Oct 21, 2013 - tions catechol can be detected in addition to salicylic acid as an in ... cylic acid such as salicylic alcohol, gentisic acid, salicyluric acid,.
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Catechol Conjugates Are In Vivo Metabolites of Salicis cortex
DOI 10.1055/s-0033-1350898 Planta Med 2013; 79: 1489–1494
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Catechol Conjugates Are In Vivo Metabolites of Salicis cortex
Authors
Susanne Knuth 1, Rania M. Abdelsalam 2, Mohamed T. Khayyal 2, Frank Schweda 3, Jörg Heilmann 1, Martin Georg Kees 4, 5, Georg Mair 6, Frieder Kees 7, Guido Jürgenliemk 1
Affiliations
The affiliations are listed at the end of the article
Key words " Salix l " Salicaceae l " catechol l " salicortin l " metabolism l " conjugates l " salicylic acid l " pharmacokinetic study l " in vivo l
Abstract
April 22, 2013 August 22, 2013 August 27, 2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1350898 Published online October 21, 2013 Planta Med 2013; 79: 1489–1494 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Guido Jürgenliemk Institute of Pharmacy Pharmaceutical Biology University of Regensburg Universitätsstr. 31 93053 Regensburg Germany Phone: + 49 (0) 94 19 43 47 58 Guido.Juergenliemk@ chemie.uni-regensburg.de
After oral administration of 100 mg/kg b. w. (235.8 µmol/kg) salicortin to Wistar rats, peak serum concentrations of 1.43 mg/L (13.0 µM) catechol were detected after 0.5 h in addition to salicylic acid by HPLC‑DAD after serum processing with β-glucuronidase and sulphatase. Both metabolites could also be detected in the serum of healthy volunteers following oral administration of a willow bark extract (Salicis cortex, Salix spec., Salicaceae) corresponding to 240 mg of salicin after processing with both enzymes. In humans, the cmax (1.46 mg/L, 13.3 µM) of catechol was reached after 1.2 h. The predominant phase-II metabolite in humans and rats was catechol sulphate, determined by HPLC analysis of serum samples processed with only one kind of enzyme. Without serum processing with glucuronidase and sulphatase, no unconjugated catechol could be detected in human and animal serum samples.
Introduction !
Salicylic alcohol derivatives are common secondary plant metabolites in the family Salicaceae. Most of these derivatives, such as salicortin, possess a 1-hydroxy-6-oxo-2-cyclohexenecarboxylate moiety, which has also been detected and connected to flavan-3-ols [1–3]. This special moiety degrades to catechol in alkaline solutions [4] or in cell culture medium [5], which has also been demonstrated for salicortin. Furthermore, catechol itself can be detected in different foods such as coffee, beer, bread crust, and fruits [6, 7]. This antioxidative compound is able to reduce LPS-induced TNF-α expression in macrophages [8], LPSinduced NF-κB activation, NO, and TNF-α production in BV-2 microglia cells [9] and TNF-α-induced ICAM-1 expression in endothelial cells [5]. Yang et al. demonstrated that among multiple
As catechol is described as an anti-inflammatory compound, these results may contribute to the elucidation of the mechanism of the action of willow bark extract.
Abbreviations !
WBE: cmax: tmax: AUC: AUC0–8 h: AUC0-∞:
willow bark extract maximum observed concentration time of cmax area under the serum concentrationtime curve area under the serum concentrationtime curve from 0 to 8 hours area under the serum concentrationtime curve from 0 to infinity
Supporting information available online at http://www.thieme-connect.de/ejournals/toc/ plantamedica
isolated substances from Salix pseudo-lasiogyne, only those with a 1-hydroxy-6-oxo-2-cyclohexenecarboxylate moiety were able to decrease the LPS-induced NO production in BV-2 cells [10]. For antioxidants, an anti-inflammatory and cell protecting effect via the activation of the Nrf2KEAP1 pathway has been described [11–13]. Recent results indicated that a willow bark extract and fractions thereof activated this Nrf2 pathway and prevented endothelial cells from experiencing oxidative stress-induced cytotoxicity [14]. These findings clearly demonstrate that catechol and other polyphenols can play an important role in the anti-inflammatory activity of Salicis cortex. Salicylic acid was already identified as a metabolite of the salicylic alcohol derivatives in animal studies and humans after oral intake of willow bark extracts (Salicis cortex, Salix spec, Salicaceae.) or single compounds such as salicin, re-
Knuth S et al. Catechol Conjugates Are …
Planta Med 2013; 79: 1489–1494
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Fig. 1 Catechol and salicylic acid concentration in glucuronidase- and sulphatase-processed serum samples of male Wistar rats after oral application of 100 mg/kg b. w. salicortin; n = 7 for each time point; mean ± SD.
spectively [15–18]. The anti-inflammatory and analgesic activity of Salicis cortex has been demonstrated in several animal models [19, 20] and human studies [21–23]. The metabolite salicylic acid cannot be responsible for these effects alone as its plasma concentration is too low after oral application of willow bark extract [17, 24]. Additional extract ingredients or metabolites must contribute to the overall effect [24]. The questions of the present study were if and in what concentrations catechol can be detected in addition to salicylic acid as an in vivo metabolite in serum after oral administration of salicortin to Wistar rats and after oral intake of willow bark extract in human volunteers.
Fig. 2 Catechol and salicylic acid concentration in glucuronidase- and sulphatase-processed serum samples of healthy volunteers after ingestion of WBE corresponding to 240 mg of salicin; n = 8; mean ± SD.
Fig. 3 Structural formulas
Results !
After oral administration of 100 mg/kg b. w. (235.8 µmol/kg b. w.) salicortin to Wistar rats, blood was withdrawn over a period of 8 h. The resulting serum samples of 7 animals for each time point were processed with sulphatase and β-glucuronidase and then analysed by HPLC‑DAD. Catechol, salicylic acid, and further me" Fig. 1, catechol is tabolites should be detected. As observed in l a metabolite of salicortin in rats. The cmax was already reached after 30 minutes with a concentration of 13.0 µM (1.4 mg/L). An AUC0–8 h of 35.4 µM·h (3.9 mg · h/L) for catechol was determined. Without processing with enzymes, no free catechol could be detected. These results demonstrate, for the first time, that catechol conjugates are relevant in vivo metabolites of salicortin after oral administration of this substance, which is one of the most common salicylic alcohol derivatives in willow bark. The appearance of salicylic acid in rat sera was different from catechol. The cmax was reached 4 hours after salicortin administration with a concentration of 10.9 µM (1.5 mg/L), and an AUC0–8 h of 54.4 µM · h (7.5 mg · h/L) was determined. Other possible metabolites of salicylic acid such as salicylic alcohol, gentisic acid, salicyluric acid, or guajacol, which is the methylated metabolite of catechol, were not detectable in the serum samples.
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In a pharmacokinetic study with 10 healthy volunteers, the appearance of catechol, salicylic acid, and further metabolites in their serum was investigated after oral intake of a standardised WBE. Eight subjects received a single dose of WBE corresponding to 240 mg of total salicin. HPLC quantification of the extract revealed that a dose of 89.8 µmol (9.9 mg) free catechol, 346.6 µmol (147.0 mg) salicortin, and 21.4 µmol (11.3 mg) tremulacin (which also possess a 1-hydroxy-6-oxo-2-cyclohexenecar" Fig. 3) was administered. Two volunteers boxylate moiety, l served as the control group. They did not take the WBE, but they received the standardised meals and blood draws during the
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Salicortin in rats
"
WBE in humans
"
"
"
salicylic acid catechol salicylic acid catechol
tmax (h)
cmax (µM)
AUC (µM · h)
t1/2 (h)
4.0 0.5 2.7 ± 1.0 1.2 ± 0.4
10.9 13.0 24.5 ± 11.9 13.3 ± 3.2
54.4# 35.4# 127.8 ± 61.6+ 60.8 ± 21.5+
2.4 1.0 2.4 ± 0.8 2.5 ± 0.5
study to determine the possible effects of diet on the detected metabolites. The human sera were processed analogously to the rat sera with enzymes to cleave the phase-II conjugates before HPLC analysis. Peak concentrations of 13.3 µM (1.5 mg/L) catechol " Fig. 2). The were obtained 1.2 hours after intake of the WBE (l area under the curve (AUC0-∞) was determined to be 60.8 µM·h (6.7 mg · h/L). With the exception of one volunteer, there was no catechol detectable after 10 h, and no catechol was found after 24 h in any serum sample. No catechol could be detected in serum samples analysed without processing with enzymes. Maximum salicylic acid concentrations of 24.5 µM (3.4 mg/L) could be observed after 2.7 hours and an AUC0-∞ of 127.8 µM · h (17.7 mg · h/L) for salicylic acid was determined. Salicylic alcohol, salicyluric acid, gentisic acid, and guajacol could not be detected in the serum samples. Catechol and salicylic acid were not detectable in both control subjects. Three animal and human serum samples with the highest concentration of catechol obtained by HPLC analysis after enzyme processing were additionally analysed without enzymes or only with one type of enzyme. Without the addition of sulphatase and β-glucuronidase, catechol could neither be detected by HPLC‑DAD in the rat serum samples nor in the human serum samples. These samples were also examined by HPLC-HRMS. In animal and human serum, the molecular mass of catechol-sulphate (188.9859 g/mol) was easily observed whereas the peak of catechol-glucuronide (285.0616 g/mol) was very small. In accordance with Schmid et al. [17], we also observed that salicylic acid occurs mainly as the free unconjugated molecule in human or rat serum, as there was no difference between the enzymatic and nonenzymatic processed samples. In the human serum samples that were only processed with β-glucuronidase, no catechol could be detected by HPLC‑DAD, whereas in the rat serum samples, there was a mean catechol concentration of 2.5 µM (0.3 mg/ L). In contrast, catechol was easily observed in the serum samples of both species when only sulphatase was added (mean concentration in the Wistar rat serum: 17.8 µM, 19.6 mg/L). These results demonstrate that free catechol or catechol glucuronide do not predominate but that catechol sulphate predominates as a " Table 1 gives an overview of the pharmacometabolite in vivo. l kinetic results.
Discussion !
Salicylic acid has already been described as a metabolite of different salicylic alcohol derivatives in animals and humans, and has been detected after oral administration of standardised willow bark extracts or purified compounds [15–18]. Pentz et al. observed a maximum salicylic acid concentration of 0.13 mg/L in humans after oral intake of a willow bark extract containing
55 mg of total salicin [18], which is a 4.36-fold lower dose than the total salicin that was applied in the present study. The highest salicylic acid concentration in plasma in the present study with 3.4 mg/L is 6-fold higher than the cmax in the study of Pentz et al. considering a 4.36-fold higher concentration of administered salicylic alcohol derivatives. The matrix effects of the different extracts and coadministration of a cola extract in the study of Pentz et al. could explain the different results. The cmax of salicylic acid in humans determined by Schmid et al. was 1.2 mg/L after oral administration of a willow bark extract containing 240 mg of total salicin [17]. However, the peak concentrations and time of peak concentrations are difficult to compare, as the WBE was administered as two equivalent dosages at 0 h and after 3 h. Nevertheless, the AUC0-∞ of salicylic acid in the present study (127.8 µM · h) is in the range of that published by Schmid et al. (99.0 µM · h). Schmid et al. also demonstrated that salicylic acid is conjugated with glycine to salicyluric acid and is oxidised to gentisic acid via further metabolisation with a very low concentration of both substances in serum and a relatively high concentration of salicyluric acid in urine. In the present study, neither salicyluric acid nor gentisic acid could be detected in the serum samples. With the present pharmacokinetic results with rats, it is clearly demonstrated that catechol is a metabolite of salicortin in vivo and predominates as catechol sulphate. This is the first report of catechol being an in vivo metabolite of compounds containing a 1-hydroxy-6-oxo-2-cyclohexenecarboxylate moiety. This finding confirms the in vitro results that catechol is a degradation product of salicortin [4, 5]. Until now, there have been no findings about the transformation in detail or whether enzymes are involved. The AUC0–8 h of salicylic acid is approximately 1.5-fold higher than that of catechol after oral administration of salicortin in rats. Pearl and Darling demonstrated that under certain acid conditions, salicortin is able to convert to salicoylsalicin, which could deliver 2 equivalents of salicylic acid [25]. This conversion could explain the higher bioavailability of salicylic acid derived from salicortin, but the in vivo relevance of this phenomenon was disproved by Buß [26]. In that study, the bioavailability of salicylic acid after oral administration of salicortin and salicin in healthy volunteers was determined. The semimolar administration of salicortin resulted in a ~ 30% bioavailability of salicylic acid compared with salicin as determined by AUC. As 1 mol salicortin could supply 1 mol salicylic acid and 1 mol catechol, the lower bioavailability of catechol could be due to its lower stability in serum, to its tissue distribution, or to the fact that the final degradation of salicortin to catechol is not as extensive as its biotransformation to salicylic acid. Further investigations about the detailed transformation of salicortin to catechol in vivo could aid in understanding the inconsistent findings.
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Planta Med 2013; 79: 1489–1494
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Table 1 Pharmacokinetic parameters of catechol and salicylic acid (after serum processing with glucuronidase and sulphatase) following the single dose administration of salicortin (100 mg/kg b. w.) in rats and WBE (4 coated tablets of WBE corresponding to 240 mg of salicin) in healthy human volunteers; values = mean ± standard deviation, + = AUC0-∞, # = AUC0–8 h. Because in rats only one measurement per individuum was available, mean pharmacokinetic parameters, but no individual values or standard deviations, could be calculated.
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In the pharmacokinetic study in humans, catechol could also be observed in serum after oral intake of a willow bark extract. The compound predominates not as the free molecule but conjugated with sulphate. As the extract also contained free catechol itself, it can be assumed, concerning the pharmacokinetic results from the rat experiments, that the observed serum catechol is a mixture of both the administered catechol and the metabolisation product of extract ingredients containing a 1-hydroxy-6-oxo-2cyclohexenecarboxylate moiety such as salicortin. In both serum samples of humans and rats, no free catechol could be detected. The predominant phase-II metabolite was catechol sulphate. Phase-II metabolites such as glucuronides or sulphates are very polar substances that often have less pharmacological activities than the parent compounds, along with a renal or biliary excretion [27]. The same effect can be observed for many polyphenols, such as the very similar catechol derivative hydroxytyrosol. This anti-inflammatory and cardioprotective compound from olive oil also has a very low bioavailability and is rapidly sulphated or glucuronidated [28, 29]. Regarding these findings, most results obtained by in vitro assays with free, unconjugated substances appear questionable and should be complemented with evaluation results of possible phase-II metabolites. For instance, it has been demonstrated that some flavonoid conjugates do have pharmacological properties in vitro [30, 31]. On the other hand, there are recent findings that the activity of deconjugating enzymes, such as β-glucuronidase, may be increased in inflamed tissue [32]. Moreover, in a study with hypertensive rats, the lowering effect of quercetin glucuronides (i. v.) on blood pressure could be neutralised by simultaneous inhibition of βglucuronidase [33]. Further studies will focus on the synthesis of catechol metabolites and their pharmacological activities in vitro. The present results identified compounds containing a 1-hydroxy-6-oxo-2-cyclohexenecarboxylate moiety such as salicortin as a potential new class of anti-inflammatory substances, as they are able to metabolise to catechol in vivo, which seems to predominate as catechol sulphate. In addition to salicylic acid, catechol and its conjugates are further metabolites that may contribute to the analgesic and anti-inflammatory efficacy of Salicis cortex. These results underline the importance of determining the stability of substances in in vitro experiments and to seek active metabolites in vivo.
Materials and Methods !
Chemicals and reagents Salicortin (purity = 96.4 %, HPLC) and tremulacin (purity = 85.7%, HPLC) were isolated from a Salicis cortex extract (see Supporting Information). Sulphatase (Helix pomatia Type H-1, 22 400 U/g), βglucuronidase (bovine liver Type B-10, 10 100 U/mg), L-ascorbic acid (purity = 99 %), catechol (purity ≥ 99 %), salicylic acid (purity ≥ 99%), 2,5-dihydroxybenzoic acid (= gentisic acid, purity = 98 %), guajacol (purity ≥ 98%), and TFA (purity ≥ 95 %) were obtained from Sigma-Aldrich. o-Hydroxyhippuric acid (= salicyluric acid, purity ≥ 96%) was obtained from Carl Roth and 4-methylcatechol (purity ≥ 95 %) from Fluka. Methanol (HPLC-grade, Lichrosolv®) and H3PO4 (p. a., 85 %) were obtained from Merck. For the WBE, Optovit acitflex® (Hermes Arzneimittel GmbH, Germany; batch No: 0 113 911) was used. The WBE was a Salicis cortex extract (70 % EtOH, 8–14 : 1), and one coated tablet is equivalent to 60 mg of salicin.
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Pharmacokinetic study in humans The pharmacokinetic study in humans (EudraCT‑No. 2011– 001 829–24) was approved by the Ethics Committee of the Bavarian Medical Association, Munich, Germany, and by the Federal Institute of Drugs and Medical Devices (BfArM, Bonn, Germany). The study was performed with 10 healthy volunteers. The volunteers were allowed to eat only food that was poor in polyphenols and consisted of carbohydrates, animal fat, salt, and mineral water from 19 h before until 24 h after drug administration. Eight volunteers (4 m/4 f, age 21–28 years, body height 156–193 cm, body weight 49–80 kg, body mass index 20.1–24.8 kg/m2) ingested 4 tablets of the WBE, corresponding to 240 mg of total salicin, which is the maximum recommended dose per day [34]. Two subjects participated in the study without taking WBE to determine the possible effects of the diet. The tablets were administered with 200 mL of water after 12 hours of fasting time. Standardised meals were served after 2, 5, and 10 hours. No comedication (except contraceptives) was permitted during the study. Venous blood was sampled before and 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, and 24 hours after drug administration. The blood was collected in Serum-Monovettes® (Sarstedt), immediately centrifuged (2800 g, 20 min; Heraeus Megafuge 1.0 R Sepatech), and the decanted serum was stored at − 80 °C until further use.
Animals Male Wistar rats, each weighing 150–200 g, were obtained from the Animal Breeding Unit of the National Ophthalmology Institute, Giza (Egypt). They were housed for one week prior to the experiments, fed a standard pellet diet, and given water ad libitum. The animals were housed at 22 °C ± 2 °C, constant humidity, and under a 12-h light/dark cycle. All experiments were performed at the Pharmacology Department, Faculty of Pharmacy, Cairo University (Egypt), following the European Communities Council Directive of 1986 (86/609/EEC) and approved by the Ethical Committee for Animal Experimentation at the Faculty of Pharmacy, Cairo University (PT 412, February 2012).
Pharmacokinetic study in animals The animals were randomly assigned to 6 groups, each consisting of 7 animals. After withdrawing food but not water for 24 h, salicortin was administered as an aqueous solution at a dose of 100 mg/kg body weight by oral gavage. One group was sacrificed before salicortin administration and the other groups after 0.5 h, 1 h, 2 h, 4 h or 8 h. The collected blood was centrifuged at 3000 rpm for 15 min in a K2015 multi-micro centrifuge (Centurion Scientific Ltd.), and the separated serum was kept at − 80 °C until further use.
Serum processing For processing, 1 U/µL sulphatase and 10 parts Na-acetate-buffer, pH 4.7, containing 100 U/µL β-glucuronidase as well as 10 mg/mL L-ascorbic acid were added to 90 parts of each serum sample. The samples were then incubated for 60 min at 37 °C and 5 % CO2 (incubator: New Brunswick Scientific). Proteins were precipitated by adding 100 parts of ice-cold internal standard solution (18 µM 4-methylcatechol in MeOH) and vortexing the samples for 5 minutes. After centrifugation (14 000 rpm, 4 °C, 30 min, BR4i multifunction; Jouan Industries SAS), the supernatant was filtered through Nanosep® filters (Pall, 30 k) by centrifugation (14 000 rpm, 4 °C, 60 min), and the filtrate was then analysed by HPLC. To look for free catechol and to investigate which phase-II metabolite predominates, three animal and human serum sam-
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Analytical HPLC A Hitachi DAD L2455, autosampler L2200, pump L2130, column oven L2350, and EZChrom Elite software (VWR) were used. The HPLC conditions were as follows: LiChrocart cartridge with Purospher®-Star RP-18e (5 µm, 4 × 250 mm; Merck); solvent system: A – 0.1 % H3PO4 in H2O, B – MeOH; 5–6.8 % B over 18 min, 6.8–20 % B over 22 min, 20–60% B over 10 min, 60% B for 5 min, 60–5 % B over 10 min, 5 % B for 5 min; flow rate: 1.0 mL/min; injection volume: 20 µL; column temperature: 40 °C; and DAD conditions: 275 nm (catechol) and 301 nm (salicylic acid). For calibration, the ratio of the areas of 0.75, 1, 2, 4, 6, 8, and 10 µM catechol, and the ratio of the areas of 0.25, 1, 2, 5, 8, 10, 10.9, and 20 µM salicylic acid to the areas of the internal standard 4-methylcatechol were determined from serum matrix. The calibration functions were as follows: catechol, y = 0.1341 x – 0.0172, R2 = 0.9986; salicylic acid, y = 0.1872 x – 0.0963, R2 = 0.9966. The recovery from serum matrix was determined for 9 µM 4-methylcatechol (88.7%) and for different concentrations of catechol [2 (99.1 %), 8 (93.3 %), 16 (83.4%), and 20 µM (79.3 %)] and salicylic acid [2 (85.3 %), 10 (76.9%), and 20 µM (80.4%)]. The limit of detection (LOD, signal to noise ratio 3 : 1) and the limit of quantification (LOQ, signal to noise ration 9 : 1) were determined with serum matrix: LOQ of catechol = 0.75 µM, LOQ of salicylic acid = 0.25 µM, LOD of catechol = 0.5 µM and LOD of salicylic acid = 0.1 µM. The intraday precision (1.52 % SD) was defined by repeated injection of the same solution with catechol (4 µM) and 4methyl-catechol (9 µM) in serum matrix, and the interday precision (2.52 % SD) was determined by injection of this solution on 5 different days. All validation experiments were repeated 5 to 6 times. Mass spectrometry of serum samples was performed with an ESI‑MS, Agilent Q‑TOF 6540 UHD with Masshunter B.05.00 software (Agilent Technologies).
Pharmacokinetic parameters All pharmacokinetic calculations were performed by standard noncompartmental analysis using WinNonlin 6.3 (Pharsight). Peak serum concentrations (cmax) and time to peak concentrations (tmax) were directly obtained from the individual serum concentration-time curves. The elimination constant λz was calculated by log-linear regression in the elimination phase. The terminal half-life was calculated according to t1/2 = ln2/λz. The area under the serum concentration-time curve to the last measured concentration (AUCt) was calculated by the linear trapezoidal rule. The measured last concentration (ct) was used for extrapolation to infinity to determine AUC0-∞ = AUCt + ct/λz. Naive pooling was used for analysis of the sparse data in rats (one data point per individuum) to generate one single pharmacokinetic profile. Therefore, only mean pharmacokinetic parameters without variability estimates could be estimated. In 5 volunteers, a certain concentration of salicylic acid was detected in samples before ingestion of WBE; this blank value was subtracted from all following concentrations.
Acknowledgements Special thanks are given to Hermes-Arzneimittel GmbH for financial support and to T. Emmer, Centre for Clinical Studies, University Hospital Regensburg, for helpful discussions. G. Wilberg and A. Seefeld from the Dept. of Pharmacology and Toxicology and M. Untergehrer, Dept. of Pharmaceutical Biology, are gratefully acknowledged for their technical support. Many thanks are also due to J. Kiermeier, Faculty of Chemistry and Pharmacy, for aid with the MS experiments and to F. M. Matysik and C. Niegel, Department of Analytical Chemistry, for their analytical support.
Supporting information The isolation process of salicortin and tremulacin, the mass spectra of the catechol conjugates in human and rat sera as well as the quantification method of catechol, salicortin, and tremulacin in the WBE are available as Supporting Information.
Conflict of Interest !
The authors declare no conflict of interest.
Affiliations 1 2 3 4 5 6 7
University of Regensburg, Institute of Pharmacy, Department of Pharmaceutical Biology, Regensburg, Germany Cairo University, Faculty of Pharmacy, Department of Pharmacology, Cairo, Egypt University of Regensburg, Institute of Physiology, Regensburg, Germany Charité University Hospital, Campus Benjamin Franklin, Department of Anesthesiology and Intensive Care, Berlin, Germany Freie Universität Berlin, Institute of Pharmacy, Department of Clinical Pharmacy and Biochemistry, Berlin, Germany Intensive Care Unit, Bezirksklinikum Regensburg, Regensburg, Germany University of Regensburg, Institute of Pharmacy, Department of Pharmacology and Toxicology, Regensburg, Germany
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ples with the highest concentration of catechol obtained by HPLC after enzyme processing were additionally not or only partially processed with enzymes before HPLC analysis. In this case, the sample preparation was the same as described above without adding the enzymes or only with the addition of one type of enzyme.
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