Identification of phase-II metabolites from human serum samples after oral intake of a willow bark extract

Identification of phase-II metabolites from human serum samples after oral intake of a willow bark extract

Accepted Manuscript Identification of phase-II metabolites from human serum samples after oral intake of a willow bark extract Monika Untergehrer , J...

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Accepted Manuscript

Identification of phase-II metabolites from human serum samples after oral intake of a willow bark extract Monika Untergehrer , Josef Kiermaier , Susanne Reintjes , Jorg ¨ Heilmann , Guido Jurgenliemk ¨ PII: DOI: Reference:

S0944-7113(18)30560-9 https://doi.org/10.1016/j.phymed.2018.11.003 PHYMED 52742

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

26 January 2018 2 November 2018 3 November 2018

Please cite this article as: Monika Untergehrer , Josef Kiermaier , Susanne Reintjes , Jorg , Identification of phase-II metabolites from human ¨ Heilmann , Guido Jurgenliemk ¨ serum samples after oral intake of a willow bark extract, Phytomedicine (2018), doi: https://doi.org/10.1016/j.phymed.2018.11.003

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ACCEPTED MANUSCRIPT Identification of phase-II metabolites from human serum samples after oral intake of a willow bark extract Monika Untergehrera, Josef Kiermaierb, Susanne Reintjesa, Jörg Heilmanna, Guido Jürgenliemka* a

Fakultät für Chemie und Pharmazie, Lehrstuhl für Pharmazeutische Biologie, Universität

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Regensburg, 93040 Regensburg, Deutschland

Fakultät für Chemie und Pharmazie, Zentrale Analytik, Universität Regensburg, 93040

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Regensburg, Deutschland

*Corresponding author.

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PD Dr. Guido Jürgenliemk

Fakultät für Chemie und Pharmazie

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Lehrstuhl für Pharmazeutische Biologie Universität Regensburg

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93040 Regensburg, Deutschland Tel.: +49 (0)9419434758; Fax.: +49 (0)9419434990 E-mail: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT

Background: Willow bark (Salicis cortex) is an herbal medicinal drug used to treat fever and pain, such as headaches and lower back pain. Until now, it has not been fully understood which compounds are responsible for the efficacy of the drug. Purpose: Although salicylic acid is known as a metabolite of salicylic alcohol derivatives of willow bark in vivo, it has been shown in previous studies that its concentration is too low to

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account for the overall efficacy of Salicis cortex. The aim this study was to broaden the knowledge regarding phenolic phase-II metabolites after oral intake of a willow bark extract. Study design/methods: Serum samples of a human pharmacokinetic study (Salicis cortex extract intake corresponding to 240 mg of total salicin, 10 volunteers, 12 h fasting time, controlled diet low in phenolics, and 12 blood withdrawals over a period of 24 h) were

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analysed by LC-ESI-MS. A library of 142 possible metabolites associated with salicylic alcohol derivatives, flavonoids, and proanthocyanidins was used to characterize possible metabolisation products. Their structures were confirmed by LC-ESI-MS experiments with reference compounds after a cleavage reaction using glucuronidase and sulfatase as well as by

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LC-MS/MS experiments.

Results: In the serum samples, phase-II metabolites of naringenin (2x glucuronides, 2x sulfates, 2x mixed glucuronide-sulfates), eriodictyol (3x glucuronides, 1x sulfate), taxifolin

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(1x sulfate), catechin (1x sulfate, 1x mixed glucuronide sulfate), ferulic acid (1x sulfate), hydroxyphenyl-propionic acid (1x sulfate), dihydroxyphenyl-valerolactone (1x sulfate),

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saligenin (1x glucuronide, 1x sulfate), salicylic acid (1x sulfate, 1x unconjugated, 1x salicyluric acid), and catechol (1x glucuronide, 1x sulfate) were characterized. Because

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taxifolin, dihydroxyphenyl-valerolactone, ferulic acid, and hydroxyphenyl-propionic acid could not be detected in the willow bark preparation, they could be metabolisation products of

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genuine flavanones and flavan-3-ols as well as coumaric acid or C-ring cleavage products of flavonoids, which were present in the extract. No phase-II metabolites of procyanidins and no genuine flavonoid glycosides were detected in all serum samples. Conclusion: This is the first study to identify human metabolites of flavonoids, proanthocyanidins and salicylic alcohol derivatives of Salicis cortex beside salicylic acid or catechol. For the most characterized metabolites, anti-inflammatory activity has been described in the literature, and the present results are an important step in understanding the anti-inflammatory efficacy of willow bark in-vivo.

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ACCEPTED MANUSCRIPT Keywords

Willow bark, Salicis cortex, metabolisation, phase-II metabolites, flavonoids, serum samples

Abbreviations NAR, naringenin; NAR-chalk, naringenin-chalcone; ERI, eriodictyol; CAT, catechin; TAX, taxifolin; FA, ferulic acid; HPPA, hydroxyphenyl-propionic acid; CL, catechol; SG, saligenin;

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SA, salicylic acid; SUA, salicyluric acid; B3, procyanidin B3; DHVL, dihydroxyphenylvalerolactone, GluA, glucuronyl-

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Introduction

Herbal remedies from willow bark (Salicis cortex, Salix spp., Salicaceae) are used against fever, pain, and inflammation, and flavanones, procyanidins and salicylic alcohol derivatives are the main groups of ingredients (Nahrstedt et al. 2007). The clinical efficacy of

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willow bark has been shown in several clinical studies (Vlachojannis et al. 2009). In previous research regarding the active substances of the drug, salicylic acid has been identified as a metabolite of salicylic alcohol derivatives in humans. It was hypothesized to be responsible

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for the efficacy of Salicis cortex until it was found that its concentration in human plasma is much too low to explain the overall efficacy of the whole drug (Nahrstedt et al. 2007; Schmid

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et al. 2001). In a recent study, catechol was also identified as a metabolite of salicortin in human serum samples at relevant concentrations (Knuth et al. 2013). Because catechol has

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demonstrated anti-inflammatory activities in vitro (Knuth et al. 2011; Ma and Kinneer 2002; Zheng et al. 2008) and thus seems to be another important bioactive metabolite of Salicis

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cortex beside salicylic acid, this finding demonstrates the necessity to include in vivo metabolisation pathways for identifying bioactive compounds from herbal medicinal plants in addition to isolating substances from extracts and testing them in vitro (Kroon et al. 2004). While the metabolisation of salicylic alcohol derivatives has been well investigated, there is limited information regarding the metabolisation of flavonoids and proanthocyanidins from Salicis cortex in vivo (Nahrstedt et al. 2007). The flavanones naringenin and eriodictyol with their corresponding glucosides are the predominant flavonoids in willow bark. Regarding flavanone metabolisation, oral application of grapefruit or orange juice has resulted in the identification of naringenin-glucuronide, naringenin-di-glucuronide, naringenin-sulfate, and 3

ACCEPTED MANUSCRIPT naringenin-glucuronide-sulfate from plasma and especially urine samples (Manach et al. 2005, Mullen et al. 2008, Bredsdorff et al. 2010, Brett et al. 2009, Zhang and Brodbelt 2004). Furthermore, eriodictyol-sulfate has been identified in urine samples after oral intake of rooibos-tea and orange juice (Stalmach et al. 2009, Pereira-Caro et al. 2014). Considering the C-ring fission of flavonoids by gut microbiota and the resulting C6-C3, C6-C2, and C6-C1 compounds, the bioavailability of flavanones is nearly 100% (Pereira-Caro et al. 2014). Additionally, proanthocyanidins are poorly absorbed but extensively metabolized by the gut

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microbiome (Rodriguez- Mateos et al. 2014).

To identify important in vivo metabolites of Salicis cortex and to broaden the knowledge regarding the complex metabolisation pathway of phenolic compounds, serum samples of a human pharmacokinetic study of willow bark (Knuth et al. 2013) were analysed

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by HPLC-DAD-MS and screened for phase-I and phase-II metabolites over a period of 24 h.

Materials and methods

Chemicals and substances

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Acetonitrile (LiChrosolv®), formic acid, DMSO, methanol (LiChrosolv®, sodium acetate, and o-phosphoric acid were obtained from Merck (Darmstadt, Germany). Eriodictyol, eriodictyol-7-O-β-D-glucoside, naringenin, and naringenin-7-O-β-D-glucoside were obtained

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from Extrasynthese (Lyon, France). Ferulic acid was purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). 3-(4-hydroxyphenyl)-propionic acid, ammonium acetate for MS

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analysis, (+/-) catechin hydrate, catechol (1,2-dihydroxybenzene), salicylic acid, salicyluric acid (2-hydroxyhippuric acid), saligenine (2-hydroxybenzylic alcohol, taxifolin, sulfatase

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(from Helix pomatia, type H-1, lyophilized, > 10000 U/g), and β-glucuronidase (from bovine liver, type B-10, 10100 U/mg) were obtained from Sigma Aldrich (Taufkirchen, Germany).

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Willow bark preparation: Optovit® ActiFLEX (Hermes Arzneimittel GmbH, Großhesselohe, Germany; 70% ethanol as extraction solvent, drug-extract-ratio = 8-14 : 1). (2R/2S)naringenin-5-O-β-D-glucoside,

6’’-O-trans-p-coumaroyl-(2R/2S)-naringenin-5-O-β-D-

glucoside, 6’’-O-trans-p-coumaroyl-isosalipurposide, dihydrokaempferol, dihydrokaempferol7-O-β-D-glucoside, and isosalipurposide were isolated (Freischmidt et al. 2012); naringenin7-O-β-D-glucuronide was synthesized (Untergehrer et al. 2015).

Pharmacokinetic study

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ACCEPTED MANUSCRIPT Serum samples from a previously described pharmacokinetic study (Knuth et al. 2013) were used to identify phase-II metabolites of phenolic compounds. Briefly, after a 9 h fasting time, 8 healthy volunteers consumed a willow bark extract corresponding to 240 mg of total salicin (information from the package insert from the herbal remedy), 29.1 mg of total naringenin and 1.6 mg of total eriodictyol (quantified with the method reported by Freischmid and colleagues 2015). Twelfe blood samples were obtained before and up to 24 h after Salicis cortex intake. Two volunteers did not take the willow bark extract, but they participated in the

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study to determine the possible effects of the diet. All ten subjects followed a controlled diet 19 h before the study and throughout the pharmacokinetic study.

Non-enzymatic sample preparation

To characterize conjugated phenolic phase-II metabolites, 30 µl of a cold sodium

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acetate buffer (2 M, adjusted to pH 4.7 with acetic acid (20 mg/ml) was added to 270 µl of the cold serum samples. After adding 900 µl of ice-cold acetonitrile, the mixture was centrifuged (14000 rpm, 4 °C, 20 min) and the clear supernatant was dried under nitrogen. The residue was resuspended in a mixture of 50 µl 95% acetonitrile and 100 µl 0.1% acetic acid and

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centrifuged again (14000 rpm, 4 °C, 5 min). After filtration, the samples were analysed by HPLC-DAD-MS.

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Enzymatic sample preparation

To cleave the conjugated phase-II metabolites and to identify the resulting phenolic

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compounds or aglycones, 3.5 mg of sulfatase and 30 µl of the 2 M sodium acetate-ascorbic acid buffer (10 mg/ml, see above) containing 10 mg/ml β-glucuronidase was added to 270 µl

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of the serum samples (final concentration of 35 U sulfatase and 3030 U β-glucuronidase).

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After 1.5 h of incubation at 37 °C, the samples were processed as described above.

Sample preparation of reference compounds For HPLC-DAD-MS analysis, all reference compounds were dissolved in DMSO at a

concentration of 100 mM and diluted (1:100) with 95% acetonitrile. An aliquot (1.5 µl) was added to 50 µl of 95% acetonitrile and 100 µl of 0.1% acetic acid. For HPLC-DAD analysis, the reference compounds were dissolved in 70% methanol at a concentration of 500 µM. To identify genuine compounds from the willow bark preparation, one dragée Optovit® ActiFLEX was pulverised and dissolved in acetonitrile using an ultrasonic bath (20 min). 5

ACCEPTED MANUSCRIPT After preparing a 1:100 dilution with acetonitrile, the solution was used for HPLC-DAD-MS analysis. The procedure was identical for HPLC-DAD analysis, but 70% methanol was used as the solvent (Figure Supp. 1a and b).

HPLC-DAD Elite LaChrom with an L2200 autosampler, L2130 pump, L2350 column oven, L2444 DAD and EZChrom Elite 3.1.7 software (Hitachi, Tokio, Japan); column: Hibar®RT 250-4

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cartridge with Purosphere®Star RP-18e (5 µm, same material pre-column; Merck, Darmstadt, Germany); injection volume: 20 µl; oven temp.: 40 °C; auto sampler temp.: 10 °C; detection wavelength: 289 nm; flow: 0.6 ml/min; A = 0.5% o-phosphoric acid 85%, B = methanol; gradient: 0-45 min 20% B → 80% B, 45-50 min 80% B, 50-51 min 80% B → 20% B, 51-60

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min 20% B

HPLC-DAD-MS

Agilent G4220A binary pump, G4226A HiP sampler, G1316C column comp. and G4212A DAD (Agilent Technologies, Santa Clara, USA); column: Phenomenex Kinetex (5

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µm), XB-C18, 100 Å, 250 x 4.6 mm (Phenomenex, Aschaffenburg, Germany); injection volume: 20 µl; oven temp.: 30 °C; auto sampler temp.: 5 °C; detection wavelength: 190-640 nm; flow: 1.0 ml/min; A = ammonium acetate (10 mM) in water, B = acetonitrile; gradient: 0-

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3 min 5% B, 3-15 min 5% B → 60% B, 15-16 min 60% B → 95% B, 16-19 min 95% B, 1920 min 95% B → 5% B, 20-24 min 5% B. All samples were filtered (Perfect-Flow RC

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membrane (0.2 µm), Wicom, Heppenheim, Germany) before injection. Agilent MS Q-TOF 6540 UHD, ion source: AJS ESI G6540A (Agilent Technologies, Santa

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Clara, USA); detection range: 80-1200 m/z; ion-polarity: negative; scan-rate: 3.00 spectra/sec; gas-temp.: 300 °C; gas-flow: 10 l/min; nebulizer: 45 psi; sheathing-gas-temp.: 300 °C;

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sheathing-gas-flow: 12 l/min; for MS/MS experiments, the collision gas was nitrogen and the collision energy 15 eV. The evaluation of all MS data was performed with Masshunter Qualitative Analysis B.05.00 G6540A (Agilent Technologies, Santa Clara, USA). The masses of the resulting signals were compared with the exact masses of 142 possible phenolic metabolites including phase-II conjugates. A table of all metabolites (comprising the exact masses) and EIC-peak (extracted ion chromatogram) area/time curves of detected metabolites are provided in the Supplementary materials (Table Supp. 1, Figures Supp. 2 & 3). As a representative example, the ESI-TIC, MS and MS/MS spectra for naringenin-glucuronide (m/z 477.0940 [M6

ACCEPTED MANUSCRIPT H]- from the serum sample of one subject at t = 0.5 are shown in Figure Supp. 4. MS chromatogram peak filter; ≥ 1000 counts, ≤ 10 ppm, molecular feature extraction: m/z range = 100-1200, ionic charge = negative, allowed ions = - H and + CH3COO.

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Results and Discussion

Metabolisation of flavonoids

After analysing all serum samples from a pharmacokinetic study of willow bark extract by HPLC-DAD-MS, -MS/MS, and HPLC-DAD experiments, 23 metabolites were characterized (Table & Figure 1) out of a library containing 142 possible metabolites (117

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different masses, Table Supp. 1). Naringenin-glucuronide was detected at two and eriodictyol-glucuronide at three different retention times. This could be due to stereochemical differences at C-2 (2-R/2-S diastereomers) or due to different glucuronidation positions. Naringenin is presumably glucuronidated at C-7 and C-4’ (Brett et al. 2009), and a

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conjugation at C-7, C-4’, and C-3’ is likely for eriodictyol. A conjugation at C-5 is unlikely due to the hydrogen bond between the C-4 carbonyl and the OH group of C-5. Sulfated monoconjugates of flavonoids were detected for naringenin (two compounds), catechin, eriodictyol,

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and taxifolin, and mixed di-conjugates (glucuronide/sulfate) were also found for catechin and naringenin (two compounds). Whereas the sulfation of eriodictyol seems to be predominant in

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contrast to its glucuronidation, one glucuronide and one sulfate of naringenin could be detected as predominant phase-II metabolites (Table 2). Glucuronide-sulfate-diconjugates

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were present in only approximately half of the subjects. Because the flavanonol taxifolin was not detected in the genuine extract, it could be an oxidized metabolite of the flavanones

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eriodictyol or naringenin (Fig. Supp. 5a). Until now, the possibility can not be excluded that the detected eriodictyol-conjugates are likely dihydrokaempferol-conjugates because both have the same molecular mass and the conjugates were neither cleaved with glucuronidase or sulfatase nor cleaved by LC-MS/MS experiments. This could only be unambiguously clarified with reference compounds. Dihydroxyphenyl-valerolactone-sulfate was identified as a ring fission product of flavan-3-ols which was also reported in previous pharmacokinetic studies for example with cranberry juice (e.g. Feliciano et al. 2017, Rodriguez-Mateos et al. 2016). Due to the lack of reference material, this expected metabolite was speculated by its fragmentation (M-sulfate). 7

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Metabolisation of phenylpropanoids As phenylpropanoid metabolites, sulfated ferulic acid and hydroxyphenyl-propionic acid were characterized in the samples. Ferulic acid could be an oxidized metabolization product of coumaroylated flavonoids which can be found in Salicis cortex (Freischmidt et al. 2012). Free ferulic acid, coumaric acid, and hydroxyphenyl-propionic acid, were not be detected in the ingested genuine extract. The latter metabolite could be a C-ring fission product of flavanones

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(Orrego-Lagaron et al. 2015) or condensed tannins (Rodriguez-Mateos et al. 2014) and ferulic acid could be again an oxidized and methylated product metabolized of it. Furthermore, it could be a deoxidized product of coumaric acid (Fig. Supp. 5a). However, it is very interesting that it was found 0.5 h after intake of the extract (Table 2). Therefore, the

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metabolisation seems to be too fast to be influenced by the colon microbiome.

Metabolisation of salicylic alcohol derivatives

The appearance of salicylic alcohol derivative metabolites has been shown in previous pharmacokinetic studies in humans (Schmid et al. 2001, Knuth et al. 2013). In contrast to

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saligenin-glucuronide, saligenin-sulfate could be found in all 8 volunteers (Table 3). Whereas Knuth and co-workers (2013) identified catechol-sulfate as the main metabolite of salicortin by HPLC-DAD analysis, in this study by HPLC-ESI-MS, catechol-glucuronide was also

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characterized in all subjects as well as salicylic acid together with its phase-II metabolites salicylic acid-sulfate and salicyluric acid (Fig. Supp. 5b). Remarkably, catechol-sulfate (8

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subjects), salicylic acid (7 subjects), salicyluric acid (1 subject), and dihydroxyphenylvalerolactone-sulfate (6 subjects) could be characterized in serum samples before the willow

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bark extract was consumed.

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Conclusion

In conclusion, after oral intake of a willow bark extract, phase-II metabolites of ten phenolic compounds could be characterized and analysed over time as follows: four flavonoids with one ring fission product, two phenylpropanoic acid derivatives and three metabolites of salicylic alcohol derivatives. No condensed tannins, neither conjugated nor unconjugated, such as dimeric procyanidins were detected in the serum samples. Structures possessing a catechol element, such as eriodictyol or taxifolin, were sulfated rather than glucuronidated. Catechol-sulfate, salicylic acid, salicyluric acid, and dihydroxyphenyl-valerolactone-sulfate were detected in serum samples before the willow bark extract was consumed. This was also 8

ACCEPTED MANUSCRIPT observed in previous studies e.g., by Baxter and co-workers (2002) or Rodriguez-Mateos and co-workers (2016). An explanation could be that the fasting time and the period of the phenolic free diet were too short. This was indicated by the the detection of dihydroxyphenylvalerolactone, saligenin, salicylic acid, and catechol as phase-II metabolites in the serum samples of 4 to 6 volunteers over 24 h after Salicis cortex extract intake in this study and the finding of C6-C3, C6-C2, C6-C1 (for example, salicyluric acid), and C6-C0 (for example, catechol) metabolites after food intake in previous studies (Rodriguez-Mateos 2014). This

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assumption corresponds to the results of the semi quantitative analysis of the metabolites over time. Phase-II metabolites of flavonoids tentatively appeared and disappeared faster than the metabolites of dihydroxyphenyl-valerolactone, salicylic acid, saligenin and catechol (Fig Supp 2 & 3). Additionally, it could be that those substances are constitutively made by the human body, as salicyluric acid could be found as a metabolite of tryptophane (Austad et al.

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1965).

For naringenin (Jin et al. 2017), eriodictyol (Lee et al. 2013), taxifolin (Manigandan et al. 2015), catechin (Abd El-Aziz 2012 et al.), dihydroxyphenyl-valerolactone (Uhlenhut and Högger 2012), ferulic acid (Zhu et al. 2014), hydroxyphenyl-propionic acid (Arnic et al.

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2016), catechol (Knuth et al. 2011) and salicylic acid (Kwon and Chae 2003), antiinflammatory activity has been shown in-vitro or in-vivo. Additionally, Mülek and coworkers (2017) found phenolic metabolites in different human tissues after intake of a maritime pine

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bark extract, which contains similar proanthocyanidins as those found in Salicis cortex. Catechin and taxifolin were mainly present in blood cells, and dihydroxyphenyl-

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valerolactone, ferulic and caffeic acid were predominantly present in the synovial fluid (Mülek et al. 2017). The discrepancy that most in-vitro activities are obtained with non-

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conjugated compounds although in-vivo phase II-metabolites (-glucuronides, and -sulfates) are predominant, can be explained by recent results showing that phase II-conjugates can be

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cleaved by human beta-glucuronidase, especially in inflamed tissues (Ishisaka et al. 2013, Bartholomé et al. 2010, O´Leary et al. 2001, Untergehrer et al. 2015). Thus, the combination of absorbed phenolic compounds from the willow bark extract and their phenolic metabolites seems to be responsible for the anti-inflammatory efficacy of the whole Salicis cortex extract. This result is important because the serum-concentration of the single metabolite salicylic acid is too low to explain this efficacy (Schmid et al. 2001). Although this study is maybe limited by an incomplete MS-library, these results, in combination with previous findings in the literature regarding the metabolisation and

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ACCEPTED MANUSCRIPT pharmacological activities of phenolic compounds, aid in understanding the antiinflammatory efficacy of Salicis cortex.

Acknowledgements Gabriele Brunner, Dept. of Pharmaceutical Biology, University of Regensburg, is gratefully acknowledged for technical assistance.

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Conflict of interest The authors declare no conflicts of interest.

Supplementary materials

A total compound chromatogram of the medication, a library of all considered metabolites

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and area-time-curves of detected metabolites from extracted ion chromatograms are given in the Supplementary materials.

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ACCEPTED MANUSCRIPT

NAR

CR IP T

ERI

CAT

AN US

HPPA

SG

SA

SUA

AC

CE

PT

ED

M

FA

CL

TAX

DHVL Fig. 1. Structures of NAR, ERI, CAT, TAX, FA, HPPA, CL, SG, SA, SUA, and DHVL

14

ACCEPTED MANUSCRIPT Graphical abstract

23 phenolic metabolites could be detected in human serum samples by LC-MS analysis

AC

CE

PT

ED

M

AN US

CR IP T

Oral intake of a willow bark extract

15

ACCEPTED MANUSCRIPT Table 1 Identification of phenolic phase-II metabolites in human serum samples after oral intake of a willow bark extract using HPLC-DAD-MS/MS fragmentation and comparison of the retention time (RT) with a referent compount (r. c.) after enzymatic hydrolysis (a. e. h.); n.d.: not detected, n.e.: not examined, exp.: experimental [M-H]exp.

fragment ions

RT (min) aglykone a. e. h.

RT (min) aglykone r. c.

possible metabolite

9.73 9.79

447.0945 447.0948

271.0625 (-GluA); 175.0258 (NAR) 271.0620 (-GluA); 175.0254 (-NAR)

14.11

14.08

NARglucuronide

7.72 9.64 10.00

463.0882 463.0874 463.0883

n.d. 287.0564 (-GluA); 287.0563 (-GluA); 175.0213 (NAR)

n.d.

8.36 9.40

527.0508 527.0508

n.d. n.d.

14.11

9.79

545.0636

369.0250 (-GluA)

11.28 11.69

351.0174 351.0199

271.0611 (-SO3) 271.0609 (-SO3)

8.57

369.0292

11.48

CR IP T

RT (min)

ERIglucuronide

14.08

NARglucuronidesulfate

9.19

CATglucuronidesulfate

14.11

14.08

NAR-sulfate

289.0718 (-SO3)

n.d.

9.19

CAT-sulfate

367.0135

287.0563 (-SO3)

n.d.

13.60

ERI-sulfate

10.04

383.0079

303.0512 (-SO3)

n.d.

11.46

TAX-sulfate

5.51

285.0616

n.d.

9.25

9.38

CLglucuronide

7.18

299.0783

9.11

9.17

SGglucuronide

8.43 7.86

PT

ED

M

n.d.

CE

AN US

13.60

123.0451 (-GluA)

137.0251 (-SO3)

7.90

7.96

SA-sulfate

188.9864

109.0302 (-SO3)

9.25

9.38

CL-sulfate

7.05

203.0028

123.0456 (-SO3)

9.11

9.17

SG-sulfate

4.91

273.0083

193.0504 (-SO3); 178.0260 (-CH3)

7.18

7.22

FA-sulfate

9.50

245.0494

165.0926 (-SO3); 79.9578 (-HPPA)

n.d.

n.d.

HPPA-sulfate

7.92

137.0241

n.e.

n.e.

7.96

SA

7.96

194.0458

n.e.

n.e.

7.99

SUA

9.26

287.0216

207.0645 (-SO3)

n.e.

n.e.

DHVL-sulfate

AC

216.9819

16

ACCEPTED MANUSCRIPT Table 2 Number of subjects (a total of 8 volunteers) in which the following flavonoid metabolites occure; 0-24 h after oral intake of a willow bark extract; b: breakfast, l: lunch, d: dinner, RT: retention time

Time of blood withdrawal after oral intake of the willow bark extract (h)

NAR-glucuronide

0.5

1

1.5

2

3

4

6

8

10

9.73

5

8

8

8

8

8

3

4

3

NAR-glucuronide

9.79

5

4

3

4

3

3

3

1

NAR-sulfate

11.28

8

7

8

8

7

6

3

2

NAR-sulfate

11.69

7

7

6

5

5

NAR-glucuronidesulfate

8.36

2

4

5

5

5

NAR-glucuronidesulfate

9.40

2

ERI-glucuronide

7.72

4

ERI-glucuronide

9.64

5

ERI-glucuronide

10.00

ERI-sulfate

TAX-sulfate

AC

CAT-glucuronidesulfate DHVL-sulfate

5

4

4

3

2

4

3

2

5

4

3

2

11.48

7

6

6

8

10.04

8

8

7

4

8.57

8

8

8

9.79

6

5

6

5

ED

M

2

PT

CE

CAT-sulfate

0

9.

6

12

24

CR IP T

RT (min)

5

1

1

1

1

5

5

1

3

AN US

Possible metabolite

4

2

7

7

2

3

8

7

2

2

1

5

2

3

1

5

6

7

7

7

5

b

17

l

d

4

ACCEPTED MANUSCRIPT Table 3 Number of subjects (a total of 8 volunteers) in which the following flavonoid, phenylpropanoid, or salicylic alcohol derivative metabolites occure; 0-24 h after oral intake of a willow bark extract; b: breakfast, l: lunch, d: dinner, RT: retention time

Possible metabolite

RT (min)

FA-sulfate

4.91

7

6

7

6

5

HPPA-sulfate

9.50

6

5

2

2

1

SG-sulfate

7.05

8

8

8

8

8

SG-glucuronide

7.18

2

4

4

5

CL-sulfate

7.86

CL-glucuronide

5.51

8

SA-sulfate

8.43

8

AN US

Time of blood withdrawal after oral intake of the willow bark extract (h)

SA

7.94

7

SUA

7.96

1

8

1.5

2

3

4

6

6

5

8

4

10

12

2

2

CR IP T

1

2

8

24

1

1

8

7

6

6

6

7

6

6

6

8

8

8

8

8

7

8

7

6

6

4

1

8

8

8

8

8

7

6

5

4

1

8

8

8

8

7

8

7

7

6

6

5

8

8

8

8

8

8

7

7

7

6

5

b

AC

CE

PT

ED

8

0.5

M

0

18

l

d