H OH metabolites OH

Article Compound Characterization and Metabolic Profile Elucidation after In Vitro Gastrointestinal and Hepatic Biotransformation of an Herniaria hirsuta Extract Using Unbiased Dynamic Metabolomic Data Analysis

Laura Peeters 1,*, Anastasia Van der Auwera 1, Charlie Beirnaert 2 , Sebastiaan Bijttebier 1, Kris Laukens 2 , Luc Pieters 1 , Nina Hermans 1 and Kenn Foubert 1 1 Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium; [email protected] (A.V.d.A.); [email protected] (S.B.); [email protected] (L.P.); [email protected] (N.H.); [email protected] (K.F.) 2 Adrem Data Lab, Department of Mathematics—Computer Sciences, University of Antwerp, Middelheimlaan 1, 2020 Antwerp, Belgium; [email protected] (C.B.); [email protected] (K.L.) * Correspondence: [email protected]; Tel.: +32-3265-9096



Received: 5 February 2020; Accepted: 12 March 2020; Published: 16 March 2020


Abstract: Herniaria hirsuta L. (Caryophyllaceae) is used for treatment of urinary stones and as a diuretic. Little is known about the active compounds and the mechanism of action. The phytochemical composition of H. hirsuta was comprehensively characterized using UHPLC-UV-HRMS (Ultrahigh-Performance Liquid Chromatography-Ultraviolet-High Resolution Mass Spectrometry) data. An in vitro gastrointestinal model was used to simulate biotransformation, which allowed the monitoring of the relative abundances of individual compounds over time. To analyze the longitudinal multiclass LC–MS data, XCMS, a platform that enables online metabolomics data processing and interpretation, and EDGE, a statistical method for time series data, were used to extract significant differential profiles from the raw data. An interactive Shiny app in R was used to rate the quality of the resulting features. These ratings were used to train a random forest model. The most abundant aglycone after gastrointestinal biotransformation was subjected to hepatic biotransformation using human S9 fractions. A diversity of compounds was detected, mainly saponins and flavonoids. Besides the known saponins, 15 new saponins were tentatively identified as glycosides of medicagenic acid, acetylated medicagenic acid and zanhic acid. It is suggested that metabolites of phytochemicals present in H. hirsuta, most likely saponins, are responsible for the pharmaceutical effects. It was observed that the relative abundance of saponin aglycones increased, indicating loss of sugar moieties during colonic biotransformation, with medicagenic acid as the most abundant aglycone. Hepatic biotransformation of this aglycone resulted in different metabolites formed by phase I and II reactions.

Keywords: Herniaria hirsuta; Caryophyllaceae; saponins; urolithiasis; machine learning; dynamic metabolomics

1. Introduction Urinary stone disease is considered as an important healthcare problem that affects 10–15% of the population in the developed world, but the incidence can be as high as 20–25% in the Middle East, with a peak at ages 20 to 40 years. In addition, the disease is characterized by its high recurrence rate, about 50% in 10 years and 75% in 20 years [1–6].

Metabolites 2020, 10, 111; doi:10.3390/metabo10030111 www.mdpi.com/journal/metabolites Metabolites 2020, 10, 111 2 of 26

Over the years, many remedies and surgical treatments have been described, varying from dietary recommendations to interventional procedures. Endoscopic management, both ureteroscopic and percutaneous, offers an efficient and efficacious way to treat stones [6]. Nowadays, extracorporeal shock wave lithotripsy (ESWL) is widely used for stone fragmentation without invasive instrumentation of the body. However, the aforementioned therapies show some significant side effects such as residual stone fragments that can act as a nidus for new stone formation [5,7]. Existing oral treatments with evidence that supports their long-term efficacy to prevent kidney stones only include a handful of drugs. Thiazide diuretics, potassium citrate and allopurinol have been used as therapy for more than 30 years. However, their adverse effects prevent long term consumption [2,8]. Phytotherapy might be useful as an alternative treatment. Some herbal remedies have been used for centuries and have been shown to be effective against urolithiasis, although the mechanism of action is often not well established through systematic pharmacological and clinical studies. Many plants have been and are still used to treat urinary stones, such as Phyllanthus niruri, Zea mays, Agropyron repens and Herniaria hirsuta [5,9]. An aqueous extract of the aerial parts of H. hirsuta (hairy rupturewort) is an herbal medicine widely used against urolithiasis and which contains diuretic properties. The Arabic name, herras lehjer, is translated as stonebreaker and refers to its use as a traditional herbal remedy for urinary stones. The beneficial effects of the extract have been demonstrated in several studies [9–14]. Even though a lot of research has been done to prove the activity of H. hirsuta against urolithiasis, little is known about the active compounds and the exact mechanism of action. Previous phytochemical research on Herniaria species revealed the presence of saponins, flavonoids and coumarins [15–18]. Four herniariasaponins have been reported in H. hirsuta: herniariasaponins E, F and G, three monodesmosidic derivatives of medicagenic acid and herniariasaponin H, a bidesmosidic derivative of medicagenic acid [17,19]. Literature suggests that the antilithiatic potential of H. hirsuta is attributed to saponins or metabolites thereof [17,20,21]. It is well known that many natural products are so-called prodrugs, which become active after biotransformation [22]. Nevertheless, this aspect is usually overlooked when searching for new therapeutic agents using classical approaches. After oral administration, an herbal extract is inevitably brought into contact with gastrointestinal enzymes and intestinal microflora, which might lead to biotransformation of the compounds. Saponins are high-molecular-weight glycosides, containing a triterpenoid or steroidal sapogenin aglycone covalently linked to one or two sugar chains via an ether or ester glycosidic bond, referred to as monodesmosidic or bidesmosidic saponins, respectively. Before absorption in the gastrointestinal tract, saponins are presumably hydrolyzed with loss of sugar chains [23]. The aglycones or metabolites thereof may be absorbed and may further be biotransformed in the liver to the ultimate active components [24]. In this study, the biotransformation of a well-characterized H. hirsuta extract was investigated in an in vitro gastrointestinal model followed by hepatic biotransformation. The longitudinal multiclass data were subjected to different data analysis workflows to screen and tentatively identify metabolites formed after in vitro biotransformation.

2. Results and Discussion

2.1. Identification of Compounds A dereplication workflow performed on the extract of H. hirsuta resulted in a molecular network containing different clusters, composed of different nodes connected by edges which define the degree of similarity between the MS/MS spectra. Compounds that are structurally related are linked to each other. Mass differences between the nodes were studied and provided information about hitherto unidentified compounds. Major clusters were analyzed to tentatively identify unknown compounds. As an example, the cluster containing herniariasaponin H is shown in Figure1. Herniariasaponin H (Figure2) is the most abundant saponin previously observed in H. hirsuta and served as starting point for identification of other compounds in this cluster. Metabolites 2020, 10, x FOR PEER REVIEW 3 of 24 hithertoMetabolites unidentified 2020, 10, x FOR PEERcompounds. REVIEW Major clusters were analyzed to tentatively identify unknown3 of 24 compounds. As an example, the cluster containing herniariasaponin H is shown in Figure 1. hitherto unidentified compounds. Major clusters were analyzed to tentatively identify unknown Herniariasaponin H (Figure 2) is the most abundant saponin previously observed in H. hirsuta and compounds. As an example, the cluster containing herniariasaponin H is shown in Figure 1. served as starting point for identification of other compounds in this cluster. Herniariasaponin H (Figure 2) is the most abundant saponin previously observed in H. hirsuta and MetabolitesStructures2020, 10 , 111were assigned to peaks only when both the mass/charge (m/z) ratios and3 ofthe 26 served as starting point for identification of other compounds in this cluster. molecular formulae of the precursor ion and product ions were in agreement. Additional information Structures were assigned to peaks only when both the mass/charge (m/z) ratios and the about the proposed structures was often provided by photodiode array (PDA) spectra and retention molecularStructures formulae were of assigned the precursor to peaks ion onlyand product when both ions the were mass in agreement./charge (m/ zAdditional) ratios and information the molecular times. However, the information was not always sufficient for full elucidation of the structural formulaeabout the of proposed the precursor structures ion was and often product provided ions wereby photodiode in agreement. array (PDA) Additional spectra information and retention about composition at an acceptable confidence level. Additional information was obtained from published thetimes. proposed However, structures the information was often was provided not always by photodiode sufficient arrayfor full (PDA) elucidation spectra of and the retention structural times. data. However,composition the at information an acceptable was confidence not always level. suffi Acientdditional for full information elucidation was of obtained the structural from published composition atdata. an acceptable confidence level. Additional information was obtained from published data.

FigureFigure 1.1. MolecularMolecular network inin negativenegative ion ion mode mode with with the the cluster cluster containing containing herniariasaponin herniariasaponin H H shownshown in in detail. detail.

Figure 2. Chemical structure of herniariasaponin H. Figure 2. ChemicalChemical structure of herniariasaponin H. All amMS and PDA data used for peak identification are listed in Table 1. The Table also specifiesAll amMS the literature and PDA consulted data used to obtain for peak additi identification identificationonal information are inlisted order in toTable confirm1 1.. The compound tableTable alsoalso specifiesspecifiesidentity. the In accordance literature withconsulted previous to obtainresearch, additionaladditi mainlyonal flavonoids information and insaponins order were to confirmconfirm identified compound [15– identity.21]. However, InIn accordanceaccordance many of with withthe identified previous previous research,compou research,nds mainly ma haveinly flavonoidsnot flavonoids been reported and and saponins saponins previously were were in identified H. identified hirsuta. [15 –[15–21]. 21].However, However, many many of the of identifiedthe identified compounds compou havends have not beennot been reported reported previously previously in H. in hirsuta H. hirsuta.. Flavonoids were mainly observed as glycoconjugates, i.e., a flavonoid aglycone attached to one or more sugar moieties. Several flavonoid compounds were identified using analytical standards. Others were tentatively identified with ddMS2. Collision-induced fragmentation resulted in product ions caused by elimination of sugar moieties, indicating glycosidic O-linkages [24]. Among others, Metabolites 2020, 10, 111 4 of 26 rutin, narcissin and quercetin-3-O-(2”-O-α-L-rhamnopyranosyl)-β-D-glucuronopyranoside were identified, in agreement with previous studies [17,20]. However, isorhamnetin-3-robinobioside and isorhamnetin-3-[3-feruloylrhamnosyl-(1 6)-galactoside], previously reported to be present in H. → fontanesii, were not detected in H. hirsuta, which is probably due to variances in phytochemical profile between different plant species [18]. A rich diversity of saponins was detected, differing in the presence of various aglycones, a different composition of sugar chains and varied linkages of sugar moieties. Fragmentation led to the distinct presentation of product ions caused by elimination of sugar moieties, indicating glycosidic O-linkages. The product ions in the MS2 spectra provide additional structural information about the sugar residues and the aglycones of the fragmented saponins. Losses of m/z 132, 146, 162 and 176 were observed, corresponding to pentose, deoxyhexose, hexose and uronic acid moieties, respectively. Three different aglycone moieties were observed with fragment ions at m/z 543.33234, 541.31787 and 501.32074, corresponding to acetylated medicagenic acid, zanhic acid and medicagenic acid, respectively [25,26]. Several other compounds, such as free phenolic and hydroxycinnamic acids, were detected. Amongst others, caffeic, p-coumaric and chlorogenic acids were identified using analytical standards. The coumarins herniarin and umbelliferone, previously reported to be present in Herniaria species, were not detected. This variation in compounds is probably due to differences in origin or regional climate [17,20]. More than one chromatographic peak was often present for the same precursor ion, indicating the presence of structural isomers with different linkage positions and/or different sugar moieties. Figure3 illustrates the identification of a hitherto unknown saponin. As shown in Figure3A, the unknown saponin has a retention time of 15.33 min. Figure3B shows a molecular ion at m/z 1703.71838 [M-H]−. Since saponins contain the elements C, H and O, 19 molecular formulas are possible within an error range of 5 ppm. The sugar residues and the aglycone were identified from the MS2 spectra as shown in Figure3C and Table2. The product ion of m/z 1381.62830 [M-H-176-146]− results from the loss of uronic acid (176 u) and deoxyhexose (146 u). The ion at m/z 879.30701 originates from a sugar chain containing two deoxyhexosyl (146 u), two pentosyl (132 u) and two hexosyl (162 u) units. The ion at m/z 823.41089 [aglycone-H+146+176]− represents the aglycone attached to a deoxyhexose unit (146 u) and a uronic acid moiety (176 u). The ion at m/z 501.32275 is characteristic for medicagenic acid as aglycone, resulting from the loss of sugar residues. The ion at m/z 439.31934 [aglycone-H-18-44]− results from an additional loss of water (18 u) and CO2 (44 u). This suggests a bidesmosidic saponin with R1 comprising uronic acid and deoxyhexose and R2 comprising two pentosyl, two deoxyhexosyl and two hexosyl moieties, with a hexosyl moiety in a terminal position. Combining all the information with the retention time and literature data, the saponin with m/z 1703.71734 supports a molecular formula of C76H120O42 and was tentatively identified as medicagenic acid attached to three deoxyhexosyl, two hexosyl, two pentosyl and a uronic acid moiety [15–21]. However, MSn does not provide enough information for absolute structural characterization. Nevertheless, the use of a hybrid orbitrap mass analyzer enabled the tentative identification of 15 saponins that have not been reported before in H. hirsuta (indicated with e in Table2). Full structural characterization is not the main goal in this project, as the complete herbal extract is subjected to biotransformation studies. Metabolites 2020, 10, 111 5 of 26 Metabolites 2020, 10, x FOR PEER REVIEW 5 of 24

RT: 0.00 - 23.00 12.22 NL: 3.59E8 100 12.08 Base Peak F: FTMS 1.52 - p ESI Full ms 90 13.03 [120.00-1800.00] MS polyphenols2016042 80 7a070

70 14.67 60 1.46 50 9.00 40 11.89

Relative Abundance 9.19 18.03 15.66 30 16.06 20.83 20.60 2.14 10.51 14.02 20 7.31 1.13 8.26 8.88 10 7.97 10.02 18.30 18.79 20.96 21.81 2.52 17.06 0.07 2.69 3.96 5.36 6.22 6.70 19.62 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 polyphenols20160427a070 #7906-7921 RT: 15.33-15.36 AV: 2 SB: 3 15.20-15.26 NL: 4.33E6 Time (min) T: FTMS - p ESI Full ms [120.00-1800.00] 1703.7173 100 H:\1. GOA\...\polyphenols20160427a070 04/28/16 15:20:50

90 RT: 13.45 - 16.89 NL: 6.02E6 100 80 Base Peak F: FTMS - p ESI d Full ms2 90 70 [email protected] [117.00-1755.00] MS 80 60 polyphenols20160427a07 0 70 50 851.3564 60 1726.7032 40 50

Relative Abundance 30 40 671.1265 1145.5395 1263.5654 Relative Abundance 20 30 461.1093 825.4291 1333.6071 341.1968 595.2689 689.3034 1087.5339 1201.5660 20 10 1644.6645 877.8121 1409.6226 727.3116 225.1128 303.0909 385.1405 495.2605 1013.4969 1557.6698 10 1756.6307 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 0 polyphenols20160427a070 #7903 RT: 15.32 AV: 1 NL: 1.69E6 m/z 13.6F: FTMS13.8 - p ESI d Full14.0 ms2 [email protected] 14.4 [117.00-1755.00]14.6 14.8 15.0 15.2 15.4 15.6 15.8 16.0 16.2 16.4 16.6 16.8 Time (min) 1703.7208 100 polyphenols20160427a070 #7903 RT: 15.32 AV: 1 NL: 1.69E6 F: FTMS - p ESI d Full ms290 [email protected] [117.00-1755.00] 1703.7208 100 80 90 70 80

70 60

60 50

50 40

Relative Abundance 40 30 Relative Abundance 30

[M-H-DeoxyHex-HexA]- 20 20 1381.6283 1381.6283 175.0242 175.0242 [Medicagenic acid-H]- [Medicagenic acid-H+DeoxyHex+HexA]- [2DeoxyHex+2Pent+2Hex-H]- 10 10 [M-H-DeoxyHex-Hex-HexA]- 221.0658 339.0930221.0658 339.0930439.3193 501.3228439.3193 501.3228 823.4109 879.3070909.3175 1130.44291130.44291219.2285 1219.2285 0 0 200 300200 400300 400500 500 600 600 700700 800800 900900 10001000 1100 11001200 12001300 14001300 15001400 1600 15001700 1600 1700 m/zm/z

Figure 3. LC–MS data for an unknown saponin. (A) Chromatogram obtained for the extract of Herniaria Figure 3. LC–MS data for an unknown saponin. (A) Chromatogram obtained for the extract of hirsuta.(B) MS scan of peak at 15.33 min in the heated electrospray ionization (HESI) negative mode. Herniaria hirsuta. (B) MS scan of peak at 15.33 min in the heated electrospray ionization (HESI) C 2 m z ( ) MSnegativefragmentation mode. (C) MS spectrum2 fragmentation of the ion spectrum at / 1703.71734. of the ion at m/z 1703.71734. 2.2. Gastrointestinal Biotransformation 2.2. Gastrointestinal Biotransformation Herbal extracts comprise a mixture of compounds, covering a wide range of bioactive constituents, Herbal extracts comprise a mixture of compounds, covering a wide range of bioactive aside from active compounds or prodrugs. As the main compounds are classified as flavonoids and constituents, aside from active compounds or prodrugs. As the main compounds are classified as saponins, extensive biotransformation after oral intake can be expected. Therefore, gastrointestinal flavonoids and saponins, extensive biotransformation after oral intake can be expected. Therefore, biotransformationgastrointestinal ofbiotransformation the lyophilized extract of the waslyophilized simulated extractin vitro wasto simulated monitor the in levelsvitro to of themonitor identified the compoundslevels of usingthe identified amMS and compounds UV data. using However, amMS gastrointestinal and UV data. enzymesHowever, and gastrointestinal fecal microflora enzymes present in theand samples fecal microflora cause a lot present of matrix in interference, the samples increasing cause a lot the of complexity matrix interference, of the data. increasing The multiclass the samplescomplexity are measured of the data. as a functionThe multiclass of time, samples adding are a longitudinal measured as aspect a function to the of complex time, adding data and a impedinglongitudinal the interpretation aspect to the of the complex data. The data concentration and impeding of precursor the interpretati compoundson of and the metabolites data. The canconcentration increase, decrease of precursor or show any compounds combination and of metabolites these patterns can during increase, biotransformation. decrease or show any combination of these patterns during biotransformation.

Metabolites 2020, 10, 111 6 of 26

Table 1. Chromatographic and Spectrometric Data of the Tentatively Identified Compounds in H. hirsuta Detected with a Generic LC-PDA-amMS Method for Moderately Polar Phytochemicals.

Com-Pound HESI Max Abs. Compound a Molecular Formula RT (min) HESI Full MS b Neutral Losses Literature Number ddMS2 (nm) neg 191.05611 - 1 quinic acid c C H O 1.54 7 12 6 pos - - neg 169.01412 - 2 gallic acid c C H O 4.06 7 6 5 pos - - neg 153.01933 109.02950 109.02950: C H O 3 protocatechuic acid [3,4- dihydroxybenzoic acid] c C H O 6.46 6 6 2 259; 294 7 6 4 pos - - 179.03439; 179.03439: C H O ; 135.04439: neg 341.08817 9 8 4 4 caffeyl hexose C15H18O9 7.68 135.04439 C9H8O4 - CO2 181.04962; 181.04962: C9H8O4; 163.03906: + 365.08380 [M+Na] ; 163.03906; C9H8O4 -H2O; 145.02861: pos + 360.12839 [M+NH4] 145.02861; C9H6O4 - 2H2O; 135.04443: 135.04443 C8H6O2 431.11975; 499.10757 329.09064; 329.09064: C14H18O9; 161.04463: neg [M-H+NaFA]−; 863.24756 5 asperulosidic acid C18H24O12 8.01 161.04463 C6H10O5 [2M-H]− 271.08188; 271.08188: C18H24O12 - 187.06091; C H O ; 187.06091: C H O ; pos 433.13428 6 10 5 8 10 5 145.04990; 145.04990: C6H8O4; 127.03919: 127.03919 C6H6O3 neg 181.05041 - 6 dihydrocaffeic acid c C H O 8.07 9 10 4 pos - - 367.10349; 435.09108 d neg - 7 1-O-feruloylquinic acid C17H20O9 8.76 [M-H+NaFA]− pos 369.11795 - 353.08685; 707.18355 chlorogenic acid [3.4-dihydroxycinnamoylquinic acid; neg 191.05545 191.05611: C H O 8 C H O 8.94 [2M-H] 7 12 6 326 5-Caffeoylquinic acid] c 16 18 9 − 355.10160; 377.08360 + pos [M+Na] ; 731.17920 163.03897 163.03897: C9H6O3 [2M+Na]+ 623.16479; 623.16479: C H O - 476.09396; 34 42 21 785.21631; 853.20349 C H O ; 315.05124: C H O ; neg 315.05124; 6 10 5 16 12 7 9 isorhamnetin-3-O-rutinoside-7-O-glucoside d C H O 9.27 [M-H+NaFA] 300.02750: C H O ; 271.02466: 34 42 21 − 300.02750; 15 9 7 C H O 271.02466 14 7 6 641.17114: C H O - 641.17114; 34 42 21 pos 787.22931 C H O ; 479.11838: C H O 479.11838 6 10 4 34 42 21 -C6H10O4 -C6H10O5 Metabolites 2020, 10, 111 7 of 26

Table 1. Cont.

Com-Pound HESI Max Abs. Compound a Molecular Formula RT (min) HESI Full MS b Neutral Losses Literature Number ddMS2 (nm) neg 353.08781 191.05611 191.05611: C H O 10 chlorogenic acid c C H O 9.36 7 12 6 16 18 9 pos 355.10236 163.03897 neg 289.07176 - 11 Catechin c C H O 11.21 279 Mbark et al. 15 14 6 pos - - neg 289.07176 - 12 epicatechin c C H O 11.25 280 15 14 6 pos - - neg 577.13515 - 13 procyanidin B2 c C H O 9.68 30 26 12 pos 579.14970 - neg 179.03498 135.04515 135.04515: C H O - CO 14 caffeic acid c C H O 9.7 9 8 4 2 9 8 4 163.03915; pos 181.04954 145.02861; 163.03897: C9H8O4 -H2O 135.04443 193.05003; 367.10358; 435.09106 193.05003: C H O ; 173.04485: neg 173.04489; 10 10 4 15 1-O-feruloylquinic acid d C H O 10.51 [M-H+NaFA] C H O 17 20 9 − 134.03622 7 10 5 145.02855; 149.05952; 145.02855: C9H4O2; 149.05952: + pos 391.10001 [M+Na] 117.03381; C9H8O2; 117.03381: C8H4O; 177.05475; 177.05475: C10H10O4 -H2O 194.05750 755.20471; 823.19204 300.02771; 300.02771: C15H9O7; 271.02499: quercetin neg [M-H+NaFA]−; 1511.41418 271.02499; 16 C33H40O20 11.05 C14H8O6; 255.02994: C14H8O5 3-rhamnosyl-(1->2)-rhamnosyl-(1->6)-glucoside or [2M-H]− 255.02994 quercetin 3-rhamninoside 465.1033: C33H40O20 - 465.1033; 2C H O ; 303.05008: pos 757.21765; 611.16085 6 10 4 303.05008 C33H40O20 - 2C6H10O4 - C6H10O5 339.12378; 329.13812; 339.12378: C20H20O5; 329.13812: 327.12405; C19H22O5; 327.12405: C19H20O5; neg 565.19336 [M-H+FA]− 324.09967; 324.09967: C19H17O5; 312.09885: 17 matairesinoside C26H32O11 11.3 355 312.09885; C18H17O5; 309.07953: C18H14O5; 309.07953; 297.07669: C17H14O5 297.07669 503.19199; 341.13672; 503.19199: C26H30O10; 341.13672: + 543.18420 [M+Na] ; 323.12738; C20H20O5; 323.12738: C20H18O4; pos + 538.22786 [M+NH4] 311.12756; 311.12756: C19H18O4; 175.07526: 175.07526; C11H10O2; 137.05983: C8H8O2 137.05983 Metabolites 2020, 10, 111 8 of 26

Table 1. Cont.

Com-Pound HESI Max Abs. Compound a Molecular Formula RT (min) HESI Full MS b Neutral Losses Literature Number ddMS2 (nm)

357.06104; 632.32239: C34H42O20 - 314.04349; C H O ; 357.06104: C H O ; 769.22040 [M-H]; 1539.44527 6 10 4 18 14 8 isorhamnetin-O-glycoside (one hexosyl and two neg 271.02512; 314.04349: C16H11O7; 299.01981: 18 C34H42O20 11.74 [2M-H]−; 632.32239 355 deoxyhexosyl units) 299.01981; C15H8O7; 285.04065: C15H10O6; 285.04065 271.02512: C14H8O6 625.17743: C34H42O20 - 625.17743; C H O ; 479.11874: C H O 771.23315; 625.17635; 6 10 4 34 42 20 pos 479.11874; - 2C H O ; 317.06540: 479.11849 6 10 4 317.06540 C34H42O20 - 2C6H10O4 - C6H10O5 623.12537; 1247.25657 301.03543: C H O -C H O quercetin 3-O-β-(2”-O-α-L- neg 301.03543 27 28 17 6 10 4 van Dooren 19 C H O 11.96 [2M-H] -C H O 255; 355 rhamnopyranosyl)-glucopyranoside 27 28 17 − 6 8 6 et al. (2016) 479.08180: C H O - 479.08240; 27 28 17 pos 625.13943; 479.08180 C H O ; 303.04999: C H O 303.04999 6 10 4 27 28 17 -C6H10O4 -C6H8O6 300.02762; 300.02755: C H O ; 271.02481: 609.14667; 677.13349 271.02481; 15 9 7 neg C H O ; 255.02990: C H O ; van Dooren 20 rutin c C H O 12.08 [M-H+NaFA] 255.02990; 14 8 6 14 8 5 256; 355 27 30 16 − 243.02990: C H O et al. (2016) 243.02990 13 8 5 465.10267; 465.10275: C H O ; 303.04993: pos 611.15973 21 22 12 303.04999 C15H12O7 neg 447.09364 - Wagner et 21 cynaroside c C H O 12.2 21 20 11 pos - - al. (2013) neg 609.18153 - 22 hesperidin c C H O 12.24 28 34 15 pos 611.19705 -

c neg 193.05063 178.02643 178.02643: C9H7O4 23 ferulic acid C10H10O4 12.34 177.0547; 177.0547: C10H10O4 -H2O; 145.02855; 149.05984: C H O ; 145.02855: pos 195.06519 9 12 2 135.04424; C9H4O2; 135.04424: C8H6O2; 117.03378 117.03378: C8H4O neg 1215.2698 - 24 herniariasaponin 1 C H O 12.59 54 56 32 pos 1239.26343 [M+Na]+ - 300.02725; 300.02725: C H O ; 271.02539: 271.02539; 15 9 7 C H O ; 255.02905: C H O ; neg 463.0882 255.02905; 14 8 6 14 8 5 25 isoquercetin c C H O 12.61 243.02859: C H O ; 151.00230: 21 20 12 243.02859; 13 8 5 C H O 151.00230 7 4 4 303.05008; 303.05008: C H O ; 287.05533: pos 465.10275 287.05533; 15 10 7 C H O ; 257.04483: C H O 257.04483 15 10 6 14 8 5 Metabolites 2020, 10, 111 9 of 26

Table 1. Cont.

Com-Pound HESI Max Abs. Compound a Molecular Formula RT (min) HESI Full MS b Neutral Losses Literature Number ddMS2 (nm)

c neg 163.04007 119.05024 119.05024: C8H8O 26 p-coumaric acid C9H8O3 12.62 pos 165.05462 147.04414 147.04414: C9H6O2 637.14125; 1275.28809 463.09000; 463.09000: C H O ; 300.02728: 2-(3.4-Dihydroxyphenyl)-5.7-dihydroxy-4- neg 21 20 12 27 oxo-4H-chromen-3-yl- 6-O-(2-O-acetyl-α- C28H30O17 12.67 [2M-H]− 300.02728 C15H9O7 493.09749: C H O – L-arabinopyranosyl)-β-D-glucopyranoside 465.10071; 28 30 17 pos 639.15543; 493.09749 C H O ; 465.10071: C H O ; 303.04972 6 10 4 21 20 12 303.04972: C15H10O7 301.03538; 301.03538: C H O ; 271.02481: 271.02481 15 10 7 C H O ; 255.02990: C H O ; neg 477.06746 255.02990; 14 8 6 14 8 5 28 miquelianin [quercetin-3-O-glucuronide]c C H O 12.89 178.99860: C H O ; 151.00368: 21 18 13 178.99860; 8 4 5 C H O 151.00368 7 4 4 303.04993; 303.04993: C H O ; 257.04445: pos 479.08202 15 10 7 257.04445 C14H8O5 477.06749: C H O - 623.16211; 477.06749; 28 32 16 neg 315.05072 C6H10O4; 315.05072: C28H32O16 van Dooren 29 narcissin [isorhametin-3-O-rutinoside] C28H32O16 12.89 1247.33026 [2M-H]− 254; 355 -C6H10O4 -C6H10O5 et al. (2016) 479.11804: C H O - Wagner et 479.11804; 28 32 16 pos 625.14606 C H O ; 317.06586: C H O al. (2013) 317.06586 6 10 4 28 32 16 -C6H10O4 -C6H10O5 neg 433.0782 301.03586 30 avicularin [quercetin-3-O-arabinoside] c C H O 13.16 256; 355 20 18 11 pos 435.09219 - neg 431.09837 268.03827 268.03827: C H O 31 apigetrin c C H O 13.27 15 9 5 21 24 9 pos 433.11292 - 284.03314; 284.03314: C H O ; 255.03024: neg 447.09381 255.03024; 15 9 6 32 astragalin [kaempferol 3-O-glucoside] c C H O 13.34 C H O ; 227.03455: C H O 265; 348 21 20 11 227.03455 14 8 5 13 8 4 pos 449.10814 - neg 447.09328 - 33 quercitrin c C H O 13.35 265; 346 21 20 11 pos 449.10784 - Metabolites 2020, 10, 111 10 of 26

Table 1. Cont.

Com-Pound HESI Max Abs. Compound a Molecular Formula RT (min) HESI Full MS b Neutral Losses Literature Number ddMS2 (nm) neg 137.02442 93.03459 93.03459: C H O 34 salicylic acid c C H O 14.07 6 6 236; 302 7 6 3 pos - - 647.29342: C H O + C H O ; 501.32303; 30 46 6 6 10 4 neg 1249.58482; 647.29342 501.32303: C30H46O6; 439.32358: Mbark et al. 35 herniariasaponin F C59H94O28 14.17 439.32358 C30H46O6 -H2O - CO2 (2000) 1273.58240 [M+Na]+; 457.33133: C H O ; 771.25336: 1268.62600 [M+NH ]+; 29 44 4 pos 4 771.25336 2C H O + 2C H O + 1122.56885; 645.27747; 6 10 5 6 10 4 C H O + Na 457.33133 5 8 4 1249.59241; 821.43506: C H O - 1231.57312; 61 96 29 2C H O -C H O ; 543.33441: 821.43506; 6 10 5 6 10 4 C H O ; 501.32294: C H O ; acetylated medicagenic acid + two hexosyl + two neg 1291.59644 543.33441; 32 48 7 30 46 6 36 C H O 14.53 483.31107: C H O -H O; deoxyhexosyl + one pentosyl moieties e 61 96 29 501.32294; 30 46 6 2 439.32251: C H O -H O- 483.31107; 30 46 6 2 CO 439.32251 2 + pos 1310.63818 [M+NH4] -

705.38928; 705.38928: C32H48O7 + 543.33337; C H O ; 543.33337: C H O ; acetylated medicagenic acid + three hexosyl + one neg 1175.549 6 10 5 32 48 7 Schröder et 37 C H O 14.61 501.32281; 501.32281: C H O ; 439.32205: deoxyhexosyl moieties e 56 88 26 30 46 6 al. (1993) 439.32205 C30H46O6 -H2O - CO2 + pos 1194.59088 [M+NH4] -

1235.56738; 1411.60317: C70H110O38 - 983.57922; C H O ; 1235.56738: medicagenic acid + two hexosyl + two deoxyhexosyl + neg 1557.6595; 1411.60317 6 10 4 Mbark et al. 38 C H O 14.89 796.23248; C H O -C H O - two pentosyl + 1 glucuronic acid moiety e 70 110 38 70 110 38 6 10 4 (2000) 439.32043 C6H8O6; 439.32043: C29H44O3 1259.56189; 741.24219; pos 1581.65795 [M+Na]+ 595.18250; 445.13116

1381.62830; 1381.62830: C76H120O42 - 823.41089; C H O -C H O ; 517.31879: 1703.71708; 851.35632 6 10 4 6 8 6 zanhic acid + one hexose + four deoxyhexoses + two neg 2 517.31879; C30H46O7; 455.31766: C30H46O7 39 e C76H120O42 14.92 [M-2H] − pentoses + one glucuronic acid moiety 455.31766; -H2O - CO2; 839.40845: 839.40845 C30H47O7 + C6H10O4 + C6H8O6 1727.71399 [M+Na]+; pos + - 1722.75891 [M+NH4] Metabolites 2020, 10, 111 11 of 26

Table 1. Cont.

Com-Pound HESI Max Abs. Compound a Molecular Formula RT (min) HESI Full MS b Neutral Losses Literature Number ddMS2 (nm) 543.33105: C H O ; 483.31180: 543.33105; 32 48 7 C H O -H O; 439.32227: 1145.5387; 1213.52771 483.31180; 30 46 6 2 C H O -H O - CO ; neg [M-H+NaFA] ; 572.26715; 439.32227; 30 46 6 2 2 Mbark et al. 40 herniariasaponin E C H O 15.03 − 601.19806: C H O + C H O + 55 86 25 595.26941 [M-2H-FA]2 601.19806; 5 8 4 6 10 4 (2000) − 2C H O ; 439.32208: C H O + 439.32208 6 10 5 5 8 4 C6H10O4 + C6H10O5 1169.53381 [M+Na]+; pos + 545.34430 545.34430: C32H48O7 1164.57869 [M+NH4] 543.33105; 483.31180; 543.33105: C H O ; 483.31180: 1029.49191; 1075.49754 32 48 7 neg 439.32227; C30H44O5; 439.32227; 791.07910: e [M-H+FA]− 41 acetylated medicagenic acid + three hexosyl moieties C50H78O22 15.06 791.07910; C29H28O26 585.34424 1048.53173 [M+NH ]+; pos 4 - 1053.48612 [M+Na]+ + pos 1398.65376 [M+NH4] - 925.48315; medicagenic acid + three deoxyhexosyl + one pentosyl + neg 1247.569 C H O 15.12 501.31976 42 one glucuronic acid moiety e 59 92 28 1127.49792; 949.47644; pos 1271.56687 [M+Na]+ 701.34851; 525.32062; 447.14737 1163.15430; zanhic acid + three deoxyhexosyl + one pentosyl + one neg 1263.5657 43 e C59H92O29 15.24 1081.94128 glucuronic acid moiety + pos 1282.60583 [M+NH4] - 1381.62830; 1219.22852; 879.30701; medicagenic acid + two hexosyl + three deoxyhexosyl + neg 1703.7174 843.52264; 44 C H O 15.33 two pentosyl + one glucuronic acid moiety e 76 120 42 823.41089; 501.32275; 439.31934 1727.71399 [M+Na]+; pos + - 1722.75891 [M+NH4]* 1365.63477; medicagenic acid + one hexosyl + four deoxyhexosyl + neg 1687.7229 998.50537; 45 C H O 15.52 two pentoses + one glucuronic acid moiety e 76 120 41 823.42078 pos 1711.71936 [M+Na]+ - Metabolites 2020, 10, 111 12 of 26

Table 1. Cont.

Com-Pound HESI Max Abs. Compound a Molecular Formula RT (min) HESI Full MS b Neutral Losses Literature Number ddMS2 (nm) 1057.52454: C H O - 1057.52454; 64 100 32 C H O -C H O ; 925.48077: 925.48077; 6 8 6 6 10 4 C H O -C H O -C H O medicagenic acid + three deoxyhexosyl + two pentosyl + neg 1379.6127 823.41821; 64 100 32 6 8 6 6 10 4 46 C H O 15.12; 15.62 –C H O ; 823.41821: C H O ; one glucuronic acid moiety d,e 64 100 32 501.32251; 5 8 4 35 68 21 501.32251: C H O ; 439.32050: 439.32050 30 46 6 C30H46O6 -H2O - CO2 1259.54272; 1081.51782; pos 1403.60791 [M+Na]+ 847.40808; 579.18964 439.32248: C H O -H O- 439.32248; 30 46 6 2 medicagenic acid + one hexosyl + two deoxyhexosyl + neg 1395.6069 CO2; 1219.58301: C64H100O33 - Charrouf et 47 e C64H100O33 15.67 1219.58301 two pentosyl + one glucuronic acid moiety C6H8O6 al. (1995) pos 1419.60059 [M+Na]+ -

1087.53479; 1087.53479: C65H102O33 - 823.41272; C H O -C H O ; 823.41272; neg 1409.6244 6 10 4 6 8 6 Charrouf et 48 herniariasaponin B C H O 15.83 501.32300; 501.32300: C H O ; 439.32343: 65 102 33 30 46 6 al. (1995) 439.32343 C30H46O6 -H2O - CO2 1433.61982 [M+Na]+; + pos 1428.66283 [M+NH4] ; - 1411.63638

501.32312; 821.43097: C47H74O19 -C5H8O4; 439.32037; 543.33167: C H O ; 501.32312: acetylated medicagenic acid + one deoxyhexosyl + two neg 941.47546 32 48 7 49 C H O 16 821.43097; C H O ; 439.32037: C H O pentosyl moieties e 47 74 19 30 46 6 30 46 6 543.33167 -H2O - CO2 + 960.51593 [M+NH4] ; 463.14227: pos + 965.47021 [M+Na] C17H27O13Na 271.02539; 271.02539: C H O ; 178.99774: 178.99774; 14 8 6 neg 301.03538 C H O ; 151.00273: C H O ; 50 quercetin c C H O 16.4 151.00273; 8 4 5 7 4 4 255; 372 15 10 7 121.02832: C H O 121.02832 7 6 2 285.03964; 257.04440; 285.03964: C15H8O6; 257.04440: 229.04947; C14H8O5; 229.04947: C13H8O4; pos 303.04993 201.05476; 201.05476: C12H8O3; 165.01811: 165.01811; C8H4O4; 153.01846: C7H4O4; 153.01846; 137.02357: C7H4O3 137.02357 Metabolites 2020, 10, 111 13 of 26

Table 1. Cont.

Com-Pound HESI Max Abs. Compound a Molecular Formula RT (min) HESI Full MS b Neutral Losses Literature Number ddMS2 (nm) 652.28625 [M-2H]2 ; 439.32321; 439.32321: C H O ; 585.20691: acetylated medicagenic acid + one hexosyl + two neg − 29 43 3 51 C61H94O30 16.76 1305.57764 585.20691 C5H8O4 + 2C6H10O4 + C6H10O5 deoxyhexosyl + one pentosyl + one glucuronic acid + e 1329.57288 [M+Na] ; moiety pos + - 1324.61719 [M+NH4] neg 271.06120 253.05069 253.05069: C H O 52 naringenin c C H O 16.8 15 10 4 15 12 5 pos 273.07575 - 225.05510; 225.05510: C H O ; 151.00255: neg 269.04555 151.00255; 14 10 3 53 apigenin c C H O 17.73 C H O 15 10 5 117.03339 7 4 4 pos 271.06010 153.01845 153.01845: C7H4O4 300.02762; 271.02399; 243.02707; c neg 315.05103 300.02762: C15H9O7 54 isorhamnetin C16H12O7 17.87 216.04128; 164.01071; 151.00258 302.04205; pos 317.06555 285.03882; 153.01846: C7H4O4 153.01846 199.03967; 199.03967: C H O ; 151.00212: neg 285.04046 151.00212; 12 8 3 55 luteolin c C H O 18.09 C H O ; 133.02821: C H O 15 10 6 133.02821 7 4 4 8 6 2 153.01787; 153.01787: C H O ; 135.04414: pos 287.05501 7 4 4 135.04414 C8H6O2 neg 285.04046 - 56 kaempferol c C H O 18.09 265; 364 15 10 6 258.05115; 258.05115: C H O ; 231.06519: 231.06519; 14 9 5 C H O ; 165.01807: C H O ; pos 287.05501 165.01807; 13 10 4 8 4 4 153.01816: C H O ; 121.02866: 153.01816; 7 4 4 C H O 121.02866 7 4 2 301.03552; 271.02505; c neg 477.0675 57 miquelianin [quercetin 3-O-glucuronide] C21H18O13 12.89–13.49 255.03020;178.99788; 257; 357 151.00258 303.04993; pos 479.0824 257.04370 Metabolites 2020, 10, 111 14 of 26

Table 1. Cont.

Com-Pound HESI Max Abs. Compound a Molecular Formula RT (min) HESI Full MS b Neutral Losses Literature Number ddMS2 (nm) 821.43842; 821.43842: C H O ; 501.32306: 1103.52829; 574.26304 36 70 20 neg 501.32306; C30H46O6; 439.32156: C30H46O6 van Dooren 58 herniariasaponin G C53H84O24 14.61 [M-2H+FA]− 439.32156 -H2O - CO2 et al. (2016) 973.50128; 811.44629: C H O -C H O - 1127.52344 [M+Na]+; 53 84 24 5 8 4 811.45264; C H O ; 503.33636: C H O ; 1122.56841 [M+NH ]+; 6 10 5 30 46 6 pos 4 649.39624; 973.50128: C H O -C H O ; 811.44629; 503.33636; 53 84 24 5 8 4 503.33704; 649.39624: C H O -C H O - 457.33087 53 84 24 5 8 4 457.33188 2C6H10O5 1235.56738; 983.57922; 1411.60317: C H O - medicagenic acid + two hexosyl + two deoxyhexosyl + neg 1557.6595; 1411.60317 70 110 38 59 C H O 14.89–15.38 796.23248; C H O two pentosyl + one glucuronic acid moiety e 70 110 38 6 10 4 439.32043 1259.56189; 741.24219; pos 1581.65795 [M+Na]+ 595.18250; 445.13116 medicagenic acid + one hexosyl + two deoxyhexosyl + neg 1395.6069 1219.583 60 C H O 14.92–15.67 two pentosyl + one glucuronic acid moiety e 64 100 33 pos - - 1219.58069: C H O - 1446.38293; 70 110 37 C H O -C H O ; 823.41711: 1219.58069; 5 8 4 6 8 6 15.12; 15.66; neg 1541.6657 C H O -C H O - van Dooren 61 herniariasaponin H d C H O 823.41711; 70 110 37 6 10 5 70 110 37 15.72 2C H O - 2C H O ; 501.32587: et al. (2016) 501.32587 5 8 4 6 10 4 C30H46O6 1243.57349; 1565.66118 [M+Na]+; pos 905.31189; 1560.70778 [M+NH ]+ 4 741.24255

191.05544; 191.05544: C7H12O6; 179.03427: d neg 353.08685 179.03427; C9H8O4; 173.04494: C7H12O6 - 62 chlorogenic acid C16H18O9 7.31; 8.88 173.04494 H2O 163.03915; 163.03915: C9H8O4 -H2O; + pos 355.1016; 377.08360 [M+Na] 145.02861; 145.02861: C9H8O4 - 2H2O; 135.04443 135.04443: C8H8O2 193.05003; 367.10349; 435.09108 neg 173.04485; Jaiswal et al. 63 1-O-feruloylquinic acid d C H O 9.20–10.62 [M-H+NaFA] 17 20 9 − 134.03622 (2011) 145.02855; 149.05952; 369.11748; 391.09949 pos 117.03381; [M+Na]+ 177.05475; 194.05750 a If not specified otherwise: tentative identification based on accurate mass b Deprotonated and protonated molecules in negative and positive mode, respectively, unless stated otherwise c Identification with an analytical standard d Tentatively identified compound or an isomer e Tentative identification of saponins that have never been reported before in H. hirsuta. Metabolites 2020, 10, 111 15 of 26

Metabolites 2020, 10, x FOR PEER REVIEW 13 of 24 Table 2. Most probable sum formula and ppm deviation for the fragment ions of m/z 1703.71734. Table 2. Most probable sum formula and ppm deviation for the fragment ions of m/z 1703.71734. m/z Sum − ∆ ppm m/z [M-H]− SumFormula Formula ∆ ppm 1703.71838 C76 H120 O42 0.141 1703.71838 C76H120O42 0.141 1381.628301381.62830 CC64 H102102 OO3232 0.1130.113 879.30701879.30701 C C34 H5656 OO2626 9.4459.445 823.41089 C H O 1.541 823.41089 C42 H6464 O1616 -1.541− 501.32275 C30H45O6 1.172 501.32275 C30 H45 O6 1.172 439.31934 C29H44O3 5.528 439.31934 C29 H44 O3 -5.528−

FigureFigure 4 4shows shows the the chromatogram chromatogram of of the the H.H. hirsuta hirsuta extractextract before before and and after after biotransformation. biotransformation. BeforeBefore biotransformation biotransformation (t0), (t 0peaks), peaks are are mostly mostly attributed attributed to tothe the tentatively tentatively identified identified compounds. compounds. AfterAfter the the colonic colonic phase phase of ofgastrointestinal gastrointestinal biotra biotransformation,nsformation, the the chromatogram chromatogram contains contains peaks peaks of of compounds,compounds, metabolites metabolites and and matrix matrix interferences, interferences, originating originating from from enzymes enzymes and and fecal fecal microflora, microflora, whichwhich are are absent absent at att0. t0 .

FigureFigure 4. 4. Total ionion chromatogram chromatogram in negativein negative ion mode ion of modeH. hirsuta of beforeH. hirsuta and after before biotransformation. and after biotransformation. Processing the chromatograms of every time point for every compound allows monitoring of theProcessing intensity overthe chromatograms time. However, of this every manual time approachpoint for isevery time-consuming compound allows and will monitoring only provide of theinformation intensity over about time. the abundance However, ofthis previously manual identifiedapproach compounds.is time-consuming Manual and screening will only for metabolites provide information about the abundance of previously identified compounds. Manual screening for metabolites is comparable to looking for a needle in a haystack because of the complexity of the chromatograms and matrix interferences.

Metabolites 2020, 10, 111 16 of 26 is comparable to looking for a needle in a haystack because of the complexity of the chromatograms andMetabolites matrix 2020 interferences., 10, x FOR PEER REVIEW 14 of 24 Generally, for most tentatively identified compounds, a decrease in relative abundance is observed mainlyGenerally, at the colonic for most phase, tentatively caused byidentified bacterial comp degradation,ounds, a decrease suggesting in formationrelative abundance of metabolites. is However,observed somemainly compounds, at the colonic mainly phase, saponins, caused showby bacterial an increase degradation, in relative suggesting abundance formation over time. of It is suggestedmetabolites. that However, this increase some iscompounds, induced by mainly conversion saponins, of one show compound an increase into in another, relative causedabundance by loss ofover one time. or more It is sugarsuggested moieties. that this For increase example, is in compoundduced by conversion46 (medicagenic of one compound acid + three into deoxyhexosyl another, +causedtwo pentosyl by loss of+ oneone or glucuronyl more sugar moiety) moieties. shows For example, an increase compound in relative 46 (medicagenic abundance acid and + reachesthree a maximumdeoxyhexosyl after + two 24 h pentosyl colon phase, + one whichglucuronyl is not moie observedty) shows in an the increase negative in controlrelative samplesabundance (NCHEX) and reaches a maximum after 24 h colon phase, which is not observed in the negative control samples (Figure5). In addition, the signal was absent in the blank (MB), as expected. At the same time, (NCHEX) (Figure 5). In addition, the signal was absent in the blank (MB), as expected. At the same a decrease of compound 45 (medicagenic acid + one hexosyl + four deoxyhexosyl + two pentosyl + time, a decrease of compound 45 (medicagenic acid + one hexosyl + four deoxyhexosyl + two pentosyl one glucuronyl moiety) is observed. The increase of compound 46 can be related to the decrease of + one glucuronyl moiety) is observed. The increase of compound 46 can be related to the decrease of compound 45, caused by the loss of a hexosyl and a deoxyhexosyl moiety. The decrease is observed compound 45, caused by the loss of a hexosyl and a deoxyhexosyl moiety. The decrease is observed in both samples and negative control during the stomach phase and the small intestinal phase. At in both samples and negative control during the stomach phase and the small intestinal phase. At thesethese time time points,points, there is is no no difference difference in in samples samples and and negative negative control control as the as thenegative negative controls controls only only lacklack fecalfecal bacteriabacteria during the the colonic colonic phase. phase. Since Since identification identification based based on onMS-data MS-data is not is notabsolute, absolute, onlyonly tentativetentative conclusionsconclusions can can be be drawn drawn from from th theseese observations. observations. However, However, the thedata data support support the the theorytheory that that gastrointestinalgastrointestinal biotransformation biotransformation le leadsads to to a aloss loss of ofsugar sugar moieties moieties in saponins. in saponins.

FigureFigure 5.5. Time profile profile of of the the biotransformation biotransformation of of compounds compounds 46 46(left)(left) and and 45 (right)45 (right) in samples in samples (HEX), (HEX), negativenegative controlscontrols (NCHEX) and and blank blank (MB). (MB).

AnAn automatedautomated datadata analysisanalysis workflow,workflow, previously described by by Peeters Peeters et et al. al. and and Beirnaert Beirnaert et et al., wasal., was used used to screen to screen for metabolitesfor metabolites [23 [23,27].,27]. The The workflow workflow resulted resulted in in 11,072 11,072 features features inin negativenegative ion mode,ion mode, of which of which 4846 4846 were were annotated annotated as isotopes.as isotopes In. positiveIn positive mode, mode, 5242 5242 features features were were detected detected after exclusionafter exclusion of isotopes. of isotopes. Firstly,Firstly, thethe metabolicmetabolic pathwaypathway ofof herniariasaponinherniariasaponin H H ( (m/zm/z 1541.6675,1541.6675, rt: rt: 17.3 17.3 min) min) was was studied studied in detailin detail to verifyto verify ifthe if the workflow workflow was was suitable suitable for for herbal herbal extracts.extracts. The time time profile profile of of herniariasaponin herniariasaponin H showsH shows a decrease a decrease in in intensity intensity over over time, time, especially especially during during thethe coloniccolonic phase, which which is is not not observed observed in thein the negative negative control control samples samples (NCHEX) (NCHEX) (Figure (Figure6). In6). addition,In addition, the the signal signal is absentis absent in thein the method method blank (MB).blank The(MB). feature The feature is rated is rated as notable as notable based based on theon the results results of tinderesting,of tinderesting, the the interactive interactive Shiny Shiny app app developed in R, with a score of 1 and a ranking of 242 out of 6226 features. During developed in R, with a score of 1 and a ranking of 242 out of 6226 features. During biotransformation, biotransformation, stepwise elimination of sugar moieties is observed resulting in formation of the stepwise elimination of sugar moieties is observed resulting in formation of the aglycone (Figure7). aglycone (Figure 7). All intermediate metabolites show a tinderesting score of at least 0.98, meaning All intermediate metabolites show a tinderesting score of at least 0.98, meaning that they are all rated that they are all rated as interesting. Finally, the aglycone medicagenic acid (m/z 501.3213; rt: 21.68) is formed, showing an increase in signal over time in the samples, with a tinderesting score of 0.998 (Figure 6).

Metabolites 2020, 10, 111 17 of 26 as interesting. Finally, the aglycone medicagenic acid (m/z 501.3213; rt: 21.68) is formed, showing an increaseMetabolites in2020 signal, 10, x overFOR PEER time REVIEW in thesamples, with a tinderesting score of 0.998 (Figure6). 15 of 24

FigureFigure 6. TimeTime profile profile of of gastrointestinal gastrointestinal biotransformation biotransformation of ofherniariasaponin herniariasaponin H (m/z H (m 1541.6675/z 1541.6675 [M- [M-H]H]−) and−) andmedicagenic medicagenic acid acid (m/z ( m501.3213/z 501.3213 [M-H] [M-H]−). −).

FeaturesFeatures withwith aa tinderesting tinderesting score score higher higher than than 0.8 0.8 were were selected selected for for in-depth in-depth exploration exploration with with the followingthe following inclusion inclusion criteria: criteria: a reasonable a reasonable intensity intensity with good-quality with good-quality MS/MS spectraMS/MS andspectra a retention and a timeretention between time 2 between and 25 min. 2 and 25 min. TheThe mainmain challenge challenge in thein analysisthe analysis of a large of a amount large ofamount analytical of dataanalytical remains data in theremains identification in the andidentification annotation and of annotation the features of ofthe interest. features of However, interest. theHowever, data analysis the data workflow analysis workflow ranks features ranks accordingfeatures according to one single to one score single taking score the taking longitudinal the longitudinal aspect and aspect the three and the sample three classes sample into classes account, into whichaccount, facilitates which facilitates selection ofselection features of of features interest. of On interest. the other On hand,the other determination hand, determination of the molecular of the formulamolecular of compoundsformula of with compound a moleculars with mass a abovemolecular 1000 Daltonmass becomesabove 1000 more Dalton challenging. becomes Therefore, more thischallenging. step in the Therefore, data analysis this step process in the is stilldata performed analysis process manually. is still performed manually. AsAs expected,expected, thethe featuresfeatures includedincluded inin thethe in-depthin-depth explorationexploration could be attributed to saponins, flavonoidsflavonoids andand theirtheir metabolites.metabolites. Saponins (compounds 35–4141,, 43–4545,, 47–4848,, 5151,, 5858,, 59 and 61)) showshow thethe samesame biotransformation biotransformation pathway pathway as theas studiedthe studied example, example, herniariasaponin herniariasaponin H. In brief, H. In the brief, intensity the ofintensity the signal of the decreases signal overdecreases time dueover to time stepwise due to elimination stepwise elimination of sugar moieties, of sugar leading moieties, to formation leading ofto theformation aglycone. of Thethe observedaglycone. biotransformationThe observed biotransfo of flavonoidsrmation is similar.of flavonoids In nature, is similar. flavonoids In mainlynature, occurflavonoids as glycosides. mainly occur Gastrointestinal as glycosides. biotransformation Gastrointestinal causes biotransformation deglycosylation, causes leading deglycosylation, to formation of theleading aglycone. to formation For example, of the quercetin aglycone. glycosides For example, are abundantly quercetin and glycosides ubiquitously are present abundantly flavonoids and inubiquitously nature. The present aglycone flavonoids is formed in after nature. biotransformation The aglycone is of formed several after precursors, biotransformation such as isoquercetin of several andprecursors, rutin. Isoquercetinsuch as isoquercetin (m/z 463.0871; and rutin. rt: Isoquercetin 11.67 min) has (m/z a 463.0871; tinderesting rt: 11.67 score min) of 0.86has a and tinderesting shows a decreasescore of 0.86 in intensity and shows over a decrease time (Figure in intensity8). The over decrease time (Figure is already 8). The observed decrease in theis already stomach observed phase andin the continues stomachin phase the small and continues intestinal in phase the small and colonic intestinal phases phase until and the colonic signal phases is completely until the absent. signal Inis completely the negative absent. control In samples, the negative the signal control stays samples, stable the during signal the stays colonic stable phase, during because the colonic there arephase, no bacteriabecause present.there are The no signalbacteria is absentpresent. in The the methodsignal is blank. absent For in quercetinthe method (m /blank.z 301.0345; For quercetin rt: 16.54 min), (m/z an301.0345; increase rt: in 16.54 intensity min), is an observed increase over in intensity time, which is observed is not observed over time, in the which negative is not control observed or method in the blanknegative (Figure control8). or The method feature blank is rated (Figure as interesting 8). The feature with is a tinderestingrated as interesting score of with 1. This a tinderesting suggests deglycosylationscore of 1. This suggests of isoquercetin deglycosylation leading to of the isoquercetin formation ofleading quercetin. to the formation of quercetin.

Metabolites 2020, 10, 111 18 of 26

Figure 7. Metabolic pathway of herniariasaponin H by in vitro gastrointestinal biotransformation.

2 Metabolites 2020, 10, x FOR PEER REVIEW 17 of 24 Metabolites 2020, 10, 111 19 of 26 Metabolites 2020, 10, x FOR PEER REVIEW 17 of 24 A feature with m/z 181.0497 and rt: 4.9 min was included in the in-depth exploration as it showed a tinderestingA feature scorewith m/z of 0.992.181.0497 The and feature rt: 4.9 showsmin was an included increase in in the intensity in-depth over exploration time, mainly as it showed during the A feature with m/z 181.0497 and rt: 4.9 min was included in the in-depth exploration as it showed colonica tinderesting phase (Figurescore of 8).0.992. The The metabolite feature shows was tentatively an increase identified in intensity as over 3,4-dihydroxyphenylpropionic time, mainly during the a tinderesting score of 0.992. The feature shows an increase in intensity over time, mainly during the acidcolonic (A) phase or 3-methoxy-4-hydroxyphenylacetic (Figure 8). The metabolite was tentatively acid (B). identified Both compounds as 3,4-dihydroxyphenylpropionic were previously described colonic phase (Figure8). The metabolite was tentatively identified as 3,4-dihydroxyphenylpropionic asacid metabolites (A) or 3-methoxy-4-hydroxyphenylacetic of quercetin [28,29]. Based on the acid obtained (B). Both spectra, compounds absolute were structural previously characterization described acidisas not metabolites (A) possible. or 3-methoxy-4-hydroxyphenylacetic of Nonetheless, quercetin [28,29]. the Basedproduct on ionthe acidobtainedcould (B). be Bothspectra, identified compounds absolute as a structuralmetabolite were previously characterization of quercetin. described This as metabolitesshowsis not possible.that despite of quercetin Nonetheless, the increase [28,29 the]. Basedproductin intensity on ion the couldof obtained quercetin, be identified spectra, the compound absoluteas a metabolite structural is further of quercetin. characterization biotransformed This is not(Figureshows possible. that9). despite Nonetheless, the increase the product in intensity ion could of quercetin, be identified the compound as a metabolite is further of quercetin. biotransformed This shows that(Figure despite 9). the increase in intensity of quercetin, the compound is further biotransformed (Figure9).

Figure 8. Time profiles of biotransformation of isoquercetin (m/z 463.0871 [M-H]− −), quercetin (m/z FigureFigure 8. TimeTime profiles profiles of ofbiotransformation biotransformation of isoquercetin of isoquercetin (m/z 463.0871 (m/z 463.0871 [M-H] ), [M-H] quercetin−), quercetin (m/z −− − − (301.0345301.0345m/z 301.0345 [M-H] [M-H])) andand−) andaa metabolitemetabolite a metabolite (m/z(m/z (m 181.0497/181.0497z 181.0497 [M-H] [M-H] [M-H]) tentatively) −)tentatively tentatively identified identified identified as 3,4-as as 3,4- 3,4- dihydroxyphenylpropionicdihydroxyphenylpropionic acid acid or oror 3- 3-methoxy-4-hydroxyphenylacetic3-methoxy-4-hydroxyphenylaceticmethoxy-4-hydroxyphenylacetic acid. acid.acid.

Figure 9. Biotransformation pathway of isoquercetin showing quercetin as intermediate metabolite which

is further transformed into 3,4-dihydroxyphenylpropionic acid (A) or 3-methoxy-4- hydroxyphenylacetic acidFigure (B ).9. Biotransformation pathway of isoquercetin showing quercetin as intermediate metabolite Figurewhich 9.is Biotransformationfurther transformed pathway into 3,4-dihydroxyphenylpropionicof isoquercetin showing quercetin acid as(A intermediate) or 3-methoxy-4- metabolite 2.3. Liverwhichhydroxyphenylacetic Biotransformation is further transformed acid (B). into 3,4-dihydroxyphenylpropionic acid (A) or 3-methoxy-4- hydroxyphenylacetic acid (B). Figure 10 shows an overview of the proposed metabolic pathway of medicagenic acid after 2.3. Liver Biotransformation S9 biotransformation. For metabolites 1–3 (M1–3), three different chromatographically separated 2.3. Liver Biotransformation peaksFigure were 10 observed shows an (rt overview 17.39; 17.60 of the and proposed 18.45 me min),tabolic suggesting pathway theof medi presencecagenic of acid three after di ffS9erent structuralbiotransformation.Figure isomers. 10 shows For This an metabolites was overview confirmed 1 of–3 the(M1 by proposed– the3), predictionthree differentmetabolic provided chromatographically pathway by Meteor. of medi Thecagenic separated software acid peaks proposed after S9 hydroxylationbiotransformation.were observed at(rt C-11,17.39; For metabolites C-24,17.60 and or C-29. 18.45 1–3 (M1 Doublemin),–3 sugge), three hydroxylationsting different the presence chromatographically (M4), oxidationof three different of a separated hydroxy structural grouppeaks (wereM5isomers.) andobserved This a combination was(rt 17.39; confirmed 17.60 of hydroxylation andby the18.45 prediction min), and sugge oxidation providedsting the(M9by presence–Meteor.10) were ofThe also three software predicted different proposed by structural Meteor. hydroxylation at C-11, C-24, or C-29. Double hydroxylation (M4), oxidation of a hydroxy group (M5) Theisomers. nontarget This screeningwas confirmed approach by confirmedthe prediction the formationprovided ofby the Meteor. predicted The metabolites, software proposed and one and a combination of hydroxylation and oxidation (M9–10) were also predicted by Meteor. The additionalhydroxylation metabolite at C-11, wasC-24, detected. or C-29. Double Metabolite hydroxylation8 (M8) shows (M4), aoxidation reduction of ofa hydroxy the carboxylic group ( acidM5) and a combination of hydroxylation and oxidation (M9–10) were also predicted by Meteor. The moiety. However, limited by the poor MS/MS fragmentation of the aglycones and lack of NMR

Metabolites 2020, 10, x FOR PEER REVIEW 18 of 24

Metabolitesnontarget2020 screening, 10, 111 approach confirmed the formation of the predicted metabolites, and20 ofone 26 additional metabolite was detected. Metabolite 8 (M8) shows a reduction of the carboxylic acid moiety. However, limited by the poor MS/MS fragmentation of the aglycones and lack of NMR data, data,identification identification remains remains tentative. tentative. Further elucidation Further elucidation of the molecular of the structures molecular was structures not possible. was The not possible.only phase The II onlyproducts phase observed II products were observed glucuronidated were glucuronidated metabolites of metabolites medicagenic of acid medicagenic (M6–7). acidSulfation (M6 –reactions7). Sulfation were not reactions observed, were although not observed, they were although predicted they by Meteor were predicted and Biotransformer. by Meteor andTo confirm Biotransformer. that the in To vitro confirm model that was the capablein vitro tomodel form wassulfated capable conjugates, to form biotransformation sulfated conjugates, of biotransformationtestosterone was ofexamined testosterone as waspositive examined contro as positivel. Testosterone control. Testosteronewas analyzed was analyzedafter hepatic after hepaticbiotransformation biotransformation and proved and proved the capability the capability of the of in the vitroin vitro modelmodel to toform form both both sulfated sulfated and glucuronidated conjugates. The phase II hydrophilic metabolites may be excretedexcreted via the kidneys and are often considered detoxifying as they usually possess lower activity compared to theirtheir parentparent aglycones.aglycones. However, a number ofof glucuronideglucuronide conjugates conjugates are are known known to beto active. be active. The 6-TheO-glucuronide 6-O-glucuronide of morphine, of morphine, a widely a usedwidely opioid used analgesic,opioid analgesic, is the most is well-knownthe most we examplell-knownof example a glucuronidated of a glucuronidated metabolite possessingmetabolite pharmacologicalpossessing pharmacological activity greater activity than greater the parent than aglyconethe parent [30 aglycone]. Based [30]. on the Based observation on the observation of in vitro formationof in vitro offormation glucuronidated of glucuronidated hepatic metabolites, hepatic metabolites, it is a possible it is hypothesis a possible thathypothesis these metabolites that these maymetabolites be responsible may be forresponsible the urolithiatic for the activity urolithiatic [31]. Aactivity possible [31]. mode A possible of action mode for prevention of action andfor dissolutionprevention ofand urinary dissolution stones couldof urinary be the emulsifyingstones could properties be the of emulsifying the glucuronidated properties metabolites, of the facilitatingglucuronidated dissolution metabolites, of crystals. facilitating dissolution of crystals.

M10 M1-3 M9

OH

M4 HO M8 O

HO

O

M5 M6-7

Figure 10. B Metabolic pathway of medicagenic acid afterafter in vitro hepatic biotransformation.

3. Materials and Methods

3.1. Chemicals 3.1. Chemicals Ultrapure water with a resistivity of 18.2 MΩ cm at 25 C was generated with a Millipore™- Ultrapure water with a resistivity of 18.2 MΩ·cm· at 25 °C◦ was generated with a Millipore™- purificationpurification system.system. Ultrahigh-Performance Ultrahigh-Performance Liquid Liquid Chromatography Chromatography (UHPLC)-grade MeOH, acetonitrile andand formic formic acid acid were were purchased purchased from from Biosolve Biosolve (Dieuze, (Dieuze, France), France), dichloromethane dichloromethane by Merck by (Darmstadt,Merck (Darmstadt, Germany). Germany). The following The following analytical analytic standardsal standards were provided were provided by Sigma–Aldrich by Sigma–Aldrich (St. Louis, MO, USA): apigenin, benzoic acid, caffeic acid, +/ catechin, chlorogenic acid, cinnamic acid, coumarin, (St. Louis, MO, USA): apigenin, benzoic acid, caffeic− acid, +/− catechin, chlorogenic acid, cinnamic emodin,acid, coumarin, epicatechin, emodin, ferulic epicatechin, acid, isorhamnetin, ferulic acid, naringenin, isorhamnetin,p-coumaric naringenin, acid, protocatechuic p-coumaric acid, β quercetin,protocatechuic quercitrin, acid, quercetin, rutin, salicylic quercitrin, acid, sinapic rutin, salicylic acid, -sitosterol, acid, sinapic stigmasterol, acid, β-sitosterol, syringic stigmasterol, acid, tannic acid,syringic taxifolin, acid, tannic theophylline acid, taxifolin, and vanillic theophylline acid. Luteolin and vanillic and procyanidin acid. Luteolin B2 and were procyanidin providedby B2 Santawere Cruz Biotechnology (Santa Cruz, CA, USA). Gallic acid and p-hydroxybenzoic acid were provided

Metabolites 2020, 10, 111 21 of 26 by Carl Roth (Karlsruhe, Germany). Human liver S9 fraction and NADPH RS (Regenerating System) were purchased from XenoTech. Medicagenic acid was provided by Phytolab (Vestenbergsgreuth, Germany). All other chemicals and biochemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

3.2. Preparation of Standard Solutions

1 Standard stock solutions of the analytical standards were prepared at a concentration of 1 mg mL− in UHPLC-grade MeOH for each analyte separately and stored in the dark at 80 C. Dilutions of these − ◦ solutions were prepared in 60:40 (v:v) MeOH:ammonium formate buffer (40 mM, aqueous). Standard stock and working solutions were stored at 80 C in the dark. − ◦ 3.3. Sample Preparation Aerial parts from H. hirsuta were collected in d’Oujda, Morocco. A voucher specimen of the plant is kept at the Muséum National d’Histoire Naturelle—Institut Scientifique-Université Mohamed V Agdal, Morocco—(Reference number: 5902). The air-dried plant material of H. hirsuta was ground prior to extraction with a PF 10 basic Microfine grinder drive (IKA-Werke GmbH & Co. KG, Staufen, Germany) using a sieve mesh size 0.5 mm. To cover the full range of constituents, a comprehensive extraction protocol was applied, previously described by Bijttebier et al. with minor adaptations [32,33]. Combination of extraction with H2O:EtOAc and CHCl3:MeOH:H2O afforded both polar and apolar compounds. Approximately 18 g of sample was subsequently wetted with 54 mL of water and 225 mL of EtOAc. The mixture was stirred for 2 h followed by 1 h of ultrasound-assisted extraction. The solvent was removed, and the residue was washed three times with 20 mL EtOAc. The extract was evaporated to dryness. CHCl3:MeOH:H2O extraction was performed by adding 18 g of sample to 180 mL CHCl3:MeOH:H2O (4:4:2). The mixture was stirred for 5 min, followed by 5 min of ultrasound-assisted extraction and stirred for 15 more min. The solvent was removed, and the residue was washed 3 times with 20 mL CHCl3:MeOH:H2O (4:4:2). The dried H2O:EtOAc extract was redissolved with the extract from the CHCl3:MeOH:H2O extraction method, leading to an apolar and a polar phase. Both phases were separated and dried.

3.4. Gastrointestinal Biotransformation Many natural products are pro-drugs that are biotransformed and activated after oral administration. A previously developed and validated in vitro gastrointestinal biotransformation model was used to mimic human biotransformation processes [34]. This model allows the study of biotransformation processes in the stomach, small intestine and colon using a culture of pooled human feces, avoiding extensive in vivo studies. The digestive juices and fecal suspension were formulated corresponding to the human conditions, previously described by Breynaert et al. and Peeters et al. [23,34]. Small adaptations to the method were made [35]. Briefly, the pepsin solution was prepared by dissolving 16% (w/v) of pepsin powder in 0.1 1 M HCl (622,000 FIP-U 100 mL− ). To obtain a pancreatin-bile mixture, 0.4% (w/v) pancreatin and 2.5% (w/v) bile were dissolved in 0.1 M NaHCO3 (32,000 FIP-U lipase, 143,600 FIP-U amylase, 16,400 FIP-U 1 protease and 58.4 mmol bile L− ). A suspension of 10% (w/v) feces was prepared by homogenizing 3 human fecal samples with a sterile phosphate-buffer solution (0.1 M, pH 7.0). The phosphate-buffer solution consisted of NaH2PO4.2H2O (1.03% w/v), sodium thioglycolate broth (3.45% v/v) and glycerol (17%). Prior to use, the fecal suspension was cultivated. To 10% (v/v) pooled fecal suspension in phosphate buffer 90% (v/v) phosphate buffer was added in an anaerobic glove-box (5% H2, 5% CO2 and 90% N2) (Jacomex Glove Box T3, TCPS, Belgium). The bacteria were cultivated in an anaerobic environment for 1 h at 37 ◦C by continuously stirring. Three groups were included in the experiment: (1) samples containing the Herniaria extract, which were treated with digestive enzymes and fecal microflora (HEX); (2) negative control samples, also containing the extract with addition of digestive enzymes but not of fecal bacteria (NCHEX); Metabolites 2020, 10, 111 22 of 26 and (3) method blanks, comprising an equal volume of solvent instead of extract and undergoing treatment with digestive enzymes and fecal bacteria (MB). For the samples, an amount of 300 mg lyophilized polar phase of the comprehensive extract of H. hirsuta was accurately weighed in triplicate. Gastrointestinal biotransformation was simulated in vitro. An aliquot of each sample was taken at several time points: before biotransformation (t0), after the gastric stage (S), after the small intestinal phase (SI) and at several time points during the colonic phase (after 2, 4, 6, 10, 14, 18, 22, 24, 32, 40, 48 and 72 h). Every sample was diluted with MeOH (1:2) and centrifuged for 5 min at 3500 rpm. The supernatant was collected, and samples were diluted 10 times with MeOH:H2O (60:40) before analysis.

3.5. Liver Biotransformation Liver biotransformation mimicking phase I and II reactions was performed using medicagenic acid, the most abundant aglycone after gastrointestinal biotransformation. Hepatic biotransformation was simulated in vitro by using pooled S9 fractions, previously described by Van den Eede et al. [36]. Small adaptations to the method were made. Briefly, a mixture of 65 mM TRIS buffer (pH adjusted to 1 7.4 at 37 ◦C), human liver S9 fraction (1 mg mL− final protein concentration) and medicagenic acid (100 µM final concentration) was prepared in a total volume of 0.5 mL and preincubated in a shaking waterbath at 37 ◦C. The reaction was initiated by addition of an NADPH regenerating system (1 mM NADP, final concentration). Cofactors were added to expose the samples to phase II glucuronidation and sulfation: UDPGA (2 mM final concentration), GSH (2 mM final concentration) and PAPS (0.1 mM final concentration). The reaction was inhibited after 1 h by adding 0.5 mL of acetonitrile and storing the tubes on ice. Thereafter, the tubes were centrifuged for 5 min at 10,000 rpm (4 ◦C). The supernatant was collected and analyzed by LC–MS-MS. Blank samples were prepared as described above, replacing substrate by solvent. Negative control samples were prepared by immediately adding acetonitrile to quench the biotransformation reactions. A positive control was included by incubating testosterone (100 µM final concentration) [36].

3.6. Instrumental Analysis For characterization of the H. hirsuta extract, an aliquot of 5 µL was injected with a CTC PAL™ autosampler (CTC Analytics) on a Waters Acquity UHPLC BEH SHIELD RP18 column (3.0 mm 150 × mm, 1.7 µm; Waters, Milford, MA, USA) and eluted with an Accela™ quaternary solvent manager. The temperature of the column was kept at 40 ◦C using a “Hot Pocket” column oven (Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase solvents consisted of water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B). Compounds were eluted using the following gradient (min/B%): 0.00/0, 9.91/26, 18.51/65, 18.76/100, 20.76/100, 20.88/0, 23.00/0. For detection, an amMS (Q Exactive™; Thermo Fisher Scientific) was used with heated electrospray ionization (HESI). Spray voltage was set at 2.5 kV, sheath gas and auxiliary gas at 47 and 15 (adimensional), respectively, and capillary − temperature at 350 ◦C. The PDA detector was set to scan from 190 to 800 nm. During the first analysis, full scan data were acquired in positive and negative ion mode over an m/z range of 120–1800 and resolving power was set at 70,000 at full width at half maximum (FWHM). Consecutively, a second analysis was performed to selectively fragment the generated ions by data-dependent fragmentation (ddMS2) in positive and negative ionization mode. Selective ion fragmentation was obtained by using a hybrid quadrupole-orbitrap MS analyzer (Q Exactive™, Thermo Fisher Scientific). The raw data were processed using XCalibur™ 3.0 software (Thermo Fisher Scientific). For the qualitative UHPLC-UV-QTOF analyses of the biotransformation samples, an aliquot of 5 µL was injected on a Waters Acquity UHPLC BEH SHIELD RP18 column (3.0 mm 150 mm, 1.7 µm; × Waters). The temperature of the column was kept at 40 ◦C. The mobile phase solvents consisted of water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B), and the gradient was set as follows 1 (min/B%): 0/2, 1/2, 14/26, 24/65, 26/100, 29/100, 31/2, 41/2. The flow rate was set at 0.4 mL min− . For detection, accurate mass measurements were done using a Xevo G2-XS QTof spectrometer (Waters, Metabolites 2020, 10, 111 23 of 26

Milford, MA, USA) coupled with an ACQUITY LC system equipped with MassLynx 4.1 software. During the first analysis, full scan data were recorded in ESI (+) and ESI ( ) mode from m/z 50 to 2000, − and the analyzer was used in sensitivity mode (approximate resolution: 22,000 FWHM). The spray 1 voltage was set at either +1.5 kV and 1.0 kV; cone gas flow and desolvation gas flow at 50.0 L h− 1 − and 1000.0 L h− respectively; source temperature and desolvation temperature at 120 ◦C and 500 ◦C respectively. Data were also recorded using MSE in positive and negative ionization modes, providing separate MS and MSE data. A ramp collision energy from 20 V to 30 V was applied to obtain additional structural information. Leucine encephalin was used as lock mass. To monitor analytical drift and assess precision, quality control (QC) samples were injected after every time point. QC samples were 1 prepared using a dilution of the standard solution mix (39 ng mL− ).

3.7. Data Analysis Dereplication was used as an untargeted workflow for rapid identification of the major compounds present in the extract, regardless of their chemical class. Global Natural Products Social Molecular Networking (GNPS, a web-based mass spectrometry open-access knowledge base) was used to facilitate identification [37]. Raw files were uploaded into the METABOLOMICS-SNETS-V2 workflow from GNPS (Global Natural Products Social Molecular Networking) in order to cluster the data based on their MS/MS fragmentation pattern. The precursor ion mass tolerance and the fragment ion mass tolerance were set at 2.0 Da and 0.5 Da respectively, and the cosine score was set at 0.7. The generated molecular network was visualized using Cytoscape 3.7.1 software. To encounter the dynamic nature of the biotransformation process, a data analysis workflow for in vitro gastrointestinal biotransformation was implemented, previously developed and validated using hederacoside C as a model compound [23,27]. Briefly, data were converted to the open-source mzData format to allow further processing [38]. XCMS was used to convert the raw data into features via peak-picking, using following parameters: ppm = 10, peakwidth = c(5, 25, snthresh = 10, noise = 1000, mzdiff = 0.01, prefilter = c(3, 5000), integrate = 1. XCMS was followed by grouping, and EDGE was used for the extraction of significant differential profiles [39,40]. Tinderesting, an interactive Shiny app developed in R, was used to rate the quality of the resulting features. These ratings were used to train a random forest model for predicting experts response. The machine learning model provided a single score for each feature, referred to as tinderesting score, which allowed ranking of all features based on the difference over time between the three groups. The maximal score of 1 corresponds to the model labeling this feature as interesting, and the minimal score of 0 defines an uninteresting feature [23,27]. Several peaks in the output correspond to the same type of sample molecule, reflecting its isotope pattern. Isotopes were removed, using following parameters: IsoMass = 1.003355, IsoMassMaxDiff = 0.05, RTdiff = 10, corrThreshold = 0.8, ppmIsoDiffThreshold = 5. The metabolic pathway of herniariasaponin H was studied since it is the most abundant saponin present in the extract and it is structurally related to hederacoside C, which was used for optimization of the workflow [23]. Both compounds contain a sapogenin linked to sugar moieties via the hydroxy groups at C-3 and C-28 of the aglycone. Herniariasaponin H comprises medicagenic acid as aglycone, which only differs from hederagenin by an additional hydroxy group at C-2 and a C-24 hydroxycarbonyl group instead of a hydroxymethyl group. However, the challenge in revealing the metabolic pathway of herniariasaponin H was that these experiments were not conducted with a single saponin, but with an herbal extract, comprising a complex mixture of compounds of high structural variety. A suspect screening method, based on in silico metabolite prediction, was combined with a nontarget screening workflow to enhance the identification of products formed by in vitro liver biotransformation [41]. A list of potential biotransformation products was generated using Meteor Nexus 2.1 (Lhasa Limited, Leeds, UK). For phase I biotransformation, all redox and non-redox biotransformations were selected. For phase II biotransformation, O-glucuronidation, O-sulfonylation, acetylation and Metabolites 2020, 10, 111 24 of 26 conjugation with amino acids were selected. The maximum depth was set at 3, and the maximum number of biotransformation products at 1000. Biotransformer was used as an additional software tool to predict phase II metabolites. The tool uses both a knowledge-based approach and a machine-learning-based approach to predict biotransformation [42]. The SMILES string of medicagenic acid was uploaded, and “Phase I Transformation” and “Phase II Transformation” were selected separately. The generated csv file contained InChIKey, synonyms, major isotope mass, molecular formula, type of biotransformation reaction and precursor ID. A nonarget screening workflow was conducted with MZmine 2.29 software (pluskal, mzmine 2) after converting the raw data files to the open-source mzXML format. Firstly, m/z features were detected using the centroid algorithm followed by a chromatogram building step. Resulting chromatograms were deconvoluted using the noise amplitude algorithm. Further data reduction occurred by deisotoping, keeping the lowest m/z value as the representative isotope. Meanwhile, chromatographic peaks were also filtered according to peak width: only peaks with a width between 0.05 and 1.00 min were retained. Next, peaks were aligned across samples using the random sample consensus (RANSAC) alignment algorithm correcting for nonlinear shifts in retention times. Finally, using the same rt and m/z range gap filler algorithm, any missing peaks were reiteratively extracted. The obtained m/z features were imported in R and processed as described by Vervliet et al. [43]. In brief, fold changes between samples and negative controls were calculated. A volcano plot was constructed to plot the p-value from a student t-test as a function of the calculated fold change for every feature. Features with a p-value lower than 0.05 and a log 10-fold change higher than 10 were selected for in depth investigation [41].

4. Conclusions Since there is no prophylactic therapy for urinary stone disease, there is an urgent need for new drugs. An integrated strategy, based on natural prodrugs and their metabolites, is developed to characterize new lead compounds derived from H. hirsuta. The combination of a comprehensive extraction method and biotransformation via in vitro gastrointestinal and hepatic models, followed by metabolomics profiling, is an innovative concept to identify and further develop new lead compounds for drugs. Comprehensive extraction led to the tentative identification of 15 saponins that have not been reported before in H. hirsuta. The automated data analysis workflow used for unbiased screening for metabolites revealed the gastrointestinal biotransformation pathway of several compounds. A decrease in relative abundance over time was observed for the majority of all identified compounds, especially during the colon phase. Additionally, the relative abundance of saponin aglycones increased, illustrating the biotransformation of possible saponin prodrugs to their respective aglycones, which was also observed for flavonoids. Further hepatic biotransformation of the most abundant saponin aglycone revealed further biotransformation of medicagenic acid to different phase I and phase II metabolites, which can be used as a possible target for in vitro activity studies against urinary stones.

Author Contributions: Conceptualization, L.P. (Laura Peeters), A.V.d.A., C.B., S.B., K.L., L.P. (Luc Pieters), N.H. and K.F.; methodology, L.P. (Laura Peeters), A.V.d.A.; software, C.B., L.P. (Laura Peeters); validation, L.P. (Laura Peeters), A.V.d.A. and C.B.; formal analysis, L.P. (Laura Peeters), A.V.d.A.; investigation, L.P. (Laura Peeters).; resources, K.L., L.P. (Luc Pieters) and N.H.; data curation, L.P. (Laura Peeters).; writing—original draft preparation, L.P. (Laura Peeters); writing—review and editing, L.P. (Laura Peeters), L.P. (Luc Pieters) and K.F.; visualization, L.P. (Laura Peeters); supervision, K.L., L.P. (Luc Pieters) and N.H.; project administration, L.P. (Luc Pieters) and N.H.; funding acquisition, L.P. (Luc Pieters) and N.H. All authors have read and agreed to the published version of the manuscript. Funding: Research was funded by Special Fund for Research of the University of Antwerp (Concerted Action), project ID no. 30732 and by the Research Foundation (FWO)—Flanders Project no. G032415N. Acknowledgments: Y. Cherrah (University of Rabbat, Morocco) is kindly acknowledged for providing the original plant material. Conflicts of Interest: The authors declare no conflict of interest. Metabolites 2020, 10, 111 25 of 26

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