Encapsulation of Polymethoxyflavones in Citrus Oil Emulsion-Based

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Encapsulation of polymethoxyflavones in citrus oil emulsion-based delivery systems Ying Yang, Chengying Zhao, Jingjing Chen, Guifang Tian, David Julian McClements, Hang Xiao, and Jinkai Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00147 • Publication Date (Web): 09 Feb 2017 Downloaded from http://pubs.acs.org on February 13, 2017

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Journal of Agricultural and Food Chemistry

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Encapsulation

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emulsion-based delivery systems

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Ying Yang1, #, Chengying Zhao1, #, Jingjing Chen2,3, Guifang Tian1, David Julian

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McClements3, Hang Xiao3,*, Jinkai Zheng1,*.

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1

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polymethoxyflavones

in

citrus

oil

Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China

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of

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China

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Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States

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Correspondence to: Jinkai Zheng; Hang Xiao.

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E-mail: [email protected] (J. Zheng); [email protected] (H. Xiao).

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#

Both authors have contributed equally to this work.

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ACS Paragon Plus Environment


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Abstract

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The purpose of this work was to elucidate the effects of citrus oil type on

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polymethoxyflavone (PMF) solubility and on the physicochemical properties of

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PMF-loaded emulsion-based delivery systems. Citrus oils were extracted from

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mandarin, orange, sweet orange, and bergamot. The major constituents were

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determined by GC-MS: sweet orange oil (97.4% d-limonene); mandarin oil (72.4%

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d-limonene); orange oil (67.2% d-limonene); and bergamot oil (34.6% linalyl acetate

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and 25.3% d-limonene). PMF-loaded emulsions were fabricated using 10% oil phase

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(containing 0.1% w/v nobiletin or tangeretin) and 90% aqueous phase (containing 1%

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w/v Tween 80) using high-pressure homogenization. Delivery systems prepared using

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mandarin oil had the largest mean droplet diameters (386 or 400 nm), followed by

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orange oil (338 or 390 nm), bergamot oil (129 or 133 nm) and sweet orange oil (122

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or 126 nm) for nobiletin or tangeretin-loaded emulsions, respectively. The optical

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clarity of the emulsions increased with decreasing droplet size due to reduced light

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scattering. The viscosities of the emulsions (with or without PMFs) were similar (1.3

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to 1.4 mPa·s), despite appreciable differences in oil phase viscosity. The loading

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capacity and encapsulation efficiency of the emulsions depended on carrier oil type,

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with bergamot oil giving the highest loading capacity. In summary, differences in the

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composition and physical characteristics of citrus oils led to PMF-loaded emulsions

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with different encapsulation and physicochemical characteristics. These results will

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facilitate the rational design of emulsion-based delivery systems for encapsulation of

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PMFs and other nutraceuticals in functional foods and beverages.

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Keywords: polymethoxyflavones; emulsions; nanoemulsions; citrus oil; tangeretin;

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nobiletin; encapsulation.

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Introduction

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Citrus fruits, such as mandarin, orange, lime, grapefruit and lemon, belong to the

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Rutaceae family and are widely cultivated around the world.1 The global production

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of citrus fruits has increased dramatically in the past few years and has now reached

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around 100 million tons per year.2 Citrus fruits are rich sources of many different

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bioactive components, which may be present in the flesh or the peel.3 As the major

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byproduct of citrus fruit processing, citrus peels are an excellent source of raw

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materials for food and dietary supplement industries.3-4 Polymethoxyflavones (PMFs),

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a class of flavonoids with more than two methoxyl groups on their chemical skeletons,

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are abundant in citrus fruits and especially in their peels.5 Consequently, there has

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been growing interest in utilizing PMFs as nutraceuticals in recent years because of

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their multiple biological activities, such as anticancer,6-7 anti-inflammatory,8-9

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antioxidative,10 and anti-lipogenic11 activities. Nobiletin and tangeretin are two major

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citrus PMFs,12 and have been the most widely studied.13-14 Despite several

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health-promoting benefits of PMFs, their utilization as nutraceuticals in food products

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is often limited by their poor solubility and high melting points.15

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Emulsion-based delivery systems, such as emulsions and nanoemulsions, are highly

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effective tools for encapsulating, protecting, and delivering lipophilic bioactive

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ingredients for food, beverage, dietary supplement, and pharmaceutical applications.16

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These systems may inhibit bioactive degradation during storage, and increase

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bioactive bioavailability after ingestion, thereby enhancing their overall bioactivity.17

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Emulsions with different compositions and structures, and therefore different

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physicochemical and functional attributes, can be fabricated by using different kinds

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of carrier oils and emulsifiers. In the current study, we focus on the impact of oil

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phase type on emulsion formation, stability, and performance. The polarity, density,

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molecular weight, viscosity and interfacial tension of the oil phase are known to have

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a significant influence on emulsion properties.18

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Triacylglycerol oils, such as corn, sunflower, safflower, rapeseed, palm, and fish

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oils, are commonly used to formulate oil-in-water emulsions.18-19 A potential 3



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drawback of using triacylglycerol oils for some applications is that their low polarity,

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high viscosity, and high interfacial tension, which lead to the formation of relatively

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large droplets during homogenization. Consequently, there is interest in utilizing

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different kinds of oil phases to overcome this limitation. Citrus oils mainly exist in the

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oil cells of citrus peels, and their content in the fresh peels is around 0.5 to 2.0%,

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depending on citrus genus and cultivation conditions.3 Citrus oils are widely used as

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flavoring agents in beverages, confectionaries, pastries and cosmetics because of their

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desirable flavor profiles.20 However, they also exhibit a number of other functional

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attributes that can be utilized in commercial products, including antioxidant,

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anti-inflammatory, antibacterial, and larvicidal effects.21-23 In our previous studies, the

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utilization of orange oil as a carrier oil to encapsulate bioactive compounds showed

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higher loading capacity, lower droplet size, and higher physical stability than other

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types of carrier oil.24-25 PMFs naturally exist in the oil gland of citrus peels, which

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suggests that citrus oils are a good solvent for PMFs. Therefore, we hypothesized that

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citrus oils would be particularly suitable for formulating emulsion-based delivery

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systems for PMFs.

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Citrus oils isolated from different citrus species have different compositions and

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physicochemical properties, which would be expected to impact their ability to

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solubilize PMFs and to form stable emulsions. In this study, four types of citrus oils

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were extracted from the peels of four different citrus species (mandarin, orange, sweet

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orange, and bergamot), and their physical properties and compositions were analyzed.

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Nobiletin and tangeretin were used as model PMFs to determine the influence of

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citrus oil types on the formation, stability, encapsulation efficiency, and

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physicochemical properties of emulsion-based delivery systems. The knowledge

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gained from this study will prove useful for the rational design of effective

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emulsion-based delivery systems for citrus-derived lipophilic nutraceuticals such as

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PMFs.

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Materials and methods

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Materials. Nobiletin (Nob) and tangeretin (Tan) powders were purchased from

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Huike Co., Ltd. (Shanxi, China). The purity of the PMFs was over 98% as determined

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by HPLC analysis. Fifty kilograms of each kind of citrus fruit (mandarin, orange,

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sweet orange and bergamot) were purchased from a local market (Xiaoying Market,

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Beijing, China). A food-grade non-ionic surfactant (Tween 80) was provided by the

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Guoxin Co. Ltd. (Shenzhen, China). Phosphate buffer solution (PBS, pH=7.00,

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0.0067 M) was purchased from HyClone Lab (South Logan, Utah, USA).

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HPLC-grade dimethylsulphoxide (DMSO), acetonitrile and chloroform were

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purchased from Fisher Scientific (Beijing, China). Calcium hydroxide was obtained

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from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).

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Extraction of citrus oils. The citrus oils used in this study were extracted using the

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cold press method as described previously with some modifications.26 The citrus fruits

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were cut and peeled. Citrus peels were soaked in a calcium hydroxide solution (1.6%

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to 2.0%, pH 12.0) for 10 hours at room temperature. Before pressing with a juice

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extractor (SKG 2075, Guangdong, China), the peels were scoured with saturated

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NaHCO3 to adjust the pH to 7.0~9.0. The juice was collected and then centrifuged at

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4000 g for 15 minutes at 4 oC (Sorvall ST 16R, Thermo Scientific, USA). About 50 g

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oil was obtained from each type of citrus fruit and stored at 4 oC prior to analysis.

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GC/MS analysis. The compositions of citrus oils were determined by gas

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chromatography coupled with a mass spectrometer (GCMS-QP2010 plus, Shimadzu,

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Japan). A Shimadzu GC was equipped with DB-WAX fused silica capillary column

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(100 m × 0.25 mm i.d., film thickness, 0.25 µm), and a flame ionization detector (FID)

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was used. The column oven temperature was programmed as follows: 40 °C for 4

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minutes, raised to 220 °C at the rate of 3 °C/minute and held for 5 minutes, then

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raised to 240 °C at the rate of 20 °C/minute. The injector and detector temperature

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was maintained at 245 °C. Helium was used as a carrier gas at a flow rate of 1.00

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ml/min. The citrus oil samples were diluted 30-fold with chloroform before analysis.

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The split ratio was 1:30. The MS was set in electron impact mode at an ionization 5



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energy of 70 eV and scanned from m/z 40 to 600 at 2 scan/s. The ionization source

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and interface temperatures were 245 and 250 °C, respectively. Compounds of interest

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were identified by comparing their retention time (RT) and mass spectra with those in

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the NIST database.

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PMF solubility in citrus oils. The solubility of nobiletin or tangeretin in different

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citrus oils was determined using a HPLC method described previously with some

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slight modifications.25 An excessive amount of powdered nobiletin or tangeretin was

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added to a weighed amount of citrus oil, then the sample was heated to 30, 40, 50, and

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60 oC under sonication at 45 Hz for 2 h, respectively. The samples were placed at

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different temperatures for 24 h to ensure equilibrium. After being centrifuged at

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10,000 g for 10 minutes at different temperatures using a centrifuge (Sorvall ST 16R,

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Thermo Scientific, USA), the supernatant was diluted with acetonitrile (1:200~1:2000,

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v/v) and analyzed by HPLC to determine the PMF content. Blank oil was used as a

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control. Samples were analyzed by HPLC with a UV-detector set at 330 nm. The

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mobile phase was comprised of 60% acetonitrile in 0.04% (v/v) formic acid. Standard

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curves were prepared by analyzing known samples containing from 2 to 32 ppm

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nobiletin or tangeretin (R2 for nobiletin and tangeretin were 0.9999 and 0.9998,

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respectively).

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Preparation of PMF-loaded citrus oil-in-water emulsions. The oil phase was

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prepared by dispersing 0.1 % (w/v) nobiletin or tangeretin powder into citrus oils,

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followed by heating to 30 oC with stirring for 2 h to ensure complete dissolution. The

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aqueous phase was prepared by adding 1 % (w/v) Tween 80 into phosphate buffer

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(pH=7.00, 0.0067 M). Coarse oil-in-water emulsions were fabricated by mixing 10%

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oil phase and 90% aqueous phase using a high-shear mixer (ULTRA-TURRAX T25

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digital, IKA, Germany) at 9,000 rpm for 3 minutes. The final emulsions were

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obtained by passing the coarse emulsions through a high-pressure homogenizer

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(APV-2000, SPX, Germany) at 600 bar for 5 times.

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Determination of particle size and ξ-potential. The mean droplet diameter,

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particle size distribution, and ξ-potential of the emulsions were determined using a 6


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dynamic light scattering / micro-electrophoresis instrument (Zetasizer Nano ZS,

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Malvern Instruments, Malvern, UK). To prevent the effect of the multiple scattering,

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each sample was diluted 1000 times using an appropriate buffer solution at room

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temperature prior to measurement. The angle of scattered light was 173o and the

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detection temperature was maintained at 25 oC. All measurements were performed in

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triplicate and results were reported as the cumulate mean diameter (size, nm) for

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particle size, polydispersity index (PDI) for size distribution, and surface potential

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(mV) for ξ-potential.

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Physical properties of citrus oils and emulsion systems. The density and

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refractive index of citrus oils were measured using a density meter (DMA 5000,

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Anton Paar, Austria) and refractometer (RX-5000, ATAGO, Japan), respectively. The

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turbidity of all emulsions was measured using a turbidimeter (2100N, HACH. Co.

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Ltd., Colorado, USA), after they had been diluted 1000 times using buffer solution.

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All measurements were performed in triplicate, and the turbidity results are reported

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in Nephelometric Turbidity Units (NTU). The viscosity of the citrus oils and

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emulsions was measured using a dynamic shear rheometer (Physia MCR 301, Anton

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Paar, Austria). About 1 mL of oil or emulsion was placed on a 50 mm-diameter

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parallel plate geometry measurement cell. Steady-state flow measurements were

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performed at 25 oC in the range from 0.01 to 300 s-1. The rheological parameters

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(shear stress, shear rate, and apparent viscosity) were recorded.

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PMF encapsulation efficiency of citrus oil emulsions. The encapsulation

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efficiency of the emulsions was determined by adding 10, 20, or 30 mg of nobiletin or

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tangeretin into different kinds of citrus oils (10 mL) and then preparing oil-in-water

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emulsions as described above. The amount of PMFs encapsulated within the oil

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droplets was determined by HPLC as described above. The encapsulation efficiency

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of PMFs was calculated using the following formula:

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where mcolloidal was the amount of PMFs in the colloidal suspension and mtotal was the 7



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total PMFs in the system. The emulsion was centrifuged (223 g, 20 minutes) to

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remove the excess PMFs before analysis by HPLC.

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Statistical analysis. All measurements were performed on freshly prepared

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samples in triplicate at least. The results are reported as means and standard

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deviations. Statistical analysis was performed by ANOVA using SPSS Statistics

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Software (IBM). A P-value of < 0.05 was considered statistically significant.

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Results and discussion

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Characterization of citrus oils extracted from different citrus species. The

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citrus oils used in this study were extracted from fresh fruits peels using a modified

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cold press method, which gave yields of 0.98%, 1.28%, 1.09% and 1.16% for

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mandarin, orange, sweet orange and bergamot, respectively. The composition of the

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citrus oils would be expected to impact the formation, stability, and performance of

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emulsion-based delivery systems, and therefore oil phase composition was determined

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by GC-MS. Constituents with an abundance above 1.00% are shown in Figure1 and

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Table 1. The compositions of mandarin and orange oil were relatively similar, while

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those of the other two citrus oils were quite different (Figure 1). The major

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components of these citrus oils could be categorized into hydrocarbons, esters,

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alcohols and aldehydes. The minor compounds in the four oils were mainly α-pinene,

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camphene, (-)-β-pinene, and γ-terpinene. The composition of sweet orange oil was the

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simplest since it consisted almost entirely of d-limonene (97.4%). The most common

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constituent of mandarin and orange oil was also d-limonene with an abundance of

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72.4% and 67.2%, respectively. The main constituents in bergamot oil were

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appreciably different from those in the other three oils. The most abundant compound

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in bergamot oil was linalyl acetate (34.6%), followed by d-limonene (25.3%) and

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β-linalool (15.4%). The nature of the main constituents of the oils might have an

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important influence on the properties of oils and their emulsions. For example, the

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density, refractive index, and viscosity of the major constituents could influence the

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physical properties of oils and emulsions, while the polarity of the major constituents 8


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could influence the solubility of PMFs in the oil phase and their encapsulation

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efficiency in the emulsions. The physical properties of the three main components

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were obtained by searching the SciFinder Database (scifinder.cas.org). The refractive

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index of linalyl acetate, β-linalool and d-limonene were about 1.456, 1.460 and 1.472,

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respectively. The densities of linalyl acetate (0.901 g/cm3) and β-linalool (0.858-0.868

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g/cm3) were higher than that of d-limonene (0.844 g/cm3). The log P values

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(logarithm of the oil-water partition coefficient) of linalyl acetate, β-linalool and

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d-limonene, which reflect the polarity of the compounds, were about 3.907, 2.795 and

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4.552, respectively, indicating that they were all predominantly non-polar compounds.

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The physical properties, including color, refractive index, density and viscosity, of

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the four oils were determined (see in Table 1). As expected, all of the physical

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properties of mandarin oil and orange oil were similar to each other due to the fact

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that the compositions of these two oils were very similar. The refractive index and

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density of sweet orange oil was almost the same as that of mandarin oil and orange oil,

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and the refractive index and density of these oils were close to that of d-limonene, the

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major component of these three types of oil. As for bergamot oil, its refractive index

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and density were significantly different from that of the other three oils, and more

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close to those of linalyl acetate and β-linalool, its major constituents. The viscosity of

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orange oil (0.968 mPa·s) and mandarin oil (0.947 mPa·s) were similar with each other

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but significantly different from the other oils, i.e., sweet orange oil (3.838 mPa·s) and

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bergamot oil (1.832 mPa·s). Overall, these results indicated that the physical

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properties of oils were closely associated to their composition.

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Solubility of PMFs in different citrus oils. One of the most important factors

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dictating the encapsulation efficacy of an emulsion-based delivery system is the

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solubility of the bioactive compounds in the oil phase. We therefore determined the

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solubility of nobiletin and tangeretin in the four different citrus oils at different

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temperatures. Figure 2 shows the maximum amount of the two PMFs soluble in

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different oils after being sonicated for 2 h and stored at 30, 40, 50, or 60 oC for 24 h.

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Each sample was centrifuged to remove undissolved PMFs prior to measurement. As 9



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expected, the solubility of the two PMFs in the different oils increased as the storage

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temperature increased.28 Thus, the solubility can be increased by raising the

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temperature. Although there was little difference in solubility at 30 and 40 oC for both

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nobiletin and tangeretin, the PMF solubility increased dramatically upon heating to 50

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o

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the solubility on temperature, which shows that the solubility should increase steeply

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once a critical temperature is exceeded.24,28 This effect can be attributed to the

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increase in entropy of mixing effects as temperature is increased. As shown in Figure

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2A, the solubility of nobiletin in the four citrus oils was higher than that of tangeretin

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at 50 and 60 oC. Interestingly, the PMF solubility in the four citrus oils decreased in

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the following order: bergamot oil > sweet orange oil > mandarin oil > orange oil. With

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the exception of bergamot oil, this trend was in agreement with the abundance of

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d-limonene in the citrus oils. As mentioned earlier, the major constituent was

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d-limonene in mandarin oil, orange oil and sweet orange oil, while linalyl acetate,

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d-limonene and β-linalool were the major constituents of bergamot oil. As reported in

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the SciFinder Database, the log P values of nobiletin and tangeretin were 1.598 and

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1.788, respectively. According to the principle of dissolution of similar materials, the

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two PMFs tended to dissolve in solvents with similar log P values. This might be the

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reason that the solubility of PMFs in bergamot oil was higher than that in the other

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three citrus oils. In this study, we also determined the effect of heat treatment on the

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solubility of the PMFs in the citrus oils. Citrus oils containing an excess of PMF were

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heated to 60 oC and then stored at 30 oC for 24 h. The other batches of samples were

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stored at 30 oC for 24 h without being heated to 60 oC first. As shown in Figure 2B,

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the heat treatment to 60 oC did not significantly change the solubility of nobiletin.

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However, there was a significant increase in tangeretin solubility after heat treatment.

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This result indicates that heat treatment is a more effective method to facilitate

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solubilization of tangeretin than nobiletin.

C. This phenomenon is consistent with theoretical predictions of the dependence of

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Effect of citrus oil type on the appearance of PMF-loaded emulsions.

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PMF-loaded emulsions prepared using different types of citrus oil were fabricated 10


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using high-pressure homogenization. Control emulsions were also prepared under

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similar conditions but without PMFs. The appearance of the citrus oils and their

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emulsions are shown in Figure 3A. The emulsions made from mandarin, orange, and

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bergamot oils were milky white due to the low color of these oils, whereas the

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emulsions made from sweet orange oil appeared yellow due to the intense yellow

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color of this oil. For certain commercial applications, the turbidity of an emulsion is

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an important parameter determining its quality attributes, e.g., beverage emulsions.29

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The emulsions prepared from mandarin oil had the highest turbidity, followed by

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those prepared from orange oil, bergamot oil and sweet orange oil. This effect can be

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attributed to differences in the size of the droplets in the different emulsions. Droplet

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concentration, particle size distribution, and refractive index are the three main factors

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affecting emulsion turbidity.29 At a fixed droplet concentration, there is a maximum in

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the turbidity versus droplet size profile, which occurs when the droplet diameter is

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fairly similar to the wavelength of light. Below this maximum value, the turbidity

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tends to decrease with decreasing droplet size, and transparent emulsions can be

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formed for systems containing very fine droplets.

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Effect of citrus oil type on the droplet size and distribution of PMF-loaded

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emulsions. The mean droplet size and polydispersity index (PDI) of each emulsion

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were determined. As shown in Figure 3B, all of the emulsions had relatively small

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mean droplet diameters in the range of 100 to 400 nm regardless (with or without

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PMFs). The largest mean droplet diameter was obtained for the emulsions prepared

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from mandarin oil (386-400 nm), followed by that of orange oil (338-390 nm),

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bergamot oil (129-133 nm) and sweet orange oil (122-126 nm). The emulsions

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fabricated from mandarin oil and orange oil can therefore be considered to be

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traditional emulsions (d > 200 nm), while those fabricated sweet orange oil and

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bergamot oil can be considered to be nanoemulsions (d < 200 nm).30 Typically,

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smaller droplet sizes lead to emulsions with improved stability to gravitational

293

separation and droplet aggregation,29-30 which is an advantage for many commercial

294

applications. The PDIs of the emulsions prepared using mandarin oil and orange oil 11



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(0.3 to 0.5) were considerably larger than the PDIs of the emulsions prepared using

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sweet orange oil and bergamot oil (0.1 to 0.2), indicating that the latter systems had

297

much narrower particle size distributions.

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The size of the droplets produced during high-pressure homogenization depends on

299

oil and aqueous phase properties, such as viscosity and interfacial tension.30 Typically,

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the more similar the viscosities of the two phases, and the lower the interfacial tension,

301

the easier it is to produce fine oil droplets during homogenization.27 Interestingly, the

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mandarin and orange oils had the lowest viscosities, which were most similar to that

303

of water, and would therefore have been expected to produce the smallest droplets.

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However, they actually produced the largest droplets after homogenization, which

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suggests that other factors must also have been important, such as differences in

306

interfacial tension or differences in droplet growth by Ostwald ripening.29 Typically,

307

the interfacial tension at an oil-water interface decreases as the concentration of polar

308

constituents in the oil phase increases. There were more polar constituents, such as

309

β-linalool and α-carene, in the bergamot oil than in the other oils, which may

310

therefore have led to a lower interfacial tension and greater droplet disruption after

311

homogenization.

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Emulsion-based products are susceptible to gravitational separation, which is

313

driven by differences in the density between the oil and water phases.18 Consequently,

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minimizing the density difference between these two phases can improve emulsion

315

stability. The density of bergamot oil was 0.877 g/cm3, which was slightly closer to

316

that of the aqueous phase (1.00 g/cm3) than the other oils (around 0.85 g/cm3), and

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therefore emulsions prepared using this oil would be expected to be slightly more

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stable to creaming at a fixed droplet size.30

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The incorporation of PMF into the emulsions did not affect the size of the droplets

320

produced by homogenization, with PMF-free and PMF-loaded emulsions having

321

similar particle size distributions. This may have been because nobiletin and

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tangeretin are relatively small molecules that were used at a relatively low

323

concentration, and so they had little impact on oil phase density, viscosity, or 12


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interfacial tension.25 The electrical charge on the droplets in oil-in-water emulsions

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may impact their physical and chemical stability.18 In principle, oil droplets coated by

326

Tween 80 should not have an electric charge because this is a non-ionic surfactant.

327

However, the droplets in all the citrus oil emulsions had a negative charge

328

(approximately -4 mV, Figure 3C), which produced electrostatic repulsion to maintain

329

stability of the emulsions. This charge may be caused by anionic impurities in the

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surfactant and/or oil phase, or due to preferential adsorption of hydroxyl ions to the

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droplet surfaces.18,25 There was no significant difference in ξ-potential among the

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emulsions made from the different oils, indicating that oil type did not influence the

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surface charge of the emulsions. As expected, the PMF-loaded emulsions had similar

334

charges as the PMF-free ones, which indicated that the non-polar neutral PMFs did

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not affect the surface charge of the oil droplets.

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Effect of citrus oil type on the viscosity of PMF-loaded emulsions. The viscosity

337

of emulsions plays an important role in determining the physicochemical properties

338

and sensory attributes of many commercial products,18 and was therefore determined

339

for the emulsion-based delivery systems prepared in this study. Figure 4 shows the

340

rheological properties of citrus oils and emulsions prepared from them with or without

341

PMFs. There were appreciable differences in the viscosities of the different citrus oils

342

(between 0.95 and 3.8 mPa·s). Sweet orange oil had the highest viscosity, followed by

343

bergamot oil, orange oil, and mandarin oil (Table 1). On the other hand, the viscosities

344

of all the emulsions were very similar to each other (between 1.3 and 1.4 mPa·s)

345

despite the differences in the viscosities of the citrus oils. This is to be expected since

346

the viscosity of an oil-in-water emulsion is mainly determined by the disperse phase

347

volume fraction (droplet concentration), rather than the viscosity of the dispersed

348

phase.18 The encapsulation of PMFs had no significant influence on the viscosity of

349

the emulsions, which can be attributed to the same reason.

350 351 352

The rheological properties of a fluid can be conveniently described using the following power-law relationship (Herschel-Bulkley model): τ = K γn 13



353

where, τ is the shear stress (Pa), γ is the shear rate (s-1), K is the consistency index

354

(Pa·s), and n is the flow behavior index. For an ideal (Newtonian) fluid, the shear

355

stress is proportional to the shear rate, and so the viscosity is independent of shear rate

356

(n = 1). Figure 4A shows that this was the case for all the emulsions prepared in this

357

study. The fluctuations observed at low shear rates can be attributed to the mechanical

358

instability of the instrument under these conditions. The Herschel-Bulkley model was

359

fit to the experimental flow curves (Figure 4B) to determine if the emulsions were

360

ideal (n = 1.0), shear thickening (n > 1), or shear thinning (n < 1). The flow behavior

361

index was n = 1.0 for all the emulsions, which indicated that they all behaved as

362

Newtonian fluids.31-32 The encapsulation of PMFs in the emulsions had no significant

363

effect on the flow behavior of the emulsions.

364

Encapsulation efficiency of PMFs. The encapsulation efficiency of a bioactive

365

compound by an emulsion is an important property of any delivery system. To

366

determine the encapsulation efficiency of PMFs in the different citrus oil emulsions,

367

different doses of PMFs were selected. One dose was selected to be in the range

368

where all of the bioactives would be expected to dissolve in the oil phase: 10 mg of

369

PMFs in 10 mL citrus oil. Two other doses were selected to be in the range where the

370

PMFs should only partly be dissolved in the oil phase: 20 and 30 mg of PMFs in 10

371

mL of oil.

372

When adding 10 mg of PMFs into oil phase, the encapsulation efficiency was

373

between 79% and 95%. Theoretically, all the PMFs should have been completely

374

dissolved in the oil phase and encapsulated within the emulsion systems when their

375

initial concentration was below the saturation concentration. Some of the PMFs may

376

have been lost during emulsion preparation due to chemical degradation, adherence to

377

homogenizer surfaces, temperature changes, or alterations in equilibrium solubility

378

caused by the decrease in particle size.16 Although the difference was relatively small,

379

the encapsulation efficiency of the two PMFs in the emulsions was in the following

380

order: mandarin oil > orange oil > bergamot oil > sweet orange oil. In terms of the

381

encapsulation efficiency, conventional emulsions of mandarin and orange oils had 14


Page 14 of 28

Page 15 of 28


382

slightly higher encapsulation efficiency than the nanoemulsions of bergamot and

383

sweet orange oils. However, conventional emulsions might be less stable during

384

long-term storage than nanoemulsions due to their larger droplet sizes.

385

When 20 mg of PMFs was added to 10 mL of oil phase, the mandarin oil and

386

bergamot oil emulsions should not have exceeded the saturation concentration, while

387

the orange oil and sweet orange oil emulsion should have exceeded it. The loading

388

capacity of the PMFs increased significantly and the encapsulation efficiency of the

389

PMFs increased slightly for the mandarin oil and bergamot oil emulsions compared to

390

the systems where only 10 mg of PMFs were added. In contrast, the loading capacity

391

increased significantly for the sweet orange oil emulsion, but did not change

392

significantly for the orange oil emulsion, while the encapsulation efficiency decreased

393

for both orange and sweet orange oil emulsions. This is mainly due to the fact PMFs

394

reached their maximum solubility when 20 mg of PMFs were added to the orange and

395

sweet orange oil systems.

396

When 30 mg of PMFs were added to 10 mL of oil phase, which exceeded the

397

maximum solubility of all oils, the results showed that the loading capacity of all

398

emulsions did not increase much further in comparison with the situation where 20

399

mg of PMFs were used. As expected the encapsulation efficiency decreased for all

400

emulsions. These results suggested that the amount of PMFs that could be loaded into

401

the emulsions should not be higher than their maximum solubility in citrus oil to

402

ensure effective encapsulation in the final emulsion in food/nutraceutical products. Of

403

the four citrus oils studied, bergamot solubilized the highest amount of PMFs and may

404

therefore be the most suitable for developing emulsion-based delivery systems with

405

high loading capacities.

406

In conclusion, PMF-loaded emulsions were successfully fabricated from four

407

different citrus oils (mandarin, orange, sweet orange, and bergamot oil), and their

408

physicochemical properties and encapsulation efficiency were systematically studied

409

in this study. The results showed that the composition of citrus oils had a strong

410

influence on the solubility of the PMFs, as well as emulsion properties. Emulsions 15



411

fabricated using bergamot oil contained relatively small droplets (d, 129~133 nm) that

412

had high PMF loading capacity. To our knowledge, this is the first published work

413

that utilizes different citrus oils to manufacture and characterize PMF-loaded

414

emulsion-based delivery systems. These emulsions could be utilized for encapsulation

415

of PMFs and other nutraceuticals for application in functional foods and beverages.

416

Future studies on the storage stability, gastrointestinal digestion, and biological

417

activity of these emulsion systems are needed to determine the usefulness in practical

418

applications.

419

Conflicts of interest

420 421

The authors declare no competing financial interests.

Acknowledgements

422

The authors would like to acknowledge the financial support of the National

423

Natural Science Foundation of China (No. 31428017 and 31401581) and Program of

424

Introducing International Advanced Agricultural Science and Technology of the

425

Ministry of Agriculture of P. R. China (948 Program, No.2016-X31).

16


Page 16 of 28

Page 17 of 28


426

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Xiao, H. Synergistic anti-inflammatory effects of nobiletin and sulforaphane in

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lipopolysaccharide-stimulated raw 264.7 cells. J. Agr. Food Chem. 2012, 60,

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in vivo, bioavailability and biological efficacy of nutraceuticals. J. Funct.

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Foods. 2014, 7, 112–128.

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(16) McClements, D. J. Advances in fabricate of emulsions with enhanced

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type on bioavailability of pterostilbene. J. Funct.Foods. 2015, 13, 61–70.

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(20) M’Hiri, N.; Ioannou, I.; Ghoul, M.; Boudhrioua, N. M. Phytochemical

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characteristics of citrus peel and effect of conventional and nonconventional

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antimicrobial and antioxidant activity of essential oil of citrus limetta var. mitha

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(sweet lime) peel in pakistan. Afr. J. Microbiol. Res. 2013, 7, 3071–3077.

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Sci. 2011, 51, 285–330.

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(31) Hou, Z.; Zhang, M.; Liu, B.; Yan, Q.; Yuan, F.; Xu, D.; Gao, Y. Effect of chitosan

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(32) Taherian, A. R.; Fustier, P.; Ramaswamy, H. S. Effect of added oil and modified

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527

20


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528

Figure Captions

529

Figure. 1 The GC/MS profiles of four citrus oils extracted from mandarin, orange,

530

sweet orange, and bergamot.

531

Figure. 2 (A) The solubility of nobiletin and tangeretin in mandarin oil, orange oil,

532

sweet orange oil, and bergamot oil at the temperatures of 30, 40, 50, and 60 oC.

533

(B) Influence of heating procedure on the solubility of nobiletin and tangeretin.

534

Nobiletin and tangeretin content were determined after the samples were heated

535

to 30°C, 60°C, as well as after cooled down to 30°C from 60°C.

536 537

Figure. 3 Appearance and turbidity (A), particle size (B), and Zeta-potential (C) of emulsions of citrus oils.

538

Figure. 4 (A) Influence of citrus oil type on the viscosity of emulsion systems; (B)

539

Flow curves of citrus oil emulsion systems loaded with or without PMFs.

540

Table 1 The physical property and composition analysis of four citrus oils.

541

Table 2 The loading capacity and encapsulation efficiency of PMFs in citrus oil-based

542

emulsion systems.

21



543

Page 22 of 28

Table 1 The physical property and composition analysis of four citrus oils. Mandarin oil

Orange oil

Sweet orange oil

Bergamot oil

Color

pale yellow

pale yellow

yellow

pale yellow

Refractive index

1.4745

1.4740

1.4730

1.4665

Density (g/cm3)

0.8460

0.8500

0.8450

0.8770

Viscosity (mPa·s)

0.9470

0.9680

3.8380

1.8320

Composition No.

RT (min)

Peak (%)

1

11.9

α-pinene

3.49

4.26

-

0.65

2

12.3

1,5,5-trimethyl-6-methylene-cyclohexene

3.47

4.74

-

-

3

13.9

camphene

6.80

9.31

-

-

4

16.0

(-)-β-Pinene

5.04

6.72

-

2.10

5

17.5

1,3,5,5-tetramethyl-1,3-cyclohexadiene

1.03

1.45

-

-

6

19.1

β-myrcene

1.65

1.52

1.62

0.59

7

21.3

d-limonene

72.44

67.18

97.38

25.26

8

24.1

γ-terpinene

3.94

2.24

-

1.73

9

26.4

1-methyl-4-(1methylethylidene)-cyclohexene -

-

-

1.94

10

42.2

β-linalool

0.19

-

0.40

15.41

11

43.0

linalyl acetate

-

-

-

34.64

12

50.0

2-carene

-

-

-

1.52

13

50.7

α-terpineol

0.26

-

-

4.70

14

52.4

α-citral

-

-

-

1.61

544

22


Page 23 of 28


545

Table 2 The loading capacity and encapsulation efficiency of PMFs in citrus oil-based

546

emulsion systems.

Oil type

10 mg PMF Nob

Tan

LC (mg)

EE (%)

LC (mg)

EE (%)

Mandarin oil

9.527 ± 0.002

95.27 ± 0.02

8.590 ± 0.002

85.90 ± 0.02

Orange oil

9.114 ± 0.003

91.14 ± 0.03

8.370 ± 0.004

83.70 ± 0.04

Sweet orange oil

8.800 ± 0.001

88.00 ± 0.01

7.900 ± 0.003

79.00 ± 0.03

Bergamot oil

9.047 ± 0.004

90.47 ± 0.04

7.930 ± 0.002

79.30 ± 0.02

Nob LC (mg)

EE (%)

Tan LC (mg)

EE (%)

Mandarin oil

19.474 ± 0.002

97.37 ± 0.02

17.300 ± 0.004

86.50 ± 0.04

Orange oil

9.400 ± 0.002

47.00 ± 0.02

8.642 ± 0.003

43.21 ± 0.03

Sweet orange oil

18.920 ± 0.001

94.60 ± 0.01

13.860 ± 0.003

69.30 ± 0.03

Bergamot oil

19.600 ± 0.003

98.00 ± 0.03

16.380 ± 0.002

81.90 ± 0.02

20 mg PMF

30 mg PMF Nob

Tan

LC (mg)

EE (%)

LC (mg)

EE (%)

Mandarin oil

20.130 ± 0.005

67.10 ± 0.05

18.300 ± 0.003

61.00 ± 0.03

Orange oil

9.534 ± 0.002

31.78 ± 0.02

9.510 ± 0.002

31.70 ± 0.02

Sweet orange oil

18.720 ± 0.003

62.40 ± 0.03

13.830 ± 0.003

46.10 ± 0.03

Bergamot oil

21.120 ± 0.002

70.40 ± 0.02

18.369 ± 0.001

61.23 ± 0.01

547 548

LC = Loading capacity; EE = Encapsulation efficiency.

23



549 550

Figure 1 The GC/MS profiles of four citrus oils extracted from mandarin, orange,

551

sweet orange, and bergamot.

24


Page 24 of 28

Page 25 of 28


552 553

Figure 2 (A) The solubility of nobiletin and tangeretin in mandarin oil, orange oil,

554

sweet orange oil, and bergamot oil at the temperatures of 30, 40, 50, and 60 oC. (B)

555

Influence of heating procedure on the solubility of nobiletin and tangeretin. Nobiletin

556

and tangeretin content were determined after the samples were heated to 30°C, 60°C,

557

as well as after cooled down to 30°C from 60°C.

25



558 559

Figure 3 Appearance and turbidity (A), particle size (B), and Zeta-potential (C) of

560

emulsions of citrus oils.

26


Page 26 of 28

Page 27 of 28


561 562

Figure 4 (A) Influence of citrus oil type on the viscosity of emulsion systems; (B)

563

Flow curves of citrus oil emulsion systems loaded with or without PMFs.

27



564

Graphical for table of contents

565

28


Page 28 of 28

Encapsulation of Polymethoxyflavones in Citrus Oil Emulsion-Based

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