoside (maybe the sources of M17–M72, M131–M139), formononetin and its glycosides (maybe the sources of M73–M78, M131–M139), astrapterocarpan-3-O-glucoside (maybe the sources of M79–M84), astraisoflavan-7-O-glucisode (maybe the sources of M85–M106, M147) and many other low content constituents such as astrapterocarpan (maybe one of the sources of M79–M84), astraisoflavan (maybe one of the sources of M85–M106, M147), daidzein (maybe one source of M107–M117, M131–M139), genistein (maybe one source of M118–M130), and so on. Hence, they were speculated to be the main sources of existence forms of ARTF. These metabolites could be classified into 9 phase I metabolites and 138 phase II metabolites. A hundred and thirty-eight phase II metabolites consisted of 7 methyl conjugates, 92 sulfates, 26 glucuronides, and 13 both sulfuric acid and glucuronic acid conjugates, which indicated that sulfates were the most important existence forms of ARTF. The structural elucidation process of some representative metabolites was described as follows. Identification of the Sulfates of the Ring Cleavage Products of Flavone (M1–M16) A total of 16 compounds were assigned as metabolites originating from flavone, which underwent ring cleavage, then conjugation with sulfuric acid, and all of them were new metabolites of ARTF. M1 showed [M − H]− at m/z 200.99 and its molecular formula was predicted as C7H6O5S, and the fragment ion at m/z 121.03 was formed by the neutral loss of 79.95 Da (SO3) in its MS2 spectra, so it was determined as hydroxyl-benzaladehyde sulfate or other isomers. M2 showed [M − H]− at m/z 219.00 and its molecular formula was predicted as C7H8O6S, and the fragment ion generated by loss 79.95 Da could be detected at m/z 139.04, which was predicted as C7H8O3 and was one CH2 more than pyrogallol, so it was tentatively identified as methyl pyrogallol sulfate [24] or other isomers. M3 and M4 showed [M − H]− at m/z 231.00, which indicated that their molecular formulae were C8H8O6S. In the MS2 spectra, fragment ions at m/z 151.04, 137.03 were formed by sequential losses of SO3 and CH2. According to literature, they were identified as hydroxy phenylacetic acid sulfate [25]. M5 and M6 showed [M − H]− at m/z 243.00, which indicated that their molecular formulae were C9H8O6S, and according to the fragment ions at m/z 163.04, 119.05 and previous report, they were identified as hydroxycinnamic acid sulfate [26]. M7 showed [M − H]− at m/z 243.03 and was predicted as C10H12O5S. And according to the fragment ions at m/z 163.08, 148.05 in MS2 spectra, it was regarded as eugenol sulfate [27]. M8 showed [M − H]− at m/z 247.00 and was predicted as C8H8O7S, and yielded a fragment ion at m/z 167.03 by neutral loss of 79.95 Da (SO3), so it was determined as vanillic acid sulfate [28]. M9 showed [M − H]− at m/z 247.03, which indicated that their molecular formulae were C9H12O6S, and according to the fragment ions at m/z 167.07, 153.09, 137.05 and previous report, it was identified as homovanillyl alcohol sulfate [29]. M10 showed [M − H]− at m/z 273.00, which indicated that its molecular formula was C10H10O7S, and according to the fragment ions at m/z 178.03, 134.04, it was identified as ferulic acid sulfate [24]. M11 and M12 showed [M − H]− at m/z 273.04, which indicated that their molecular formulae were C11H14O6S. And the fragment ions at m/z 193.09, 178.06, 163.03 were observed in MS2 spectra, which were 30.01 Da (OCH2) lager than that of the aglycon of M7. Therefore, they were tentatively determined as methoxyeugenol sulfate. M16 showed [M − H]− at m/z 303.02, which indicated that its molecular formula was C11H12O8S, and the fragment ions at m/z 223.06, 208.04, 164.05, 149.02, were similar to the fragment ions of 4-hydroxy-3,5-dimethoxycinnamic acid, so it was determined as 4-hydroxy-3,5-dimethoxycinnamic acid sulfate [30]. Identification of the Calycosin-Related Metabolites (M17–M72, M146) M17 showed [M − H]− at m/z 283.06 and its molecular formula was predicted as C16H12O5. The fragment ions at m/z 269.04, 268.04, 195.04 were detected in MS2, which were like those of calycosin, so it was identified as calycosin isomer. M18–M20 were identified as calycosin isomer owing that their predicted molecular formulae and fragment ions were like those of calycosin in the positive ion mode. M21 showed [M + H]+ at 301.06 and its molecule formula was predicted as C16H12O6, which had one more oxygen atom than that of calycosin, and the fragment ions at m/z 270.08, 197.08 were similar to those of calycosin in positive ion mode, so it was determined as hydroxycalycosin. The fragment ion of [aglycon−H]− at m/z 283.06 could be detected in the MS2 spectra of M25–M34, which was predicted as C16H12O5, and other fragment ions were like those of calycosin, so they were determined as calycosin metabolites. M25–M30 were predicted as C16H12O8S according to [M − H]− at m/z 363.02, and the [aglycon − H]− formed by the neutral loss of 79.95 Da (SO3) was detected. Therefore, they were determined as calycosin sulfate and its isomers. In the same way, M30 was determined as calycosin-7,3’-O-disulfate. M31 and M146 showed [M − H]− at m/z 459.09 and their molecular formulae were predicted as C22H20O11, and the fragment ion of [aglycon − H]− were formed by the neutral loss of 176.03 Da (C6H8O6) in the MS2 spectra, so they were determined as calycosin glucuronide. There are two positions (C-7-OH and C-3’-OH) in calycosin that could be linked with glucuronic acid, and C-3’-OH was the major position according to a previous study [31]. In addition, M31 was the main metabolite, hence it was determined as calycosin-3’-O-glucuronide, and M146 was determined as calycosin-7-O-glucuronide. In the MS2 spectra of M35–M39, the fragment ion of [aglycon − H]− at m/z 299.05 can be detected, which were predicted as C16H11O6 and was the same as M21, so they were tentatively determined as metabolites of hydroxycalycosin. And according to molecule formulae and characteristic neutral losses, they were tentatively determined as hydroxycalycosin sulfate or glucuronide, respectively. M42 showed [M − H]− at m/z 621.15 and its molecular formula was predicted as C28H30O16. The fragment ion at m/z 283.06 predicted as C16H11O5 was detected in MS2 which was generated by sequential loss of 162.05 Da (C6H10O5), and 176.03 Da (C6H8O6). Since calycosin-7-O-glucoside was the main constituent of ARTF, so it was determined as calycosin-7-O-glucoside-3’-O-glucuronide. The fragment ion of [aglycon − H]− at m/z 285.08 could be detected in the MS2 spectra of M43–M46, which was predicted as C16H13O5 and had 2H (2.01 Da) more than that of calycosin, so they were tentatively regarded as metabolites of dihydrocalycosin. The fragment ion of [aglycon − H]− at m/z 287.09 could be detected in the MS2 spectra of M54–M63, which were predicted as C16H15O5 and had 4H more than that of calycosin, so they were tentatively regarded as metabolites of tetrahydrocalycosin. In the MS2 spectra of M64–M69, the fragment ion of [aglycon − H]− at m/z 303.08 could be detected, which were predicted as C16H15O6 and had one more oxygen atom than that of tetrahydrocalycosin, so they were tentatively regarded as hydroxy tetrahydrocalycosin metabolites. Identification of the Formononetin-Related Metabolites (M73–M78) M73 showed [M − H]− at m/z 347.02 and its molecular formula was predicted as C16H12O7S. The fragment ion of m/z 267.07 in the MS2 spectra was formed by the neutral loss of 79.95 (SO3), which was predicted as C16H11O4, and its fragment ion m/z 252.03 was like that of formononetin. Because only C-7-OH could be sulfated, so M73 was determined as formononetin-7-O-sulfate. In the same way, M74 was determined as formononetin-7-O-glucuronide. M75–M77 showed [M − H]− at m/z 351.05 and their molecular formulae were predicted as C16H16O7S. The fragment ions at m/z 271.09 formed by neutral loss of 79.95 Da (SO3) was determined as C16H15O4, which had 4H more than that of formononetin, so they were tentatively determined as tetrahydroformononetin sulfate. Identification of the Astrapterocarpan-Related Metabolites (M79–M84) M79 showed [M − H]− at m/z 379.05 and its molecular formula was predicted as C17H16O8S. The fragment ions m/z 299.08 formed by a natural loss of 176.03 Da (C6H8O6) was predicted as C17H15O5, which was the same to that of astrapterocarpan. Owing that only C-3-OH of astrapterocarpan could be linked to sulfuric acid, it was determined as astrapterocarpan-3-O-sulfate. In the same way, M80 was determined as astrapterocarpan-3-O-glucuronide. M82–M84 showed [M − H]− at m/z 491.12 and their molecular formulae were predicted as C23H24O12. The fragment ions at m/z 315.09 generated by neutral loss of 176.03 Da (C6H8O6) was predicted as C17H15O6, and had one more O than that of astrapterocarpan, so they were tentatively determined as hydroxyastrapterocarpan glucuronide. Identification of the Astraisoflavan-Related Metabolites (M85–M106, M147) M85 showed [M + H]+ at m/z 303.13 and its molecular formula was predicted as C17H18O5. The fragment ions at m/z 167.10, 149.09, and 125.07 could be detected in MS2 spectra and were like those of astraisoflavan in positive ion mode. So, M85 was determined as astraisoflavan isomer. M102–M103 showed [M − H]− at m/z 477.14 and their molecular formulae were predicted as C23H26O11. The fragment ions at m/z 301.10 in MS2 spectra formed by a neutral loss of 176.03 Da (C6H8O6) was the same to that of astraisoflavan. Therefore, they were determined as astraisoflavan glucuronide. Because there are only two glucuronidation sites (C-7-OH, C-2’-OH) in astraisoflavan, and a larger CLogP value means a lower polarity and a larger retention time in reversed phase HPLC, M102 (CLogP = 3.6083, tR = 58.702 min) was determined as astraisoflavan-7-O-glucuronide and M103 (CLogP = 3.2673, tR = 57.802 min) was determined as astraisoflavan-2’-O-glucuronide. Identification of the Daidzein-Related Metabolites (M107–M117) M107 and M108 showed [M − H]− at m/z 333.00 and their molecular formulae were predicted as C15H10O7S. The fragment ion at m/z 253.05 in MS2 spectra formed by the neutral loss of 79.95 Da (SO3), and it was predicted as daidzein owing that the fragment was predicted as C15H9O4 and m/z 225.05, 197.06, 135.01 were detected in MS3 spectra. Because there are two sulfation sites (C-7-OH, C-4’-OH) in daidzein, and a larger CLogP value means a lower polarity and a larger retention time in reversed phase HPLC, M107 (CLogP = 0.4985, tR = 41.557 min) was determined as daidzein-4’-O-sulfate and M108 (CLogP = 0.3050, tR = 28.925 min) was determined as daidzein-7-O-sulfate. M112 showed [M − H]− at m/z 335.02 and its molecular formula was predicted as C15H12O7S. In MS2 spectra, the fragment ion at m/z 255.06 was formed by the neutral loss of 79.95 Da (SO3), which was predicted as C15H11O4 and had 2H more than that of daidzein. Therefore, it was tentatively determined as dihydrodaidzein sulfate. M113–M116 showed [M − H]− at m/z 337.04 and their molecular formulae were predicted as C15H14O7S. The fragment ion at m/z 257.08 in MS2 spectra formed by the neutral loss of 79.95 Da (SO3), which was predicted as C15H13O4 and had 4H more than that of daidzein. Therefore, they were tentatively determined as tetrahydrodaidzein sulfate. Identification of the Genistein-Related Metabolites (M118–M130) M118 showed [M + H]+ at m/z 271.06 and its molecule formula was predicted as C15H10O5. The fragment ions at m/z 253.01, 225.06, 215.07 which were like those of genistein in reported literature [32], so it was determined as genistein. M123–M125 showed [M − H]− at m/z 351.02 and their molecular formulae were predicted as C15H12O8S. The fragment ion at m/z 271.06 was formed by a neutral loss 79.95 Da (SO3), which was predicted as C15H11O5 and had 2H more than that of genistein, so they were tentatively determined as dihydrogenistein sulfate. M126 showed [M − H]− at m/z 273.07 and its molecular formula was predicted as C15H14O5, which was 4H more than that of genistein, so it was tentatively determined as tetrahydrogenistein. M130 showed [M − H]− at m/z 515.09 and its molecules formula was predicted as C21H24O13S. The fragment ion at m/z 273.07 which was predicted as C15H13O5 formed by a neutral loss of 162.05 Da (C6H10O5) and 79.95 Da (SO3), so it was tentatively determined as tetrahydrogenistin sulfate. Identification of the Equol-Related Metabolites (M131–M139) In the MS2 spectra of M131–M138, the fragment ion of [aglycon − H]− at m/z 241.09 could be detected, which were predicted as C15H13O3 and its fragment ions of m/z 135.05, 121.04, 119.06 were like those of equol [33], so they were regarded as equol metabolites. M136–M138 showed [M − H]− at m/z 497.07 and their molecules formulae were predicted as C21H22O12S, and the [aglycon − H]− formed by the neutral loss of 176.03 Da (C6H8O6), 79.97 Da (SO3). Therefore, they were determined as equol glucuronide sulfate. M139 showed [M − H]− at m/z 323.06 and its molecular formula was predicted as C15H16O6S. The fragment ion at m/z 243.10 formed by a neutral loss of 79.95 Da (SO3), which was predicted as C15H15O3 and 2H (2.01Da) more than that of equol. Hence, it was tentatively determined as dihydroequol sulfate. Identification of the other Metabolites (M140–M145) M140 showed [M + H]+ at m/z 303.09 and its molecular formula was predicted as C16H14O6, which had 2H more than that of pratensein. Therefore, it was tentatively determined as dihydropratensein. M141 and M145 showed [M − H]− at m/z 555.05 and their molecular formulae were predicted as C22H20O15S. The fragment ions at m/z 299.05 predicted as C16H11O6 was formed by a neutral loss of 176.03 Da (C6H8O6) and 79.95 Da (SO3). Therefore, it was tentatively determined as pratensein glucuronide sulfate. 2.2. Distribution of Original Constituents and Metabolites of ARTF in Rats Organs 2.2.1. Distribution of Original Constituents Nineteen original constituents were detected in the organs, with zero in brain, three in heart, five in liver, four in spleen, three in lung, five in kidney, seventeen in stomach, seven in small intestine, seven in colon intestine, and six in thymus, respectively (Table S2, Figure S17a–S25a). Six (F18–F23) were only detected in the organs and were not detected in the urine, plasma, and feces. Calycosin (F1), formononetin (F6), daidzein (F11), and naringin (F18) were widely distributed, which could be detected in seven and even more organs, and these compounds may be important material basis for the efficacy of ARTF. 2.2.2. Distribution of Metabolites Sixty-five metabolites were identified in the organs, of which three metabolites (M145–M147) were only detected in the organs, and 0, 3, 18, 5, 6, 22, 30, 46, 21, and 6 were identified in the brain, heart, liver, spleen, lung, kidney, stomach, small intestine, colon, and thymus (Table S3, Figures S17b–S25b), respectively. Twelve metabolites containing seven sulfates and five glucuronides were distributed widely in five and even more tissues. Seven sulfates were calycosin sulfate (M26), tetrahydrocalycosin sulfate (M57, M58), hydroxyastraisoflavan sulfate (M96), daidzein-4’-O-sulfate (M107), equol sulfate (M132, M133), respectively. Five glucuronides consisted of calycosin-3’-O-glucuronide (M32), dimethoxy hydroxytetrahydrocalycosin glucuronide (M72), astrapterocarpan-3-O-glucuronide (M80), astraisoflavan-2’-O-glucuronide (M103), and astraisoflavan-7-O-glucoside-2’-O-glucuronide (M106). These widely distributed metabolites may play an important role in the efficacies of ARTF. 2.3. Identification of Metabolites Isolated from Rat Urine MI-1 (M108) was obtained as a white powder and assigned a molecular formula of C15H10O7S based on its HR-ESI-MS mass spectrum, which showed a quasi-molecular ion peak [M − H]− at m/z 333.0076 (calcd. for C15H10O7S 333.0069). The main fragment ion was m/z 253.0505 [M − SO3 − H]− in MS2 spectra, so it was regarded as a sulfate. MI-1 (M108):13C-NMR (DMSO-d6, 100MHz) ppm: 153.6 (C-2), 122.5 (C-3), 175.0 (C-4), 129.7 (C-5), 118.0 (C-6), 158.1 (C-7), 107.1 (C-8), 156.6 (C-9), 119.0 (C-10), 123.7 (C-1’), 130.2 (C-2’, C-6’), 115.08 (C-3’, C-5’), 157.3 (C-4’), which were in consistent with daidzein [34]. 1H-NMR spectra of MI-1 (M108) (DMSO-d6, 400MHz) ppm: 8.38 (1H, s, H-2), 8.03 (1H, d, J = 8.8Hz, H-5), 7.43 (1H, d, J = 2.1Hz, H-8), 7.40 (2H, d, J = 8.3Hz, H-2’ and H-6’), 7.25 (1H, dd, J = 8.8Hz, 2.1Hz, H-6), 6.81 (2H, d, J = 8.3Hz, H-3’ and H-5’), which were like those of daidzein-7-O-sulfate reported in literature [35].Given all of this, MI-1 was determined as daidzein-7-O-sulfate. Its structure and NMR spectroscopy were shown in Figure 3a and Figure S26, respectively. MI-2 (M32) was obtained as a white powder and assigned a molecular formula of C22H20O11 based on its HR-ESI-MS mass spectrum, which showed a quasi-molecular ion peak [M − H]− t m/z 459.0943 (calcd. for C22H20O11 459.0935). The main fragment ion was m/z 283.0609 [M − C6H8O6 − H]− in MS2 spectra, so it was regarded as a glucuronide. MI-2 (M32): 1H-NMR spectra (DMSO-d6, 400MHz) ppm: 8.31 (1H, s, H-2), 7.97 (1H, d, J = 8.8Hz, H-5), 6.94 (1H, dd, J = 8.8Hz, 2.2Hz, H-6), 6.87 (1H, d, J = 2.2Hz, H-8), 7.29 (1H, d, J = 2.0Hz, H-2’), 7.04 (1H, d, J = 8.5Hz, H-5’), 7.23 (1H, dd, J = 8.5Hz, 2.0Hz, H-6’), 3.79 (3H, s, C-4’-OCH3), 10.88 (1H, s, C-7-OH). 13C-NMR (DMSO-d6, 100MHz) ppm: 153.5 (C-2), 123.3 (C-3), 174.7 (C-4), 127.4 (C-5), 115.4 (C-6), 162.7 (C-7), 102.3 (C-8), 157.5 (C-9), 116.4 (C-10), 124.5 (C-1’), 116.7 (C-2’), 145.7 (C-3’), 149.2 (C-4’), 112.6 (C-5’), 123.3 (C-6’), 55.9 (C-4’-OCH3), which were similar to 13C-NMR of calycosin reported in literature [36]. The characteristic signals of six carbons in glucuronide were 100.2 (C-1’’), 73.1 (C-2’’), 76.3 (C-3’’), 71.6 (C-4’’), 75.6 (C-5’’), 170.2 (C-6’’), and all were consisted with calycosin-3’-O-glucuronide [31]. Based on the above analysis, MI-2 (M32) was determined as calycosin-3’-O-glucuronide. Its structure and NMR spectroscopy were shown in Figure 3b and Figure S27, respectively. MI-3 was obtained as a white powder and assigned a molecular formula of C23H22O11 based on its HR-ESI-MS mass spectrum, which showed a quasi-molecular ion peak [M − H]− at m/z 473.1113 (calcd. for C23H22O11 473.1084). The main fragment ion was m/z 283.0601 [M − C7H10O6 − H]− in MS2 spectra, so it was predicted as a glucuronide methyl ester. 1H-NMR (DMSO-d6, 400MHz) ppm: 8.31 (1H, s, H-2), 7.97 (1H, d, J = 8.7Hz, H-5), 6.94 (1H, dd, J = 8.7Hz, 2.2Hz, H-6), 6.87 (1H, d, J = 2.2Hz, H-8), 7.29 (1H, d, J = 2.0Hz, H-2’), 7.05 (1H, d, J = 8.5Hz, H-5’), 7.23 (1H, dd, J = 8.5Hz, 2.0Hz, H-6’), 3.79 (3H, s, C-4’-OCH3), 10.77 (1H, s, C-7-OH), 3.62 (3H, s, C-6’’-OCH3). 13C-NMR (DMSO-d6, 100MHZ) ppm: 153.4 (C-2), 123.2 (C-3), 174.5 (C-4), 127.3 (C-5), 115.3 (C-6), 162.8 (C-7), 102.1 (C-8), 157.4 (C-9), 116.2 (C-10), 124.5 (C-1’), 116.6 (C-2’), 145.5 (C-3’), 149.0 (C-4’), 112.4 (C-5’), 123.0 (C-6’), 55.8 (C-4’-OCH3), were carbon signals of calycosin [31], 99.9 (C-1’’), 73.0 (C-2’’), 75.8 (C-3’’), 71.4 (C-4’’), 75.2 (C-5’’), 169.2 (C-6’’) were carbon symbols of glucuronide, which were similar to those of calycosin-3’-O-glucuronide [31]. Compared with that, an additional methoxy carbon signal at δ52.0 was observed, and H signal of this methoxy at δ3.62 (3H, s, C-6’’-OCH3) was in correlated with carbonyl carbon signal of glucuronide at δ169.2 (C-6’’) in HMBC spectra, indicating that the methoxy was linked to the carbonyl of glucuronide. According to all above analysis, MI-3 was identified as calycoisn-3’-O-glucuronide methyl ester (Figure 3c). It was a new compound. And its NMR spectroscopy was shown in Figure S28. But unfortunately, MI-3 could not be detected in the bio-samples using the HPLC-DAD-ESI-IT-TOF-MSn technique. Therefore, MI-3 was maybe produced during the isolation process. MI-4 (M106) was obtained as a faint yellow powder and assigned a molecular formula of C29H36O16 based on its HR-ESI-MS mass spectrum, which showed a quasi-molecular ion peak [M − H]− at m/z 639.1959 (calcd. for C22H20O11 639.1925). The main fragment ion was m/z 463.1650 [M − C6H10O5 − H]−, 301.1066 [M − C6H10O5 − C6H8O8 − H]− in MS2 spectra, so it was regarded as glucoside and glucuronide. 1H-NMR (DMSO-d6, 400MHz) ppm: 3.86 (1H, t, H-2a), 4.23 (1H, d, J = 8.0Hz, H-2b), 3.59 (1H, m, H-3), 2.74 (1H, m, H-4a), 2.84 (1H, m, H-4b), 6.98 (1H, d, J = 8.4Hz, H-5), 6.54 (1H, dd, J = 8.4 Hz, 2.4Hz, H-6), 6.48 (1H, d, J = 2.4Hz, H-8), 6.81 (1H, d, J = 8.8Hz, H-5’), 6.92 (1H, d, J = 8.8Hz, H-6’), 3.72 (3H, s, C-3’-OCH3), 3.77 (3H, s, C-4’-OCH3). 13C-NMR (DMSO-d6, 100MHZ) ppm: 69.8 (C-2), 30.0 (C-3), 30.9 (C-4), 130.0 (C-5), 108.8 (C-6), 156.8 (C-7), 104.0 (C-8), 115.9 (C-9), 154.6 (C-10), 128.4 (C-1’), 147.2 (C-2’), 141.1 (C-3’), 152.1 (C-4’), 121.8 (C-5’), 103.2 (C-6’), 60.5 (C-3’-OCH3), 55.8 (C-4’-OCH3), 100.8 (C-1’’), 73.7 (C-2’’), 76.6 (C-3’’), 69.5 (C-4’’), 77.1 (C-5’’), 60.8 (C-6’’) were carbon signal of astraisoflavan-7-O-glucoside [36]. And 100.9 (C-1’’’), 73.3 (C-2’’’), 75.8 (C-3’’’), 71.5 (C-4’’’), 75.7 (C-5’’’), 170.1 (C-6’’’) were carbon signal of glucuronide. According to HMBC spectra, δ4.90, which was the terminal hydrogen signal of glucuronide, related to the δ147.2 (C-2’), which indicated that glucuronide was linked to C-2’. At the same time, the terminal hydrogen of glucoside δ4.79 was in correlated with glucoside carbon at δ156.8 (C-7), which indicated that glucoside was linked to C-7. Considering all of the above, MI-4 (M106) was determined as astraisoflavan-7-O-glucoside-2’-O-glucuronide (Figure 3d). It was a compound that isolated and identified by NMR for the first time. And its NMR spectroscopy was shown in Figure S29. 2.4. ARTF-Related Pharmacological Effect of Compounds In Vivo The pharmacological literature of over 40 existence forms of ARTF which have specific or potential structure were retrieved from SciFinder and then analyzed. We found that 13 existence forms showed related pharmacological effect to ARTF, such as cardiovascular protective, neuroprotective, anti-inflammatory, and so on (Table S4). Six of them were original constituents, namely calycosin (F1), calycosin-7-O-glucoside (F4), ononin (F5), formononetin (F6), daidzein (F11), naringin (F18); seven of them were metabolites, namely daidzein-4’-O-sulfate (M107), daidzein-7-O-sulfate (M108), daidzein-7-O-glucuronide or daidzein-4’-O-glucuronide (M110), genistein (M118), genistein-7-O-sulfate and genistein-4′-O-sulfate (two of M119–M121), and equol-7-O-sulfate (M132 or M133). From Table S4, we could find that the metabolites in Table S4, especially phase Ⅱ metabolites, could activate estrogen receptor (ER). ER activation is associated with cardiovascular protective [37] and anti-inflammatory [38] effects, which is the main pharmacological effect of ARTF. In addition, we predicted that sulfate of flavonoids might have an effect on ER by molecular docking technique (data not shown). Furthermore, phase Ⅱ metabolites, especially sulfates, were the main existence forms of ARTF, so it could be speculated that some existence forms in vivo might be the material bases of the efficacies of ARTF, i.e., its effective forms. 3. Materials and Methods 3.1. Chemicals and Materials ARTF (lot: 20170730) was obtained from Shanxi Baoji Herbest Biotech Co., Ltd. (Baoji, Shanxi, China) in August 2017 and its content was 62.7%, and the content of six main flavonoids namely calycosin-7-O-glucoside, calycosin, ononin, formononetin, astrapterocarpan-3-O-glucoside, and asisoflavan-7-O-glucoside was 11.3, 6.3, 5.8, 5.4, 7.2, and 1.6%, respectively, which was detected by HPLC-DAD-ELSD and calculated by using area normalization of ELSD (Evaporative Light Scattering Detector) chromatogram (Figure S30). The analysis of its constituents was conducted by HPLC-DAD-ESI-IT-TOF-MSn, and 69 constituents had been identified (Table S5). Calycosin (lot: MUST-16031110), calycosin-7-O-glucoside (lot: MUST-16031205) were brought from Chengdu Must Biotech Co., Ltd. (Chengdu, Sichuang, China). Formononetin (lot: LS60Q22) was supplied by Beijing J&K Scientific Co., Ltd. (Beijing, China). Astrapterocarpan (lot: PRF7042221), astrapterocarpan-3-O-glucoside (lot: PRF7043003), astraisoflavan (lot: PRF7120221) were provided by Chengdu Biopurify Phytochemicals Ltd. (Chengdu, Sichuang, China). Astraisoflavan-7-O-glucoside (lot: 160217) was purchased from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, Sichuang, China). Ononin was isolated in our laboratory [36]. The purities for all reference compounds were over 98%. Acetonitrile (HPLC grade, lot: 184866), formic acid (LC/MS grade, lot: 182088) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Methanol was supplied by Tianjing Damao Co., Ltd. (Tianjing, China). Sodium carboxymethyl cellulose (CMC-Na, Analytical grade) was purchased from Tianjing Guangfu Fine Chemical Research Institute (Tianjin, China). XAD-2 macroporous resins (lot: 94664), ODS (lot:9833), sephadex LH-20 were applied by Supelco (Bellefonte, PA, USA), YMC (Kyoto, Japan), and Amersham Biociences (Boston, MA, USA), respectively. Ultra-pure water was obtained by a Millipore Milli-Q Integral 3 Ultrapure water system (Billerica, MA, USA). 3.2. Animals and ARTF Administration Fifty male Sprague-Dawley (SD) rats (250–300g) were obtained from the Experimental Animal Center of Peking University Health Science Center (Beijing, China) and 40 of them were kept in metabolic cages (Type: DXL-DL, Suzhou Fengshi Laboratory Animal Equipment Co. Ltd. (Suzhou, Jiangsu, China)) with two rats in each cage, and the other 10 rats were kept in normal cages, for free water and food twice a day. All the animals were maintained in an environmentally controlled breeding room for two days. Then, the next two days, urine and feces samples were collected twice a day and combined as blank samples. After that, the 40 rats in metabolic cages were orally administered with ARTF at 200 mg/kg (the suspension of ARTF at 25 mg/kg was prepared with 0.5% CMC-Na in an ultrasonic bath), twice a day (8:00 am and 8:00 pm) for 31 days, and the 10 rats in normal cages weren’t administered anything except for water and food. The animal experiments were approved by the Biomedical Ethical Committee of Peking University (approval No. LA2016205). 3.3. Bio-Samples Collection and Pre-Treatment 3.3.1. Urine Collection and Pre-Treatment Urine samples were collected twice a day (8:00 am and 8:00 pm) after administration of ARTF from metabolic cages and merged together, then filtered to remove impurities such as hair and dried in vacuum at 50 °C using a Heidolph Laborota 4001 rotatory evaporator (Heidolph Instruments GmhH & Co., Schwabach, Germany). After that, at a ratio of 1.0 g, dried samples were reconstituted in 10 mL methanol followed by 30 min ultrasonic extraction and filtered, then the filtrate was dried in vacuum (ca 25 g/day obtained) and 1.0 g sample was added 1 mL methanol to resuspended and stored at −20 °C. After 31 days, all the processed urine samples were mixed together as one sample and dried in vacuum. g). 1.0 g of the urine extract was taken out and redissolved in 5 mL methanol, then centrifuged at 15,000 rpm for 15 min and the supernatant was filtered through 0.45 μm nylon filter (Tianjin jinteng Experiment Co. Ltd., Tianjin, China). Finally, the filtrate was transferred into sample injection vial waiting for LC/MS analysis. 3.3.2. Feces Collection and Pre-Treatment Feces samples were collected twice a day (8:00 am and 8:00 pm) from metabolic cages. Feces samples of each day were dried in 50 °C for 48 h and were crushed into powder. A pulverized sample of 1.0 g was extracted with 5 mL methanol for 30 min in an ultrasonic bath three times. Afterward, the filtrates were combined and concentrated to dryness, then redissolved in 10 folds methanol to store at −20 °C. All feces samples were combined after processing, and concentrated to dryness as ARTF-containing feces extract; 0.3 g was taken out to resuspended in 3 mL methanol and centrifuged at 15,000 rpm for 15 min, and filtered through 0.45 μm nylon filter before LC/MS analysis. 3.3.3. Plasma Collection and Pre-Treatment At 32nd day, blood samples from rats in metabolic cages were collected by heart puncture technique under anesthesia at 10 min, 30 min, 1 h, 2 h, and 4 h (eight rats were sacrificed at each time point), and centrifuged at 5000 rpm for 15 min to obtain ARTF-containing plasma. Blank plasma was collected and processed in the same way from the rats in normal cages. All ARTF-containing and blank plasma were merged respectively, and 10 folds volume of methanol was added, and ultrasonically vibrated for 30 min to precipitate protein. Then, the mixture was centrifuged at 5000 rpm for 15 min, and the supernatant was condensed and dissolved in methanol, and the volume of methanol was 1% of the initial volume of plasma, and then centrifuged at 15,000 rpm for 15 min, finally filtered through 0.45 μm nylon filter before LC/MS analysis. 3.3.4. Organs Collection and Pre-Treatment After blood collection, the heart, liver, spleen, lung, kidney, stomach, small intestine, colon, thymus, and brain were quickly removed and flushed clearly with stroke-physiological saline solution until there was no obvious blood or content in the surface or cavity. All the organs were stored at –80℃. All the organs were shredded and suspended in deionized water at a ratio of 1.0 g to 4 mL, then homogenized by ultrasound homogenizer (Ultra-Turrax T8, Ika-werke Gmbh & Co. KG, Staufen, Germany). After that, 8 mL homogenates were extracted with 10-folds volume methanol in an ultrasonic bath for 30 min. The mixture was centrifuged at 5000 rpm for 15 min, and the supernatant was separated, dried, and resuspended in 2 mL methanol and filtered through 0.45 μm nylon filter before LC/MS analysis. 3.4. Isolation and Identification of Metabolites from ARTF-Containing Urine Isolation procedure: ARTF-containing urine extract obtained in Section 3.3 (ca. 750 g), was dissolved in 1.5 L deionized water, filtered, and then subjected to XAD-2 macroporous resins column chromatography. Water, 20% methanol-water, 60% methanol-water, and 100% methanol were used to elute the column and get Fraction 1 to Fraction 4, respectively. The four metabolites, MI-1 (M108) (2.67 mg), MI-2 (M32) (818.30 mg), MI-3 (16.66 mg), MI-4 (M106) (34.62 mg), were isolated and purified from Fraction-3 by ODS column chromatography, Sephadex LH-20 column chromatography, and a Shimadzu preparative HPLC system sequentially, and their purity was above 90% determined by an Agilent 1200 HPLC. Structure identification using NMR: MI-1 and the other three metabolites were dissolved in 0.15 mL and 0.5 mL DMSO-d6, respectively. Their 1H, 13C, heteronuclear singular quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC). NMR spectra were recorded on a Bruker DRX-400 NMR spectrometer (Bruker, Rheinstetten, Germany), using tetramethylsilane (TMS) as internal standard. All chemical shifts were reported in parts per million (ppm, δ), and coupling constants (J) in Hertz. UV spectra (200–400 nm) and HRMS data were recorded on the LC-MS-IT-TOF instrument with a PDA detector. 3.5. Instruments and Conditions HPLC-DAD-ESI-IT-TOF-MSn analyses were performed on a Shimadzu HPLC instrument (two LC-20AD pumps, an SIL-20AC autosampler, a CTO-20A column oven, an SPD-M20A PDA detector, a CBM-20A system controller) coupled with an IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan) through an ESI interface. All data were processed by Shimadzu software, specifically, LCMS solution Version 3.60, Formula Predictor Version 1.2. and Accurate Mass Calculator. The chromatography separations were performed on an Industries Epic C18 column (250mm × 4.6 mm, 5 μm) (New Brunswick, NJ, USA) protected with an Agilent ZORBAX SB C18 column (12.5 mm × 4.6 mm, 5 μm) (Santa Clara, CA, USA). The mobile phase consisted of water-formic acid (100:0.1, v/v) (A) and acetonitrile (B) at a flow rate of 10,000 mL/min. A gradient elution program was adopted, specifically as 5% B at 0–10 min, 5–16% B at 10–12 min, 16–20% B at 12–25 min, 20–22% B at 25–45 min, 22–35% B at 45–60 min, 35–60% B at 60–85 min, 60–100% at 85–90 min. At the end of each run, 100% B was used to flush the column for 20 min. For mass detection, the mass spectrometer was programmed to carry out a full scan over m/z 100–1500 (MS1) with 30 ms accumulation time and m/z 50–1500 (MS2 and MS3) with 20 ms accumulation in both positive ion (PI) and negative ion (NI) detection mode; the flow rate was 0.2000 mL/min; the heat block and curved desolvation line temperature was 200 °C; the nebulizing nitrogen gas flow was 1.5 L/min; the interface voltage was (+), 4.5 kV; (−), 3.5 kV; the detector voltage was 1.7 kV; the relative collision-induced dissociation energy was 50%. 4. Conclusions In summary, 170 kinds of compound (23 original constituents and 147 metabolites) were identified after administration of ARTF to rats, which included three newly detected original constituents and 89 new metabolites of ARTF, and 12 were regarded as new compounds (they are all metabolites) by retrieving information from the Sci inder database. Nineteen original constituents and 65 metabolites were detected and characterized in 10 organs. Four metabolites, including a new compound (calycoisn-3’-O-glucuronide methyl ester) and a first-isolated compound (astraisoflavan-7-O-glucoside-2’-O-glucuronide), along with two known compounds (daidzein-7-O-sulfate and calycosin-3’-O-glucuronide) were isolated from ARTF-containing urine and identified by NMR. Although the bioactivity studies of phase Ⅱ metabolites were little, 13 compounds (six original constituents, one phase I metabolite, six phase Ⅱ metabolites) in vivo were reported to possess similar pharmacological effects with ARTF, which indicated that they were effective forms of ARTF, and phase II metabolites might contribute to the efficacies of ARTF in vivo. In the future, firstly, more kinds of phase I and phase II metabolites of ARTF should be obtained by synthesis or biotransformation. Then, the bioactivities of these metabolites should be determined to clarify the effective forms of ARTF. After that, the action mechanism of the effective forms can be studied. Finally, a new strategy to evaluate and control the quality of AR can be established. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online. Detailed information on the determination of the contents of ARTF and its major constituents and the isolation procedure of compounds from urine, with Tables S1–S5 and Figures S1–S35 are available. References [39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69] are cited in the Supplementary Materials. Click here for additional data file. Author Contributions Conceptualization, S.-Q.C. and F.X.; methodology, F.X., H.-F.L.; validation, S.-Q.C., F.X.; formal analysis, L.-J.L.; investigation, L.-J.L., H.-Y.W., Y.-F.Z.; writing—original draft preparation, L.-J.L; writing—review and editing, L.-J.L., H.-F.L., F.X., G.-X.L., M.-Y.S., X.W., S.-Q.C.; supervision, S-Q.C., F.X.; project administration, S-Q.C., F.X., L.-J.L.; funding acquisition, S.-Q.C., F.X. All authors have read and agreed to the published version of the manuscript. Funding This research was funded by the National Natural Science Foundation of China (NO. 81673595) and the National Science and Technology Major Project for Significant New Drugs Development (2019ZX09201004). Conflicts of Interest The authors declare no conflict of interest. Sample Availability Samples of the compounds are not available from the authors. Figure 1 The extracted ion chromatograms (EICs) of original constituents (F1–F17) in rat urine after administration of Astragali Radix total flavonoid (ARTF). Figure 2 The EICs of 106 metabolites in rat urine after administration of ARTF. (a) EICs of M2–M41; (b) EICs of M43–M93; (c) EICs of M96–M144. Figure 3 The structures of isolated metabolites from drug-containing urine. (a) MI-1 (M108); (b) MI-2 (M32); (c) MI-3; (d) MI-4 (M106). molecules-25-05560-t001_Table 1Table 1 Original constituents in vivo after administration of ARTF to rats. No. tR (min) Formula (M) Ion Meas. (m/z) Pred. (m/z) Diff (ppm) DBE Identification Plasma Urine Feces F1 ♥ 58.602 C16H12O5 [M − H]− 283.0623 283.0612 3.89 11 Calycosin ▲ ▲ F2 77.588 C16H12O5 [M + H]+ 285.0753 285.0758 −1.75 11 Calycosin isomer 1 ▲ F3 * 56.485 C16H12O5 [M − H]− 283.0608 283.0612 −1.41 11 Calycosin isomer 2 ▲ F4 ♥ 27.200 C22H22O10 [M + HCOO]− 491.1215 491.1195 4.07 12 Calycosin-7-O-glucoside ▲ ▲ F5 ♥ 47.743 C22H22O9 [M + H]+ 431.1322 431.1337 −3.48 12 Ononin ▲ ▲ F6 ♥ 71.750 C16H12O4 [M − H]− 267.0673 267.0663 3.74 11 Formononetin ▲ ▲ ▲ F7 * 66.802 C16H12O6 [M − H]− 299.0569 299.0561 2.68 11 Pratensein/Rhamnocitrin/5,7,4’-trihydroxy-3’-methoxyisoflavone ▲ F8 * 68.197 C16H12O6 [M − H]− 299.0576 299.0561 5.02 11 Pratensein/Rhamnocitrin/5,7,4’-trihydroxy-3’-methoxyisoflavone ▲ F9 65.018 C15H12O5 [M − H]− 271.0622 271.0612 3.69 10 Trihydroxy-dihydroisoflavone/flavone ▲ F10 44.750 C15H14O5 [M − H]− 273.0777 273.0768 3.30 9 Trihydroxy-tetrahydroisoflavone/flavone ▲ F11 54.377 C15H10O4 [M − H]− 253.0514 253.0506 3.16 11 Daidzein ▲ F12 55.485 C15H12O4 [M − H]− 255.0675 255.0663 4.70 10 Trihydroxychalcone ▲ ▲ F13 65.627 C15H10O5 [M − H]− 269.0452 269.0455 −1.12 11 Trihydroxyisoflavone/flavone ▲ F14 60.827 C17H14O6 [M + H]+ 315.0882 315.0863 6.03 11 Odoratin isomer ▲ F15 57.460 C17H18O5 [M + H]+ 303.1216 303.1227 −3.63 9 Astraisoflavane isomer ▲ F16 65.852 C17H16O6 [M + H]+ 317.1040 317.1020 6.31 10 Dihydroxy dimethoxyisoflavone/flavone ▲ F17 ♥ 73.328 C17H16O5 [M + H]+ 301.1052 301.1071 −6.31 10 Astraptercarpan ▲ ▲ F18 35.440 C27H32O14 [M − H]− 579.1748 579.1719 5.01 12 Naringin F19 34.635 C28H36O13 [M − H]− 579.2103 579.2083 3.45 11 Dihydrocalycosin pentose glucoside F20 51.837 C28H34O14 [M − H]− 593.1899 593.1876 3.88 12 Astrapterocarpan pentose glucoside F21 63.302 C16H14O5 [M + H]+ 287.0927 287.0914 4.53 10 3,10-dihydroxy-9-methoxypterocarpan F22 60.812 C22H22O11 [M − H]− 461.1114 461.1089 5.42 12 Pratensein glucoside/5,7’,4’-trihydroxy-3’-methoxyisoflavone glucoside F23 22.332 C22H22O11 [M − H]− 461.1112 461.1089 4.99 12 Pratensein glucoside/5,7’,4’-trihydroxy-3’-methoxyisoflavone glucoside Sum 3 17 4 Note: tR: Retention time; Meas.: measured; Pred.: predicted; Diff: difference; DBE: double bone equivalents. ♥ These constituents were identified by comparison with reference compounds; * New original constituents found in vivo after administration of ARTF. ▲ Detected. molecules-25-05560-t002_Table 2Table 2 Metabolites in vivo after administration of ARTF to rats. NO. tR(min) Formula (M) Ion Meas. (m/z) Pred. (m/z) Diff (ppm) DBE Identification Urine Plasma Feces M1 △ 19.500 C7H6O5S [M − H]− 200.9862 200.9863 −0.50 5 Hydroxyl-benzaladehyde sulfate or isomer ▲ M2 △ 10.948 C7H8O6S [M − H]− 218.9967 218.9969 −0.91 4 Methyl pyrogallol sulfate or isomer ▲ M3 △ 20.550 C8H8O6S [M − H]− 230.9961 230.9969 −3.46 5 Hydroxyphenylacetic acid sulfate ▲ M4 △ 28.350 C8H8O6S [M − H− 230.9963 230.9969 −2.6 5 Hydroxyphenylacetic acid sulfate isomer ▲ M5 △ 21.008 C9H8O6S [M − H]− 242.9978 242.9969 3.7 6 Hydroxycinnamic acid sulfate 1 ▲ ▲ M6 △ 23.533 C9H8O6S [M − H]− 242.9960 242.9969 −3.70 6 Hydroxycinnamic acid sulfate 2 ▲ M7 △ 55.018 C10H12O5S [M − H]− 243.0345 243.0333 4.94 5 Eugenol sulfate ▲ M8 △ 36.100 C8H8O7S [M − H]− 246.9921 246.9918 1.21 5 Vanillic acid sulfate ▲ M9 △ 19.617 C9H12O6S [M − H]− 247.0281 247.0282 −0.40 4 Homovanillyl alcohol sulfate ▲ M10 △ 20.383 C10H10O7S [M − H]− 273.0080 273.0074 2.20 6 Ferulic Acid sulfate ▲ M11 △ 58.818 C11H14O6S [M − H]− 273.0436 273.0438 −0.73 5 Methoxyeugenol sulfate 1 ▲ M12 △ 55.593 C11H14O6S [M − H]− 273.0433 273.0438 −1.83 5 Methoxyeugenol sulfate 2 ▲ ▲ M13 △ 43.617 C11H12O7S [M − H]− 287.0237 287.0231 2.09 6 C11H12O4 sulfate ▲ M14 △ 23.750 C11H14O7S [M − H]− 289.0393 289.0387 2.08 5 Ethylhomovanillic acid sulfate 1 ▲ ▲ M15 △ 26.642 C11H14O7S [M − H]− 289.0397 289.0387 3.46 5 Ethylhomovanillic acid sulfate 2 ▲ ▲ M16 △ 27.558 C11H12O8 S [M − H]− 303.0173 303.018 −2.31 6 3’,5’-dimethoxy-4’-hydroxycinnamic acid sulfate ▲ ▲ M17 80.230 C16H12O5 [M − H]− 283.0604 283.0612 −2.83 11 Calycosin isomer 1 ▲ M18 △ 65.833 C16H12O5 [M + H]+ 285.0767 285.0758 3.16 11 Calycosin isomer 2 ▲ M19 △ 31.508 C16H12O5 [M + H]+ 285.0738 285.0758 −7.02 11 Calycosin isomer 3 ▲ ▲ M20 △ 79.592 C16H12O5 [M + H]+ 285.0777 285.0758 6.66 11 Calycosin isomer 4 ▲ M21 51.610 C16H12O6 [M + H]+ 301.0688 301.0707 −6.31 11 Hydroxycalycosin ▲ M22 54.902 C17H14O6 [M + H]+ 315.0883 315.0863 6.35 11 Methoxycalycosin 1 ▲ M23 34.517 C17H14O6 [M + H]+ 315.0836 315.0863 −8.57 11 Methoxycalycosin 2 ▲ M24 61.477 C17H14O6 [M + H]+ 315.0840 315.0863 −7.3 11 Methoxycalycosin 3 ▲ M25 31.483 C16H12O8S [M − H]- 363.0178 363.0180 −0.55 11 Calycosin sulfate 1 ▲ ▲ M26 47.512 C16H12O8S [M − H]− 363.018 363.0180 0 11 Calycosin sulfate 2 ▲ ▲ M27 51.168 C16H12O8S [M − H]− 363.0187 363.0180 1.93 11 Calycosin sulfate isomer 1 ▲ ▲ M28 △ 67.248 C16H12O8S [M − H]− 363.0174 363.0180 −1.65 11 Calycosin sulfate isomer 2 ▲ M29 △ 44.867 C16H12O8S [M − H]− 363.0189 363.0180 2.48 11 Calycosin sulfate isomer 3 ▲ ▲ M30 △ 79.697 C16H12O8S [M − H]− 363.0193 363.0180 3.58 11 Calycosin sulfate isomer 4 ▲ M31 △ 33.158 C16H12O11S2 [M − H]− 442.9767 442.9748 4.29 11 Calycosin-7,3’-O-disulfate ▲ ▲ M32 △ 36.133 C22H20O11 [M − H]− 459.0952 459.0933 4.14 13 Calycosin-3’-O-glucuronide ▲ ▲ M33 △ 23.417 C22H20O14S [M − H]− 539.0538 539.0501 6.86 13 Calycosin glucuronide sulfate 1 ▲ M34 △ 31.245 C22H20O14S [M − H]− 539.0519 539.0501 3.34 13 Calycosin glucuronide sulfate 2 ▲ ▲ M35 71.298 C16H12O9S [M − H]− 379.0137 379.0129 2.11 11 Hydroxycalycosin sulfate 1 ▲ ▲ M36 57.743 C16H12O9S [M − H]- 379.0153 379.0129 6.33 11 Hydroxycalycosin sulfate 2 ▲ M37 61.243 C16H12O9S [M − H]− 379.0151 379.0129 5.80 11 Hydroxycalycosin sulfate 3 ▲ M38 △ 26.125 C22H20O12 [M − H]− 475.0847 475.0882 −7.37 13 Hydroxycalycosin glucuronide 1 ▲ M39 △ 51.610 C22H20O12 [M − H]− 475.0918 475.0882 7.58 13 Hydroxycalycosin glucuronide 2 ▲ M40 △ 39.042 C23H22O12 [M − H]− 489.1063 489.1038 5.11 13 Methoxycalycosin glucuronide ▲ ▲ M41 △ 33.123 C23H22O15S [M − H]− 569.0634 569.0607 4.74 13 Methoxycalycosin glucuronide sulfate ▲ ▲ M42 △ 20.108 C28H30O16 [M − H]− 621.1451 621.1461 −1.61 14 Calycosin-7-O-glucoside-3’-O-glucuronide ▲ M43 48.258 C16H14O8S [M − H]− 365.0341 365.0337 1.1 10 Dihydrocalycosin sulfate 1 ▲ M44 43.285 C16H14O8S [M − H]− 365.0346 365.0337 2.47 10 Dihydrocalycosin sulfate 2 ▲ M45 45.652 C16H14O8S [M − H]− 365.0356 365.0337 5.2 10 Dihydrocalycosin sulfate 3 ▲ ▲ M46 △ 38.098 C22H22O11 [M − H]− 461.1111 461.1089 4.77 12 Dihydrocalycosin glucuronide ▲ M47 57.285 C16H14O9S [M − H]− 381.0282 381.0286 −1.05 10 Hydroxy dihydrocalycosin sulfate 1 ▲ ▲ M48 93.980 C16H14O9S [M − H]− 381.0296 381.0286 2.62 10 Hydroxy dihydrocalycosin sulfate 2 ▲ M49 49.422 C16H14O9S [M − H]− 381.0300 381.0286 3.67 10 Hydroxy dihydrocalycosin sulfate 3 ▲ ▲ M50 39.792 C18H18O8S [M − H]− 393.0665 393.065 3.82 10 Dimethyl dihydrocalycosin sulfate ▲ M51 △ 52.118 C17H16O9S [M − H]− 395.0434 395.0442 −2.03 10 Methoxy dihydrocalycosin sulfate 1 ▲ ▲ M52 △ 68.430 C17H16O9S [M − H]− 395.0457 395.0442 3.80 10 Methoxy dihydrocalycosin sulfate 2 ▲ M53 66.472 C16H14O10S [M − H]− 397.0251 397.0235 4.03 10 Dihydroxyl dihydrocalycosin sulfate ▲ M54 25.252 C16H16O8S [M − H]− 367.0477 367.0493 −4.36 9 Tetrahydrocalycosin sulfate 1 ▲ M55 19.342 C16H16O8S [M − H]− 367.0496 367.0493 0.82 9 Tetrahydrocalycosin sulfate 2 ▲ M56 21.443 C16H16O8S [M − H]− 367.0500 367.0493 1.91 9 Tetrahydrocalycosin sulfate 3 ▲ M57 50.593 C16H16O8S [M − H]− 367.0516 367.0493 6.27 9 Tetrahydrocalycosin sulfate 4 ▲ ▲ M58 67.630 C16H16O8S [M − H]− 367.0517 367.0493 6.54 9 Tetrahydrocalycosin sulfate 5 ▲ ▲ M59 57.860 C16H16O8S [M − H]− 367.0518 367.0493 6.81 9 Tetrahydrocalycosin sulfate 6 ▲ M60 △ 34.182 C22H24O11 [M − H]− 463.1257 463.1246 2.38 11 Tetrahydrocalycosin glucuronide 1 ▲ ▲ M61 △ 54.027 C22H24O11 [M − H]− 463.1269 463.1246 4.97 11 Tetrahydrocalycosin glucuronide 2 ▲ ▲ M62 △,★ 26.975 C22H24O14S [M − H]− 543.0792 543.0814 −4.05 11 Tetrahydrocalycosin glucuronide sulfate ▲ M63 △,★ 32.350 C28H32O17 [M − H]− 639.1607 639.1567 6.26 13 Tetrahydrocalycosin diglucuronide ▲ M64 48.375 C16H16O9S [M − H]− 383.0449 383.0442 1.83 9 Hydroxy tetrahydrocalycosin sulfate 1 ▲ M65 25.200 C16H16O9S [M − H]− 383.046 383.0442 4.70 9 Hydroxy tetrahydrocalycosin sulfate 2 ▲ ▲ M66 64.843 C16H16O9S [M − H]− 383.0462 383.0442 5.22 9 Hydroxy tetrahydrocalycosin sulfate 3 ▲ M67 40.192 C16H16O9S [M − H]− 383.0467 383.0442 6.53 9 Hydroxy tetrahydrocalycosin sulfate 4 ▲ M68 30.608 C16H16O9S [M − H]− 383.0444 383.0442 0.52 9 Hydroxy tetrahydrocalycosin sulfate 5 ▲ M69 △,★ 27.617 C16H16O12S2 [M − H]− 463.0031 463.0010 4.75 9 Hydroxy tetrahydrocalycosin disulfate ▲ M70 △,★ 45.733 C18H20O10S [M − H]− 427.0716 427.0704 2.81 9 Dihydroxy dihydrocalycosin sulfate ▲ M71 △ 56.593 C24H28O12 [M − H]− 507.1535 507.1508 5.32 11 Dimethyl hydroxy tetrahydrocalycosin glucuronide 1 ▲ ▲ M72 △ 54.727 C24H28O12 [M − H]− 507.1539 507.1508 6.11 11 Dimethyl hydroxy tetrahydrocalycosin glucuronide 2 ▲ ▲ M73 △ 71.300 C16H12O7S [M − H]− 347.0228 347.0231 −0.86 11 Formononetin-7-O-sulfate ▲ ▲ M74 △ 49.357 C22H20O10 [M − H]- 443.0977 443.0984 −1.58 13 Formononetin-7-O-glucuronide ▲ M75 21.960 C16H16O7S [M − H]− 351.0537 351.0544 −1.99 9 Tetrahydroformononetin sulfate 1 ▲ M76 59.352 C16H16O7S [M − H]− 351.0554 351.0544 2.85 9 Tetrahydroformononetin sulfate 2 ▲ ▲ M77 59.860 C16H16O7S [M − H]− 351.0564 351.0544 5.70 9 Tetrahydroformononetin sulfate 3 ▲ M78 △ 49.257 C22H24O10 [M − H]− 447.1322 447.1297 5.59 11 Tetrahydroformononetin glucuronide ▲ ▲ M79 △ 69.797 C17H16O8S [M − H]− 379.0509 379.0493 4.22 10 Astrapterocarpan-3-O-sulfate ▲ M80 △ 56.043 C23H24O11 [M − H]− 475.1276 475.1246 6.31 12 Astrapterocarpan-3-O-glucuronide ▲ ▲ M81 31.018 C18H18O6 [M − H]− 329.1027 329.1031 −1.22 10 Methoxyastrapterocarpan ▲ M82 △ 53.593 C23H24O12 [M − H]− 491.1225 491.1195 6.11 12 Hydroxyastrapterocarpan glucuronide 1 ▲ M83 △ 40.915 C23H24O12 [M − H]− 491.1194 491.1195 −0.20 12 Hydroxyastrapterocarpan glucuronide 2 ▲ M84 △ 43.618 C23H24O12 [M − H]− 491.1173 491.1195 −4.48 12 Hydroxyastrapterocarpan glucuronide 3 ▲ M85 △ 35.667 C17H18O5 [M +H]+ 303.1205 303.1227 −7.26 9 Astraisoflavan isomer ▲ M86 △ 32.868 C18H20O5 [M − H]− 315.1228 315.1238 −3.17 9 Methoxyastraisoflavan ▲ M87 △ 37.802 C18H20O5 [M − H]− 315.1235 315.1238 −0.95 9 Methoxyastraisoflavan isomer ▲ M88 △ 34.368 C19H22O6 [M − H]- 345.1360 345.1344 4.64 9 Hydroxy dimethoxyastraisoflavan ▲ M89 59.918 C17H18O8S [M − H]− 381.0639 381.0650 −2.89 9 Astraisoflavan-7-O-sulfate ▲ M90 62.510 C17H18O8S [M − H]− 381.0660 381.0650 2.62 9 Astraisoflavan-2’-O-sulfate ▲ ▲ M91 34.427 C17H18O8S [M − H]− 381.0662 381.0650 3.15 9 Astraisoflavan sulfate isomer ▲ M92 △ 34.993 C18H20O8S [M − H]− 395.0795 395.0806 −2.78 9 Methyoxyastraisoflavan sulfate 1 ▲ M93 △ 30.460 C18H20O8S [M − H]− 395.0819 395.0806 3.29 9 Methyoxyastraisoflavan sulfate 2 ▲ ▲ M94 20.252 C17H18O9S [M − H]− 397.0602 397.0599 0.76 9 Hydroxyastraisoflavan sulfate 1 ▲ M95 49.543 C17H18O9S [M − H]− 397.0603 397.0599 1.01 9 Hydroxyastraisoflavan sulfate 2 ▲ M96 44.008 C17H18O9S [M − H]− 397.0608 397.0599 2.27 9 Hydroxyastraisoflavan sulfate 3 ▲ ▲ M97 △ 51.435 C17H18O9S [M − H]− 397.0614 397.0599 3.78 9 Hydroxyastraisoflavan sulfate 4 ▲ M98 △ 68.372 C17H18O9S [M − H]− 397.0620 397.0599 5.29 9 Hydroxyastraisoflavan sulfate 5 ▲ M99 △ 18.817 C17H18O9S [M − H]− 397.0622 397.0599 5.79 9 Hydroxyastraisoflavan sulfate 6 ▲ M100 △ 62.393 C18H20O9S [M − H]− 411.0743 411.0755 −2.92 9 Methoxyastraisoflavan sulfate 1 ▲ M101 △ 60.210 C18H20O9S [M − H]− 411.0758 411.0755 0.73 9 Methoxyastraisoflavan sulfate 2 ▲ M102 △ 58.702 C23H26O11 [M − H]− 477.1429 477.1402 5.66 11 Astraisoflavan-7-O-glucuronide ▲ M103 △ 56.868 C23H26O11 [M − H]− 477.1430 477.1402 5.87 11 Astraisoflavan-2’-O-glucuronide ▲ ▲ M104 △,★ 38.250 C23H26O14S [M − H]− 557.0997 557.0970 4.85 11 Astraisoflavan glucuronide sulfate 1 ▲ M105 △,★ 52.510 C23H26O14S [M − H]− 557.1001 557.0970 5.56 11 Astraisoflavan glucuronide sulfate 2 ▲ M106 △ 35.633 C29H36O16 [M − H]− 639.1949 639.1931 2.82 12 Astraisoflavan-7-O-glucoside-2’-O-glucuronide ▲ ▲ M107 41.557 C15H10O7S [M − H]− 333.0074 333.0074 0 11 Daidzein-4’-O-sulfate ▲ ▲ M108 28.925 C15H10O7S [M − H]− 333.0080 333.0074 1.80 11 Daidzein-7-O-sulfate ▲ M109 △ 28.400 C15H10O10S2 [M − H]− 412.9658 412.9643 3.63 11 Daidzein-7,4’-O-disulfate ▲ ▲ M110 △ 23.817 C21H18O10 [M − H]− 429.0835 429.0827 1.86 13 Daidzein glucuronide ▲ M111 △ 20.167 C21H18O13S [M − H]− 509.0403 509.0395 1.57 13 Daidzein glucuronide sulfate ▲ M112 △ 44.292 C15H12O7S [M − H]− 335.0212 335.0231 −5.67 10 Dihydrodaidzein sulfate ▲ M113 53.852 C15H14O7S [M − H]− 337.0389 337.0387 0.59 9 Tetrahydrodaidzein sulfate 1 ▲ M114 39.333 C15H14O7S [M − H]− 337.0400 337.0387 3.86 9 Tetrahydrodaidzein sulfate 2 ▲ M115 57.002 C15H14O7S [M − H]− 337.0402 337.0387 4.45 9 Tetrahydrodaidzein sulfate 3 ▲ ▲ M116 60.560 C15H14O7S [M − H]− 337.0409 337.0387 6.53 9 Tetrahydrodaidzein sulfate 4 ▲ M117 △ 34.458 C15H14O10S2 [M − H]− 416.9988 416.9956 7.67 9 Tetrahydrodaidzein disulfate ▲ M118 40.017 C15H10O5 [M + H]+ 271.0617 271.0601 5.90 11 Gensitein ▲ M119 41.833 C15H10O8S [M − H]− 349.0033 349.0024 2.58 11 Genistein sulfate 1 ▲ M120 57.918 C15H10O8S [M − H]− 349.0039 349.0024 4.30 11 Genistein sulfate 2 ▲ M121 37.233 C15H10O8S [M − H]− 349.0030 349.0024 1.72 11 Genistein sulfate 3 ▲ M122 △ 25.042 C21H18O14S [M − H]− 525.0367 525.0370 −0.57 9 Genistein glucuronide sulfate ▲ M123 33.658 C15H12O8S [M − H]− 351.0180 351.0180 0 10 Dihydrogenistein sulfate 1 ▲ M124 △ 74.098 C15H12O8S [M − H]− 351.0184 351.0180 1.14 10 Dihydrogenistein sulfate 2 ▲ M125 △ 51.688 C15H12O8S [M − H]− 351.0189 351.018 2.56 10 Dihydrogenistein sulfate 3 ▲ M126 44.758 C15H14O5 [M − H]− 273.0757 273.0768 −4.03 9 Tetrahydrogenistein ▲ M127 63.985 C15H14O8S [M − H]− 353.0359 353.0337 6.23 9 Tetrahydrogenistein sulfate 1 ▲ ▲ M128 65.410 C15H14O8S [M − H]− 353.0360 353.0337 6.51 9 Tetrahydrogenistein sulfate 2 ▲ M129 △,★ 49.090 C20H22O12S [M − H]− 485.0787 485.0759 5.77 10 Tetrahydrogenistein pentose sulfate ▲ M130 △,★ 39.850 C21H24O13S [M − H]− 515.0911 515.0865 8.93 10 Tetrahydrogenistin sulfate ▲ M131 62.235 C15H14O6S [M − H]− 321.0439 321.0438 0.31 9 Equol sulfate isomer ▲ M132 56.152 C15H14O6S [M − H]− 321.0444 321.0438 1.87 9 Equol sulfate 1 ▲ ▲ M133 58.943 C15H14O6S [M − H]− 321.0454 321.0438 4.98 9 Equol sulfate 2 ▲ M134 △ 46.017 C21H22O9 [M − H]− 417.1185 417.1191 −1.44 11 Equol glucuronide 1 ▲ ▲ M135 △ 47.975 C21H22O9 [M − H]− 417.1209 417.1191 4.32 11 Equol glucuronide 2 ▲ ▲ M136 △,★ 32.293 C21H22O12S [M − H]− 497.0786 497.0759 5.43 11 Equol glucuronide sulfate 1 ▲ M137 △,★ 28.058 C21H22O12S [M − H]− 497.0797 497.0759 7.64 11 Equol glucuronide sulfate 2 ▲ M138 △ 37.317 C21H22O12S [M − H]− 497.0757 497.0759 -0.4 11 Equol glucuronide sulfate 3 ▲ M139 △ 57.977 C15H16O6S [M − H]− 323.0616 323.0595 6.5 8 Dihydroequol sulfate ▲ M140 28.817 C16H14O6 [M + H]+ 303.0878 303.0863 4.95 10 Dihydropratensein ▲ M141 △ 24.925 C22H20O15S [M − H]− 555.0487 555.0450 6.67 13 Pratensein glucuronide sulfate ▲ M142 △,★ 26.300 C27H30O17 [M − H]− 625.1462 625.1410 8.32 13 Tetrahydro trihydroxyisoflavone diglucuronide 1 ▲ M143 △,★ 26.867 C27H30O17 [M − H]− 625.1467 625.1410 9.12 13 Tetrahydro trihydroxyisoflavone diglucuronide 2 ▲ M144 △ 41.650 C15H14O9S [M − H]− 369.0303 369.0286 4.61 9 Tetrahydro-tetrahydroxyisoflavone sulfate ▲ M145 △ 41.930 C22H20O15S [M − H]− 555.0478 555.0450 6.76 13 Pratensein glucuronide sulfate M146 △ 61.012 C22H20O11 [M − H]− 459.0925 459.0933 −1.23 13 Calycosin-7-O-glucuronide M147 △ 69.988 C17H18O8S [M − H]− 381.0652 381.0650 0.52 9 Astraisoflavan sulfate isomer Sum 106 64 17 Note: tR: Retention time; Meas.: measured; Pred.: predicted; Diff: difference; DBE: double bone equivalents. △ New metabolites found in vivo after administration of ARTF; ★ Potential New compound by retrieving information from SciFinder database. ▲ Detected. ==== Refs References 1. 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