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Absorption and metabolism of milk thistle flavanolignans in humans
Phytomedicine 20 (2012) 40–46
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Phytomedicine journal homepage: www.elsevier.de/phymed
Absorption and metabolism of milk thistle flavanolignans in humans Luca Calani, Furio Brighenti, Renato Bruni, Daniele Del Rio ∗ The Laboratory of Phytochemicals in Physiology, Department of Food Science, University of Parma, Italy
a r t i c l e Keywords: Flavanolignans Urinary metabolites Bioavailability Mass spectrometry Silymarin Silybum marianum
i n f o
a b s t r a c t This study evaluated the absorption and metabolism of milk thistle flavonolignans silychristin, silydianin, silybin and isosilybin isomers (all together known as silymarin) in humans. Fourteen volunteers consumed an extract of milk thistle and urine was collected up to 48 h after consumption. Thirty-one metabolites were identified in urine by means of HPLC–MS/MS, monoglucuronides being the most common excreted form, followed by sulphate–glucuronides and diglucuronides, respectively. The excretion of monoglucuronides peaked 2 h after consumption, whereas sulphate–glucuronide and diglucuronide excretion peaked at 8 h. The bioavailability of milk thistle flavanolignans was 0.45 ± 0.28% (mean ± SD). In conclusion, milk thistle flavonolignans are extensively modified after ingestion and recovered in urine as sulpho- and glucuronyl-conjugates, indicating a strong affinity for hepatic phase II enzymes. All future studies (in vitro and in vivo) dealing with the effects of milk thistle should start by considering the modification of its flavonolignans after ingestion by humans. © 2012 Elsevier GmbH. All rights reserved.
Introduction Milk thistle (Silybum marianum [L.] Gaertn.) is a member of the Asteraceae family with a long phytotherapic history as hepatoprotectant, nowadays representing the herbal product most frequently suggested for patients with chronic liver disease (Mayer et al. 2005; Strader et al. 2002). This plant, and its fruit in particular, contains a unique mixture of polyphenolic compounds, among which the main subclass comprises the flavonolignans silychristin, silydianin, silybin and isosilybin (all together known as silymarin, Fig. 1). Some of these molecules, including silybin and isosilybin, are naturally present as diastereomers (A and B), but silychristin may also exist in the diastereomeric form (Lee et al. 2006; Shibano et al. 2007). Several in vitro studies have focussed on the bioactivity of these molecules, showing that they exert anti-inflammatory and antiviral effects against hepatitis C virus (HCV), thus making them good candidates for the management of patients with chronic hepatitis C (Wagoner et al. 2010; Morishima et al. 2010; Trappoliere et al. 2009). In vivo, daily treatment with 600 mg of silymarin for 12 months reduced hyperinsulinaemia, exogenous insulin
Abbreviations: HCV, hepatitis C virus; ESI, electrospray interface; MRM, multiple reaction monitoring; UDP, uridine diphosphate; UGTs, UDPglucuronosyltransferases; SULTs, sulphotransferases; EHR, entero-hepatic recirculation; amu, atomic mass unit. ∗ Corresponding author at: Medical Building C, Via Volturno 39, 43125, Parma, Italy. Tel.: +39 0521 903830; fax: +39 0521 903832. E-mail address: [email protected] (D. Del Rio). 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.09.004
requirements and malondialdehyde levels in 30 cirrhotic diabetic patients (Velussi et al. 1997), whereas daily consumption of 150 mg silymarin for 6 months slightly increased the total erythrocyte glutathione level and decreased lipid peroxidation in peripheral blood cells in 60 subjects with alcoholic liver cirrhosis (Lucena et al. 2002). However, the beneficial effects of milk thistle extract for patients with liver injury remain unclear, as only low-quality trials have suggested beneficial effects. Therefore, high-quality randomized clinical trials are needed to demonstrate the real efficacy of this natural product in the context of liver pathologies and in the management of physiological perturbations of the liver function (Rambaldi et al. 2005, 2007; Jacobs et al. 2002). Despite the many liver-health benefits attributed to silymarin, few studies have investigated the metabolism of its bioactive components within the human body. In fact, following ingestion by humans, (poly)phenolic compounds undergo extensive metabolism during their passage through the gastrointestinal tract and, with very few exceptions, only metabolites of the parent compounds enter the circulatory system. Chemical modifications initially occur in the lumen of the small intestine with cleavage of sugar moieties (when present), after which the aglycones that are released undergo glucuronidation, sulphation and/or methylation (Crozier et al. 2009). After entry of these metabolites into the circulatory system, phase II metabolism may also occur in the liver and other organs (Del Rio et al. 2010). The molecules that eventually reach the internal compartments of the human body are therefore different from those present in planta. Investigating the biological effects of the parent polyphenolic compounds instead of those of their human metabolites could lead to misleading or wrong
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Fig. 1. Chemical structure of milk thistle flavonolignans.
conclusions. Although the identification of polyphenolic catabolites was until recently confined to very well equipped laboratories, the necessary analytical facilities are now readily available in most research laboratories and are increasingly being used to investigate the metabolism of polyphenols by humans. The identification of a vast array of phenolic metabolites in vivo has also allowed the re-interpretation of bioavailability, which, for this specific class of components has always been considered extremely low (Crozier et al. 2010). To fully understand the biological and putatively beneficial effects of this very peculiar array of flavonolignans in vivo, one cannot ignore their bioavailability and metabolism. Therefore, the aim of this study was to investigate silymarin absorption and metabolism in human volunteers by means of high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) as an essential step on the road to finally verifying the putative health-related effects of milk thistle.
Materials and methods Milk thistle-based product and chemicals A water-soluble formulation of milk thistle was supplied by Sofar (Trezzano Rosa, Milano, Italy), in sealed bags containing 4 g of powder. Each bag contained 190 mg of milk thistle extract (silymarin), standardized with a minimum silybin content of 25%. In addition to silymarin, each bag contained: sugar, citric acid, aroma, colloidal silica and vitamin E. Pure silybin (A and B) was purchased from Sigma (St. Louis, MO, USA). Pure silydianin, silychristin,
isosilybin (A and B) and quercetin-3-O-glucuronide were purchased from PhytoLab GmbH & Co (Vestenbergsgreuth, Germany). All solvents and reagents were purchased from Carlo Erba Reagents (Milano, Italy).
Human feeding study The feeding study was carried out on 15 healthy human volunteers (8 males and 7 females), selected in compliance with established exclusion criteria including diabetes mellitus, cardiovascular events, chronic liver diseases or nephropathies, cancer, organ failure and intake of antioxidant or vitamin integrators. The volunteers were 24 ± 5 years old (mean ± SD) with an average BMI of 24 ± 4 kg/m2 . One subject was excluded from the study, as they did not complete urine collection. Each volunteer provided signed informed consent, and the study protocol was approved by the Ethics Committee for Human Research of the University of Parma. For 24 h before and 48 h after ingestion of milk thistle extract, the subjects followed a diet containing minimal phenolic compounds, by avoiding fruits, vegetables, all kinds of herbal tea, dietary antioxidant supplements, alcohol and drugs. To check for compliance, the volunteers were asked to complete a 3-day weighed food record starting the day before the study began. On the first day of the study, after an overnight fast, each subject drank a definite amount of milk thistle formulation (8 g, equivalent to 380 mg of silymarin extract) dissolved in 400 ml of water. Urine was collected at time 0 and during 0–2, 2–4, 4–8, 8–15, 15–24, 24–28, 28–32, 32–39, and 39–48 h collection periods after ingestion. The volume of urine collected during each period was measured and samples were
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Table 1 Flavonolignan content of two bags of milk thistle product consumed by volunteers (mol ± standard deviation (n = 5)). Compound
Content
Silychristin Silydianin Silybin A Silybin B Isosilybin A and B
75.7 70.9 125.3 144.4 68.2
Total
484.5 ± 51.3
± ± ± ± ±
7.4 12.9 13.3 19.3 6.9
stored at −80 ◦ C until analysis. Suitable aliquots of urine samples were filtered through 0.45 m nylon filter (Waters, Milford, MA, USA) and directly injected into the HPLC–MS/MS without further processing. HPLC–ESI-MS/MS analysis Milk thistle flavonolignans and their metabolites in the extract and urine samples were analysed using a Waters 2695 Alliance separation module equipped with a Micromass Quattro Micro Api mass spectrometer fitted with an electrospray interface (ESI) (Waters, Milford, MA, USA). Separation was performed using a Waters Atlantis dC18 (3 m, 2.1 mm × 150 mm) reverse phase column (Waters, Milford, MA, USA). To analyse the milk thistle extract, the mobile phase was pumped at a flow rate of 0.17 ml/min using a 15-min isocratic gradient of 25% acetonitrile in 1% aqueous formic acid immediately followed by a 15-min isocratic gradient of 30% acetonitrile in 1% aqueous formic acid. For urine sample analysis, a 30-min linear gradient of 15–35% acetonitrile in 1% aqueous formic acid was used. The ESI source and the collision energy conditions were tuned by infusing a standard of silybin (mixture of A and B diastereomers) together with 5% acetonitrile in 1% aqueous formic acid at a flow rate of 30 l/min. The ESI source worked in negative ionisation mode; the source temperature was 120 ◦ C, the desolvation temperature was 350 ◦ C, the capillary voltage was 2.8 kV, and the cone voltage was 40 V. The collision energy was set at 30 eV, the desolvation gas (nitrogen) at 750 l/h, and the cone gas (nitrogen) at 50 l/h; the collision gas used was argon. The flavonolignans and their metabolites were identified and quantified using HPLC with the MS operating in the multiple reaction monitoring (MRM) mode. The native flavonolignans in the extract and the unmetabolised compounds recovered in urine were quantified using calibration curves of the appropriate standard compound, whereas urinary metabolites (glucuronide, sulphate–glucuronide and diglucuronide) were quantified as quercetin-glucuronide equivalents, monitoring the loss of the glucuronyl moiety. Results Analysis of milk thistle extract Several flavanolignans were identified in the milk thistle extract based on their chromatographic retention times, and MS in-source fragmentations and identifications were confirmed by comparison with the commercial standards. In detail, the following flavonolignans were identified: silychristin, silydianin, silybin A and B, isosilybin A and B and their abundance was in agreement with previous reports (Lee et al. 2006; Wang et al. 2010). The total amount of silymarin flavonolignans consumed by volunteers is summarised in Table 1.
Table 2 Mass spectral characteristics of flavanolignans, phase II metabolites, and number of isomers of detected metabolites. Compound
[M−H]− (m/z) Qualifier ions (m/z)
Silychristin Silydianin Silybin A and B Isosilybin A and B Silymarin glucuronides Silymarin sulphate glucuronides Silymarin diglucuronide
481 481 481 481 657 737 833
Isomers
125 125 125 125 481, 125 16 657, 561, 481, 125 7 657, 481, 125 8
Identification of flavanolignans and their metabolites in urine The HPLC–MS/MS analysis enabled identification of unmetabolised urinary flavonolignans and urinary phase II metabolites, such as glucuronides, sulphate–glucuronides and diglucuronides, of which the mass spectral characteristics and the number of detected isomers are reported in Table 2. The metabolites derived by the action of human UDP-glucuronosyltransferases (UGTs) and sulphotransferases (SULTs) were identified via the loss of the conjugating groups (i.e. glucuronic acid and sulphate) to give the aglycone fragment ion (Prasain and Barnes 2007). The aglycone flavonolignans had a negatively charged molecular ion ([M−H]− ) at m/z 481, which on MS/MS (MS2 ) produced a main fragment ion at m/z 125, derived from A-ring fission, as previously reported (Lee et al. 2006). The silymarin glucuronides had a [M−H]− at m/z 657, which on MS2 produced a fragment ion corresponding to silymarin aglycones at m/z 481, generated by the loss of a glucuronide moiety equal to 176 atomic mass units (amu). More precise identification was possible through the concomitant fragmentation of the aglycones formed, which produced a main fragment ion at m/z 125. The flavanolignan sulphate–glucuronides had a [M−H]− at m/z 737, which on MS2 produced different fragment ions corresponding to (i) silymarin glucuronides at m/z 657 derived from the loss of sulphate groups equal to 80 amu, (ii) silymarin sulphates at m/z 561 derived from the loss of a glucuronide moiety or (iii) silymarin aglycones at m/z 481 derived from the loss of both conjugations. Once again, further fragmentation generated ions at m/z 125, in keeping with the presence of one of the silymarin flavonolignans. The flavanolignan diglucuronides, with a [M−H]− at m/z 833, produced different fragment ions on MS2 at m/z 657 and 481, derived from the loss of one or two glucuronide moieties, respectively, and fragment ions at m/z 125 derived from flavanolignan ring fission. Milk thistle flavonolignans have the same molecular weight and, therefore, mass-to-charge ratio, but they also share fragmentation patterns (corresponding to the fission of the A ring), making chromatographic separation essential for distinguishing each specific compound by comparison with the pure standard. However, this approach cannot be applied to single metabolites (e.g. glucuronides), since the relevant pure standards are not available. Therefore, although 31 different silymarin metabolites (including isomers of the same aglycone) generated after milk thistle ingestion were identified, it was impossible to attribute individual metabolites to specific flavanolignan molecules. The flavonolignans were preferentially metabolised by UGTs, forming 16 and 8 different glucuronide and diglucuronide conjugates, respectively, whereas only 7 sulphoglucuronides were identified (Table 2). The number of silymarin metabolites identified in human urine was very high with respect to the native flavanolignans present in the milk thistle extract. This is due to the multiple phenolic hydroxyl groups available for conjugation by UGTs and SULTs, resulting in the formation of a multitude of possible conjugates. An example of a LC–MS2 trace
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Fig. 2. Chromatographic profile of urinary metabolites of silymarin. Total Ion Current (TIC) of multiple reaction monitoring (MRM) chromatograms. Example of urine sample from a subject from the second collection period (2–4 h) after milk thistle extract ingestion. The main isomer metabolites of silymarin are reported. (A) Silymarin glucuronides; (B) silymarin sulphate glucuronides; (C) silymarin diglucuronides.
showing the most relevant identified catabolites in the 2–4 h urine collection of one subject is shown in Fig. 2.
Quantitative analysis of urinary catabolites and their excretion profiles As for the qualitative analysis, urine samples were quantitatively analysed by liquid chromatography–tandem mass spectrometry in the multiple reaction monitoring (MRM) mode, in order to calculate the total flavonolignan excretion and estimate
their bioavailability in humans. The quantitative excretion kinetic of flavonolignans and their metabolites in urine is shown in Fig. 3. Urinary flavonolignans were mainly recovered in their conjugated form, which greatly exceeded the aglycones. The glucuronides were the major excreted form, with a total excretion equal to 1.37 mol, compared to 0.53 and 0.25 mol of sulphate–glucuronides and diglucuronides, respectively. The amount of aglycone flavonolignans excreted was 0.02 mol, accounting for the excretion of both diastereomeric forms of silybin and isosilybin. No trace of silydianin and silychristin aglycones was detected in urine.
Fig. 3. Excretion profiles of flavonolignans in urine over 48 h in 14 volunteers. (A) Silymarin glucuronides; (B) silymarin sulphate glucuronides; (C) silymarin diglucuronides; (D) aglycone flavonolignans (silybin A and B, isosilybin A and B). Data expressed as mean values with their standard errors depicted by vertical bars.
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Monoglucuronides showed a consistent excretion kinetic, indicating that all flavonolignans underwent this conjugative step simultaneously. Their profile differed from that of sulphate–glucuronides and diglucuronides, which were also consistent within their category. In fact, the peak excretion of monoglucuronides occurred 2 h after consumption of the extract, whereas the sulphate–glucuronides and diglucuronides shared an excretion profile, reaching a peak 8 h after silymarin ingestion. Unmetabolised silybin and isosilybin diastereomers had a different excretion profile, peaking at 15 and 24 h after extract intake, respectively. It was interesting to note that all silymarin conjugate excretion slightly but consistently increased between 24 and 28 h and all conjugated and unconjugated forms showed a subsequent minor relative peak between 32 and 39 h. Most of the subjects showed complete excretion within 48 h, with only trace amounts excreted in the last urine collection. Finally, the mean bioavailability of milk thistle flavonolignans, calculated as the ratio between total silymarin excretion (conjugates and aglycones) and the total intake of silymarin, was 0.45 ± 0.28% (mean ± SD), ranging from 0.12 to 1.16% among subjects.
Discussion Milk thistle extract is widely used in the United States and Europe as a herbal supplement for the treatment of liver-related disorders. Many clinical trials have been carried out to evaluate the efficacy of its putatively bioactive fraction, namely silymarin, against liver cirrhosis (viral or alcoholic) and liver inflammation. Ferenci et al. (2009) assessed the effect of silymarin on the outcome of cirrhotic patients and found that treatment was effective in patients with alcoholic cirrhosis and in patients initially rated ‘Child A’. Another study investigated the effect of 1–2 weeks intravenous administration of silybin in patients with HCV infection, showing an antiviral effect in patients who were unresponsive to pegylated-interferon/ribavirin treatment (Ferenci et al. 2008). On the contrary, Parés et al. (1998) observed no effect on the survival of alcoholics with liver cirrhosis after chronic supplementation of 150 mg of silymarin three times daily for 2 years. Thus, based on human clinical trials, the potential benefits of milk thistle silymarin in the treatment of liver disorders remain controversial. However, the putative hepatoprotective effects of these flavonolignans have been confirmed by several in vitro studies. Silymarin was able to inhibit proliferation and pro-inflammatory cytokine secretion by peripheral blood mononuclear cells and T cells (Morishima et al. 2010). Silybin alone inhibited the production of pro-inflammatory cytokines, showed a clear antioxidant effect and reduced the direct and indirect pro-fibrogenic potential of hepatic stellate cells (Trappoliere et al. 2009). Moreover, silymarin polyphenols have been shown to be involved in blocking the entry and transmission of HCV virus in host cells (Wagoner et al. 2010). Several other in vitro studies confirmed the bioactivity observed, at least in certain cases, in human interventions (Saliou et al. 1998), but none of these studies considered the possible modifications occurring to phenolic compounds during their passage through the gastrointestinal tract (Crozier et al. 2010). The milk thistle flavonolignans are extensively modified by phase II human enzymes, this process resulting in the formation of intestinal and hepatic metabolites that considerably exceed the native aglycone molecules in biological fluids, as shown in the present study and reported elsewhere (Wen et al. 2008; Hawke et al. 2010). However, to date, none of the in vitro studies present in the scientific literature have investigated the bioactivity of silymarin metabolites, the molecules that actually come into contact with the internal compartments of the human body.
Few studies have investigated milk thistle flavonolignan catabolism in detail or accurately estimated their bioavailability in humans. This work confirms that silymarin phenolics undergo extensive metabolism, as do almost all flavonoids, forming sulphate and glucuronide conjugates through the action of UGTs and SULTs in the small intestine and liver. Table 2 shows how 24 of the 31 identified metabolites are derived by the action of UGTs, and that only 7 metabolites are formed through the concurrent action of SULTs. This specificity for glucuronidation contrasts with the behaviours of several other flavonoidic compounds, like green tea and cocoa catechins, which are preferentially sulphated (Stalmach et al. 2009). This is the first study, to our knowledge, to describe such a huge number of conjugated flavonolignans in vivo, and to follow their excretory profile in human volunteers for 2 complete days and until complete clearance. Previous studies failed to identify metabolites by treating biological samples (plasma or urine) with deconjugating enzymes, namely -glucuronidases and sulphatases, allowing the detection of the resulting aglycones with spectrophotometric devices (Wen et al. 2008; Hawke et al. 2010; Hoh et al. 2006, 2007). However, deconjugating enzymes show variation in their specificity and such treatment does not enable full understanding of the metabolic processes undergone by these phenolic compounds within the human body (Donovan et al. 2006). Fig. 3 shows that silymarin compounds are mainly excreted within 24 h after ingestion, but substantial amounts of glucuronides, sulpho-glucuronides and diglucuronides are excreted for up to 48 h. There was substantial interindividual variation in the excretion rate and quality among the volunteers, but on average, sulphoglucuronides and diglucuronides, despite their more hydrophilic nature, showed delayed excretion with respect to monoglucuronides. In fact, peak excretion of these compounds occurred 8 h after silymarin consumption, whereas monoglucuronides peaked in the first urine collection. This may be due to the non-marginal entero-hepatic recirculation (EHR) that preferentially occurs for silymarin sulphate–glucuronides and diglucuronides. EHR could also explain the secondary peaks in the excretion profile, which were consistent with the plasma pharmacokinetics previously described by Wen et al. (2008). The aglycones silybin and isosilybin showed high interindividual variation among volunteers, and secondary peaks were also present during the 48 h after consumption (Fig. 3). However, although 2.17 mol of total flavanolignans were excreted on average, only 0.02 mol were recovered in urine as unconjugated, making the contribution of aglycones to the final bioavailability insignificant. Although polyphenols are drastically modified by the colon microbiota, giving rise to a plethora of smaller phenolic compounds that are then absorbed and usually exceed the circulating and excreted concentration of human phase II metabolites (Crozier et al. 2010), it was impossible to detect any of these microbially derived compounds in this study. These molecules may have been missed by the analysis, being chemically different from those identified as being most frequently derived from other polyphenolic compounds, but the hypothesis that these flavanolignans are somehow catabolised through different pathways or stored within the human body was not ruled out. It is noteworthy that the calculated bioavailability, equal to 0.45% of the ingested dose, was higher than that of other flavonoids with a similar molecular weight, like anthocyanins and galloylated catechins (Manach et al. 2005). While one study reported a urinary excretion of silymarin equal to 3.1% of the ingested dose (Hawke et al. 2010), the approach used on that occasion was very different from that applied in the present study. In fact, because only a few flavonoid metabolites are commercially available, the analytical approach used to study polyphenol bioavailability almost invariably involves the treatment of samples with hydrolytic enzymes
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– namely glucuronidases and sulphatases – followed by quantification of the released aglycones by HPLC (Henning et al. 2005; Lee et al. 2002). Quantification of partially identified metabolites by MS using MRM or selected ion monitoring (SIM) should be based on calibration curves of standards that, as mentioned above, are not generally commercially available. To overcome this drawback, a related available compound may be used, which in the current study was quercetin-O-glucuronide. Quantification was therefore based on a common reaction in the mass spectrometry system, namely cleavage of one glucuronic acid from a flavonoidic structure, and this was the first and only study to our knowledge in which flavonolignan metabolites were quantified without applying enzymatic hydrolysis before the analysis, thus providing an actual fingerprint of their urinary excretion. In general, the response of glucuronidase/sulphatase sources used during sample preparation are not constant and there may be substantial batch-to-batch variation in their specificity (Donovan et al. 2006). As the enzymatic deconjugation approach is generally linked to the unavailability of MS facilities, there is a general lack of information about the efficiency by which the enzymes hydrolyse individual metabolites and release the aglycones. This introduces a variable and unquantified error factor that makes bioavailability estimates doubtful. In summary, the present study shows that milk thistle flavonolignans are extensively modified by human enzymes, as 99% of the absorbed fraction was recovered in urine as sulphoand glucuronyl-conjugates. A total of 31 different metabolites were detected, indicating a strong affinity of these molecules for hepatic phase II enzymes, which may be related to their putative liver healing potential. Moreover, the hypothesised entero-hepatic recirculation might allow biologically relevant concentrations of flavonolignan metabolites to be reached at the hepatobiliary level and this could be taken into account when establishing total silymarin dose and frequency of administration. The urinary excretion of these molecules was followed to complete clearance (48 h) and no microbial catabolites were identified, leaving room for intriguing hypotheses about their actual catabolism or storage. In conclusion, future studies dealing with the physiological effects of milk thistle should start by considering the modifications that occur to its polyphenolic components once ingested by humans. The term “phytocomplex” is customary to describe the elaborate mixtures of plant secondary metabolites administered by means of herbal medicines. However, as in the case of milk thistle, such complexity reverberates in, and is often amplified by, the behaviour of human metabolism and should be somehow redefined. Whether this remarkable phase II metabolism is related to the putative hepatoprotective effect of silymarin is not yet known, but any future in vitro study or dose response in vivo investigation will have to apply or quantify flavanolignan metabolites, given that, in this study, 31 such metabolites were identified. Finally, future studies evaluating the effect of the human faecal microbiota on silymarin flavonolignans will probably enable the bioavailability data of these particular polyphenolic compounds to be updated. Acknowledgements The authors would like to thank all the volunteers who participated in the present study. References Crozier, A., Del Rio, D., Clifford, M.N., 2010. Bioavailability of dietary flavonoids and phenolic compounds. Molecular Aspects of Medicine 31, 446–467. Crozier, A., Jaganath, I.B., Clifford, M.N., 2009. Dietary phenolics: chemistry, bioavailability and effects on health. Natural Products Reports 26, 1001–1043. Del Rio, D., Calani, L., Cordero, C., Salvatore, S., Pellegrini, N., Brighenti, F., 2010. Bioavailability and catabolism of green tea flavan-3-ols in humans. Nutrition 26, 1110–1116.
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Metabolism and bioavailability of flavonoids in chemoprevention: current analytical strategies and future prospects. Molecular Pharmaceutics 4, 846–864. Rambaldi, A., Jacobs, B.P., Gluud, C., 2007. Milk thistle for alcoholic and/or hepatitis B or C virus liver diseases. Cochrane Database of Systematic Reviews 17, CD003620. Rambaldi, A., Jacobs, B.P., Iaquinto, G., Gluud, C., 2005. Milk thistle for alcoholic and/or hepatitis B or C liver diseases – a systematic Cochrane hepato-biliary group review with meta-analyses of randomized clinical trials. American Journal of Gastroenterology 100, 2583–2591. Saliou, C., Rihn, B., Cillard, J., Okamoto, T., Packer, L., 1998. Selective inhibition of NF-kappaB activation by the flavonoid hepatoprotector silymarin in HepG2. Evidence for different activating pathways. FEBS Letters 440, 8–12. Shibano, M., Lin, A.S., Itokawa, H., Lee, K.H., 2007. Separation and characterization of active flavonolignans of Silybum marianum by liquid chromatography connected with hybrid ion-trap and time-of-flight mass spectrometry (LC–MS/IT-TOF). Journal of Natural Products 70, 1424–1428. Stalmach, A., Troufflard, S., Serafini, M., Crozier, A., 2009. Absorption, metabolism and excretion of Choladi green tea flavan-3-ols by humans. Molecular Nutrition and Food Research 53 (Suppl. 1), S44–S53. Strader, D.B., Bacon, B.R., Lindsay, K.L., La Brecque, D.R., Morgan, T., Wright, E.C., Allen, J., Khokar, M.F., Hoofnagle, J.H., Seeff, L.B., 2002. Use of complementary and alternative medicine in patients with liver disease. American Journal of Gastroenterology 97, 2391–2397. Trappoliere, M., Caligiuri, A., Schmid, M., Bertolani, C., Failli, P., Vizzutti, F., Novo, E., di Manzano, C., Marra, F., Loguercio, C., Pinzani, M., 2009. Silybin, a component
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of silymarin, exerts anti-inflammatory and anti-fibrogenic effects on human hepatic stellate cells. Journal of Hepatology 50, 1102–1111. Velussi, M., Cernigoi, A.M., De Monte, A., Dapas, F., Caffau, C., Zilli, M., 1997. Longterm (12 months) treatment with an anti-oxidant drug (silymarin) is effective on hyperinsulinemia, exogenous insulin need and malondialdehyde levels in cirrhotic diabetic patients. Journal of Hepatology 26, 871–879. Wagoner, J., Negash, A., Kane, O.J., Martinez, L.E., Nahmias, Y., Bourne, N., Owen, D.M., Grove, J., Brimacombe, C., McKeating, J.A., Pécheur, E.I., Graf, T.N., Oberlies, N.H., Lohmann, V., Cao, F., Tavis, J.E., Polyak, S.J., 2010. Multiple effects of silymarin on the hepatitis C virus lifecycle. Hepatology 51, 1912–1921.
Wang, K., Zhang, H., Shen, L., Du, Q., Li, J., 2010. Rapid separation and characterization of active flavonolignans of Silybum marianum by ultraperformance liquid chromatography coupled with electrospray tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 53, 1053–1057. Wen, Z., Dumas, T.E., Schrieber, S.J., Hawke, R.L., Fried, M.W., Smith, P.C., 2008. Pharmacokinetics and metabolic profile of free, conjugated, and total silymarin flavonolignans in human plasma after oral administration of milk thistle extract. Drug Metabolism and Disposition: The Biological Fate of Chemicals 36, 65–72.
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Phytomedicine journal homepage: www.elsevier.de/phymed
Absorption and metabolism of milk thistle flavanolignans in humans Luca Calani, Furio Brighenti, Renato Bruni, Daniele Del Rio ∗ The Laboratory of Phytochemicals in Physiology, Department of Food Science, University of Parma, Italy
a r t i c l e Keywords: Flavanolignans Urinary metabolites Bioavailability Mass spectrometry Silymarin Silybum marianum
i n f o
a b s t r a c t This study evaluated the absorption and metabolism of milk thistle flavonolignans silychristin, silydianin, silybin and isosilybin isomers (all together known as silymarin) in humans. Fourteen volunteers consumed an extract of milk thistle and urine was collected up to 48 h after consumption. Thirty-one metabolites were identified in urine by means of HPLC–MS/MS, monoglucuronides being the most common excreted form, followed by sulphate–glucuronides and diglucuronides, respectively. The excretion of monoglucuronides peaked 2 h after consumption, whereas sulphate–glucuronide and diglucuronide excretion peaked at 8 h. The bioavailability of milk thistle flavanolignans was 0.45 ± 0.28% (mean ± SD). In conclusion, milk thistle flavonolignans are extensively modified after ingestion and recovered in urine as sulpho- and glucuronyl-conjugates, indicating a strong affinity for hepatic phase II enzymes. All future studies (in vitro and in vivo) dealing with the effects of milk thistle should start by considering the modification of its flavonolignans after ingestion by humans. © 2012 Elsevier GmbH. All rights reserved.
Introduction Milk thistle (Silybum marianum [L.] Gaertn.) is a member of the Asteraceae family with a long phytotherapic history as hepatoprotectant, nowadays representing the herbal product most frequently suggested for patients with chronic liver disease (Mayer et al. 2005; Strader et al. 2002). This plant, and its fruit in particular, contains a unique mixture of polyphenolic compounds, among which the main subclass comprises the flavonolignans silychristin, silydianin, silybin and isosilybin (all together known as silymarin, Fig. 1). Some of these molecules, including silybin and isosilybin, are naturally present as diastereomers (A and B), but silychristin may also exist in the diastereomeric form (Lee et al. 2006; Shibano et al. 2007). Several in vitro studies have focussed on the bioactivity of these molecules, showing that they exert anti-inflammatory and antiviral effects against hepatitis C virus (HCV), thus making them good candidates for the management of patients with chronic hepatitis C (Wagoner et al. 2010; Morishima et al. 2010; Trappoliere et al. 2009). In vivo, daily treatment with 600 mg of silymarin for 12 months reduced hyperinsulinaemia, exogenous insulin
Abbreviations: HCV, hepatitis C virus; ESI, electrospray interface; MRM, multiple reaction monitoring; UDP, uridine diphosphate; UGTs, UDPglucuronosyltransferases; SULTs, sulphotransferases; EHR, entero-hepatic recirculation; amu, atomic mass unit. ∗ Corresponding author at: Medical Building C, Via Volturno 39, 43125, Parma, Italy. Tel.: +39 0521 903830; fax: +39 0521 903832. E-mail address: [email protected] (D. Del Rio). 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.09.004
requirements and malondialdehyde levels in 30 cirrhotic diabetic patients (Velussi et al. 1997), whereas daily consumption of 150 mg silymarin for 6 months slightly increased the total erythrocyte glutathione level and decreased lipid peroxidation in peripheral blood cells in 60 subjects with alcoholic liver cirrhosis (Lucena et al. 2002). However, the beneficial effects of milk thistle extract for patients with liver injury remain unclear, as only low-quality trials have suggested beneficial effects. Therefore, high-quality randomized clinical trials are needed to demonstrate the real efficacy of this natural product in the context of liver pathologies and in the management of physiological perturbations of the liver function (Rambaldi et al. 2005, 2007; Jacobs et al. 2002). Despite the many liver-health benefits attributed to silymarin, few studies have investigated the metabolism of its bioactive components within the human body. In fact, following ingestion by humans, (poly)phenolic compounds undergo extensive metabolism during their passage through the gastrointestinal tract and, with very few exceptions, only metabolites of the parent compounds enter the circulatory system. Chemical modifications initially occur in the lumen of the small intestine with cleavage of sugar moieties (when present), after which the aglycones that are released undergo glucuronidation, sulphation and/or methylation (Crozier et al. 2009). After entry of these metabolites into the circulatory system, phase II metabolism may also occur in the liver and other organs (Del Rio et al. 2010). The molecules that eventually reach the internal compartments of the human body are therefore different from those present in planta. Investigating the biological effects of the parent polyphenolic compounds instead of those of their human metabolites could lead to misleading or wrong
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Fig. 1. Chemical structure of milk thistle flavonolignans.
conclusions. Although the identification of polyphenolic catabolites was until recently confined to very well equipped laboratories, the necessary analytical facilities are now readily available in most research laboratories and are increasingly being used to investigate the metabolism of polyphenols by humans. The identification of a vast array of phenolic metabolites in vivo has also allowed the re-interpretation of bioavailability, which, for this specific class of components has always been considered extremely low (Crozier et al. 2010). To fully understand the biological and putatively beneficial effects of this very peculiar array of flavonolignans in vivo, one cannot ignore their bioavailability and metabolism. Therefore, the aim of this study was to investigate silymarin absorption and metabolism in human volunteers by means of high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) as an essential step on the road to finally verifying the putative health-related effects of milk thistle.
Materials and methods Milk thistle-based product and chemicals A water-soluble formulation of milk thistle was supplied by Sofar (Trezzano Rosa, Milano, Italy), in sealed bags containing 4 g of powder. Each bag contained 190 mg of milk thistle extract (silymarin), standardized with a minimum silybin content of 25%. In addition to silymarin, each bag contained: sugar, citric acid, aroma, colloidal silica and vitamin E. Pure silybin (A and B) was purchased from Sigma (St. Louis, MO, USA). Pure silydianin, silychristin,
isosilybin (A and B) and quercetin-3-O-glucuronide were purchased from PhytoLab GmbH & Co (Vestenbergsgreuth, Germany). All solvents and reagents were purchased from Carlo Erba Reagents (Milano, Italy).
Human feeding study The feeding study was carried out on 15 healthy human volunteers (8 males and 7 females), selected in compliance with established exclusion criteria including diabetes mellitus, cardiovascular events, chronic liver diseases or nephropathies, cancer, organ failure and intake of antioxidant or vitamin integrators. The volunteers were 24 ± 5 years old (mean ± SD) with an average BMI of 24 ± 4 kg/m2 . One subject was excluded from the study, as they did not complete urine collection. Each volunteer provided signed informed consent, and the study protocol was approved by the Ethics Committee for Human Research of the University of Parma. For 24 h before and 48 h after ingestion of milk thistle extract, the subjects followed a diet containing minimal phenolic compounds, by avoiding fruits, vegetables, all kinds of herbal tea, dietary antioxidant supplements, alcohol and drugs. To check for compliance, the volunteers were asked to complete a 3-day weighed food record starting the day before the study began. On the first day of the study, after an overnight fast, each subject drank a definite amount of milk thistle formulation (8 g, equivalent to 380 mg of silymarin extract) dissolved in 400 ml of water. Urine was collected at time 0 and during 0–2, 2–4, 4–8, 8–15, 15–24, 24–28, 28–32, 32–39, and 39–48 h collection periods after ingestion. The volume of urine collected during each period was measured and samples were
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Table 1 Flavonolignan content of two bags of milk thistle product consumed by volunteers (mol ± standard deviation (n = 5)). Compound
Content
Silychristin Silydianin Silybin A Silybin B Isosilybin A and B
75.7 70.9 125.3 144.4 68.2
Total
484.5 ± 51.3
± ± ± ± ±
7.4 12.9 13.3 19.3 6.9
stored at −80 ◦ C until analysis. Suitable aliquots of urine samples were filtered through 0.45 m nylon filter (Waters, Milford, MA, USA) and directly injected into the HPLC–MS/MS without further processing. HPLC–ESI-MS/MS analysis Milk thistle flavonolignans and their metabolites in the extract and urine samples were analysed using a Waters 2695 Alliance separation module equipped with a Micromass Quattro Micro Api mass spectrometer fitted with an electrospray interface (ESI) (Waters, Milford, MA, USA). Separation was performed using a Waters Atlantis dC18 (3 m, 2.1 mm × 150 mm) reverse phase column (Waters, Milford, MA, USA). To analyse the milk thistle extract, the mobile phase was pumped at a flow rate of 0.17 ml/min using a 15-min isocratic gradient of 25% acetonitrile in 1% aqueous formic acid immediately followed by a 15-min isocratic gradient of 30% acetonitrile in 1% aqueous formic acid. For urine sample analysis, a 30-min linear gradient of 15–35% acetonitrile in 1% aqueous formic acid was used. The ESI source and the collision energy conditions were tuned by infusing a standard of silybin (mixture of A and B diastereomers) together with 5% acetonitrile in 1% aqueous formic acid at a flow rate of 30 l/min. The ESI source worked in negative ionisation mode; the source temperature was 120 ◦ C, the desolvation temperature was 350 ◦ C, the capillary voltage was 2.8 kV, and the cone voltage was 40 V. The collision energy was set at 30 eV, the desolvation gas (nitrogen) at 750 l/h, and the cone gas (nitrogen) at 50 l/h; the collision gas used was argon. The flavonolignans and their metabolites were identified and quantified using HPLC with the MS operating in the multiple reaction monitoring (MRM) mode. The native flavonolignans in the extract and the unmetabolised compounds recovered in urine were quantified using calibration curves of the appropriate standard compound, whereas urinary metabolites (glucuronide, sulphate–glucuronide and diglucuronide) were quantified as quercetin-glucuronide equivalents, monitoring the loss of the glucuronyl moiety. Results Analysis of milk thistle extract Several flavanolignans were identified in the milk thistle extract based on their chromatographic retention times, and MS in-source fragmentations and identifications were confirmed by comparison with the commercial standards. In detail, the following flavonolignans were identified: silychristin, silydianin, silybin A and B, isosilybin A and B and their abundance was in agreement with previous reports (Lee et al. 2006; Wang et al. 2010). The total amount of silymarin flavonolignans consumed by volunteers is summarised in Table 1.
Table 2 Mass spectral characteristics of flavanolignans, phase II metabolites, and number of isomers of detected metabolites. Compound
[M−H]− (m/z) Qualifier ions (m/z)
Silychristin Silydianin Silybin A and B Isosilybin A and B Silymarin glucuronides Silymarin sulphate glucuronides Silymarin diglucuronide
481 481 481 481 657 737 833
Isomers
125 125 125 125 481, 125 16 657, 561, 481, 125 7 657, 481, 125 8
Identification of flavanolignans and their metabolites in urine The HPLC–MS/MS analysis enabled identification of unmetabolised urinary flavonolignans and urinary phase II metabolites, such as glucuronides, sulphate–glucuronides and diglucuronides, of which the mass spectral characteristics and the number of detected isomers are reported in Table 2. The metabolites derived by the action of human UDP-glucuronosyltransferases (UGTs) and sulphotransferases (SULTs) were identified via the loss of the conjugating groups (i.e. glucuronic acid and sulphate) to give the aglycone fragment ion (Prasain and Barnes 2007). The aglycone flavonolignans had a negatively charged molecular ion ([M−H]− ) at m/z 481, which on MS/MS (MS2 ) produced a main fragment ion at m/z 125, derived from A-ring fission, as previously reported (Lee et al. 2006). The silymarin glucuronides had a [M−H]− at m/z 657, which on MS2 produced a fragment ion corresponding to silymarin aglycones at m/z 481, generated by the loss of a glucuronide moiety equal to 176 atomic mass units (amu). More precise identification was possible through the concomitant fragmentation of the aglycones formed, which produced a main fragment ion at m/z 125. The flavanolignan sulphate–glucuronides had a [M−H]− at m/z 737, which on MS2 produced different fragment ions corresponding to (i) silymarin glucuronides at m/z 657 derived from the loss of sulphate groups equal to 80 amu, (ii) silymarin sulphates at m/z 561 derived from the loss of a glucuronide moiety or (iii) silymarin aglycones at m/z 481 derived from the loss of both conjugations. Once again, further fragmentation generated ions at m/z 125, in keeping with the presence of one of the silymarin flavonolignans. The flavanolignan diglucuronides, with a [M−H]− at m/z 833, produced different fragment ions on MS2 at m/z 657 and 481, derived from the loss of one or two glucuronide moieties, respectively, and fragment ions at m/z 125 derived from flavanolignan ring fission. Milk thistle flavonolignans have the same molecular weight and, therefore, mass-to-charge ratio, but they also share fragmentation patterns (corresponding to the fission of the A ring), making chromatographic separation essential for distinguishing each specific compound by comparison with the pure standard. However, this approach cannot be applied to single metabolites (e.g. glucuronides), since the relevant pure standards are not available. Therefore, although 31 different silymarin metabolites (including isomers of the same aglycone) generated after milk thistle ingestion were identified, it was impossible to attribute individual metabolites to specific flavanolignan molecules. The flavonolignans were preferentially metabolised by UGTs, forming 16 and 8 different glucuronide and diglucuronide conjugates, respectively, whereas only 7 sulphoglucuronides were identified (Table 2). The number of silymarin metabolites identified in human urine was very high with respect to the native flavanolignans present in the milk thistle extract. This is due to the multiple phenolic hydroxyl groups available for conjugation by UGTs and SULTs, resulting in the formation of a multitude of possible conjugates. An example of a LC–MS2 trace
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Fig. 2. Chromatographic profile of urinary metabolites of silymarin. Total Ion Current (TIC) of multiple reaction monitoring (MRM) chromatograms. Example of urine sample from a subject from the second collection period (2–4 h) after milk thistle extract ingestion. The main isomer metabolites of silymarin are reported. (A) Silymarin glucuronides; (B) silymarin sulphate glucuronides; (C) silymarin diglucuronides.
showing the most relevant identified catabolites in the 2–4 h urine collection of one subject is shown in Fig. 2.
Quantitative analysis of urinary catabolites and their excretion profiles As for the qualitative analysis, urine samples were quantitatively analysed by liquid chromatography–tandem mass spectrometry in the multiple reaction monitoring (MRM) mode, in order to calculate the total flavonolignan excretion and estimate
their bioavailability in humans. The quantitative excretion kinetic of flavonolignans and their metabolites in urine is shown in Fig. 3. Urinary flavonolignans were mainly recovered in their conjugated form, which greatly exceeded the aglycones. The glucuronides were the major excreted form, with a total excretion equal to 1.37 mol, compared to 0.53 and 0.25 mol of sulphate–glucuronides and diglucuronides, respectively. The amount of aglycone flavonolignans excreted was 0.02 mol, accounting for the excretion of both diastereomeric forms of silybin and isosilybin. No trace of silydianin and silychristin aglycones was detected in urine.
Fig. 3. Excretion profiles of flavonolignans in urine over 48 h in 14 volunteers. (A) Silymarin glucuronides; (B) silymarin sulphate glucuronides; (C) silymarin diglucuronides; (D) aglycone flavonolignans (silybin A and B, isosilybin A and B). Data expressed as mean values with their standard errors depicted by vertical bars.
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Monoglucuronides showed a consistent excretion kinetic, indicating that all flavonolignans underwent this conjugative step simultaneously. Their profile differed from that of sulphate–glucuronides and diglucuronides, which were also consistent within their category. In fact, the peak excretion of monoglucuronides occurred 2 h after consumption of the extract, whereas the sulphate–glucuronides and diglucuronides shared an excretion profile, reaching a peak 8 h after silymarin ingestion. Unmetabolised silybin and isosilybin diastereomers had a different excretion profile, peaking at 15 and 24 h after extract intake, respectively. It was interesting to note that all silymarin conjugate excretion slightly but consistently increased between 24 and 28 h and all conjugated and unconjugated forms showed a subsequent minor relative peak between 32 and 39 h. Most of the subjects showed complete excretion within 48 h, with only trace amounts excreted in the last urine collection. Finally, the mean bioavailability of milk thistle flavonolignans, calculated as the ratio between total silymarin excretion (conjugates and aglycones) and the total intake of silymarin, was 0.45 ± 0.28% (mean ± SD), ranging from 0.12 to 1.16% among subjects.
Discussion Milk thistle extract is widely used in the United States and Europe as a herbal supplement for the treatment of liver-related disorders. Many clinical trials have been carried out to evaluate the efficacy of its putatively bioactive fraction, namely silymarin, against liver cirrhosis (viral or alcoholic) and liver inflammation. Ferenci et al. (2009) assessed the effect of silymarin on the outcome of cirrhotic patients and found that treatment was effective in patients with alcoholic cirrhosis and in patients initially rated ‘Child A’. Another study investigated the effect of 1–2 weeks intravenous administration of silybin in patients with HCV infection, showing an antiviral effect in patients who were unresponsive to pegylated-interferon/ribavirin treatment (Ferenci et al. 2008). On the contrary, Parés et al. (1998) observed no effect on the survival of alcoholics with liver cirrhosis after chronic supplementation of 150 mg of silymarin three times daily for 2 years. Thus, based on human clinical trials, the potential benefits of milk thistle silymarin in the treatment of liver disorders remain controversial. However, the putative hepatoprotective effects of these flavonolignans have been confirmed by several in vitro studies. Silymarin was able to inhibit proliferation and pro-inflammatory cytokine secretion by peripheral blood mononuclear cells and T cells (Morishima et al. 2010). Silybin alone inhibited the production of pro-inflammatory cytokines, showed a clear antioxidant effect and reduced the direct and indirect pro-fibrogenic potential of hepatic stellate cells (Trappoliere et al. 2009). Moreover, silymarin polyphenols have been shown to be involved in blocking the entry and transmission of HCV virus in host cells (Wagoner et al. 2010). Several other in vitro studies confirmed the bioactivity observed, at least in certain cases, in human interventions (Saliou et al. 1998), but none of these studies considered the possible modifications occurring to phenolic compounds during their passage through the gastrointestinal tract (Crozier et al. 2010). The milk thistle flavonolignans are extensively modified by phase II human enzymes, this process resulting in the formation of intestinal and hepatic metabolites that considerably exceed the native aglycone molecules in biological fluids, as shown in the present study and reported elsewhere (Wen et al. 2008; Hawke et al. 2010). However, to date, none of the in vitro studies present in the scientific literature have investigated the bioactivity of silymarin metabolites, the molecules that actually come into contact with the internal compartments of the human body.
Few studies have investigated milk thistle flavonolignan catabolism in detail or accurately estimated their bioavailability in humans. This work confirms that silymarin phenolics undergo extensive metabolism, as do almost all flavonoids, forming sulphate and glucuronide conjugates through the action of UGTs and SULTs in the small intestine and liver. Table 2 shows how 24 of the 31 identified metabolites are derived by the action of UGTs, and that only 7 metabolites are formed through the concurrent action of SULTs. This specificity for glucuronidation contrasts with the behaviours of several other flavonoidic compounds, like green tea and cocoa catechins, which are preferentially sulphated (Stalmach et al. 2009). This is the first study, to our knowledge, to describe such a huge number of conjugated flavonolignans in vivo, and to follow their excretory profile in human volunteers for 2 complete days and until complete clearance. Previous studies failed to identify metabolites by treating biological samples (plasma or urine) with deconjugating enzymes, namely -glucuronidases and sulphatases, allowing the detection of the resulting aglycones with spectrophotometric devices (Wen et al. 2008; Hawke et al. 2010; Hoh et al. 2006, 2007). However, deconjugating enzymes show variation in their specificity and such treatment does not enable full understanding of the metabolic processes undergone by these phenolic compounds within the human body (Donovan et al. 2006). Fig. 3 shows that silymarin compounds are mainly excreted within 24 h after ingestion, but substantial amounts of glucuronides, sulpho-glucuronides and diglucuronides are excreted for up to 48 h. There was substantial interindividual variation in the excretion rate and quality among the volunteers, but on average, sulphoglucuronides and diglucuronides, despite their more hydrophilic nature, showed delayed excretion with respect to monoglucuronides. In fact, peak excretion of these compounds occurred 8 h after silymarin consumption, whereas monoglucuronides peaked in the first urine collection. This may be due to the non-marginal entero-hepatic recirculation (EHR) that preferentially occurs for silymarin sulphate–glucuronides and diglucuronides. EHR could also explain the secondary peaks in the excretion profile, which were consistent with the plasma pharmacokinetics previously described by Wen et al. (2008). The aglycones silybin and isosilybin showed high interindividual variation among volunteers, and secondary peaks were also present during the 48 h after consumption (Fig. 3). However, although 2.17 mol of total flavanolignans were excreted on average, only 0.02 mol were recovered in urine as unconjugated, making the contribution of aglycones to the final bioavailability insignificant. Although polyphenols are drastically modified by the colon microbiota, giving rise to a plethora of smaller phenolic compounds that are then absorbed and usually exceed the circulating and excreted concentration of human phase II metabolites (Crozier et al. 2010), it was impossible to detect any of these microbially derived compounds in this study. These molecules may have been missed by the analysis, being chemically different from those identified as being most frequently derived from other polyphenolic compounds, but the hypothesis that these flavanolignans are somehow catabolised through different pathways or stored within the human body was not ruled out. It is noteworthy that the calculated bioavailability, equal to 0.45% of the ingested dose, was higher than that of other flavonoids with a similar molecular weight, like anthocyanins and galloylated catechins (Manach et al. 2005). While one study reported a urinary excretion of silymarin equal to 3.1% of the ingested dose (Hawke et al. 2010), the approach used on that occasion was very different from that applied in the present study. In fact, because only a few flavonoid metabolites are commercially available, the analytical approach used to study polyphenol bioavailability almost invariably involves the treatment of samples with hydrolytic enzymes
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– namely glucuronidases and sulphatases – followed by quantification of the released aglycones by HPLC (Henning et al. 2005; Lee et al. 2002). Quantification of partially identified metabolites by MS using MRM or selected ion monitoring (SIM) should be based on calibration curves of standards that, as mentioned above, are not generally commercially available. To overcome this drawback, a related available compound may be used, which in the current study was quercetin-O-glucuronide. Quantification was therefore based on a common reaction in the mass spectrometry system, namely cleavage of one glucuronic acid from a flavonoidic structure, and this was the first and only study to our knowledge in which flavonolignan metabolites were quantified without applying enzymatic hydrolysis before the analysis, thus providing an actual fingerprint of their urinary excretion. In general, the response of glucuronidase/sulphatase sources used during sample preparation are not constant and there may be substantial batch-to-batch variation in their specificity (Donovan et al. 2006). As the enzymatic deconjugation approach is generally linked to the unavailability of MS facilities, there is a general lack of information about the efficiency by which the enzymes hydrolyse individual metabolites and release the aglycones. This introduces a variable and unquantified error factor that makes bioavailability estimates doubtful. In summary, the present study shows that milk thistle flavonolignans are extensively modified by human enzymes, as 99% of the absorbed fraction was recovered in urine as sulphoand glucuronyl-conjugates. A total of 31 different metabolites were detected, indicating a strong affinity of these molecules for hepatic phase II enzymes, which may be related to their putative liver healing potential. Moreover, the hypothesised entero-hepatic recirculation might allow biologically relevant concentrations of flavonolignan metabolites to be reached at the hepatobiliary level and this could be taken into account when establishing total silymarin dose and frequency of administration. The urinary excretion of these molecules was followed to complete clearance (48 h) and no microbial catabolites were identified, leaving room for intriguing hypotheses about their actual catabolism or storage. In conclusion, future studies dealing with the physiological effects of milk thistle should start by considering the modifications that occur to its polyphenolic components once ingested by humans. The term “phytocomplex” is customary to describe the elaborate mixtures of plant secondary metabolites administered by means of herbal medicines. However, as in the case of milk thistle, such complexity reverberates in, and is often amplified by, the behaviour of human metabolism and should be somehow redefined. Whether this remarkable phase II metabolism is related to the putative hepatoprotective effect of silymarin is not yet known, but any future in vitro study or dose response in vivo investigation will have to apply or quantify flavanolignan metabolites, given that, in this study, 31 such metabolites were identified. Finally, future studies evaluating the effect of the human faecal microbiota on silymarin flavonolignans will probably enable the bioavailability data of these particular polyphenolic compounds to be updated. Acknowledgements The authors would like to thank all the volunteers who participated in the present study. References Crozier, A., Del Rio, D., Clifford, M.N., 2010. Bioavailability of dietary flavonoids and phenolic compounds. Molecular Aspects of Medicine 31, 446–467. Crozier, A., Jaganath, I.B., Clifford, M.N., 2009. Dietary phenolics: chemistry, bioavailability and effects on health. Natural Products Reports 26, 1001–1043. Del Rio, D., Calani, L., Cordero, C., Salvatore, S., Pellegrini, N., Brighenti, F., 2010. Bioavailability and catabolism of green tea flavan-3-ols in humans. Nutrition 26, 1110–1116.
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