The study of silymarin release kinetic in free and hydrogel bound micellar forms: a qualitative and quantitative analysis using RP-HPLC

Document Type : Research Paper


1 Student research committee, Birjand University of medical sciences, Birjand, Iran

2 Cellular and molecular research center, Birjand University of medical sciences, Birjand, Iran

3 Department of chemistry, Faculty of science, University of Birjand, Birjand, Iran



Silymarin is a safe herbal medicine; however, it has some undesirable properties such as short half-life and poor aqueous solubility.
To the best of our knowledge, this study is the first to report utilizing a dual-drug delivery system (DDDS) to enhance the release profile of silymarin from both micelles and hydrogels.
In this experimental study, the release profile of micellar silymarin and micelle-hydrogel bounded silymarin during 21 days was examined using Knauer K2600A liquid chromatography.
The calibration curve was plotted using the peak-areas of the silymarin at different concentrations.
The RP-C18 column allowed a good separation of the components of standard silymarin.
LOD and LOQ were 16.5 and 55.02 μg/ml, respectively. The in vitro release profiles of the two compounds showed a rapid release of silymarin, especially in the absence of hydrogel. The cumulative release graph revealed that the hydrogel-bound form has more constant release kinetics than the free micelle form; this means that the hydrogel-bound form may sustain for longer durations.
In this study, a dual-drug delivery system based on hydrogel/micelle composites was introduced.
The results showed that Puramatrix hydrogel plays an important role in the constant release of silymarin. Furthermore, the RP-HPLC method presented in this study can be used by other researchers to overcome the difficulties associated with the in-vitro separation and quantification of silymarin.


[1] Anthony K, Subramanya G, Uprichard S, Hammouda F, Saleh M. Antioxidant and anti-hepatitis c viral activities of commercial milk thistle food supplements. Antioxidants. (2013) 2 (1): 23-36.
[2] Vargas-Mendoza N, Madrigal-santillan E, Morales-Gonzalez A, Esquivel-soto J, Esquivel-Chirni C,Gonzalez-Rubio M, Gayosso-de-Lucio J, et al. Hepatoprotective effect of silymarin. World J. Hepatol. (2014) 6 (3): 144-9.
[3] Wu W, Wang Y, Que L. Enhanced bioavailability of silymarin by self-microemulsifying drug delivery system. Eur. J. Pharm. Biopharm. (2006) 63 (3): 288-94.
[4] El-Far YM, Zakaria MM, Gabr MM, El Gayar AM, El-Sherbiny IM, Eissa LA. A newly developed silymarin nanoformulation as a potential antidiabetic agent in experimental diabetes. Nanomedicine. (2016)11(19): 2581-602.
[5] Sun H-p, Su J-H, Meng Q-Sh, Yin Q, Zhang Z-W, Yu H-J, Zhang P-C, et al. Silibinin and indocyanine green-loaded nanoparticles inhibit the growth and metastasis of mammalian breast cancer cells in vitro. Acta  Pharmacol. Sin. (2016) 37 (7): 941-9.
[6] Bonepally CR, Gandey SJ, Bommineni K, Gottumukkala KM, Aukunuru J. Preparation, characterisation and in vivo evaluation of silybin nanoparticles for the treatment of liver fibrosis. Trop.  J. Pharm.  Res. (2013) 12 (1):1-6.
[7] Mata-Santos HA, Lino FG, Rocha CC, Paiva CN, Branco MTLC, Pyrrho ADS. Silymarin treatment reduces granuloma and hepatic fibrosis in experimental schistosomiasis. Parasitol. Res. (2010) 107 (6): 1429-34.
[8] Theodosiou E, Purchartová K, Stamatis H, Kolisis F, Křen V. Bioavailability of silymarin flavonolignans: drug formulations and biotransformation. Phytochem. Rev. (2014) 13 (1):1-18.
[9] El-Far M, Salah N, Essam A, Abd El-Azim AO, El-Sherbiny. Silymarin nanoformulation as potential anticancer agent in experimental Ehrlich ascites carcinoma-bearing animals. Nanomedicine. (2018) 13 (15):1865-58.
[10]         Cheng K-C, Asakwa A, Li Y-X, Chung H-H, Amitani H, Ueki T, Cheng J-T, et al. Silymarin induces insulin resistance through an increase of phosphatase and tensin homolog in Wistar rats. Plos One. (2014) 9 (1): e84550.
[11]         Quaglia M, Bossu E, Donati E, Mazzanti G, Brandt A. Determination of silymarin in the extract from the dried silybum marianum fruits by high performance liquid chromatography and capillary electrophoresis. J. Pharm. Biomed. Anal. (1999) 19 (3-4): 435-42.
[12]         Lorenz D, Lücker P, Mennicke W, Wetzelsberg N. Pharmacokinetic studies with silymarin in human serum and bile. Methods. Find. Exp. Clin. Pharmacol. (1984) 6 (10): 655-61.
[13]         Martinelli E, Morazzoni P, Livio S, Uberti E. Liquid chromatographic assay of silybin in human plasma and urine. J. Liq. Chromatogr. (1991) 14 (7): 1285-96.
[14]         Mascher H, Kikuta C, Weyhenmeyer R. Diastereomeric separation of free and conjugated silibinin in plasma by reversed phase HPLC after specific extraction. J. Liq. Chromatogr. Relat. Technol. (1993) 16 (13): 2777-89.                           [15]              Rickling B, Hans B, Kramarczyk R, Krumbiegel G, Weyhenmeyer R. 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 Biomed.  Sci. Appl. (1995) 670 (2): 267-77.
[16]         El-Ridy MS, Badawi AA, Safar MM, Mohsen MA.  Niosomes as a novel pharmaceutical formulation encapsulating the hepatoprotective drug silymarin. Int. J. Pharm. Pharm. Sci. (2012) 4 (1): 549-59.
[17]         Khorram M, Vasheghani-Farahani E, Ebrahimi NG. Fast responsive thermosensitive hydrogels as drug delivery systems. IRAN. POLYM. J. (2003) 12 (4): 315-22.
[18]         Wei L, Cai C, Lin J, Chen T. Dual-drug delivery system based on hydrogel/micelle composites. Biomaterials. (2009) 30 (13): 2606-13.
[19]         Xiao N-Y, Li A-L, Liang H, Lu J. A well-defined novel aldehyde-functionalized glycopolymer: synthesis, micelle formation, and its protein immobilization. Macromolecules. (2008) 41 (7): 2374-80.
[20]         Lee AL, Wang Y, Ye W-H, Yoon HS, Chan SY, Yang Y-Y. Efficient intracellular delivery of functional proteins using cationic polymer core/shell nanoparticles. Biomaterials. (2008) 29 (9): 1224-32.
[21]         Korany MA, Haggag RS, Ragab MA, Elmallah OA. A validated stability-indicating HPLC method for simultaneous determination of Silymarin and Curcumin in various dosage forms. Arab. J. Chem. (2017) 10 (2): S1711-S25.
[22]         Ding T-m, Tian S-j, Zhang Z-x, Gu D, Chen Y, Shi Y, Sun Z. Determination of active component in silymarin by RP-LC and LC/MS. J. Pharm. Biomed. Ana. (2001) 26 (1): 155-61.
[23]         Graf TN, Cech NB, Polyak SJ, Oberli Oberlies NH. A validated UHPLC-tandem mass spectrometry method for quantitative analysis of flavonolignans in milk thistle (Silybum marianum) extracts. J. Pharm. Biomed. Anal. (2016) 126: 26-33.
[24]         Kim N-C, Graf TN, Sparacino CM, Wani MC, Wall ME. Complete isolation and characterization of silybins and isosilybins from milk thistle (Silybum marianum). Org. Biomol. Chem. (2003) 1 (10): 1684-9.
[25]         Nishimura A, Hayakawa T, Yamamoto Y, Hamori M, Tabata K, Seto K,Shibata N. Controlled release of insulin from self-assembling nanofiber hydrogel, PuraMatrix™: application for the subcutaneous injection in rats. Eur. J. Pharm. (2012) 45 (1-2): 1-7.
[26]         Zhang S, Holmes TC, dipersio CM, Richard O, Hynes XS, Rich A. Self-complementary oligopeptide matrices support mammalian cell attachment. Biometerials. (1995) 16 (18): 1385-1393.
[27]         Holmes TC, LacalleS de, Xing Su, Liu G, Rich A, Zhang sh. Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. Proc. Natl. Acad. Sci. U.S.A. (2000) 97(12): 6728-6733.
[28]         KisidayJ, JinM, KurzB, Hung H, Semino C, Zhang S, Grodzinsky J. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair.Proc. Natl. Acad. Sci. U.S.A. (2002) 99 (15): 9996-10001.
[29]         TangC, ShaoX, SunB, W, Huang W, Zhao X. The effect of self-assembling peptide RADA16-I on the growth of human leukemia cells in vitro and in nude mice. Int. J. Mol. Sci. (2009) 10 (5): 2136-2145.