Article pubs.acs.org/JAFC
Preparation, Characterization, and Preliminary Antibrowning Evaluations of Norartocarpetin Microemulsions Zong-Ping Zheng,† Xue Dong,† Kun Yuan,† Shan Lan,† Qin Zhu,§ Mingfu Wang,# and Jie Chen*,†,‡ †
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang, People’s Republic of China # School of Biological Sciences, The University of Hong Kong, Hong Kong, People’s Republic of China ‡ Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China §
S Supporting Information *
ABSTRACT: Norartocarpetin is a flavone widely distributed in Moraceae plants with strong tyrosinase inhibitory activity. However, its poor solubility in aqueous systems and in food grade solvents (oils) limits its extensive applications. The aim of this study was to improve the solubility of norartocarpetin by developing an oil-in-water (o/w) microemulsion with food grade components. A microemulsion was developed and characterized, and its chemical and physical stabilities were also evaluated within 8 weeks. Using the construction of pseudoternary phase diagrams, the optimized formulation of 2% w/w of ethyl oleate, 12% w/w of Tween 80, 6% w/w of polyethylene glycol 400, and 80% w/w of water was obtained, with a maximum solubility of norartocarpetin up to 1.73 ± 0.21 mg/mL, at least a 1000-fold increase in solubility. The microemulsion base and norartocarpetin-loaded microemulsion were demonstrated to be stable after accelerated and long-term conditions (8 weeks). The norartocarpetin microemulsion still showed strong tyrosinase inhibitory activity and antibrowning effect on fresh-cut apple slices. These combined results indicated that norartocarpetin microemulsion may be suitable as an antibrowning agent for fresh-cut fruits. KEYWORDS: norartocarpetin, microemulsion, tyrosinase inhibition, antibrowning
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additives through delivery systems such as microemulsions,8−11 nanoemulsions,12−14 solid lipid nanoparticles,15 complexation with cyclodextrin,16 and liposome encapsulation.17 Among them, microemulsions (ME) have attracted much interest in recent years as drug delivery systems because they are not only highly dispersed, stable, and transparent systems and easily prepared but also help to improve the solubility and stability of many food and medical bioactive components.8−11 In general, ME consist of oil, water, and surfactant, frequently in combination with cosurfactant.18,19 The appearance of ME is clear or translucent due to their dispersed phase size usually in the range of 10−100 nm.19,20 Previous studies have revealed that ME can enhance the solubility and bioavailability of some flavonoids.21,22 The aim of this study is to formulate an oil-in-water (o/w) microemulsion of norartocarpetin to increase its solubility as antibrowning agent, using food available components. With the optimization of norartocarpetin solubility in selected ME, the chemical and physical stabilities were also investigated as well as their antibrowning effects in apple slices.
INTRODUCTION Tyrosinase (EC 1.14.18.1) plays a vital role in the processes of undesirable browning. The browning caused by tyrosinase usually impairs the color and sensory properties of various food products, especially fruits and vegetables, which eventually leads to the loss of essential amino acids, with lower nutritional quality, digestibility, and market values.1 Tyrosinase inhibitors have received great attention due to their abilities to suppress undesirable browning reactions and thus help maintain the quality attributes of food products. One of the most important requirements for an effective tyrosinase inhibitor is the safety for application in food products without causing significant negative influence on the appearance and nutritional quality. Therefore, tyrosinase inhibitors from natural sources, especially those from plants, are considered promising candidates.2 Up to now, numerous tyrosinase inhibitors have been identified from natural origins exhibiting strong tyrosinase inhibitory activities. However, few of them have been used as antibrowning agents in the food industry because of safety concerns and other factors, such as limited sources, poor solubility, and instability. Norartocarpetin is a flavone widely distributed in Moraceae plants, especially very rich in the Artocarpus heterophyllus fruit tree; it showed strong tyrosinase inhibitory activity but exhibited poor solubility in aqueous system and in food grade solvents (oils).3−7 How to improve the solubility of insoluble natural inhibitors such as norartocarpetin has become an important issue in the food industry. Many methods have been applied to increase the solubility and bioavailability of phytochemicals, nutraceuticals, and food © 2015 American Chemical Society
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MATERIALS AND METHODS
Materials. Tween 80, Tween 40, Tween 20, ethyl oleate, propylene glycol, ethyl butyrate, limonene, glycerol, polyethylene glycol 400
Received: Revised: Accepted: Published: 1615
October 9, 2014 December 29, 2014 January 20, 2015 January 20, 2015 DOI: 10.1021/jf5048805 J. Agric. Food Chem. 2015, 63, 1615−1621
Article
Journal of Agricultural and Food Chemistry
Characterization of MEs. The appearance of ME was determined by visual inspection of clarity, turbidity, phase separation, and color. The conductivity, mean particle size, and polydispersity index (PI) were determined by three independent experiments. Physical Properties. Physical properties of blank ME and NorA ME were characterized for pH and conductivity using a pH meter (Mettler Toledo, Shanghai, China) and conductivity meter (Mettler Toledo) at 25 ± 1 °C. Particle Size Analysis. The mean particle diameter of the ME was determined by photon correlation spectroscopy using a Zetasizer Nano ZS (Marvern Instruments, Worcestershire, UK). Each size analysis lasted 180 s and was performed with an angle detection of 90°. The measurements of samples without dilution were performed at 25 ± 0.1 °C. Morphology. Morphology of NorA ME was observed using a transmission electron microscope (TEM, JEOL JEM2100, Eindhoven, The Netherlands). One drop of NorA ME sample was deposited on a formvar film-coated copper grid and allowed to dry before TEM observation. Stability Studies. The thermodynamic stability of ME systems was investigated visually for phase separation after their centrifugation at 10000g for 20 min at room temperature (25 ± 1 °C). Stability tests using accelerated condition by cooling−heating wee conducted at 4 and 40 °C for 24 h for six cycles. Long-term stability testing at room temperature (20−37 °C) for 8 weeks was also performed. Their chemical and physical stabilities were studied and recorded by monitoring the occurrence of phase separation, dispersed phase size, and NorA content before and after the stability tests. The particle size of the ME was evaluated by laser light scattering technique at 25 ± 0.1 °C. NorA content was measured by HPLC-DAD method. Tyrosinase Inhibition Activity of NorA ME. The tyrosinase inhibitory activity of NorA ME was determined by spectrophotometric method (UV-5300PC spectrophotometer, Metash Instrument Co., Ltd., Shanghai, China) as described in our previous study.3 Briefly, the prepared ME were first diluted to 20, 10, 5, 2.5, 1.25, and 0.625 μg/mL with deionized water. Each of the sample solutions (300 μL) was diluted with 700 μL of 0.05 mM sodium phosphate buffer (pH 6.8) in the test tubes, followed by the addition of 1 mL of 0.1 mg/mL Ltyrosine and finally 1.0 mL of mushroom tyrosinase solution (200 units/mL). Thirty microliters of DMSO and a kojic acid solution was used as the blank reference and positive control, respectively. The reaction mixtures (3.0 mL) were mixed by vortex, and the initial absorbance at 492 nm was measured. After incubation for 20 min at 37 °C, the final absorbance at the same wavelength was taken. The IC50 values, which represent the concentrations of ME at which 50% of the tyrosinase activity was inhibited, were determined. The percent inhibition of tyrosinase activity was calculated as follows:
(PEG 400), dimethyl sulfoxide (DMSO), ethanol (EtOH), methanol (MeOH), sodium dihydrogen orthophosphate (NaH2PO4·2H2O), anhydrous disodium hydrogen phosphate (Na2HPO4), and dichloromethane (CH2Cl2) were all purchased from Sinopharm Chemical Reagent Co., Ltd. (China). (+)-Limonene was purchased from J&K Scientific (New Jersey, USA). Mushroom tyrosinase (3130 units/mg), L-tyrosine, kojic acid, ascorbic acid (VC), and 4-hexylresorcinol (4-HR) were purchased from Sigma Chemical Co (St. Louis, MO, USA). HPLC grade solvents were purchased from J&K Scientific. Norartocarpetin (NorA) was isolated from the wood of A. heterophyllus in our previous study.3 HPLC-DAD Analysis of NorA. The analytical HPLC system consisted of a Shimadzu LC-20AT series pumping system, an SIL-20A automatic injector, an SPD-M20A UV−visible detector, and Class-Vp chromatography data station software. All samples were analyzed by HPLC using a reverse-phase GraceSmart column (4.6 μm, 2.1 × 250 mm, Ryss Tech Ltd., China) at 25 °C with a Shimadzu HPLC system. Norartocarpetin was detected at 352 nm by a gradient elution with water containing 0.5% formic acid (solvent A) and methanol (solvent B) as a mobile phase. For the elution program, the gradient elution was as follows: initial, 20% B; 0−10 min, 20−60% B; 10−20 min, 60− 100% B; 20−25 min, 100% B; 25−30 min, 100−20% B. The flow rate was set at 1.0 mL/min. The sample injection volume was 10 μL. Three injections were performed for each sample. The calibration curve of NorA in the concentration range of 3.125−50.0 μg/mL was linear with a correlation coefficient of 0.9995. Solubility Studies. The solubility of NorA was determined in different oils, surfactants, and cosurfactants to find the appropriate constituents of microemulsions. An excess amount of NorA was added in 2 mL of ethyl butyrate, ethyl oleate, (+)-limonene, peppermint oil, Tween 80, Tween 40, Tween 20, ethanol, glycerin, PEG 400, 1,2propanediol, and n-octanol. The mixtures were shaken reciprocally at 25 °C for 48 h to reach equilibrium using a magnetic stirrer (Genius 3 vortex and RW 20D magnetic stirrer, IKA Laboratory Technology, Germany). Each sample was centrifuged at 10000g for 20 min (Jintanshi Jinchengguosheng Shiyanyiqichang, Jiangsu, China), followed by filtering through a 0.45 μm membrane filter. The concentration of NorA was determined by HPLC analysis at appropriate dilution with methanol. The solubility was determined in triplicate. Construction of Pseudoternary Phase Diagrams. Pseudoternary phase diagrams were constructed to obtain the concentration of all components of ME. Pseudoternary phase diagrams were constructed according to the water titration method.18 Different weight ratios (Smix) of surfactants and cosurfactants were mixed first, the Smix ratio varying from 1:1 to 2:1 to 3:1. For each Smix ratio, a pseudoternary phase diagram was elaborated by testing the weight ratios of oil/Smix of 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 95:5. Water was added drop by drop to each oily mixture under magnetic stirring (Genius 3 vortex and RW 20D magnetic stirrer, IKA Laboratory Technology) at room temperature. After being equilibrated, each sample was visually checked and determined as a clear microemulsion. The pseudoternary phase diagram was drawn by OriginPro for Windows version 9.0. Preparation and Solubility Determination of NorA-Loaded ME. The selected formulas based on phase diagram and NorA solubility were prepared by mixing ethyl oleate, Tween 80, and PEG 400 together, and then the required quantity of water was added and stirred to form a clear and transparent dispersion. All ME were stored to achieve equilibrium at room temperature for at least 24 h before further investigation. To determine the maximum loading capacity of NorA in ME, increasing amounts of NorA were loaded to the base ME.18,23 The mixture was stirred for 24 h at 25 ± 1 °C. The undissolved NorA was removed by centrifugation at 10000g (DL-5-B table model large capacity centrifuge, Shanghai Anting Scientific Instrument Factory, Shanghai, China) for 20 min, and then supernatant was taken and the encapsulation efficiency was quantified by HPLC-DAD at 352 nm after 100-fold dilution with methanol. The analyses were performed in triplicate.
% inhibition = [(A 2 − A1) − (B2 − B1)]/(A 2 − A1) × 100 A1 is the absorbance at 492 nm of the blank at 0 min, A2 is the absorbance at 492 nm of the blank at 20 min, B1 is the absorbance at 492 nm of test sample at 0 min, and B2 is the absorbance at 492 nm of test sample at 20 min. Antibrowning Effects of NorA ME on Fresh-Cut Apple Slices. Apples of comparable size were cleaned and cut into 4 mm thick slices and then treated by dipping in 500 mL of the corresponding test solutions for 5 min and drained. Samples were then placed in plastic Petri dishes, sealed with parafilm, and stored at room temperature for 24 h. Replicate samples were prepared for each treatment, and the experiment was repeated three times. Test solutions used for the above samples included aqueous solutions of blank ME, 0.5% VC, 0.01% NorA ME, 0.01% 4-HR + 0.5% VC, and 0.01% NorA ME + 0.5% VC. Visual assessment of color development in the samples was performed with a digital camera, whereas the relative extents of browning were measured with a tristimulus reflectance colorimeter. The center of the apple slices was in touch with the lens of the Minolta CR-400 Chroma meter (Konica Minolta Sensing, Inc., Japan) when the readings (a* values) were taken. Measurements were made immediately following each treatment and at timed intervals of 3, 6, 12, and 24 h thereafter. 1616
DOI: 10.1021/jf5048805 J. Agric. Food Chem. 2015, 63, 1615−1621
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Journal of Agricultural and Food Chemistry Statistical Analysis. All of the measurements were carried out in triplicate. An analysis of variance (ANOVA) of the data was performed using the SPSS 13.0 statistical analysis system. The treatments were considered to be significantly different at p < 0.05.
relatively large droplets of the system, whereas higher concentrations of Tween 80 (>15% w/w) led to homogeneous transparent liquids and very small particles.24 Although the content of Tween 80 is not clearly defined, it is necessary to reduce the content of surfactant to obtain ideal ME without potential side effects.20,25 The content of Tween 80 was controlled in the range of 10−20% in this study to form the transparent ME. Among all of the cosurfactants, PEG 400 has the best solubility values of NorA. PEG 400 is a low-molecularweight grade of polyethylene glycol. It is a clear, colorless, and odorless liquid, it is strongly hydrophilic, and it can be soluble in water, acetone, alcohols, benzene, glycerin, glycols, and aromatic hydrocarbons.26 Due to its low toxicity, it is widely used in foods, pharmaceuticals, and personal and home care products as a solvent or solubilizing agent.27 On the basis of these advantages and great solubility for NorA, PEG 400 was chose as cosurfactant for NorA ME. On the basis of solubility results, ethyl oleate, Tween 80, and PEG 400, which showed maximal solubilizing capacity for NorA, were selected for further NorA ME formulations. Pseudoternary Phase Diagram Study and Determination of ME Formulation. To figure out the optimal proportion of the components in ME, pseudoternary phase diagrams were constructed using a water titration method. The pseudoternary phase diagrams of ME systems contained ethyl oleate, Tween 80, PEG 400 at fixed ratios (w/w, Smix) of 1:1, 2:1, and 3:1), and water. In the pseudoternary phase diagrams, the left area along the curve was referred to as the turbid region, whereas the right region was the ME region (Figure 1). All ME were isotropic, transparent dispersions, and no phase separation could be observed with all the ratios of oil/ surfactant−cosurfactant/water after centrifugation at 10000g for 20 min at 25 °C. The previous study showed that the cosurfactant was arranged exactly among surfactant molecules and resulted in the formation of ME with maximum solubilizing capacity at the optimum ratio of surfactant and cosurfactant.28 Therefore, the ratio of surfactant and cosurfactant (Smix) was very important in the formation of stable ME. In this study, when the same oil phase (ethyl oleate) was fixed, the area of ME regions existing in various ratios of surfactant and cosurfactant in pseudoternary phase diagrams were in the order 2:1 > 3:1 > 1:1 (Figure 1). On the basis of the above optimization, the formulation of NorA ME was composed of ethyl oleate (2% w/w), Tween 80 (12% w/w), PEG 400 (6% w/w), with a ratio of 2:1 of
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RESULTS AND DISCUSSION Solubility Study in Various Vehicles. The solubility of NorA in different vehicles was determined and is listed in Table 1. On the basis of the results, NorA showed the highest Table 1. Solubility of Norartocarpetin in Various Vehicles (25 °C)a vehicle water oils ethyl butyrate ethyl oleate (+)-limonene peppermint oil surfactants Tween 20 Tween 40 Tween 80 cosurfactants ethanol glycerol PEG 400 1,2-propanediol n-octanol a
solubility ± SD (mg/mL) NDb 0.22 0.36 0.16 0.28
± ± ± ±
0.03 0.07 0.04 0.05
2.59 ± 0.03 0.89 ± 0.01 3.63 ± 0.02 24.46 (14.50 72.72 6.93 6.09
± ± ± ± ±
0.18 0.03) × 10−3 0.09 0.03 0.02
Results are presented as the mean ± SD (n = 3). bNot detected.
solubility in ethyl oleate among four oils. It is considerably higher than its solubility in water, although the solubility in oils is limited. Among the three surfactants, Tween 80 showed the best solubility for NorA relative to Tween 20 and Tween 40, up to 3.63 mg/mL. The solubility values of NorA in surfactants are much higher compared to oils. Tween 80 is one of the polysorbates (ethoxylated sorbitan esters) belonging to a nonionic surfactant; it is widely used as an emulsifier and dispersing agent for pharmaceutical products and as an emulsifier in food products. Meanwhile, it is reported that the concentration of Tween 80 in the systems greatly affected the appearance and stability of the systems.24 Low concentrations of Tween 80 (1−4% w/w) resulted in a milky white layer and
Figure 1. Construction of pseudoternary phase diagram of ethyl oleate/Tween 80/PEG 400/water system at a ratio of surfactant and cosurfactant (w/w) of 1:1, 2:1, or 3:1. 1617
DOI: 10.1021/jf5048805 J. Agric. Food Chem. 2015, 63, 1615−1621
Article
Journal of Agricultural and Food Chemistry
area to w/o, o/w, and the bicontinuous structure between them.29 The conductivity values of blank ME and NorA ME were 126.13 ± 0.21 and 129.13 ± 0.06 μS/cm, respectively. The results of high conductivity suggested that the ME were in o/w systems.18,29 TEM results confirmed the presence of droplets with size
