Gram-Scale Purification of Flavonolignan Diastereoisomers from Silybum marianum (Milk Thistle) Extract in Support of Preclinical in vivo Studies for Prostate Cancer Chemoprevention

Abstract

Extracts of milk thistle (Silybum marianum, Asteraceae), termed ”silymarin,” are used worldwide, primarily for hepatoprotective applications and recently for prostate cancer chemoprevention. Silymarin is a mixture of at least eight compounds, and four major constituents are a group of structurally related flavonolignans: silybin A, silybin B, isosilybin A, and isosilybin B. The initiation of in vivo studies to compare the respective preclinical activities of each compound required that gram quantities of these diastereoisomers be prepared. Procedures were developed and optimized to produce multigram-scale quantities of each of these in > 97 % purity. A hybrid chromatographic/precipitative technique was developed, whereby mixtures were chromatographed at high concentrations so as to induce formation of a precipitate in the column fractions, yielding samples that were more enriched in the desired compounds than would be obtained solely by the chromatographic steps alone.

Key words

Silybum marianum - Asteraceae - milk thistle - silymarin - silybin - isosilybin - silibinin - isosilibinin

References

• 1 Wellington K, Jarvis B. Silymarin: A review of its clinical properties in the management of hepatic disorders.  Biodrugs. 2001;  15 465-89.
• 2 Deep G, Agarwal R. Chemopreventive efficacy of silymarin in skin and prostate cancer.  Integr Cancer Ther. 2007;  6 130-45.
• 3 Agarwal R, Agarwal C, Ichikawa H, Singh R P, Aggarwal B B. Anticancer potential of silymarin: From bench to bed side.  Anticancer Res. 2006;  26 4457-98.
• 4 Zi X L, Mukhtar H, Agarwal R. Novel cancer chemopreventive effects of a flavonoid antioxidant silymarin: Inhibition of mRNA expression of an endogenous tumor promoter TNF alpha.  Biochem Biophys Res Commun. 1997;  239 334-9.
• 5 Kroll D J, Shaw H S, Oberlies N H. Milk thistle nomenclature: why it matters in cancer research and pharmacokinetic studies.  Integr Cancer Ther. 2007;  6 110-9.
• 6 Singh R P, Dhanalakshmi S, Tyagi A K, Chan D C, Agarwal R, Agarwal C. Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels.  Cancer Res. 2002;  62 3063-9.
• 7 Morazzoni P, Bombardelli E. Silybum marianum (Carduus marianus) .  Fitoterapia. 1995;  66 3-42.
• 8 Kim N -C, Graf T N, Sparacino C M, Wani M C, Wall M E. Complete isolation and characterization of silybins and isosilybins from milk thistle (Silybum marianum).  Org Biomol Chem. 2003;  1 1684-9.
• 9 Lee D YW, Liu Y Z. Molecular structure and stereochemistry of silybin A, silybin B, isosilybin A, and isosilybin B, isolated from Silybum marianum (milk thistle).  J Nat Prod. 2003;  66 1171-4.
• 10 Kroll D J, Oberlies N H. The impact of newly proposed dietary supplement manufacturing guidelines on patient safety and clinical trials outcomes.  Focus Altern Complement Ther. 2003;  8 302-6.
• 11 Davis-Searles P R, Nakanishi Y, Kim N C, Graf T N, Oberlies N H, Wani M C. et al . Milk thistle and prostate cancer: differential effects of pure flavonolignans from Silybum marianum on antiproliferative end points in human prostate carcinoma cells.  Cancer Res. 2005;  65 4448-57.
• 12 Deep G, Oberlies N H, Kroll D J, Agarwal R. Isosilybin B and isosilybin A inhibit growth, induce G1 arrest and cause apoptosis in human prostate cancer LNCaP and 22Rv1 cells.  Carcinogenesis. 2007;  28 1533-42.
• 13 Flaig T W, Gustafson D L, Su L J, Zirrolli J A, Crighton F, Harrison G F. et al . A phase I and pharmacokinetic study of silybin-phytosome in prostate cancer patients.  Invest New Drugs. 2007;  25 139-4.6
• 14 Simanek V, Kren V, Ulrichova J, Vicar J, Cvak L. Silymarin: What is in the name...? An appeal for a change of editorial policy.  Hepatology. 2000;  32 442-4.
• 15 During the review process, one referee noted that there was an inconsistency in the mass balance for the fractions from 1PS (1PSP and 1PSS). Indeed, we double checked our notebook, and the values given in the text are consistent. However, we believe that there could be a mathematical or empirical error resulting in this inconsistency, and we suspect that the actual weight of 1PSS may have been closer to 6 g. 
• 16 Hoh C SL, Boocock D J, Marczylo T H, Brown V A, Cai H, Steward W P. et al . Quantitation of silibinin, a putative cancer chemopreventive agent derived from milk thistle (Silybum marianum), in human plasma by high-performance liquid chromatography and identification of possible metabolites.  J Agric Food Chem. 2007;  55 2532-5.
• 17 Lee J I, Narayan M, Barrett J S. Analysis and comparison of active constituents in commercial standardized silymarin extracts by liquid chromatography-electrospray ionization mass spectrometry.  J Chromatogr B. 2007;  845 95-103.
• 18 Wen Z M, Dumas T E, Hawke R L, Smith P C, Fried M W. Characterization of free, sulfated, and glucuronidated silymarin flavonoligans in human plasma after oral administration of milk thistle extract.  Drug Metab Rev. 2006;  38 166-7.
• 19 Smith W A, Lauren D R, Burgess E J, Perry N B, Martin R J. A silychristin isomer and variation of flavonolignan levels in milk thistle (Silybum marianum) fruits.  Planta Med. 2005;  71 877-80.
• 20 Guz N R, Stermitz F R, Johnson J B, Beeson T D, Willen S, Hsiang J. et al . Flavonolignan and flavone inhibitors of a Staphylococcus aureus multidrug resistance pump: structure-activity relationships.  J Med Chem. 2001;  44 261-8.