Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET

User menu

  • My alerts
  • Log in
  • Log out
  • My Cart

Search

  • Advanced search
Drug Metabolism & Disposition
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET
  • My alerts
  • Log in
  • Log out
  • My Cart
Drug Metabolism & Disposition

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Visit dmd on Facebook
  • Follow dmd on Twitter
  • Follow ASPET on LinkedIn
Research ArticleArticle

Pharmacokinetics and Metabolic Profile of Free, Conjugated, and Total Silymarin Flavonolignans in Human Plasma after Oral Administration of Milk Thistle Extract

Zhiming Wen, Todd E. Dumas, Sarah J. Schrieber, Roy L. Hawke, Michael W. Fried and Philip C. Smith
Drug Metabolism and Disposition January 2008, 36 (1) 65-72; DOI: https://doi.org/10.1124/dmd.107.017566
Zhiming Wen
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Todd E. Dumas
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sarah J. Schrieber
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Roy L. Hawke
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michael W. Fried
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Philip C. Smith
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF
Loading

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.

Fig. 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 1.

Chemical structures of the silymarin flavonolignans and naringenin (IS).

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).

Fig. 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 2.

Representative HPLC-UV chromatogram for the determination of silymarin flavonolignans in milk thistle extracts. HPLC-UV (288 nm) analysis was performed as described under Materials and Methods. 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.

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).

Fig. 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 3.

Representative LC-ESI-MS chromatograms for the determination of silymarin flavonolignans in human plasma. Standardized milk thistle extracts (2.5 μg extract/ml in final) from Sigma-Aldrich were spiked into 100 μl of pooled blank human plasma and analyzed by the LC-ESI-MS assay as described under Materials and Methods. 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. A, SIM chromatogram for the six silymarin flavonolignans with [M - H]- (m/z 481). B, SIM chromatogram for NG (IS) with [M - H]- (m/z 271).

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.

View this table:
  • View inline
  • View popup
TABLE 1

Estimation of the silymarin flavonolignans in various commercial sources of milk thistle extracts

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.

View this table:
  • View inline
  • View popup
TABLE 2

Pharmacokinetic parameters of the free (unconjugated), conjugated (sulfated and glucuronidated), and total (free and conjugated) silymarin flavonolignans in human plasma Plasma samples were obtained from three healthy volunteers after a single oral administration of 600 mg of standardized milk thistle extracts at 0 (predose) and 0.25 to 24 h. Concentrations of the free (without enzyme hydrolysis), total (mixed enzyme hydrolysis with sulfatase and β-glucuronidase), and conjugated (sulfated and glucuronidated) silymarin flavonolignans were measured or calculated as described under Materials and Methods. Data are expressed as mean ± S.D. (n = 3).

Fig. 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 4.

MS spectra of the silymarin flavonolignans in the negative ion mode. LC-ESI-MS conditions were described under Materials and Methods.

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).

View this table:
  • View inline
  • View popup
TABLE 3

Metabolic profiles of the free (unconjugated), conjugated (sulfated and glucuronidated), and total (free and conjugated) silymarin flavonolignans in human plasma near Tmax Plasma samples were pooled from three healthy volunteers near the peak times (1–3 h) for each flavonolignans after a single oral administration of 600 mg of standardized milk thistle extracts. Enzyme hydrolysis and quantification of the free, sulfated, glucuronidated, and total silymarin flavonolignans were performed as described under Materials and Methods. Data are expressed as the mean of duplicates. The value for silymarin is the sum of six flavonolignans.

Fig. 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 5.

Plasma concentration-time profiles of the free (unconjugated) and total (free and conjugated) silymarin flavonolignans after a single oral administration of 600 mg of standardized milk thistle extracts to three healthy volunteers. Quantification of silymarin flavonolignans was performed by the LC-ESI-MS assay as described under Materials and Methods. Free-1, Free-2, and Free-3, the free silymarin flavonolignans of the individual healthy volunteers; Total-1, Total-2, and Total-3, the total silymarin flavonolignans of the individual healthy volunteers.

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.

Fig. 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 6.

Representative LC-ESI-MS chromatograms (SIM) of the free (unconjugated), conjugated (sulfated and glucuronidated), and total (free and conjugated) silymarin flavonolignans in plasma pooled from three healthy volunteers near the peak times (1–3 h) after a single oral administration of 600 mg of standardized milk thistle extracts. Enzyme hydrolysis and LC-ESI-MS analysis were performed as described under Materials and Methods. A, free silymarin (without enzyme hydrolysis); B, after sulfatase hydrolysis (containing d-saccharic acid 1,4-lactone); C, after β-glucuronidase hydrolysis; D, after mixed enzyme hydrolysis (sulfatase + β-glucuronidase).

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

  • This research was supported, in part, by Grants R21AT001376 and U01AT003506 from the National Center for Complementary and Alternative Medicine.

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

  • doi:10.1124/dmd.107.017566.

  • 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

References

  1. ↵
    Ball KR, and Kowdley KV (2005) A review of Silybum marianum (milk thistle) as a treatment for alcoholic liver disease. J Clin Gastroenterol 39: 520-528.
    OpenUrlCrossRefPubMed
  2. ↵
    Barzaghi N, Crema F, Gatti G, Pifferi G, and Perucca E (1990) Pharmacokinetic studies on IdB 1016, a silybin-phosphatidylcholine complex, in healthy human subjects. Eur J Drug Metab Pharmacokinet 15: 333-338.
    OpenUrlPubMed
  3. ↵
    Crocenzi FA and Roma MG (2006) Silymarin as a new hepatoprotective agent in experimental cholestasis: new possibilities for an ancient medication. Curr Med Chem 13: 1055-1074.
    OpenUrlCrossRefPubMed
  4. ↵
    D'Andrea V, Perez LM, and Pozzi EJS (2005) Inhibition of rat liver UDP-glucuronosyltransferase by silymarin and the metabolite silibinin-glucuronide. Life Sci 77: 683-692.
    OpenUrlCrossRefPubMed
  5. ↵
    Desplaces A, Choppin J, Vogel G, and Trost W (1975) The effects of silymarin on experimental phalloidine poisoning. Arzneimittelforschung 25: 89-96.
    OpenUrlPubMed
  6. ↵
    Dryden GW, Song M, and McClain C (2006) Polyphenols and gastrointestinal diseases. Curr Opin Gastroenterol 22: 165-170.
    OpenUrlPubMed
  7. ↵
    Flora K, Hahn M, Rosen H, and Benner K (1998) Milk thistle (Silybum marianum) for the therapy of liver disease. Am J Gastroenterol 93: 139-143.
    OpenUrlCrossRefPubMed
  8. ↵
    Fraschini F, Demartini G, and Esposti D (2002) Pharmacology of silymarin. Clin Drug Invest 22: 51-65.
    OpenUrlCrossRef
  9. ↵
    Gatti G and Perucca E (1994) Plasma concentrations of free and conjugated silybin after oral intake of a silybin-phosphatidylcholine complex (silipide) in healthy volunteers. Int J Clin Pharmacol Ther 32: 614-617.
    OpenUrlPubMed
  10. ↵
    Han YH, Lou HX, Ren DM, Sun LR, Ma B, and Ji M (2004) Stereoselective metabolism of silybin diastereoisomers in the glucuronidation process. J Pharm Biomed Anal 34: 1071-1078.
    OpenUrlCrossRefPubMed
  11. ↵
    Hoofnagle JH (2005) Milk thistle and chronic liver disease. Hepatology 42: 4.
    OpenUrlCrossRefPubMed
  12. ↵
    Hruby K, Csomos G, Fuhrmann M, and Thaler H (1983) Chemotherapy of Amanita phalloides poisoning with intravenous silibinin. Hum Toxicol 2: 183-195.
    OpenUrlPubMed
  13. ↵
    Jacobs BP, Dennehy C, Ramirez G, Sapp J, and Lawrence VA (2002) Milk thistle for the treatment of liver disease: a systematic review and meta-analysis. Am J Med 113: 506-515.
    OpenUrlCrossRefPubMed
  14. ↵
    Kim NC, Graf TN, Sparacino CM, Wani MC, and Wall ME (2003) Complete isolation and characterization of silybins and isosilybins from milk thistle (Silybum marianum). Org Biomol Chem 1: 1684-1689.
    OpenUrlCrossRefPubMed
  15. ↵
    Kren V, Ulrichova J, Kosina P, Stevenson D, Sedmera P, Prikrylova V, Halada P, and Simánek V (2000) Chemoenzymatic preparation of silybin β-glucuronides and their biological evaluation. Drug Metab Dispos 28: 1513-1517.
    OpenUrlPubMed
  16. ↵
    Ladas EJ and Kelly KM (2003) Milk thistle: is there a role for its use as an adjunct therapy in patients with cancer? J Altern Complement Med 9: 411-416.
    OpenUrlCrossRefPubMed
  17. ↵
    Lee DYW and Liu Y (2003) Molecular structure and stereochemistry of silybin A, silybin B, isosilybin A, and isosilybin B, isolated from Silybum marianum (milk thistle). J Nat Prod 66: 1171-1174.
    OpenUrlPubMed
  18. ↵
    Lee JI, Hsu BH, Wu D, and Barrett JS (2006) Separation and characterization of silybin, isosilybin, silydianin and silychristin in milk thistle extract by liquid chromatography-electrospray tandem mass spectrometry. J Chromatogr A 1116: 57-68.
    OpenUrlCrossRefPubMed
  19. ↵
    Mascher H, Kikuta C, and Weyhenmeyer R (1993) Diastereomeric separation of free and conjugated silibinin in plasma by reversed phase HPLC after specific extraction. J Liq Chromatogr 16: 2777-2789.
    OpenUrlCrossRef
  20. ↵
    Mayer KE, Myers RP, and Lee SS (2005) Silymarin treatment of viral hepatitis: a systematic review. J Viral Hepat 12: 559-567.
    OpenUrlCrossRefPubMed
  21. ↵
    Morazzoni P, Montalbetti A, Malandrino S, and Pifferi G (1993) Comparative pharmacokinetics of silipide and silymarin in rats. Eur J Drug Metab Pharmacokinet 18: 289-297.
    OpenUrlPubMed
  22. ↵
    Rambaldi A, Jacobs BP, Iaquinto G, and Gluud C (2005) Milk thistle for alcoholic and/or hepatitis B or C liver diseases—a systematic Cochrane hepato-biliary group review with meta-analysis of randomized clinical trials. Am J Gastroenterol 100: 2583-2591.
    OpenUrlCrossRefPubMed
  23. ↵
    Rickling B, Hans B, Kramarczyk R, Krumbiegel G, and Weyhenmeyer R (1995) Two high-performance liquid chromatographic assays for the determination of free and total silibinin diastereomers in plasma using column switching with electrochemical detection and reversed-phase chromatography with ultraviolet detection. J Chromatogr B 670: 267-277.
    OpenUrlCrossRefPubMed
  24. ↵
    Schandalik R and Perucca E (1994) Pharmacokinetics of silybin following oral administration of silipide in patients with extrahepatic biliary obstruction. Drugs Exp Clin Res 20: 37-42.
    OpenUrlPubMed
  25. ↵
    Simánek V, Kren V, Ulrichova J, Vicar J, and Cvak L (2000) Silymarin—what is in the name? Hepatology 32: 442-443.
    OpenUrlCrossRefPubMed
  26. ↵
    USP (2006) The United States Pharmacopoeia and The National Formulary. United States Pharmacopeial Convention, Rockville, MD.
  27. ↵
    Venkataramanan R, Komoroski B, and Strom S (2006) In vitro and in vivo assessment of herb drug interactions. Life Sci 78: 2105-2115.
    OpenUrlCrossRefPubMed
  28. ↵
    Vogel G, Tuchweber B, and Trost W (1984) Protection by silibinin against Amanita phalloides intoxication in beagles. Toxicol Appl Pharmacol 73: 355-362.
    OpenUrlCrossRefPubMed
PreviousNext
Back to top

In this issue

Drug Metabolism and Disposition: 36 (1)
Drug Metabolism and Disposition
Vol. 36, Issue 1
1 Jan 2008
  • Table of Contents
  • Table of Contents (PDF)
  • About the Cover
  • Index by author
  • Back Matter (PDF)
  • Editorial Board (PDF)
  • Front Matter (PDF)
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Drug Metabolism & Disposition article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Pharmacokinetics and Metabolic Profile of Free, Conjugated, and Total Silymarin Flavonolignans in Human Plasma after Oral Administration of Milk Thistle Extract
(Your Name) has forwarded a page to you from Drug Metabolism & Disposition
(Your Name) thought you would be interested in this article in Drug Metabolism & Disposition.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Research ArticleArticle

Pharmacokinetics and Metabolic Profile of Free, Conjugated, and Total Silymarin Flavonolignans in Human Plasma after Oral Administration of Milk Thistle Extract

Zhiming Wen, Todd E. Dumas, Sarah J. Schrieber, Roy L. Hawke, Michael W. Fried and Philip C. Smith
Drug Metabolism and Disposition January 1, 2008, 36 (1) 65-72; DOI: https://doi.org/10.1124/dmd.107.017566

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
Research ArticleArticle

Pharmacokinetics and Metabolic Profile of Free, Conjugated, and Total Silymarin Flavonolignans in Human Plasma after Oral Administration of Milk Thistle Extract

Zhiming Wen, Todd E. Dumas, Sarah J. Schrieber, Roy L. Hawke, Michael W. Fried and Philip C. Smith
Drug Metabolism and Disposition January 1, 2008, 36 (1) 65-72; DOI: https://doi.org/10.1124/dmd.107.017566
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Materials and Methods
    • Results
    • Discussion
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • In Vivo Functional Effects of CYP2C9 M1L
  • Clearance pathways: fevipiprant with probenecid perpetrator
  • Predicting Volume of Distribution from In Vitro Parameters
Show more Articles

Similar Articles

  • Home
  • Alerts
Facebook   Twitter   LinkedIn   RSS

Navigate

  • Current Issue
  • Fast Forward by date
  • Fast Forward by section
  • Latest Articles
  • Archive
  • Search for Articles
  • Feedback
  • ASPET

More Information

  • About DMD
  • Editorial Board
  • Instructions to Authors
  • Submit a Manuscript
  • Customized Alerts
  • RSS Feeds
  • Subscriptions
  • Permissions
  • Terms & Conditions of Use

ASPET's Other Journals

  • Journal of Pharmacology and Experimental Therapeutics
  • Molecular Pharmacology
  • Pharmacological Reviews
  • Pharmacology Research & Perspectives
ISSN 1521-009X (Online)

Copyright © 2021 by the American Society for Pharmacology and Experimental Therapeutics