Silymarin liposomes improves oral bioavailability of silybin besides targeting hepatocytes, and immune cells

doi:10.1016/j.pharep.2014.04.007

Pharmacological Reports

Volume 66, Issue 5, October 2014, Pages 788-798

https://doi.org/10.1016/j.pharep.2014.04.007 Get rights and content

Abstract

Background

Silymarin, a hepatoprotective agent, has poor oral bioavailability. However, the current dosage form of the drug does not target the liver and inflammatory cells selectively. The aim of the present study was to develop lecithin-based carrier system of silymarin by incorporating phytosomal–liposomal approach to increase its oral bioavailability and to make it target-specific to the liver for enhanced hepatoprotection.

Methods

The formulation was prepared by film hydration method. Release of drug was assessed at pH 1.2 and 7.4. Formulation was assessed for in vitro hepatoprotection on Chang liver cells, lipopolysaccharide-induced reactive oxygen species (ROS) production by RAW 267.4 (murine macrophages), in vivo efficacy against paracetamol-induced hepatotoxicity and pharmacokinetic study by oral route in Wistar rat.

Results

The formulation showed maximum entrapment (55%) for a lecithin–cholesterol ratio of 6:1. Comparative release profile of formulation was better than silymarin at pH 1.2 and pH 7.4. In vitro studies showed a better hepatoprotection efficacy for formulation (one and half times) and better prevention of ROS production (ten times) compared to silymarin. In in vivo model, paracetamol showed significant hepatotoxicity in Wistar rats assessed through LFT, antioxidant markers and inflammatory markers. The formulation was found more efficacious than silymarin suspension in protecting the liver against paracetamol toxicity and the associated inflammatory conditions. The liposomal formulation yielded a three and half fold higher bioavailability of silymarin as compared with silymarin suspension.

Conclusions

Incorporating the phytosomal form of silymarin in liposomal carrier system increased the oral bioavailability and showed better hepatoprotection and better anti-inflammatory effects compared with silymarin suspension.

Introduction

Silymarin, a hepatoprotective agent, obtained from single herb Silybum marianum, is widely used in the treatment of liver diseases. A mixture of flavolignan isomers, namely silybin, isosilybin, silydianin, silychristin is collectively expressed as silymarin [1]. Among these isomers, the most active component is silybin, which accounts for 60–70% of the total content of silymarin and is considered as the marker of silymarin [2].

Many experimental studies have proved the hepatoprotective activity of silymarin [3]. One of the major limitations of silymarin is poor oral-bioavailability. The oral absorption of silymarin is only about 23–47% [4], leading oral bioavailability to 0.73% [5]. Therefore, a higher dose of silymarin is required to improve therapeutic efficacy. The reasons suggested for its poor bioavailability includes the following: poor enteral absorption [6], instability in gastric environment [4] and poor solubility [4]. Thus, enhancement of bioavailability of silymarin is a challenging task. Although substantial advancement has been made in improving the bioavailability of silymarin through various dosage forms, little information is available on the measures adopted by researchers to make silymarin target specific to promote hepatocytes’ regeneration and to prevent inflammation in liver [7].

The process of repair in the liver is largely by the regeneration of hepatocytes. However, inflammation in liver is one of the major problems associated with hepatocyte toxicity. If inflammation is not controlled sufficiently, the cellular phase of inflammation through macrophages (Kupffer cells) and fibroblast (stellate cells) promote fibrosis to replace dead cells [8]. Therefore, a formulation of silymarin that would target the liver in general and inflammation in particular would be beneficial over a formulation of silymarin that would just enhance the bioavailability of silymarin. In this context, the present study is aimed at developing a formulation of silymarin with the help of liposomal and phytosomal combination. This is based on the fact that phytosomal silymarin is more stable in the gastric environment [9] to enhance the bioavailability of silymarin while the liposomal silymarin is having the highest ability to get captured by macrophages, Kupffer cells and infiltrated WBC viz., neutrophil, monocytes, etc. through phagocytosis process and modulate their actions [10]. This phenomenon makes silymarin in formulation to target inflammation.

Section snippets

Materials

Triton-X 100, trypsin and dithiothreitol (DTT) were purchased from Himedia lab Pvt. Ltd. (Mumbai, India). Lecithin (Soya L-α-Phosphatidylcholine or SPC), 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Dulbecco's modified eagle's medium (DMEM), minimum essential medium (MEM), fetal bovine serum (FBS) and silymarin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used in the study were of analytical grade.

Cell lines

Chang liver cells and RAW 264.7 were

Morphology

Liposomes were suspended in water and particle shape was visualized by transmission electron microscope (TEM), TECNAI 200 Kv TEM from Sophisticated analytical instrument facility, All India Institute of Medical Sciences, New Delhi, India.

Preformulation studies

Physical mixtures (silymarin and lipids) did not show any drug-lipid interactions. They were stable, physically and chemically as no changes were observed in the data of DSC (Fig. 1a).

Preparation of silymarin-liposomes

The encapsulation percentage of silymarin was found to be maximum for formulation containing SPC and cholesterol at molar ratio 6:1 (Table 1). Particle size obtained from sonication and high-pressure homogenizer (HPH) was compared. More homogeneous and uniform particles were obtained by HPH compared to sonication

Discussion

The optimized liposome of silymarin was spherical in shape and homogeneous in particle size distribution. Zeta potential of formulation (−70 mV) was found to be more than optimum for physical stability of the formulation [23]. DSC thermogram of liposomal formulation showed that there was an interaction between the lipid and drug, as the peak of silymarin shifted significantly by more than 40 °C. This interaction suggested the formation of phytosomes. The formulation had increased the solubility

Conclusion

Incorporating phytosomal form of silymarin in liposomal carrier system showed better in vitro and in vivo hepatoprotection besides showing better anti-inflammatory effects and improvement in histopathological changes as compared to silymarin suspension. These effects were further supported by increase in AUC and C_max of silybin by silymarin-liposomes compared to silymarin suspension.

Role of funding

We would like to thank AICTE-MODROB scheme (Ref. No.: 9-126/RIFD/MODROB/Policy-1/2013-14(Pvt.)) for providing funding support.

AICTE sanctioned the grant after reviewing our proposal. As such, it has no role in drafting and designing the plan of work. Prior permission for the publication of manuscript is not required.

Conflict of interest

There authors declare there are no conflicts of interest.

Acknowledgment

We would like to thank AICTE-MODROB scheme for providing funding support.

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¹: Present address: Faculty of Pharmacy, AIMST University, Malaysia.

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Original research articleSilymarin liposomes improves oral bioavailability of silybin besides targeting hepatocytes, and immune cells

Abstract

Background

Methods

Results

Conclusions

Introduction

Section snippets

Materials

Cell lines

Morphology

Preformulation studies

Preparation of silymarin-liposomes

Discussion

Conclusion

Role of funding

Conflict of interest

Acknowledgment

J Pharm Biomed Anal

Biochem Pharmacol

Int J Pharm

Methods Enzymol

Int Immunopharmacol

Methods Enzymol

J Biol Chem

Eur J Pharmacol

J Pharmacol Toxicol Methods

Dissolution test for silymarin tablets and capsules

Drug Dev Ind Pharm

Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine

Indian J Med Res

Original research article
Silymarin liposomes improves oral bioavailability of silybin besides targeting hepatocytes, and immune cells