ARTICLE pubs.acs.org/JAFC
Preparation of a Tea Polyphenol Nanoliposome System and Its Physicochemical Properties Qun Lu, Di-Cai Li, and Jian-Guo Jiang* College of Food and Bioengineering, South China University of Technology, Guangzhou, 510640, China ABSTRACT: Tea polyphenol is rich in green tea with diverse biological activities. However, its application in the food industry is limited due to its instability toward oxygen and light. In this study, the preparation of tea polyphenol liposome by the thin film ultrasonic dispersion method was performed in order to enhance the bioavailability of tea polyphenol. The process conditions were optimized using response surface analysis, and the optimal parameters were as follows: ratio of tea polyphenol to lecithin, 0.125:1; ratio of lecithin to cholesterol, 4:1; phosphate buffered saline (PBS) pH, 6.62; ultrasonic time, 3.5 min. The theoretical and practical entrapment efficiency were 60.36% and 60.09 ( 0.69%, respectively. Furthermore, physicochemical properties including size distribution, zeta potential, permeability, infrared spectrum and in vitro release of liposomal formulations were determined. The mean size of tea polyphenol liposome was 160.4 nm, and the ζ-potential value was 67.2. The tea polyphenol liposome was formed by physical interaction, and the in vitro release process followed a first-order equation. The results indicated that the prepared tea polyphenol liposome was stable and suitable for more widespread application. KEYWORDS: tea polyphenol, liposome, thin film ultrasonic dispersion method, physicochemical properties
’ INTRODUCTION Tea polyphenol is abundant in green tea and has excellent biological and pharmacological actions, such as antioxidation, antibacteria, anticancer, lowering the blood lipids, etc.14 It is widely used in food, health products and cosmetics.5 Tea polyphenol is often used to act as an antioxidant by donation of a hydrogen atom, as an acceptor of free radicals, interrupting chain oxidation reactions, or by chelating metals. However, the sensibility of tea polyphenol toward oxygen and light reduces its stability in processing and storage and limits its application. Entrapment systems including liposome, nanoparticle and microcapsule are considered applicable to overcome this problem because of their favorable characteristics as biopolymeric carriers.6,7 Microcapsule and microemulsions have been adopted in the encapsulation of tea polyphenols. Mixed calcium carbonate and phosphate microparticles were employed in the encapsulation of green tea polyphenols,8 and the preparation of tea polyphenol loaded solid lipid nanoparticles based on the phase behaviors of hot microemulsions was developed.9 However, few studies have been reported on the liposomal system for tea polyphenol. Tea polyphenol and vitamin E loaded liposome was investigated in order to offer a new approach to entrap aqueous soluble drug and insoluble drug together,10 and Fang et al.11 prepared tea catechin liposome for local delivery, including skin and tumor deposition. Liposomes, microscopic bilayer vesicles from dispersion of membrane-like lipids in aqueous solvents, are gaining increasing popularity for their ability to act as delivery vehicles by protecting reactive or sensitive compounds.12 Liposomes are stable against various environmental and chemical changes, and are able to enhance the performance of products by increasing ingredient solubility, improving ingredient bioavailability and in vitro and in vivo stability, which have been useful in industries of food, cosmetics and drugs.13 The food applications of liposomes include encapsulation of nutrients, nutraceuticals, food flavors, food r 2011 American Chemical Society
additives, and food antimicrobials.1417 Liposomes can be formulated from a variety of lipids and lipid mixtures with different compositions. High-purity lecithins are generally used for preparing liposomes.18 The materials of conventional liposomes are lecithin plus cholesterol, which have hydrophilic groups and lipophilic groups to form a lipid bilayer that encapsulates an aqueous phase.19 The common technologies for the preparation of liposome are thin film dispersion, reverse evaporation, pH gradient method, high pressure homogenization, emulsification, freeze-drying, centrifugation, pressure extrusion and melting technologies.2022 The thin film dispersion method, the most widely used technology in the preparation of liposome, is simple and easy to carrying out. Commonly, membranous materials such as phospholipid and cholesterol together with fat-soluble compounds are dissolved in an adequate amount of organic solvent. The solvent is removed by vacuum rotation, and a thin film is formed and then dispersed using mechanical means.23 According to different dispersion methods, there are the film ultrasonic dispersion method, the film homogenization method and the film oscillation method. The thin film dispersion ultrasonic method is a treatment to obtain 0.251 μm unilamellar liposome. The methodology of liposome with thin film ultrasonic dispersion technology is adopted in this research for its potential advantages to improve the oxidation resistance and extend the storage time of tea polyphenol. Operational process conditions were optimized, and the physicochemical properties of tea polyphenol liposome were investigated by transmission electron microscopy, Nano-ZS particle size analyzer, MPT-2 automatic titrators and FT-IR spectrograph to estimate its stability and applicability. Received: August 9, 2011 Accepted: November 16, 2011 Revised: October 13, 2011 Published: November 16, 2011 13004
dx.doi.org/10.1021/jf203194w | J. Agric. Food Chem. 2011, 59, 13004–13011
Journal of Agricultural and Food Chemistry
ARTICLE
Figure 1. Effects of different ratio of tea polyphenol to lecithin (A), different ratio of lecithin to cholesterol (B), different PBS pH (C) and different ultrasonic time (D) on entrapment efficiency and mean size of tea polyphenol liposome.
’ MATERIALS AND METHODS Materials and Apparatus. Green tea (Huang Keng tea factory, Jiaoling County, Guangdong, China), lecithin (Beijing Shuangxuan microbe culture medium products factory, Beijing city, China), cholesterol (Wuhan life Co., Ltd., Wuhan city, Hubei, China), centrifugal molecular distillation apparatus (CMS-180-1, Guangzhou Pu source of Biochemical Technology Co., Ltd., Guangzhou city, Guangdong, China), visible-infrared spectrometer (WFJ2100, Shanghai Unico Instrument Co., Ltd., Shanghai city, China), pH meter (S-3D, Shanghai Precision and Scientific Instrument Co., Ltd., Shanghai city, China), high-voltage transmission electron microscope (Philips Co., Ltd., Shanghai city, China), particle size analyzer (Nano-ZS, Malvern Instruments Co., Ltd., England), automatic titrator (MPT-2, Malvern Instruments Co., Ltd., England), Fourier transform infrared spectrometers (Nicolet 380, Thermo Electron Co., Ltd., Waltham, MA, USA). Preparation of Tea Polyphenol Liposome. The tea polyphenol was extracted from green tea by microwave-assisted extraction. The extraction process conditions and the identification of tea polyphenol were described in our previous study.24 An appropriate amount of tea polyphenol was dissolved in 10 mL of ethanol, while an adequate amount of lecithin and cholesterol was dissolved in 10 mL of ether. The two parts were mixed together with vibration. The organic solvent was removed in a rotary evaporator at 35 °C to obtain a thin film. The residual solvent in the film was removed at room temperature under vacuum. The film was washed with 20 mL of phosphate buffer, kept in a water bath at 40 °C for 2 h and treated with ultrasound for 4 min. The liposomal suspension was kept overnight in a
refrigerator and, on the following day, filtered through a sintered glass filter and then kept in a refrigerator. The lipid recovery in all cases was well over 95%.
Determination of Tea Polyphenol Content, Entrapment Efficiency and Mean Size. Preparation of standard curve: the reference standard of tea polyphenol weighed precisely was dissolved in anhydrous ethanol to form 5, 10, 15, 20, 25, 30, 35, 40 μg/mL standard solutions. The absorbance of these standard solutions was determined at 275 nm respectively with ethanol as the blank. The linear regression of absorbance on concentration was made and the regression equation was calculated. The regression equation of the standard curve was y = 0.0265x 0.0195, R2 = 0.9983. A certain amount of sample was put into a brown capacity bottle and diluted with anhydrous ethanol. After being treated in an ultrasonic water bath for 15 min, the sample was cooled to room temperature and then fixed to the calibration with anhydrous ethanol. Finally, the sample was mixed and filtered to measure its absorbance, which was substituted into the regression equation to calculate the content of tea polyphenol. Entrapment efficiency is an important indicator in the evaluation of prepared tea polyphenol liposome. The entrapment efficiency (EE) was determined as follows:25 EE % ¼
m1 m2 100% m1
ð1Þ
where m1 is the amount of total tea polyphenol and m2 is the amount of free tea polyphenol in the liposome solution. A volume of 400 μL of the liposome solution was mixed with distilled water to 2 mL in a centrifuge tube, followed by centrifugation at 13000 rpm for 30 min. 13005
dx.doi.org/10.1021/jf203194w |J. Agric. Food Chem. 2011, 59, 13004–13011
Journal of Agricultural and Food Chemistry
ARTICLE
Table 1. The Experimental Design and Response Results
Table 2. Analysis of Variance for the Experimental Results sum of
level source
mean
squares
df
square
F-value
prob > F significanta
A
B
C
D
model
652.33
14 46.59
15.94
