Abstract
Silymarin, a mixture of polyphenolic flavonoids extracted from milk thistle (Silybum marianum), is composed mainly of silychristin, silydianin, silybin A, silybin B (SBB), isosilybin A (ISBA), and isosilybin B. In this study, the plasma concentrations of free (unconjugated), conjugated (sulfated and glucuronidated), and total (free and conjugated) silymarin flavonolignans were measured using liquid chromatography-electrospray ionization-mass spectrometry, after a single oral dose of 600 mg of standardized milk thistle extracts to three healthy volunteers. Pharmacokinetic analysis indicated that silymarin flavonolignans were rapidly eliminated with short half-lives (1–3 and 3–8 h for free and conjugated, respectively). The AUC0→∞ values of the conjugated silymarin flavonolignans were 4- to 30-fold higher than those of their free fractions, with SBB (mean AUC0→∞ = 51 and 597 μg · h/l for free and conjugated, respectively) and ISBA (mean AUC0→∞ = 30 and 734 μg · h/l for free and conjugated, respectively) exhibiting higher AUC0→∞ values in comparison with other flavonolignans. Near the plasma peak times (1–3 h), the free, sulfated, and glucuronidated flavonolignans represented approximately 17, 28, and 55% of the total silymarin, respectively. In addition, the individual silymarin flavonolignans exhibited quite different plasma profiles for both the free and conjugated fractions. These data suggest that, after oral administration, silymarin flavonolignans are quickly metabolized to their conjugates, primarily forming glucuronides, and the conjugates are primary components present in human plasma.
Silymarin, a mixed extract of polyphenolic flavonoids isolated from milk thistle (Silybum marianum), is composed mainly of six flavonolignans including silychristin (SC), silydianin (SD), silybin A (SBA), silybin B (SBB), isosilybin A (ISBA), and isosilybin B (ISBB) (Fig. 1). As an herbal remedy, silymarin is widely used for the self-treatment of liver disease and cancer (Flora et al., 1998; Jacobs et al., 2002; Fraschini et al., 2002; Ladas and Kelly, 2003). Silymarin is also used for the treatment of Amanita phalloides mushroom poisoning (Desplaces et al., 1975; Hruby et al., 1983; Vogel et al., 1984). In vitro and animal studies have demonstrated the hepatoprotective properties of silymarin or silybin (a mixture of SBA and SBB) (Fraschini et al., 2002; Hoofnagle, 2005; Crocenzi and Roma, 2006). Several clinical trials have shown an excellent safety profile for silymarin in humans (Fraschini et al., 2002; Ball and Kowdley, 2005; Dryden et al., 2006). However, the clinical efficacy and dose-exposure relationships in humans remain unclear, because of the small number of participants and the lack of information on the exposure levels of the major silymarin flavonolignans with administration of standardized dosage regimens (Jacobs et al., 2002; Ball and Kowdley, 2005; Mayer et al., 2005; Rambaldi et al., 2005).
Silymarin is believed to be metabolized primarily to conjugates (e.g., sulfates and glucuronides) both in vitro and in vivo (Rickling et al., 1995; Kren et al., 2000; Han et al., 2004; D'Andrea et al., 2005). Some previous studies have reported the plasma concentrations of the free and total silybin isomers in animals and humans after an oral dose of silybin or silymarin (Barzaghi et al., 1990; Mascher et al., 1993; Morazzoni et al., 1993; Gatti and Perucca, 1994; Schandalik and Perucca, 1994; Rickling et al., 1995). However, the pharmacokinetics and metabolism of individual silymarin flavonolignans in humans have not been reported previously. To better understand the elimination and metabolic profile of the six major active isomers of silymarin, we investigated the pharmacokinetics and metabolic profile of the free (unconjugated), conjugated (sulfated and glucuronidated), and total (free and conjugated) silymarin flavonolignans in plasma after a single oral administration of 600 mg of standardized milk thistle extracts to healthy volunteers, using a rapid and sensitive liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS) assay. The contents of the six silymarin flavonolignans in several commercial milk thistle products were also estimated using a simple high-performance liquid chromatography (HPLC)-UV assay to provide a better understanding of the relationship between the composition of the major flavonolignans in the administered product and their relative exposures in blood.
Materials and Methods
Chemicals. Silybin (Silibinin) was purchased from Sigma-Aldrich (St. Louis, MO). The composition of silybin was confirmed to be a mixture of SBA and SBB by LC-ESI-MS, and the contents of SBA and SBB were analyzed to be 48 and 52%, respectively, by an HPLC-UV assay. The measured ratios of SBA and SBB in silybin were used for the qualitative and quantitative analysis in this study. SC was obtained from ChromaDex (Santa Ana, CA). SD and powdered milk thistle extracts were purchased from U.S. Pharmacopoeia (USP) (Rockville, MD). Standardized silymarin, naringenin (NG) (the internal standard for quantification), sulfatase (EC 3.1.6.1; type H-1 from Helix pomatia), β-glucuronidase (EC 3.2.1.31; type B-10 from bovine liver), d-saccharic acid 1,4-lactone (d-SL) (a specific β-glucuronidase inhibitor), and glacial acetic acid (HAc) were purchased from Sigma-Aldrich. Silymarin Plus tablets (labeled to contain 237 mg of milk thistle extracts per tablet) were obtained from Source Naturals (Scotts Valley, CA). Capsule 1 (labeled to contain 300 mg of milk thistle extracts per capsule) was from Nutraceutical Sciences Institute (Boynton Beach, FL). Capsule 2 (labeled to contain 175 mg of milk thistle extracts per capsule) was from Nature's Way (Springville, UT). Pooled human plasma was obtained from Valley Biomedical (Winchester, VA). Acetonitrile (HPLC grade) and methanol (MeOH) (HPLC grade) were obtained from Mallinckrodt (Phillipsburg, NJ). All other chemicals and reagents used were of analytical grade.
Analysis of Silymarin Flavonolignans in Commercial Milk Thistle Products. Milk Thistle extracts are currently marketed as dietary supplements in the United States and are not regulated by the Food and Drug Administration as drugs. Because the Food and Drug Administration has little control over the quality of herbal products such as silymarin, it is necessary to quantitatively estimate the contents of the potentially active ingredients in botanically derived therapies before use. Determination of the six silymarin flavonolignans in various commercial milk thistle extracts was performed with a simple HPLC system using NG as the internal standard (IS). Chromatographic separation was performed using an Agilent 1100 LC system (Palo Alto, CA) with a BrownLee RP-C18 guard column (15 mm × 3.2 mm i.d., 7 μm; PerkinElmer Life and Analytical Sciences, Shelton, CT) and an Axxiom ODS analytical column (150 mm × 4.6 mm i.d., 5 μm; Thomson Instrument, Clear Brook, VA). HPLC conditions were as follows: mobile phase, MeOH-0.1% HAc (pH 3) (46:54, v/v) with isocratic elution; detection wavelength, 288 nm; flow rate, 1.5 ml/min; injection volume, 20 μl; and run time, 20 min. Typical retention times of SC, SD, NG, SBA,SBB, ISBA, and ISBB under the experimental conditions used were 4.1, 4.8, 9.1, 10.8, 12.1, 15.0, and 16.0 min, respectively (Fig. 2). Stock standard solutions of SC, SD, SBA, and SBB were separately prepared in MeOH and diluted with 50% MeOH. Calibration curves were set up using mixed standard solutions containing SC, SD, SBA, and SBB. Concentrations of silymarin flavonolignans in the samples were estimated with 1/x2 weighted least-squares regression equations derived from the peak area ratios of individual silymarin flavonolignans to that of NG. Because ISBA and ISBB were not commercially available during this study, their concentrations were initially calculated using the calibration curves of SBA and SBB, assuming that ISBA and ISBB have the similar quantitative responses as those of SBA and SBB, respectively. These analytical responses were later evaluated and corrected using purified standards of ISBA and ISBB obtained from Madaus (Köln, Germany).
Sample Preparation for Commercial Milk Thistle Products. Ten tablets or capsules were weighed and finely pulverized. Appropriate amounts of the powder corresponding to one tablet or capsule were separately weighed and transferred to a 25-ml volumetric flask and then mixed with 20 ml of MeOH. The mixture was sonicated for 15 min at room temperature and diluted to 25 ml with MeOH. The mixture was filtered by a Millex-HX Nylon syringe filter (0.45 μm, 25 mm; Millipore, Bedford, MA) to remove any particles. The first 5 ml of the filtrates was discarded, and the following filtrates were collected. Appropriate aliquots of the filtrates were diluted with 50% MeOH, as well as the addition of NG (final concentration 5 μg/ml), and analyzed by the HPLC-UV assay as described above. Standardized silymarin (Sigma-Aldrich) and powdered milk thistle extracts (USP) were directly dissolved in MeOH and then diluted with 50% MeOH, and determined by the HPLC-UV assay.
Analysis of Silymarin Flavonolignans in Human Plasma. Identification and quantification of the six silymarin flavonolignans in human plasma required more sensitivity than UV detection could provide; thus, it was performed by LC-ESI-MS. Separation of the six silymarin flavonolignans was performed using an Agilent HP 1050 LC system (Palo Alto, CA) with a C18 SecurityGuard cartridge (4 × 2.0 mm i.d.; Phenomenex, Torrance, CA) and a Luna C18(2) analytical column (50 × 2.0 mm i.d., 3 μm; Phenomenex). HPLC conditions were as follows: mobile phase, MeOH-1% HAc (pH 2.8) (44:56, v/v) with isocratic elution; flow rate, 0.3 ml/min; injection volume, 25 μl; and run time, 12 min. Typical retention times of SC, SD, SBA, NG, SBB, ISBA, and ISBB, under the experimental conditions used, were 1.9, 2.4, 5.2, 5.5, 5.9, 8.4, and 9.2 min, respectively (Fig. 3). MS analysis and detection were performed with an API 100 LC/MS system (PerkinElmer Sciex, Toronto, ON, Canada) with a TurboIonspray interface in the negative ESI ionization mode. MS parameters used for qualitative analysis were ionspray voltage, -3100 V; ionspray temperature, 450°C; orifice voltage, -30 V; focusing ring voltage, -200 V; nebulizer gas, 10 liters/min; curtain gas, 8 liters/min; dwell time, 1 ms; and scan mode, full scan at the range of 100 to 800 m/z. MS parameters used for quantitative analysis were ionspray voltage, -3100 V; ionspray temperature, 450°C; orifice voltage, -30 V; focusing ring voltage, -200 V; nebulizer gas, 10 liters/ml; curtain gas, 8 liters/ml; dwell time, 300 ms; and scan mode, selective ion monitoring (SIM) with [M - H]- for silymarin flavonolignans (m/z 481), silymarin sulfates (m/z 561), silymarin glucuronides (m/z 657), and NG (m/z 271), respectively. Calibration curves were set up using standards of SC, SD, SBA, and SBB. Mixed standard solutions containing SC, SD, SBA, and SBB were spiked into pooled blank human plasma and then treated as described under Sample Preparation for Human Plasma. Concentrations of silymarin flavonolignans in the samples were estimated with 1/x2 weighted least-squares regression equations derived from the peak area ratios of individual silymarin flavonolignans to that of NG (described above).
Sample Preparation for Human Plasma. Plasma samples were treated with and without enzyme hydrolysis. The free (unconjugated) silymarin flavonolignans were directly determined without enzyme hydrolysis. The total (free and conjugated) silymarin flavonolignans were measured after hydrolysis using a mixed enzyme solution containing sulfatase and β-glucuronidase. Concentrations of the conjugated (sulfated and glucuronidated) silymarin flavonolignans were calculated from the differences between total and free concentrations. Sulfated or glucuronidated silymarin flavonolignans were calculated from the differences between the measured concentrations of free and those after hydrolysis with sulfatase (containing d-SL, a specific β-glucuronidase inhibitor) or β-glucuronidase. In brief, 100-μl aliquots of plasma were treated with sulfatase (80 U/ml in the final incubation) containing d-SL (10 mM in the final incubation), β-glucuronidase (8000 U/ml in the final incubation), and a mixture of sulfatase (80 U/ml in the final incubation) and β-glucuronidase (8000 U/ml in the final incubation), respectively. Plasma samples with different hydrolytic enzymes were buffered using sodium acetate (pH 5.0, 0.125 M in the final incubation) and incubated (final volume 120 μl) at 37°C with gentle shaking for 4 h. Preliminary experiments demonstrated that, after each hydrolysis, no peaks corresponding to the conjugated (sulfated or glucuronidated) silymarin flavonolignans were found by LC-ESI-MS, indicating that the enzyme hydrolyses were complete. After the addition of 0.6 ml of ice-cold ACN containing 1% HAc and NG (20 ng) into the incubations or plasma (for free silymarin flavonolignans), the mixture was centrifuged at 15,000g for 15 min at 4°C. The supernatants were transferred and then evaporated with a gentle stream of nitrogen at 45°C in a water bath. The residue was reconstituted in 100 μl of HPLC mobile phase (MeOH-1% HAc, 44:56, v/v) and then centrifuged at 10,000g for 10 min at 4°C, and 25 μl of the reconstituted supernatants was introduced for LC-ESI-MS analysis.
Pharmacokinetics of Silymarin Flavonolignans in Human Plasma. Plasma samples were obtained from three healthy volunteers after a single oral dose of 600 mg of standardized milk thistle extracts (capsule 1, labeled to contain 300 mg of milk thistle extracts per capsule) at 0 (predose) and 0.25 to 24 h. Aliquots (100 μl) of plasma at each time point were prepared with and without a mixed enzyme hydrolysis (80 U/ml sulfatase and 8000 U/ml β-glucuronidase) and measured by LC-ESI-MS as described above. Pharmacokinetic parameters of individual silymarin flavonolignans were estimated by a noncompartmental analysis using WinNonlin (Pharsight, Mountain View, CA). The maximum plasma concentration (Cmax) and time to maximum plasma concentration (Tmax) were obtained directly from the plasma concentration-time data. The terminal elimination rate constant (λz) was estimated by linear least-squares regression of the terminal portion of the plasma concentration-time curve, and the corresponding elimination half-live (t1/2) was then obtained by t1/2 = 0.693/λz. The area under the plasma concentration-time curve from time 0 to infinity (AUC0→∞) was calculated according to the linear trapezoidal rule.
Results
Estimation of Silymarin Flavonolignans in Milk Thistle Extracts. The limit of detection (signal/noise ratio >3:1) and linear quantitative range for the determination of each silymarin flavonolignans in milk thistle extracts using the HPLC-UV assay were 20 ng/ml and 0.05 to 200 μg/ml, respectively. The intraday and interday precisions, expressed as the relative standard deviations (n = 5) were 0.74 to 10 and 1.9 to 12%, respectively. The contents of the six silymarin flavonolignans in various commercial milk thistle extracts are summarized in Table 1. This composition allowed us to select a marketed silymarin formulation for the clinical study with reasonably high purity and known content. The relative percentages of individual silymarin flavonolignans in the standardized milk thistle extracts measured were very similar, with SC at 22 to 24%, SD at 9 to 15%, SBA at 18 to 22%, SBB at 30 to 35%, ISBA at 8 to 9%, and ISBB at 3 to 4%, respectively. These results indicated that SC, SBA, and SBB were the three predominant constituents (total 70–80%), whereas SD, ISBA, and ISBB were the minor components. Using the sum of six flavonolignans as the total content of silymarin, the measured contents of silymarin ranged from 57 to 71%. Standardized milk thistle extracts are generally considered to contain approximately 70 to 80% as silymarin (Flora et al., 1998; Simánek et al., 2000; Jacobs et al., 2002; Venkataramanan et al., 2006), whereas the content of silybin (SBA + SBB) represents approximately 40 to 70% of the total amounts of silymarin (Jacobs et al., 2002; USP, 2006; Venkataramanan et al., 2006). As shown in Table 1, the relative percentages of silybin (SBA + SBB) were found to be 48 to 56% in standardized milk thistle extracts (Sigma-Aldrich and USP) and 54 to 57% in commercial milk thistle tablets and capsules. However, there were substantial differences in silymarin content between the various commercial milk thistle products measured. The total silymarin contents of USP and Sigma-Aldrich standardized milk thistle extracts were found to be 69 and 54%, respectively, whereas the contents of the three marketed milk thistle products were actually 63% (tablet), 57% (capsule 1), and 71% (capsule 2), well below their labeled values (80%). This apparent discrepancy may be due to the use of nonspecific or colorimetric assays for determining silymarin content by the manufacturers.
Determination of Silymarin Flavonolignans in Human Plasma. Identification of silymarin flavonolignans was based on their chromatographic retentions and MS in-source fragmentations and were confirmed by comparing the standardized milk thistle extracts (Sigma-Aldrich or UPS). The six flavonolignans of silymarin exhibited different chromatographic retentions (Fig. 3), but very similar MS fragmentations (Fig. 4), with the base peak at m/z 481 ([M - H]-, deprotonated molecule ion of silymarin), and less abundant multiple subfragmentation ions (e.g., m/z 463, [M - H - H2O]-; m/z 453, [M - H - CO]-; m/z 301, [M - H - C8O5H4]-) in the negative ion mode. The MS spectra of individual silymarin flavonolignans (Fig. 4) were in agreement with those in published literature (Kim et al., 2003; Lee and Liu, 2003; Lee et al., 2006). Quantification of the six silymarin flavonolignans in human plasma was performed with LC-ESI-MS with a SIM detection at m/z 481 in the negative ion mode, which has higher sensitivity than in the positive ion mode. Although identification and quantification of the individual sulfated (m/z 561) and glucuronidated (m/z 657) silymarin flavonolignans was attempted, it was not possible to separate the conjugated products from each other with current analytical conditions (data not shown), because each silymarin flavonolignan has multiple phenolic and alcoholic hydroxyl sites for conjugation (Fig. 1), resulting in the formation of a multitude of possible conjugates. Thus, the total sulfated and glucuronidated silymarin flavonolignans were indirectly determined with enzyme hydrolysis using sulfatase and β-glucuronidase, respectively. To avoid the potential for simultaneous cleavage of glucuronides during desulfation, the addition of d-saccharic acid 1,4-lactone (a specific β-glucuronidase inhibitor) was used. The limit of detection and linear quantitative range of the six silymarin flavonolignans by the LC-ESI-MS assay were 2 and 5 to 1000 ng/ml, respectively. The intraday and interday precisions (relative standard deviations, n = 4) were 1.7 to 11 and 4.5 to 14%, respectively.
Pharmacokinetic Parameters of Silymarin Flavonolignans in Human Plasma. Pharmacokinetic analysis indicated that, after oral administration, silymarin flavonolignans were rapidly eliminated with short half-lives (1–3, 3–6, and 3–5 h for the free, conjugated, and total silymarin flavonolignans, respectively) (Fig. 5; Table 2). Conjugated SC exhibited a relatively longer half-life (∼8 h) than the other flavonolignans. Free SC and SD were not detectable or at very low concentrations. The Cmax values of free SBA,SBB, ISBA, and ISBB ranged from 9 to 23 ng/ml, whereas the Cmax values of total silymarin flavonolignans were 2- to 12-fold higher than the free fractions, with total SBB (Cmax = 131 ng/ml), and ISBA (Cmax = 113 ng/ml) exhibiting the highest peak plasma concentrations. The Cmax values of the conjugated silymarin flavonolignans were similar (SC and SD) or slightly lower (SBA,SBB, ISBA, and ISBB) in comparison with those of total silymarin flavonolignans (Table 2). The AUC0→∞ values of the conjugated and total silymarin flavonolignans were 4- to 30-fold higher than those for the free fractions, with SBB (AUC0→∞ = 51 and 597 μg · h/l for free and conjugated, respectively) and ISBA (AUC0→∞ = 30 and 734 μg · h/l for free and conjugated, respectively) exhibiting the highest AUC values, suggesting that conjugated silymarin flavonolignans, particularly with SBB and ISBA, were the major metabolites in human plasma.
Metabolic Profile of Free, Conjugated, and Total Silymarin Flavonolignans in Human Plasma. Plasma samples near the peak times (1–3 h) for each flavonolignan were pooled from all three healthy volunteers. The relative proportions of the free (unconjugated), conjugated (sulfated and glucuronidated), and total (free and conjugated) silymarin flavonolignans in human plasma are shown in Fig. 6 and Table 3. Near the peak times (1–3 h), the fractions of the free, sulfated, and glucuronidated silymarin were 17, 28, and 55% of the total silymarin, respectively. These data suggested that, after oral administration, silymarin flavonolignans were metabolized to their conjugates (sulfates and glucuronides), which represented approximately 83% of the total silymarin measured at the plasma peak times in healthy volunteers. In addition, the individual flavonolignans of silymarin exhibited quite different plasma profiles for the parents and conjugates. The major isomeric flavonolignans found in human plasma were SBB (∼30% of the total silymarin) and ISBA (∼21% of the total silymarin). Free and sulfated SDs were not detectable in this study. SBA mainly remained in the free form (∼∼60% of total), whereas SBB and SD were predominantly in their glucuronides (∼71 and 100% of total, respectively). Based on plasma exposure at the time of peak plasma concentration, ISBA preferred the formation of sulfates (∼60% of total) to glucuronides (∼35% of total), whereas SC and ISBB preferred the formation of glucuronides (∼49 and ∼60% of total, respectively) to sulfates (∼37 and ∼21% of total, respectively).
Discussion
Silymarin is considered to be primarily conjugated and excreted into bile and urine and appears to have minimal phase 1 metabolism (Flora et al., 1998; Fraschini et al., 2002). However, limited data exist for phase 2 metabolic pathways and the role of transporters in vivo (Venkataramanan et al., 2006). Silymarin metabolism in vivo may have a role in herbal-drug interactions, especially as doses for silymarin are increased. Before systematic evaluation of the safety, efficacy, and tolerability of orally administrated silymarin in patients with liver disease and other disorders, it is necessary to estimate the actual contents of the six principal isomers in the standardized silymarin extracts and obtain some preliminarily information about the pharmacokinetics of silymarin in humans. The plasma pharmacokinetics and metabolism of silybin (a mixture of SBA and SBB) in humans (Barzaghi et al., 1990; Mascher et al., 1993; Gatti and Perucca, 1994; Schandalik and Perucca, 1994; Rickling et al., 1995) and rats (Morazzoni et al., 1993; Rickling et al., 1995) have been reported previously, showing the fast elimination of both free and conjugated silybin. In this study, we investigated the pharmacokinetics and metabolic profile of the free (unconjugated), conjugated (sulfated and glucuronidated), and total (free and conjugated) silymarin flavonolignans in human plasma after a single oral dose of 600 mg of standardized milk thistle extracts to healthy volunteers. The results demonstrated that all six silymarin flavonolignans were rapidly eliminated, and the conjugated silymarin flavonolignans had relatively longer half-lives and much higher AUC0→∞ values than their free forms (Table 2). These results are in agreement with the previous observations after administration of silybin to humans ((Barzaghi et al., 1990; Mascher et al., 1993; Gatti and Perucca, 1994; Schandalik and Perucca, 1994; Rickling et al., 1995), although the individual silymarin flavonolignans were not completely characterized in the earlier studies. Our data also demonstrate that, at the peak times (1–3 h), the fractions of the free, sulfated, and glucuronidated silymarin in human plasma were approximately 17, 28, and 55% of the total (Table 3), respectively. These results confirmed the fact that silymarin flavonolignans are rapidly metabolized to their conjugates (83% of the total silymarin at Cmax), mainly present as glucuronides in plasma.
It was noted that the Cmax and AUC0→∞ values of total SBB and ISBA were 2- to 6-fold higher than those of other flavonolignans (Table 2). These data suggest that the conjugated SBB and ISBA are the major metabolites in plasma of healthy volunteers. According to the analysis of silymarin flavonolignans in the standardized milk thistle extracts (600 mg) orally dosed in this study, the estimated contents of SC, SD, SBA, SBB, ISBA, and ISBB were 78, 31, 75, 117, 30, and 13 mg (Table 1), respectively. Obviously, ISBA exhibited a relatively higher accumulation of conjugates in plasma despite a lower dose than other flavonolignans, with 60% of sulfates and 35% of glucuronides (Table 3). This relatively high metabolite content of ISBA in human plasma is consistent with a previous finding in rats (Morazzoni et al., 1993), although the underlying accumulation mechanism for this isomer is still unknown.
In vitro studies showed that the glucuronidation of silybin (a mixture of SBA and SBB) was stereoselective, and SBB was more efficient and faster than its diastereoisomer, SBA (Kren et al., 2000; Han et al., 2004). In this study, higher plasma percentage of glucuronidated SBB (71%) was observed in comparison with that of glucuronidated SBA (25%). Thus, these in vivo data are supported by the in vitro results regarding the stereoselective glucuronidation of SBA and SBB. Similarly, the in vivo glucuronidation of ISBA and ISBB also showed different stereoselectivities of metabolite present in plasma at the peak time (Table 3). Sulfated SBA and SBB had very similar plasma percentages (16 and 14%), whereas the plasma percentage of sulfated ISBA (60%) was higher than that of ISBB (21%), which may suggest that the sulfation of ISBA and ISBB is also stereoselective.
The conjugates in plasma exhibited secondary peaks and irregular profiles for some of the silymarin isomers, which varied among individual subjects (Fig. 5). Within an individual the secondary peaks were not consistent across isomers, which would suggest that irregular absorption from the dosage form was not a factor. Secondary peaks were also apparent in some of the free profiles, indicative of enterohepatic recycling. The fairly rapid absorption of silymarin, early peak times of the conjugates, and much higher maximum concentrations of the metabolites support rapid metabolism and low bioavailability. However, the expected lower volume of distribution of the conjugates relative to the parent silymarin isomers makes it difficult to provide any estimate of the fraction of the dose reaching the systemic circulation as conjugates relative to the parent silymarin isomer.
In conclusion, after oral administration of standardized milk thistle extracts, silymarin flavonolignans, the major biologically active components in milk thistle, are rapidly metabolized and measurable in plasma, mainly in the form of glucuronides. The individual silymarin flavonolignans exhibited quite different plasma profiles for the parents and metabolites, with longer half-lives for the metabolites and conjugated SBB and ISBA as the major metabolites in the plasma of healthy volunteers. The role of silymarin or flavonoid metabolism in potential herbal-drug interactions, as well as that in modulating flavonoid disposition and pharmacological activity, is still poorly understood. This study provides a basic understanding of silymarin disposition and metabolism in healthy individuals that should help in clarification and understanding of these complex relationships and provide a basis for comparison with studies of silymarin in patients that are ongoing.
Footnotes
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This research was supported, in part, by Grants R21AT001376 and U01AT003506 from the National Center for Complementary and Alternative Medicine.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.017566.
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ABBREVIATIONS: SC, silychristin; SD, silydianin; SBA, silybin A; SBB, silybin B; ISBA, isosilybin A; ISBB, isosilybin B; LC, liquid chromatography; ESI, electrospray ionization; MS, mass spectrometry; HPLC, high-performance liquid chromatography; USP, U.S. Pharmacopoeia; NG, naringenin; d-SL, d-saccharic acid 1,4-lactone; HAc, glacial acetic acid; MeOH, methanol; IS, internal standard; SIM, selective ion monitoring.
- Received July 6, 2007.
- Accepted October 1, 2007.
- The American Society for Pharmacology and Experimental Therapeutics