Summary of Health Canada’s safety assessment of milk thistle seed extract for use as a supplemental ingredient

Abstract

Health Canada's Food and Nutrition Directorate assessed milk thistle seed extract (MTE) for safety, based on publicly available information. The Food and Nutrition Directorate concluded that there was sufficient information to establish conditions under which standardized MTE could be consumed safely as a supplemental ingredient in supplemented foods. Consequently, Health Canada will permit the use of milk thistle seed extract (silymarin)^{Footnote 1} as a supplemental ingredient in supplemented foods under certain conditions. The conditions are outlined in the Notice of Modification.

Introduction

Between 2004 and 2012, after the Natural Health Products Regulations were put in place, Health Canada approved a number of products that had characteristics of both foods and natural health products (NHPs) as NHPs. This inadvertently created confusion among consumers and in 2012, following extensive consultations, Health Canada began regulating products that look like foods and are consumed as foods, as foods, noting that this would allow Canadians to make more informed choices due to consistent nutrition information and labelling requirements.

During the transition between regulatory frameworks, certain herbal and non-herbal ingredients in these products were identified as not having a history of safe food use, or were being used in these products at a level that was inconsistent with food use. As an interim measure, Health Canada used Temporary Marketing Authorizations to permit the sale of products, on a case-by-case basis and under specific conditions, while regulations were being developed for these types of products (that is, supplemented foods containing supplemental ingredients). Supplemental ingredients have historically been marketed as providing specific physiological or generally beneficial health effects. However, they can pose health risks if overconsumed by the general population, or if consumed by certain vulnerable populations.

Some of these ingredients, including MTE, were listed in Appendix 2 of Health Canada's Category Specific Guidance for Temporary Marketing Authorization: Supplemented Food, for further assessment to determine conditions (for example, use levels, specifications, and/or labelling statements), if any, under which they would be safe for use as supplemental ingredients.

The scientific information/studies that serve as the basis of the safety assessments were obtained from a search of publicly available primary literature, web searches on specific topics, and citations noted in other articles. This document summarizes the safety considerations that informed Health Canada's decision to allow the use of milk thistle seed extract (silymarin) as a supplemental ingredient.

Approach

The acceptability of a food ingredient typically considers its safety for the general population, over a lifetime of exposure with no limits on consumption; however, the safety assessment approach for supplemental ingredients gave further consideration to the potential use of cautionary labels to mitigate risks, when unlimited consumption may not be safe.

Based on the Food and Nutrition Directorate's safety assessment approach, only limited cautionary labelling is considered appropriate for foods containing supplemental ingredients (such as MTE). This labelling is designed to mitigate potential risk(s) identified for sensitive subpopulations, and to help ensure that the intake of the supplemental ingredient, through the diet, remains within acceptable (that is, safe) levels. Ingredients that require more extensive cautionary labelling (such as contraindications) to protect the consumer are not considered appropriate for food use. More information about labelling of supplemented foods is available in the Guidance Document: Supplemented Foods Regulations.

Safety assessment

The safety assessment included a review of the available information on MTE for the purposes of evaluating toxicological, nutritional and allergenicity endpoints. Based on this information, a Recommended Maximum Daily Intake (RMDI) was derived for the use of MTE as a supplemental ingredient.

Characterization/standardization of the supplemental ingredient

Silymarin, a mixture of eight substances with similar structures, is the primary active component of MTE. Silibinin is the most abundant (approximately 60%) substance in silymarin. This safety assessment applies to milk thistle seed extracts derived from the seeds of milk thistle (Silybum marianum (L.) Gaertn.), containing a silymarin (the primary constituent of MTE) content ranging between 65% (minimum) and 80% (maximum). Acceptable preparations include dry extracts, fluid extracts, tinctures, decoctions and infusions obtained via water and/or food grade organic solvent extraction techniques. This safety assessment does not apply to modified forms of MTE or those with special carrier systems that could alter its bioavailability, distribution, metabolism or excretion when ingested.

Dietary exposure

The seeds of milk thistle can be roasted and used as a coffee substitute, but this use is considered to be rare. Therefore, exposure to MTE (or silymarin) through the diet is not common, and there is also insufficient information to quantify background dietary intake.

Requirements for complying with the Supplemented Food Regulations

As with any food, the onus is on the food manufacturer or distributor to ensure that a food offered for sale in Canada complies with all regulatory provisions, including but not limited to requirements under the Food and Drugs Act (FDA) and the Safe Food for Canadians Act, and the Regulations associated with these Acts. This includes, for example, ensuring compliance with food labelling requirements, provisions for the use of food additives, and the general prohibitions in section 4 of the FDA, which prohibits selling a food that contains a poisonous or harmful substance.

More information on other requirements for supplemented foods is available in the Guidance Document: Supplemented Foods Regulations. These requirements are independent from the safety review of MTE itself, as described in this document.

Toxicological safety

Absorption, distribution, metabolism and excretion studies

Metabolic studies evaluating the fate and behaviour of MTE almost always report the results for individual constituents, namely, silibinin. Silibinin is rapidly absorbed following oral exposure; however, it has low bioavailability due to low aqueous solubility, low permeability across intestinal epithelial cells, extensive conjugation (when absorbed), and rapid excretion (of both unchanged and conjugated forms) into the bile, and subsequently into the faeces (Javed et al., 2011). The metabolic fate of MTE (and its constituents) has been confirmed in several studies (Barzaghi et al., 1990; Wen et al., 2008; Wu et al., 2007, 2008). There is no evidence of significant bioaccumulation.

Toxicology studies

Acute, subchronic, chronic, carcinogenicity, genotoxicity, reproductive, and developmental endpoints were considered to determine the potential toxicity of MTE, where available. Other endpoints considered included the effects of MTE on enzymatic activity. Clinical studies and trials were also reviewed for evidence of toxicity. Results are summarized below (N.B. studies cited below are not exhaustive, but rather represent the studies that are critical to the conclusions of the safety assessment of MTE).

The critical toxicity study was conducted over a 2-year period in both rats and mice, by the United States National Toxicology Program (NTP, 2011) using good laboratory practice (GLP). The MTE test substance was well-characterized, and provided via the diet at concentrations of 0, 12,500, 25,000 or 50,000 parts per million (ppm). There was no evidence of a carcinogenic response up to the highest concentration tested, and notable non-carcinogenic effects reported in this study are described below. The study was considered adequate for hazard characterization, and was used as the critical study for derivation of the RMDI (discussed below).

In rats, there was no effect on survival, clinical/behavioural endpoints, body weights or feed consumption. In mice, there was also no effect on survival or clinical/behavioural endpoints; however, significant reductions in body weight gain were observed in both sexes, while feed consumption was comparable to controls throughout the exposure regimen. The reductions in body weight gain were observed during the first half of the animals' lifespans (that is, up to 365 days of exposure), and persisted to the end of the 2-year exposure period. The effect on body weight gain was concentration-responsive, and resulted in reductions compared to controls of up to 19% in males, and up to 30% in females at the end of the 2-year exposure period, at the highest concentration of MTE administered.

The available data from the NTP study suggests that there is a decrease in food efficiency as the concentration of MTE increased in the diet of mice. However, the animals were in otherwise good health. Without additional information, the effect on body weight gain is considered a treatment-related, toxicologically significant effect.

Given these observations, a no-observed-adverse-effect concentration (NOAEC) of 12,500 ppm (lowest concentration tested) was identified. This NOAEC was based on body weight (both final and after one year of exposure) in mice, which was maintained within 10% of controls at this concentration and not considered a toxicologically significant change, as well as the fact that the mice were otherwise healthy at the end of the 2-year exposure period. The concentration of 12,500 ppm is equivalent to 1500 milligram (mg)/kilogram (kg) body weight (bw) per day for female mice, which was the more sensitive sex.

Multiple genotoxicity studies were also conducted as part of the NTP 2-year study (2011). These were primarily standard bacterial reverse mutation assays (that is, Ames), using multiple bacterial strains and metabolic conditions, and were conducted on five separate MTE formulations, as well as silymarin. Although there were some positive results (primarily in S. typhimurium TA98, in the presence of rat liver S9 exogenous microsomal metabolic activation) for two of the five MTE samples, as well as for silymarin, the results are not consistent. Considering the low bioavailability of MTE constituents, and the lack of neoplastic effects following lifetime administration of MTE in the diet of both rats and mice, the sporadic positive in vitro results are not considered toxicologically significant.

No reproductive or developmental toxicity studies, meeting international standards for toxicological testing, including the fulsome reporting of a standard battery of reproductive and developmental endpoints, were identified in the literature. While a 90-day NTP study in rats and mice was not designed to measure reproductive parameters, a limited set of endpoints were reported (NTP, 2011). In mice, no significant differences were observed between test and control groups for sperm parameters, estrous cyclicity, reproductive organ weights, or histopathologic lesions in any reproductive tissue. Additionally, no effects were observed in female rats. In contrast, non-concentration-responsive decreases in sperm motility and spermatid heads per testis were observed in male rats. However, these types of sperm measurements are subject to significant variation, and a definitive conclusion based on measurements taken at one time point is considered unreliable. Sperm morphology is also generally understood to be a poor predictor of fertility. The lack of well-conducted developmental toxicity studies in animals, as well as the paucity of relevant clinical data (see Clinical studies, below), supports cautionary labelling for supplemental use of MTE for certain sensitive subpopulations (that is, children and adolescents under 18 years of age, pregnant/breastfeeding individuals).

The results of an in vitro study suggested that silymarin may inhibit triiodothyronine (T3) hormone uptake into target cells (Johannes et al., 2016). However, these results are not replicated in in vivo test systems. There were no histopathologic changes (neoplastic and non-neoplastic) to the thyroid of male and female mice and rats following lifetime exposure (hormone levels and activity were not measured) (NTP, 2011). In addition, the results of a developmental neurotoxicity study conducted in mice (OECD 426 test guideline) administered MTE during gestation and lactation found no effects in the offspring following various sensory-motor reflex development testing (Barbosa et al., 2020). Given that the inhibition of thyroid hormone uptake (for example, T3 and T4) can lead to severe psychomotor retardation, the lack of effects observed in the offspring indicate that the effects observed in the in vitro assay are likely not relevant in vivo.

Recommended maximum daily intake (RMDI) derivation

The critical NOAEC for MTE of 12,500 ppm (equivalent to 1500 mg/kg bw per day) from the 2-year study in mice is equivalent to 975 mg/kg bw per day of silymarin, based on the 65% silymarin content of the MTE test article used in the study (NTP, 2011). Applying an uncertainty factor of 100 to account for inter- and intra-species differences results in an RMDI of 9.75 mg/kg bw per day of silymarin, which is equivalent to 683 mg per day of silymarin for a 70 kg adult.^{Footnote 2}

The aforementioned RMDI was rounded down to 600 mg per day silymarin, which aligns with the maximum daily therapeutic dose cited by the Health Canada NHP monograph (NNHPD, 2018). The monograph further recommends a maximum single-dose limit of 200 mg of silymarin. This additional limit, related to a single-serving, was also incorporated as part of the conditions of MTE consumption from supplemented foods to help ensure that the intake, through the diet, remains within acceptable (that is, safe) levels.

Clinical studies

In general, clinical studies testing MTE were conducted on populations with pre-existing health conditions (primarily liver hepatitis or cirrhosis) as part of a therapeutic treatment regimen. Due to the presence of pre-existing health conditions, the different and/or unstandardized test materials, the differing methodologies and administration regimens (including study length), and the lack of measured toxicological parameters (including haematological, clinical chemistry, urinalysis), these studies do not provide sufficient evidence of long-term safety for the general population, including sensitive subpopulations (such as, children, pregnant women). However, these studies did provide basic information on the tolerability of MTE and its constituents, as well as any adverse events (Rambaldi et al., 2007; Saller et al., 2001; Saller et al., 2008; Jacobs et al., 2002).

The frequency of adverse events reported from numerous clinical trials, testing a variety of MTE formulations (including silymarin and silybinin), in thousands of patients, is low (EMA, 2018). Most clinical trials reported no adverse events or no differences between placebo and treated groups. When adverse events were observed, they were generally considered minor, and included mild gastrointestinal symptoms (for example, dry mouth, nausea, upset stomach, gastric irritation, diarrhoea), headache, and/or sensitization reaction (such as, dermatitis, urticaria, skin rash, pruritus).

While there is some evidence of inhibitory effects of MTE (or silymarin/silibinin) on cytochrome P450 (CYP450) and uridine 5'-diphospho-glucuronosyltransferase (UGT) activity (examining a variety of isozymes), in vitro, several reviews have concluded that the concentrations at which inhibition is observed are extremely high, and generally not achievable with oral intake. Furthermore, these reviews concluded that, in clinical settings, there is limited influence of MTE on the pharmacokinetics of several drugs known to be metabolized by these enzymes, indicating no substantive interaction of MTE (or silymarin/silibinin) on several drug-metabolizing enzymes (Coates et al., 2010; Xie et al., 2019).

None of the available clinical studies addressed the safety of MTE in children or pregnant/breastfeeding individuals. The lack of well-conducted developmental toxicity studies in animals, as well as the paucity of relevant clinical data, supports cautionary labelling for supplemental use of MTE for these sensitive subpopulations (that is, children and adolescents under 18 years of age, pregnant/breastfeeding individuals).

Allergenicity

The United States National Institutes of Health suggests that people with known allergy to members of the aster (Asteraceae) family, which includes daisies, artichokes, common thistle, and kiwi fruit, should avoid milk thistle.^{Footnote 3} Hypersensitivity to the active substance, and to plants of the Asteraceae (Compositae) family, is also stated as a contraindication by the European Medicines Agency (EMA, 2018).

Clinical trials support the low frequency of adverse events, and indicate that the majority of reactions that did occur were mild (specifically, gastrointestinal). However, there are a few case reports in the literature describing severe allergic reactions to products containing silymarin. In addition, minor sensitization reactions (such as, dermatitis, urticarial, pruritus) were observed in clinical trials.

Sources of exposure to silymarin and/or MTE in the diet are not common, and consumers who have known allergy to members of the aster family may not be aware of the potential for sensitization/allergic reactions associated with MTE; therefore, cautionary labelling is recommended.

Nutritional safety

MTE is not present in foods in significant amounts, and does not have a nutritional role.

It has been shown that the flavanolignan, silibinin, the major component of silymarin, can bind non-chelated iron to form a silybin–Fe(III) complex in acidic pH (Borsari et al. 2001), which may interfere with dietary iron absorption. The iron-chelating properties of silymarin have been investigated in randomized controlled trials in patients with iron overload also receiving standard therapy, with inconsistent results. In larger studies, silymarin (420 mg per day, divided in 3 doses), administered in combination with standard therapy, for 24 weeks (Hagag et al. 2013) or 36 weeks (Hagag et al. 2015; Moayedi et al. 2013), resulted in a significant decrease in serum iron and ferritin when compared to the control group, also receiving standard therapy. However, some studies showed no effect (Balouchi et al. 2014; Darvishi-Khezri et al. 2017; Gharagozloo et al. 2009), including one study that measured liver iron, which is considered the best indicator of iron status (Adibi et al. 2012). The inhibitory effect of MTE on iron absorption, suggested by some of the studies, could be explained by other mechanisms. For example, the anti-inflammatory properties of MTE may have contributed to the observed reduction in serum ferritin (ferritin is an acute phase protein known to increase with inflammation). Furthermore, potentiation of the effect of the iron chelator (used as the standard therapy) by the MTE cannot be excluded.

In another small pilot study in patients with hereditary haemo-chromatosis, 140 mg of silibinin was shown to reduce serum iron following the consumption of a single meal containing non-haeme iron and ascorbic acid (vitamin C); however, the effect was only marginally statistically significant (Hutchinson et al. 2010). The authors of this study noted that this was a preliminary finding, the small number of patients is a limiting factor, and a type I statistical error cannot be ruled out. Based on the serum iron curves, another explanation could be that the silibinin may have slowed (not reduced) the absorption of iron since the curve for silibinin patients did not plateau like the curves for controls.

Overall, the available evidence does not demonstrate that MTE inhibits iron absorption.

Conclusion and decision

Health Canada's Food and Nutrition Directorate determined there to be sufficient information to establish conditions under which standardized MTE would be safe for use as a supplemental ingredient in supplemented foods. Consequently, Health Canada will permit the use of milk thistle seed extract (silymarin) as a supplemental ingredient in supplemented foods under certain conditions. These conditions are outlined in the Notice of Modification.

Supplemental ingredient submissions for MTE

To propose future, additional changes to the conditions of use for milk thistle seed extract (silymarin) as a supplemental ingredient, stakeholders can submit a pre-market request to the Food and Nutrition Directorate as described in the Guidance document: Pre-market submission process for supplemented foods. Manufacturers and distributors are encouraged to request a pre-submission consultation with the Food and Nutrition Directorate to seek additional guidance so that a complete submission can be filed at the outset, potentially reducing the number of requests to the applicant for clarification or additional information, or preventing the submission from being rejected for incompleteness. Pre-submission consultations on supplemental ingredients may be arranged by contacting the Submission Management and Information Unit (smiu-ugdi@hc-sc.gc.ca).

The information set out below is recommended to be included in the submission, if relevant to the nature of the request.

For requests to include MTE formulations outside of the current milk thistle seed extract (silymarin) characterization, the submission must characterize in detail the MTE that is requested for supplemental use. It must also clearly demonstrate that the safety information in the submission applies to the requested MTE. For example, the submission must explain why the results of toxicity testing of a particular MTE formulation apply to the MTE to be used as a supplemental ingredient.

In general, any submitted safety information should be of good quality (for example, GLP/GCP and OECD compliant), and contain full study reports, not summaries. The reports should provide clear, detailed characterization of the MTE test material, and a full description of the study design, including methods, the type and number of animals treated, the doses administered, and the toxicological endpoints measured. Studies should also provide a detailed documentation of the test results. Similarly, the submission of clinical studies should provide fulsome details of the study design, providing toxicologically focused endpoints that contribute information to the assessment of the safety of the ingredient.

Future requests to modify milk thistle seed extract (silymarin) conditions, such as the RMDI or labelling, may require addressing data gaps that were identified in the current toxicological and nutritional assessments, such as the following:

Toxicological data gaps/uncertainties

1. Reproductive and developmental toxicity

Nutritional data gaps/uncertainties

1. Information that would provide adequate evidence that MTE would not affect digestion or absorption of other nutrients, especially in the intestines, and/or information that it would not pose nutritional safety concerns if foods supplemented with MTE were to be consumed frequently over a long period of time in different food matrices.

Health Canada may ask for additional data or other information related to the safety of MTE for use as a supplemental ingredient after reviewing the above information.

References

• Adibi, A., Azin, S.H., Behjat, S.M. 2012. Therapeutic effects of deferoxamine and silymarin versus deferox-amine alone in β-thalassemia major based on findings of liver MRI. J. Res. Med. Sci. (1): 73-78.
• Barbosa, C.C., Nishimura, A.N., Lopes Dos Santos, M., Dias, W.J., Andersen, M.L., Mazaro-Costa, R. 2020. Silymarin administration during pregnancy and breastfeeding: evaluation of initial development and adult behavior of mice. Neurotoxicology 78: 64-70.
• Barzaghi, N., Crema, F., Gatti, G., Pifferi, G., Perucca, E. 1990. Pharmacokinetic studies on IdB 1016, a silybin – phosphatidylcholine complex, in healthy human subjects. Eur. J. Drug Metab. Pharmacokinet. 15(4): 333-338.
• Balouchi S, Gharagozloo M, Esmaeil N, Mirmoghtadaei M, Moayedi B. 2014. Serum levels of TGFβ, IL-10, IL-17, and IL-23 cytokines in β-thalassemia major patients: the impact of silymarin therapy. Immunopharmacol. Immunotoxicol. 36(4): 271-274.
• Borsari, M., Gabbi, C., Ghelfi, F., Grandi, R., Saladini, M., Severi, S., Borella, F. 2001. Silybin, a new iron-chelating agent. J. Inorg. Biochem. 85(2): 123-129.
• Coates, P.M., Betz, J.M., Blackman, M.R., Cragg, G.M., Levine, M., Moss, J., White, J.D. [eds.]. 2010. Encyclopedia of Dietary Supplements, Second Edition. Ladas, E., Kroll, D.J., Kelly, K.M. Milk Thistle. pp. 550-561. Informa Healthcare: New York, NY.
• [EMA] European Medicines Agency. 2018. Assessment report on Silybum marianum (L.) Gaertn., fructus. Final. 5 June 2018. Committee on Herbal Medicinal Products (HMPC). EMA/HMPC/294188/2013. Available from: https://www.ema.europa.eu/en/documents/herbal-report/final-assessment-report-silybum-marianum-l-gaertn-fructus_en.pdf. [accessed 19 Feb 2021].
• Darvishi-Khezri H, Salehifar E, Kosaryan M, Karami H, Alipour A, Shaki F, et al. 2017. The impact of silymarin on antioxidant and oxidative status in patients with beta-thalassemia major: A crossover, randomized controlled trial. Complement. Ther. Med. 35: 25-32.
• Gharagozloo M, Moayedi B, Zakerinia M, Hamidi M, Karimi M, Maracy M, et al. 2009. Combined therapy of silymarin and desferrioxamine in patients with β-thalassemia major: a randomized double-blind clinical trial. Fundam. Clin. Pharmacol. 23(3): 359-365.
• Hagag, A.A., Elfrargy, M.S., Gazar, R.A, El-Lateef, A.E. 2013. Therapeutic value of combined therapy with deferasirox and silymarin on iron overload in children with Beta thalassemia. Mediterr. J. Hematol. Infect. Dis. 5(1):e2013065.
• Hagag, A.A., Elfrargy, M.S., Gazar, R.A, El-Lateef, A.E. 2015. Therapeutic value of combined therapy with Defer-iprone and Silymarin as iron chelators in Egyptian Children with Beta Thalassemia major. Infectious Disorders-Drug Targets 15(3): 189-195.
• Hutchinson, C., Bomford, A., Geissler, CA. 2010. The iron-chelating potential of silybin in patients with hereditary haemochromatosis. Eur. J. Clin. Nutr. 64: 1239-1241.
• Jacobs, B.P., Dennehy, C., Ramirez, G., Sapp, J., Lawrence, V.A. 2002. Milk Thistle for the Treatment of Liver Disease: A Systematic Review and Meta-Analysis. Am. J. Med. 113(6): 506-515.
• Javed, S., Kohli, K., Ali, M. 2011. Reassessing Bioavailability of Silymarin. Altern. Med. Rev. 16(3): 239-249.
• Johannes, J., Jayarama-Naidu, R., Meyer, F., Wirth, E.K., Schweizer, U., Schomburg, L., Köhrle, J., Renko, K. 2016. Silychristin, a flavonolignan derived from the milk thistle, is a potent inhibitor of the thyroid hormone transporter MCT8. Endocrinology 157(4): 1694-1701.
• Moayedi, B., Gharagozloo, M., Esmaeil, N., Maracy, M.R., Hoorfar, H., Jalaeikar, M. 2013. A randomized double‐blind, placebo‐controlled study of therapeutic effects of silymarin in β‐thalassemia major patients receiving desferrioxamine. Eur. J. Haematol. 90(3): 202-209.
• [NNHPD] Natural and Non-prescription Health Products Directorate. 2018. Natural Health Product: Milk Thistle – Silybum Marianum Monograph. Available from: http://webprod.hc-sc.gc.ca/nhpid-bdipsn/monosReq.do?lang=eng. [accessed 15 Feb 2021].
• [NTP] National Toxicology Program. 2011. NTP Technical Report on the Toxicology and Carcinogenesis Studies of Milk Thistle Extract (CAS No. 84604-20-6) in F344/N Rats and B6C3F1 Mice (feed studies). NTP TR 565. NIH Publication No. 11-5907. National Toxicology Program, National Institutes of Health, Public Health Service, U.S. Department of Health and Human Services. Research Triangle Park, NC. Available from: https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr565.pdf?utm_source=direct&utm_medium=prod&utm_campaign=ntpgolinks&utm_term=tr565. [accessed 23 Feb 2021].
• 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. Issue 4. Article No: CD003620. DOI: 10.1002/14651858.CD003620.pub3.
• Saller, R., Meier, R., Brignoli, R. 2001. The use of silymarin in the treatment of liver diseases. Drugs 61(14): 2035-2063.
• Saller, R., Brignoli, R., Melzer, J., Meier, R. 2008. An updated systematic review with meta-analysis for the clinical evidence of silymarin. Forschende Komplementärmedizin 15(1):9-20. [obtained from Zurich Open Repository and Archive, University of Zurich]. Available from: https://www.zora.uzh.ch/id/eprint/12530/1/113648.pdf
• 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 Metab. Dispos. 36(1): 65-72.
• Wu, J-W., Lin, L-C., Hung, S-C., Chi, C-W., Tsai, T-H. 2007. Analysis of silibinin in rat plasma and bile for hepatobiliary excretion and oral bioavailability application. J. Pharm. Biomed. Anal. 45(4): 635-641.
• Wu, J-W., Lin, L-C., Hung, S-C., Lin, C-H., Chi, C-W., Tsai, T-H. 2008. Hepatobiliary excretion of silibinin in normal and liver cirrhotic rats. Drug Metab. Dispos. 36(3): 589-596.
• Xie, Y., Zhang, D., Zhang, J., Yuan, J. 2019. Metabolism, Transport and Drug–Drug Interactions of Silymarin. Molecules 24(20): 3693.