Nanostructured lipid carriers for parenteral delivery of silybin: Biodistribution and pharmacokinetic studies

Colloids and Surfaces B: Biointerfaces 80 (2010) 213–218

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Nanostructured lipid carriers for parenteral delivery of silybin: Biodistribution and pharmacokinetic studies Lejiao Jia a , Dianrui Zhang a,∗ , Zhenyu Li b , Cunxian Duan a , Yancai Wang a , Feifei Feng a , Feihu Wang a , Yue Liu a , Qiang Zhang c a b c

Department of Pharmaceutics, College of Pharmacy, Shandong University, 44 Wenhua Xilu, Jinan 250012, PR China Department of Medicinal Chemistry, College of Pharmacy, Shandong University, 44 Wenhua Xilu, Jinan 250012, PR China State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Bei Xueyuan Road, Beijing 100083, PR China

a r t i c l e

i n f o

Article history: Received 17 March 2010 Received in revised form 14 June 2010 Accepted 15 June 2010 Available online 23 June 2010 Keywords: Silybin Nanostructured lipid carriers Pharmacokinetics Tissue distribution In vitro release

a b s t r a c t The objective of the present study was to explore the potential of nanostructured lipid carriers (NLCs) for the intravenous delivery of silybin, a poorly water-soluble antihepatopathy agent. Silybin-NLC was prepared by the method of emulsion evaporation at a high temperature and solidification at a low temperature. The resultant NLC had a mean size 232.1 nm and a zeta potential of −20.7 mV. The differential scanning calorimetry (DSC) analysis indicated that silybin was not in crystalline state in the NLC. In vitro data for release of the drug from silybin-NLC was fitted to a two-stage exponential kinetic model. The pharmacokinetics and tissue distribution of silybin-NLC were studied after intravenous administration using New Zealand rabbits and Kunming mice as experimental animals. A silybin control solution was studied parallelly. Silybin-NLC showed higher AUC (area under tissue concentration–time curve) values and circulated in the blood stream for a longer time compared with silybin solution. The tissue distribution demonstrated a high uptake of silybin-NLC in RES organs particularly in liver. These results indicate that NLC is a potential sustained release and targeting system for silybin. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Silymarin is a polyphenolic substance extracted from the seeds and fruits of the milk thistle plant, Carduus marianus (L.) Geartn. The extract is a mixture of four isomeric flavonolignans, i.e., silybin, isosilybinin, silydianin and silychristin [1]. Among them, silybin is the major biologically active component and largely responsible for the antihepatotoxic activity [2]. Pharmacology research has demonstrated that silybin has antioxidative, anti-lipid-peroxidative, antifibrotic, anti-inflammatory, immunoregulatory and liver regenerating effects [3–5]. In clinic, silybin has been widely applied in the treatment of toxic hepatitis, fatty liver, cirrhosis, radiation toxicity and viral hepatitis [5–7]. Due to its advantages of high efficacy and low toxicity [2,8], silybin has drawn the attention of the medical community since its discovery. However, the bioavailability of silybin is quite low owing to degradation by gastric fluid [9] and its poor aqueous solubility [10,11]. Pharmacokinetic studies showed that only 23–47% of silymarin was absorbed from the gastrointestinal tract after being administered orally [12,13]. In order to improve the efficacy

∗ Corresponding author. Tel.: +86 531 88382015; fax: +86 531 88382015. E-mail addresses: zhang [email protected], zhang [email protected] (D. Zhang). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.06.008

and bioavailability of silymarin or silybin, many approaches were employed, such as silymarin ␤-cyclodextrin inclusion complexes [14], silymarin/polyvinylpyrrolidone solid dispersion pellets [11], silymarin proliposome [15], silymarin self-microemulsifying drug delivery system [12,16] and silybin–phospholipid complex [17]. However, there has been little research on the parenteral delivery system of silybin. The highest availability, avoiding GI degradation and fast effect may be obtained via administering silybin intravenously. The nano-drug delivery system such as nanomicelles, nanoemulsions and nanoparticles has displayed a great potential in improved parenteral delivery of the hydrophobic agents since last two decades [18,19]. Therefore, the utility of the above system in the parenteral delivery of herbal agents such as hydroxycamptothecin [20] and oridonin [21] has been investigated widely. In the present work, we utilize the nanostructured lipid carriers (NLC) for intravenous delivery of silybin. The nanostructured lipid carriers belonging to the second generation of lipid nanoparticles, can be prepared by blending solid-lipids and liquid-lipids which could result in the less ordered inner structure [22]. The NLC has been considered as an alternative to liposomes and emulsions due to improved properties such as ease of manufacture, high drug loading, and more flexibility in modulating the drug release profile [23,24]. Furthermore, the aqueous nature of NLC, their nanostructure and the biocompatibility of the excipients would enable

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intravenous delivery of the drug with passive targeting ability and easy abolishment. Additionally, the ability of NLC to sustain the delivery of drugs could be favorable for the short half-life drugs, for example silybin [15]. In view of this, NLC appears to be a novel approach for improving the delivery of silybin. The purpose of this study was to develop the nanostructured lipid carriers with targeting and prolonged release properties for intravenous delivery of silybin and evaluate its characteristics in vitro and in vivo. 2. Materials and methods 2.1. Materials Silybin (99%) was kindly donated by Panjin Green Bio Co. Ltd., China. Glycerol monostearate (GMS, Beijing Chemistry Reagent Company, China) was used as solid-lipid material of NLC. Medium chain triglycerides (MCT, Tieling North Asia Officinal Oil Co. Ltd., China) was chosen as liquid-lipid material of NLC. Lecithin (Injection grade) was provided by Shanghai Taiwei Pharmaceutics Co. Ltd., China. Pluronic F68 was purchased form Sigma, USA. Mannitol, sodium hydroxide and potassium dihydrogen phosphate were purchased from Shanghai Chemical Agent Co., Ltd. (China). Nitrogen gas was provided by Gas Company of Jinan (China). Water used in the experiment was doubly distilled and deionized. The methanol (Shanghai Siyou Co., Ltd., China) was of HPLC grade. Ethanol and other chemicals were analytical reagent grade. 2.2. Preparation of NLC Silybin-NLCs were prepared by the method of emulsion evaporation at a high temperature and solidification at a low temperature. 5 wt.% silybin (weight percentage of drug to the total amount of lipids), 600 mg of lipids (consisting of GMS and MCT) and 300 mg of lecithin were weighted precisely and then completely co-dissolved into ethanol (5 mL) in water bath at 75 ◦ C. The resultant organic solution was added dropwise into 20 mL of aqueous phase containing 1.5% pluronic F68 under mechanical agitate (RCT Basic, IKA, Germany) with 1000 rpm in water bath at 75 ◦ C for 4 h. The obtained warm nanoemulsion was quickly dispersed into 20 mL of cold distilled water (0 ∼ 2 ◦ C) under stirring at 1000 rpm for 2 h in order to acquire the drug-loaded NLC dispersions. Mannitol (6%, w/v) was used in the freeze-drying process as cryoprotectant. First the obtained NLC dispersions were filled into glass flasks and pre-freezed using an ultracold freezer (MDF382E, SANYO, Japan) at −80 ◦ C for 24 h, later the samples were freeze-dried using a lyophilizer (LGJ0.5, Beijing Sihuan Instrument Company, China) at temperature −40 ◦ C and pressure 0.10 mbar for 48 h. The NLC freeze-dried powders were collected for further experiments.

were diluted with 0.1 mM NaCl and then placed in the measurement cell. The zeta potential was calculated with Smolochowski equation. The pH value of silybin-NLC redispersion was determined at 20 ◦ C using a digital pH meter (PHS-25, Shanghai, China). Each sample was determined in triplicate. 2.5. Differential scanning calorimetry (DSC) analysis Thermograms were recorded with DSC (CDR-4P, Shanghai, China). For DSC measurement, 10 mg of samples was put in an open aluminium pan, and then heated at the scanning rate of 10 ◦ C/min between 0 and 400 ◦ C temperature range. Magnesia was used as the standard reference material to calibrate the temperature and energy scale of the DSC apparatus. 2.6. In vitro release assay The release studies were carried out as following. Briefly, the nanoparticles were added directly into dissolution medium and the filtrate was determined after ultrafiltration–centrifugation [25]. The silybin-NLC freeze-dried powder (containing 4 mg silybin) and 200 mL of dissolution medium (pH 7.4 phosphate buffer saline) were filled in a well-closed tube which was placed in a dissolution test analyzer (RC-3B, Tianjing Instrument Company, China) shaking horizontally with 75 strokes/min at 37 ◦ C. At definite time intervals, 2 mL dispersion sample was withdrawn out and the fresh dissolution medium was placed to maintain constant volume (200 mL). Withdrawn samples were added into Amicon Ultra-4 ultrafiltration device and centrifuged at 3500 rpm for 15 min. The filtrate was analyzed by Agilent 1100 series HPLC system (Agilent, USA). 2.7. Animals New Zealand white rabbits (2.5 ± 0.2 kg) and Kunming strain mice (25 ± 2 g), supplied by the Experimental Animal Center of Shandong University (Jinan, China), were used for pharmacokinetic and biodistribution studies, respectively. At first, the animals were acclimatized at a temperature of 25 ± 2 ◦ C and a relative humidity of 70 ± 5% under natural light/dark conditions for one week and were fed with food and water ad libitum. Prior to experiment the animals were kept under fasting overnight. All experimental procedures are abided by the ethics and regulations of animal experiments of Pharmaceutical Sciences, Shandong University, China. 2.8. Pharmacokinetics studies

The morphology of NLC was determined by TEM (H-7000, Hitachi, Japan). A drop of NLC redispersion was spread on a 200mesh copper grid and negatively stained with 2% phosphotungstic acid for 30 s. The grid was dried at room temperature and then observed by TEM.

Pharmacokinetics of silybin-NLC was compared with silybin solution. Since silybin was practically insoluble in water, its solution was prepared by dissolving silybin in polyethylene glycol (PEG) 400/ethanol/water (4/1/5, v/v/v) mixture solvent. Eight rabbits were allocated at random to two groups and administered silybin-NLC and silybin solution, respectively. After administration of a dose of silybin solution or silybin-NLC (4 mg/kg, expressed as silybin equivalents) via the left auricular vein, about 1 mL of blood sample was collected through the right auricular vein into heparinized tubes at 0.08, 0.17, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h. Blood samples were centrifuged at 5000 × g for 10 min and plasma samples were withdrawn and stored at −20 ◦ C.

2.4. Evaluation of particle size, zeta potential and pH value

2.9. Tissue distribution studies

The particle size analysis of silybin-NLC redispersion was performed by Zetasizer (3000HS, Malvern Instruments, UK). The electrophoretic mobility and the zeta potential were analyzed by television micro-electrophoresis apparatus (DVD-2, Jiangsu, China). For the measure of the electrophoretic mobility, the samples

The Kunming strain mice were randomly divided into two groups. Silybin-NLC and silybin solution were intravenously administrated via tail vein at a dose of 12 mg/kg to mice. At determined time points (for silybin solution at 0.17, 0.5, 1, 2, 4, 6, 8, 10, 12, 24 h and for silybin-NLC at 0.17, 0.5, 1, 2, 4, 6, 8, 10, 12, 24,

2.3. Transmission election microscope (TEM) examination

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Fig. 1. Transmission electron microscopy micrograph of silybin-NLC (magnification 20,000×).

36 h), the blood of mice was collected from postorbital vein into heparinized tubes and was centrifuged to get the plasma samples. Tissues of interest (heart, liver, spleen, lung, and kidney) were collected immediately after lightly rinsed with normal saline and dried with tissue paper. The plasma and tissue samples were frozen at −20 ◦ C until analysis.

Fig. 2. Particle size distribution of silybin-NLC.

2.10. Plasma and tissue sample analysis Liquid–liquid plasma extraction procedure was as follows: in a 10 mL glass screw-capped conical tube was added 500 ␮L (200 ␮L for mouse) plasma followed by 100 ␮L of an internal standard (1-naphthol) solution, 200 ␮L of 0.1 M Na2 HPO4 and 0.6 mL acetonitrile. After vortex mixing for 2 min, 5 mL ether anhydrous was added and vortexed for 5 min. After centrifuging at 3500 g for 15 min, the organic layer was transferred to another tube and evaporated under a light stream of nitrogen at 40 ◦ C. The residue was redissolved by 200 ␮L mobile phase and filtered through a 0.22 ␮m filter; 20 ␮L of the filtrate was injected for HPLC analysis. Quantification was based on area ratio (Asilybin /AIS ) and the area of silybin isomers was calculated as a whole. Tissue sample was weighed accurately and homogenized using a glass tissue homogenizer after addition of 1 mL physiologic saline. Tissue homogenates were processed similarly as plasma samples and analyzed by HPLC. The plasma and tissue samples were all analyzed by an Agilent 1100 high performance liquid chromatography (HPLC) analysis system (Agilent, USA), detection conditions were set as follows: Phenomenex-ODS column (150 mm × 4.60 mm, 5 ␮m); mobile phase: methanol:2% (v/v) acetic acid solution (48:52); flow rate of the mobile phase: 1.0 mL min−1 ; measured wavelength: 288 nm. The detection limit was 0.03 mg kg−1 for each tissue.

F68 ) in the formulation. However, despite a zeta potential below the critical value of −30 mV, nanoparticles can have the same longterm stability, in case the sterically stabilizing layer is sufficiently thick [26,27]. DSC measurements offer a close look at the crystallization and thermal behavior of the lipid nanoparticles [28–30]. Thermoanalytical studies were performed and the main purpose of this test was to inspect whether or not the crystallinity was different in the lipid nanoparticles compared with the raw materials. Fig. 3 shows the DSC thermograms of the raw materials, their physical mixture and silybin-NLC. There were peaks resulting from simple superposition of their separated component DSC curves in the thermogram of the physical mixture, for instance, the melting peak of silybin at 148 ◦ C, the melting peak of GMS at 63 ◦ C. In contrast, no melting peak corresponding to the fusion of silybin was observed in the curve of silybin-NLC, indicating that silybin was not in crystalline state in the nanoparticles. Besides, the melting peak of GMS was weak and shifted to lower temperature . The decrease of melting peak could be attributed to the small size effect, having then a high specific surface area and explained by the Thomson equation [31]. In addition, adding MCT to nanoparticles perhaps disturbed the crystal order

2.11. Statistics Statistical significance was determined using Student’s t-test with P < 0.05 indicating significant difference. Pharmacokinetic parameters were obtained using drug and statistics (DAS) version 2.1.1 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). 3. Results and discussion 3.1. Characterization of silybin-NLC Fig. 1 shows the TEM image of silybin-NLC. It displayed that the NLC were in round shape and non-adherent among each other. The mean size of silybin-NLC was 232.1 ± 8.3 nm (n = 3). The size distribution of silybin-NLC was shown in Fig. 2 that all the particles were smaller than 766.2 nm, in which 90% were less than 383.9 nm. The zeta potential of NLC was −20.7 ± 1.5 mV (n = 3), which attributed to the application of non-ionic surfactants (lecithin and pluronic

Fig. 3. Differential scanning calorimetry curves: (A) F68 ; (B) GMS; (C) silybin; (D) the physical mixture of GMS, silybin and F68 ; (E) silybin-NLC.

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Fig. 5. The mean blood concentration–time curve of silybin in rabbits following i.v. administration of silybin solution and silybin-NLC at a dose of 4 mg kg−1 (data were given as mean ± S.D., n = 4).

Fig. 4. In vitro release profile of silybin-NLC (n = 3).

of the nanoparticles, and therefore for the less ordered crystal, the melt of the substance required less energy [32]. 3.2. In vitro release of drug In vitro release profile of silybin-NLC is shown in Fig. 4. For the nanoparticles, a biphasic drug release pattern was observed. The relative burst drug release was found at the initial 6 h, and about 58% of total silybin was discharged during this time period. Then the release continued slowly and 86% of the silybin was released in 36 h. In vitro data for release of the drug from silybinNLC was fitted to a two-stage exponential kinetic model and the

equation was as follows: 1 −Q% = 0.7925 e−0.1059t + 0.5020 e−0.0359t (r˛ = 0.9991, rˇ = 0.9965). The underlying mechanism for this phenomenon might be explained by the form process of silybin-NLC. The obtained nanoparticles were prepared by the method of emulsion evaporation at a high temperature and solidification at a low temperature. During the solidification at a low temperature, due to the solid-lipid (GMS) owing higher melting point, it would rapidly solidify to form solid-lipid core in which liquid-lipid was randomly distributed. When the liquid-lipid content was higher, liquid-lipids would be located at the outer shell of the nanoparticles besides distributed in solid-lipid core. The liquid-lipid-enriched outer layers possessed a soft and considerable higher solubility for lipophilic drugs character, in which the drug was easily loaded to higher amount and could be easily released as well by the drug diffusion or the matrix erosion manners. Therefore, the nanoparticles

Fig. 6. Mean silybin concentration–time profiles in organs after intravenous administration of silybin solution and silybin-NLC to mice with dose of 12 mg kg−1 (data were given as mean ± S.D., n = 4).

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Table 1 Mean pharmacokinetic parameters of silybin after i.v. administration of silybin solution and silybin-NLC (each data was from four rabbits). Parameter

Unit

Silybin-NLC

Silybin solution

AUC(0–t) AUC(0–∞) MRT t1/2 Vd CL

mg L−1 h mg L−1 h h h L kg−1 L h−1 kg−1

6.267a 6.271 7.895a 2.747b 2.529 0.638

2.979 2.981 1.620 1.233 2.388 1.342

a b

P < 0.05, statistical significance compared with silybin solution group. P > 0.05, no statistical significance compared with silybin solution group.

showed the burst release at the initial stage. With erosion or degradation of lipid matrix, the drug dispersed or incorporated into the nanoparticle core was released in a prolonged way [24,33,34]. 3.3. Pharmacokinetic study The silybin blood concentration–time curves after intravenous (i.v.) administration of two formulations in rabbits are shown in Fig. 5. The results indicated that although the initial drug concentration for silybin solution was higher than that of silybin-NLC, silybin solution was quickly removed from the circulation system. On the contrary, the i.v. administration of silybin-NLC resulted in prolonged residence of silybin in systemic blood circulation. In addition, the double-peak of drug concentration–time curve was exhibited for silybin-NLC, which could be explained by two factors. Firstly, silybin appears to undergo the enterohepatic circulation in vivo [6,16]. Silybin excreted by bile, was reabsorbed from small intestine and then returned to the systemic circulation via the hepatic portal vein system. Secondly, the hepatic capillary is one of the most permeable capillary in the body and the liver sinusoids are spontaneously permeable [35]. Silybin-NLC in the capillary system could transfer to the interstitial perisinusoidal space through the liver sinusoids and accumulated in the liver. However, some smaller nanoparticles could also leak from the interstitial perisinusoidal space and returned to the blood circulation. Thus the double-peak was found in the drug concentration–time curve. Due to double-peak in the drug concentration–time curve, the compartment model is not a suitable analysis method and the statistical moment analysis is appropriate. Mean pharmacokinetic parameters for silybin-NLC and silybin solution are shown in Table 1. It was exhibited that the silybin-NLC had a 2-fold increase in AUC value compared with the control solution. Moreover, the mean residence time of silybin-NLC was significantly longer than silybin solution. For the half-time, there was no significant difference between silybin-NLC and silybin solution (P > 0.05). It was deduced that silybin-NLC could improve the availability of silybin by prolonging drug retention in vivo. 3.4. Biodistribution study 0.1 ∼ 50 ␮g mL−1

Within silybin concentration, the standard curves showed good linearity and correlation coefficients were 0.9949–0.9997 for all measured organs. The plasma and tissue silybin concentrations versus time after i.v. administration of silybin solution and silybin-NLC are shown in Fig. 6A–F. The results indicated that the multi-peak phenomenon existed in drug concentration–time curves of all tissues after i.v. administration of silybin-NLC or silybin solution. For the silybin solution, it could be deduced that multi-peak of drug concentration–time curves were the result of enterohepatic circulation. Silybin was reabsorbed from small intestine and returned to the systemic circulation leading to the drug redistribution in the body. For the silybin-NLC, this phenomenon might be attributed to the enterohepatic circulation,

Fig. 7. Silybin tissue concentration percentage of liver, spleen, lung, kidney and heart at 12 h following i.v. administration of silybin solution and silybin-NLC.

the nanoparticle size and the capillary permeability. Intravenously injected particulate substances or drug carriers with an average size below 7 ␮m are normally taken up by the reticulo endothelial system (RES) [36,37]. Therefore, the obtained silybin-NLC could be abounded in the RES organs. Capillary systems in some tissues are permeable, thus the nanoparticles can move to the interstitial perisinusoidal space of the organs through the capillary systems after i.v. administration, which enables nanoparticles to assemble in tissues. However, some smaller nanoparticles in organs could also return to the blood circulation due to the permeability of capillary and redistribute in the body resulting in the multi-peak of drug concentration–time curves. Fig. 7 showed silybin tissue concentration percentage of liver, spleen, lung, kidney and heart at 12 h following i.v. administration of silybin solution and silybin-NLC. For silybin-NLC, 34.1% and 21.9% silybin were distributed in liver and spleen, which was higher than the control solution. The result indicated that silybin-NLC could remain in liver for a longer time that was beneficial to therapy of liver disease. AUC(0–t) and MRT(0–t) values of tested organs for silybin solution and silybin-NLC were given in Table 2. AUC(0–t) values of silybin-NLC for liver, spleen and lung were found higher than that of silybin solution. The nanoparticles might be recognized as foreign matters and uptaken by phagocytic cells of mononuclear phagocyte system (MPS) which abounded in special tissues and organs, such as liver, lung and spleen [38]. Therefore, for silybin-NLC, silybin had higher AUC(0–t) values and re values of liver, lung and spleen were all greater than 1 suggesting good target to these tissues especially to liver. In the pharmacokinetics and biodistribution studies, multi-peak of drug concentration was found in blood and other tissues, which was attributed to the small size of silybin-NLC and the permeability Table 2 Average pharmacokinetic parameters (n = 4) of silybin after intravenous administration of silybin solution and silybin-NLC to mice with dose of 12 mg kg−1 . Organ

AUCa (0–t) (mg L−1 h)

Silybin solution Plasma Heart Liver Spleen Lung Kidney

18.006 30.507 13.845 21.570 20.248 23.588

1.566 7.841 5.986 7.172 6.160 4.081

Silybin-NLC Plasma Heart Liver Spleen Lung Kidney

28.322 19.853 52.941 27.039 30.463 18.694

6.728 7.876 12.386 9.599 9.728 4.521

a b c

MRTb (0–t) (h)

AUC: area under tissue concentration–time curve. MRT: mean residence time. re : relative uptake rate equivalent AUC(NLC) /AUC(sol) .

re c

1.6 0.6 3.8 1.3 1.5 0.8

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of capillary system. The distributed silybin-NLC could release from some particular organs into systemic circulation and redistribute, therefore, the drug might circulate for a long time in the blood system which could contribute to the efficacy enhancement. However, adverse drug reaction can be caused by multi-peak of drug blood concentration, so it is disadvantageous for the drug with narrow therapeutic window. As for silybin, clinical studies have shown that it has an excellent safety profile at therapeutic dosages. High dosages (>1500 mg/day) may only produce loose stools because of increased bile flow and secretion [6,8]. It would be reasonable to assume that multi-peak of drug blood concentration caused by small size of nanoparticle rarely gives rise to side effects for the drug with wide therapeutic window. Besides the small size effect makes nano-drug delivery system have the potential to enhance drug bioavailability, enable drug targeting and penetrate biologic barriers, but on the other hand, small size effect could cause adverse reaction due to multi-peak of drug blood concentration. Hence multi-peak of drug blood concentration for nano-drug delivery systems should be taken into account by researchers and then treating diseases with nano-drug delivery system could be safer and well tolerated. 4. Conclusion Nanostructured lipid carriers for parenteral delivery of silybin were successfully prepared in this study. In vitro release test, silybin-NLC exhibited a biphasic drug release pattern with burst release at the initial stage and sustained release afterwards. Compared with silybin solution, silybin-NLC showed higher AUC values and a prolonged residence time of drug in the blood circulation. Besides, silybin-NLC was taken up by the RES with good targeting to the RES organs especially to liver. Therefore, nanostructured lipid carriers may hold some promise to deliver silybin for therapy of liver disease. Acknowledgement The authors are thankful to the National Basic Research Program of China (973 Program) for the financial support, the program number is 2009CB930300. References [1] F. Kvasnicka, B. Biba, R. Sevcik, M. Voldrich, J. Kratka, Analysis of the active components of silymarin, J. Chromatogr. A 990 (2003) 239– 245. [2] J. Yang, Y.M. Liu, Y.Z. Liu, Advances in the pharmaceutical research on the silymarin, Nat. Prod. Res. Dev. 16 (2004) 185–187. [3] H. Basaga, G. Poli, C. Tekkaya, I. Aras, Free radical scavenging and antioxidative properties of ‘silibin’ complexes on microsomal lipid peroxidation, Cell Biochem. Funct. 15 (1997) 27–33. [4] G. Boigk, L. Stroedter, H. Herbst, Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats, Hepatology 26 (1997) 643–649. [5] S. Luper, A review of plants used in the treatment of liver disease, Part 1, Altern. Med. Rev. 3 (1998) 410–421. [6] J. Pepping, Milk thistle: silybum marianum, Am. J. Health Syst. Pharm. 56 (1999) 1195–1197. [7] K. Flora, M. Hahn, H. Rosen, K. Benner, Milk thistle (silybum marianum) for the therapy of liver disease, Am. J. Gastroenterol. 93 (1998) 139–143. [8] B.P. Jacobs, C. Dennehy, G. Ramirez, J. Sapp, V.A. Lawrence, Milk thistle for the treatment of liver disease: a systematic review and meta-analysis, Am. J. Med. 113 (2002) 506–515. [9] M.S. El-Samaligy, N.N. Afifi, E.A. Mahmoud, Increasing bioavailability of silymarin using a buccal liposomal delivery system: preparation and experimental design investigation, IpHarm 308 (2006) 140–148.

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