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Liposomes as delivery system for carotenoids: comparative antioxidant activities of carotenoids measured by ferric reducing antioxidant power, DPPH assay and lipid peroxidation Chen Tan, Jin Xue, Shabbar Abbas, Biao Feng, Xiaoming Zhang, and Shuqin Xia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf405622f • Publication Date (Web): 20 Apr 2014 Downloaded from http://pubs.acs.org on May 13, 2014
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Journal of Agricultural and Food Chemistry
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Liposome as a delivery system for carotenoids: comparative
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antioxidant activity of carotenoids as measured by ferric reducing
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antioxidant power, DPPH assay and lipid peroxidation
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Chen Tan, Jin Xue, Shabbar Abbas, Biao Feng, Xiaoming Zhang, Shuqin Xia∗
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State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan
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University, Lihu Road 1800, Wuxi, Jiangsu 214122, China
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∗Corresponding
author
(E-mail:
[email protected];
telephone
86-510-85884496 ACS Paragon Plus Environment
86-510-85884496;
fax
Journal of Agricultural and Food Chemistry 9
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ABSTRACT
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This study was conducted in order to understand how carotenoids exerted antioxidant activity after
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encapsulation in a liposome delivery system, for food application. Three assays were selected to achieve a
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wide range of technical principles, including 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging, ferric
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reducing antioxidant powder (FRAP), and lipid peroxidation inhibition capacity (LPIC) during liposome
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preparation, auto-oxidation, or when induced by ferric iron/ascorbate. Antioxidant activity of carotenoids
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was measured either after they were mixed with preformed liposomes or after their incorporation into the
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liposomal system. Whatever the antioxidant model was, carotenoids displayed different antioxidant activities
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in suspension and in liposomes. The encapsulation could enhance the DPPH scavenging and FRAP activities
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of carotenoids. The strongest antioxidant activity was observed with lutein, followed by β-carotene,
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lycopene and canthaxanthin. Furthermore, lipid peroxidation assay revealed a mutual protective relationship:
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the incorporation of either lutein or β-carotene not only exert strong LPIC but also protect themselves
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against pro-oxidation elements; however, the LPIC of lycopene and canthaxanthin on liposomes was weak
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or even pro-oxidation effect appeared, concomitantly, leading to the considerable depletion of these
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encapsulated carotenoids. The antioxidant activity of carotenoids after liposome encapsulation was not only
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related to their chemical reactivity, but also to their incorporation efficiencies into liposomal membrane and
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modulating effects on the membrane properties.
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KEYWORDS: liposome, carotenoids, antioxidant activity, lipid peroxidation, protect
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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The biological functions of carotenoids in humans and animals have been largely investigated and
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reviewed by experimental and epidemiological studies. It has been demonstrated that the carotenoids offer
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beneficial effects in preventing some types of cancer, cardiovascular and degenerative diseases (i.e. cataract,
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age related macular degeneration).1, 2, 3 The health benefits of carotenoids are most probably due to the
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antioxidant activities of their electron-rich conjugated system, both, by quenching singlet oxygen,4 and by
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scavenging radicals to terminate the chain reactions.5 For these reasons, carotenoids have become very
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popular nutritional supplements in the food and pharmaceutical industries. However, being highly
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unsaturated, carotenoids are prone to isomerization, oxidation and degradation, so it is essential to protect
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them from negative environmental factors which could affect their structural integrity and function.
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Liposomes are composed of a lipid bilayer with the hydrophobic chains of the lipids forming the bilayer
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and the polar head groups of the lipids oriented towards the extravesicular solution and inner cavity6. They
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have received higher attention in academic and industrial research owing to their biocompatibility and
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appealing ability to carry hydrophobic and hydrophilic substance. Food application of these colloidal
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structures greatly increased in the last decade for encapsulating food related substances including enzymes,7
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antimicrobial agents,8, 9 vitamins10 and functional peptides.9, 11 The application for carotenoid is mainly
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found in β-carotene,12 lutein13 and astaxanthine14 encapsulation. It has been shown that lipid bilayer can not
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only provide the physicochemical barrier to incorporated molecules against pro-oxidant elements, but also
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make them water-soluble and possibly able to be dispersed in aqueous food formulations and increase their
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bioavailability.15 Nevertheless, a question of what happens to the antioxidant activities of carotenoid after
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liposome encapsulation in food formulation yet has to be answered. Virtually all previous studies on the
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carotenoid antioxidation have been focused primarily on the physiological role of carotenoids using
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liposome as a model membrane system. It has been indicated that the antioxidant capacities of various
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carotenoids in the formulation of liposomes were different from those in solution. One generally accepted ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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conclusion is that the factors that determined the protective effect of membranes against oxidation are not
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only the chemical reactivity of their own, but also the position and orientation of the carotenoids in the
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bilayer (as depicted in Figure 1).16 Additionally, the reported antioxidant activities of carotenoids were
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correlated with the experimental conditions such as type of lipid used, preparation method of liposomes,
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concentration and oxygen tension. In the food industry, the lipid used for liposome preparation is commonly
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refers to egg yolk phosphatidylcholine (EYPC) which is isolated from natural chicken egg yolk.17 It is
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generally recognized as safe (GRAS) food ingredient that is biocompatible, biodegradable, and nontoxic. To
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our knowledge, there is a little consideration regarding the antioxidant activity of carotenoids in liposomes
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composed of mixed lipids including EYPC and nonionic surfactant Tween 80.
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Moreover, it is noted that when liposome is utilized as a delivery system in the nutraceutical and
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functional food, one has to consider, primarily, the stability of liposomes during processing and storage.
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Therefore, encapsulated carotenoids should not only be able to exert antioxidant effect, but also maintain the
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physicochemical stability of liposomes. However, carotenoids have contrasting effects on the membrane
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properties, owing to their various chemical structures. For instance, polar carotenoids incorporation can
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rigidify the lipid membranes and increase their stability, whereas, the effects of apolar carotenoids are
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negligible or opposite.18 On the other hand, the encapsulated concentration of carotenoids into liposomes
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may be much greater than that normally found in a natural membrane, since high loading contents are
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preferred for food application. When exceeding a certain concentration, carotenoids probably display
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pro-oxidation properties. In turn, the liposomal membrane damaged by the lipid peroxidation would not
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effectively
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concentration-dependent capacity of lutein to adjust the penetration of oxygen into lipid bilayer and
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consequently, the liposomes stability.19 Taking these factors into consideration, a compromise of carotenoids
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encapsulated concentration should be found in the design of carotenoids-loaded liposomal systems.
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protect
encapsulated
carotenoids.
Our
recent
study
has
also
demonstrated
the
Further understanding of the influence of liposome encapsulation on the antioxidant activity of carotenoid ACS Paragon Plus Environment
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will be crucial for the health benefits of encapsulated carotenoid. Herein, both carotenes (lycopene and
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β-carotene) and xanthophylls (lutein and canthaxanthin) were studied that are known antioxidants commonly
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found in human diet (Figure 1). The in vitro assays, including 2, 2-diphenyl-1-picrylhydrazyl (DPPH)
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scavenging ability and ferric reducing antioxidant power (FRAP), were carried out to investigate the
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antioxidant activities of carotenoids; either after they were mixed with liposomes or after they were
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incorporated into liposomes. The lipid peroxidation inhibition capacity (LPIC) of these carotenoids during
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preparation, auto-oxidation, or induced by ferric iron/ascorbate was also evaluated by determining the lipids
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oxidation product, the thiobarbituric acid-reactive substance (TBARS). Meanwhile, the depletion of
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carotenoid after oxidation was determined. Based on the experimental data, the probable mechanism of
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LPIC as well as the self-protection of carotenoids were correlated to the effects of carotenoids on membrane
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properties (dynamic and structure).
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MATERIALS AND METHODS
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Materials. Egg yolk phosphatidylcholine (EYPC) was purchased from Chemical Reagent Plant of East
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China Normal University (Shanghai, China). Carotenoids including lycopene, β-carotene, lutein and
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canthaxanthin (all 98 % purity) were a gift from Zhejiang Medicine Co., Ltd (Zhejiang, China). Petroleum
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ether (30-60 oC) and polyoxyethylene sorbitan monooleate (Tween 80) were purchased from China
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Medicine (Group) Shanghai Chemical Reagent Co. (Shanghai, China). 2, 2-diphenyl-1-picrylhydrazyl
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(DPPH) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used were of
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analytical grade.
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Preparation of carotenoid-loaded liposomes. Liposomes loading carotenoids were prepared by the
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thin-film evaporation method according to our recent report.20 Carotenoid was dissolved in 2 mL chloroform
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together with the lipids composed of EYPC and Tween 80 at the fixed mass ratio of 1:0.72. After dissolution,
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the liposomal system brought to dryness by the use of a rotary evaporator at 55 ˚C (organic solvent removal).
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The sample was further vacuum-dried in oven (at 50 ˚C ) to ensure complete removal of the solvent, ACS Paragon Plus Environment
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followed by hydration with 40 mL of 0.01 M phosphate buffer solution, 150 mM NaCl, PBS, pH 7.4, under
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vortexing for 60 min at 55 ˚C. The liposomal suspension was then subjected to a probing sonication process
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in an ice bath for 10 min at 240 W with a sequence of 5 s of sonication and 5 s of rest using a sonicator
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(Sonics & Materials , Inc., 20 kHz). The final sample preparations were transferred in vials under nitrogen
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bed and stored in the refrigerator (at 4 ˚C in dark) until use.
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Encapsulation Efficiency and Loading Content. The determination of encapsulation efficiency (EE)
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was performed by extraction according to our earlier method with a slight modification.21 Briefly, aliquots
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of 0.5 mL carotenoid-loaded liposomes and 3 mL petroleum ether were mixed by vortexing vigorously for 3
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min at an ambient temperature. The mixed sample was centrifuged at 2000 r/min for 5 min and supernatant
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was collected. The above operation was repeated twice and the collected supernatant was combined together
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in a tube, followed by dilution to 10 mL with petroleum ether. The free amount of carotenoids were
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quantified spectrophotometrically (UV-1600 spectrophotometer; Mapada Instruments Co., Ltd, China) at
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470, 455, 440, and 460 nm for lycopene, β-carotene, lutein, and canthaxanthin, respectively, with petroleum
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ether as a blank. Each experiment was carried out in triplicate.
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The total amount of carotenoid was expressed relatively to the mass of lipids through the initial carotenoid
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concentration (IC=mcarotenoid/mlipids, % wt/wt). The amount of carotenoid loaded into the liposomes was
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calculated as the difference between the total amount used to prepare loading liposomes and that recovered
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by extraction. The carotenoid encapsulation efficiency (EE, %) and loading content (LC, % wt/wt) was
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respectively calculated using the following equations: × 100
= × 100
=
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DPPH Radical-scavenging Activity. DPPH radical-scavenging activity of samples was measured
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according to the work published by our research group with a slight modification.22 In the assay, 0.7 mL of
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carotenoid-loaded liposomes (or the direct mixture of carotenoid-DMSO solution and pure liposomes) was ACS Paragon Plus Environment
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mixed with 0.2 mL of DPPH solution (0.4 mM in ethanol). The carotenoid in DMSO solution was adjusted
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to the same concentration to the carotenoid-loaded liposomes. Then, the solution was incubated at 37 oC in
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the dark for 40 min. Distilled water mixing with DPPH solution instead of the sample mixing was served as
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control. Sample blank was prepared by replacing the DPPH with ethanol. The absorbance of the sample after
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incubation was measured at 525 nm using a UV-1600 spectrophotometer (Meipuda Co., Shanghai, China). A
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low absorbance of the reaction mixture indicates a high free radical scavenging activity. The percentage of
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DPPH-scavenging activity was calculated as follows: (%) = (1 −
#$%&'( − #$%&'( )*+,-*' ) × 100 .'$+/
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Ferric Reducing Antioxidant Power (FRAP) Assay. Either the carotenoid-loaded liposomes or mixture
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between carotenoid-DMSO solution and pure liposomes (1 mL) was mixed with 1 mL of potassium
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ferricyanide (2.5%, w/v) followed by incubation at 50 oC for 20 min. After incubation, 5 mL of
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trichloroacetic acid (10%, w/v) was added to the mixture, which was centrifuged at 5000 r/min for 2min.
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The obtained supernatant (1 mL) was treated with 2 mL of distilled water and 0.5 mL of FeCl3 (0.1%, w/v)
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followed by incubation for 10 min. The absorbance of the reaction mixture was measured at 700 nm with a
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UV-1600 spectrophotometer (Meipuda Co., Shanghai, China). The blank was the sample using water instead
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of potassium ferricyanide. The larger the absorbance is, the higher the reducing power is.7
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Lipid Peroxidation Analysis. The lipid peroxidation inhibition capacity (LPIC) was determined by
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thiobarbituric acid reactive substance (TBARS). The measurements were based on our recent study:23 a
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solution containing thiobarbituric acid (15%, w/v) (TBA), trichloroacetic acid (0.37%, w/v) (TCA), and
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hydrochloric acid (1.8%, v/v) (HCl) was added to 1 mL of liposomal sample and mixed, followed by heating
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at 100 ˚C water bath for 30 minutes to promote the formation of a pink pigment resulting from the reaction
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with malondyaldehyde [(MDA)2-TBA]. Afterwards, the mixture was cooled rapidly with ice bath,
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centrifuged for 5 minutes at 2000 r/min and filtrated. The absorbance of the filtrate was measured by
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spectrophotometer at 535 nm (A535nm). Ac and As were the absorbance at 535 nm of carotenoid-free ACS Paragon Plus Environment
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liposomes and carotenoid-loaded liposomes, respectively. The inhibition activity of TBARS during initial
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sample preparation procedure was calculated by following equation: 0123 ℎ5 (%) =
) − 6 × 100 )
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When evaluating the LPIC of carotenoid induced by Fe3+/ascorbate, the liposomal samples were first
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mixed with FeCl3 (1 mL, 400 µmol/L) and ascorbic acid (1 mL, 400 µmol/L). After incubation at 37 oC for
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60 min, the TBA-TCA-HCl solution was added. When evaluating the auto-oxidation of liposomal membrane,
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all samples were incubated at 37 oC in an uncovered, shaking water bath. At proper time intervals, an aliquot
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(1 mL) of each sample was removed and combined with 5 mL TBA-TCA-HCl solution. The determination
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procedure of TBARS was the same as described above. The amount of retained carotenoid during oxidation
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treatment was measured by determining total and free amount of carotenoid in the same way for the
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determination of EE as described above. The change in TBARS induced by Fe3+/ascorbate or during
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auto-oxidation and retention rate (RR, %) were calculated respectively as following equations: ℎ 0123 (%) 0123 7 − 0123 8 × 100 0123 7
229+)* = × 100 8 0 7 7
22:;
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canthaxanthin. Due to the vertical orientation in the opposite polar membrane zones, lutein and β-carotene
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are accessible to react with DPPH at the membrane surface. However, it is difficult for lycopene to trap
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DPPH because of the deep location in the bilayer hydrophobic core, far from the membrane surface (Figure
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1). The poor scavenging ability of canthaxanthin was mainly due to the very low degrees of its incorporation
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into the lipid bilayer. It was interesting to find that the DPPH scavenging of each carotenoid decreased ACS Paragon Plus Environment
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slightly when the concentration was >1.25%. It may be the consequence of the extent of carotenoid
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aggregation, causing the decrease of antioxidant efficiency.4
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FRAP Assay. Some reducing substances in the sample could provide electron to reduce Fe3+ to Fe2+. The
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Fe2+ can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. Figure 4 shows that
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when mixed with liposomes, lutein seemed to display relatively stronger FRAP activity than other
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carotenoids. FRAP activity was not only affected by the CDB number but also by the steric hindrance
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between carotenoids and ferric di -TPTZ complex. Despite having 10 CDB, the presence of hydroxyl
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functions in the 3 (3,)-position at the ring system can reduce the steric hindrance, and consequently
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improved the FRAP activity of lutein.29 However, the differences of FRAP activity between other
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carotenoids was not apparent and also no concentration-dependence was detected. This observation was a
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little different from the conclusion of previous observation.29 The contradiction might be due to the reaction
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conditions, such as reaction time, solvents, concentration and used wavelength.29,
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explanation was the inhomogeneous dispersion of carotenoids in their direct mixture with liposomes.
30
Another probable
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When incorporated into the lipid bilayer, the FRAP activity of each carotenoid was enhanced. It was
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probably due to the reduced steric hindrance of molecules after they were encapsulated. The highest FRAP
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activity was observed with lutein, followed by β-carotene, lycopene and canthaxanthin. This trend was well
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in agreement with their activities to scavenge DPPH. On the other hand, ferricyanide is also an oxidizing
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agent impermeable through lipid bilayers and membrane, and therefore extensively used to assess the
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exposition of redox groups to the aqueous medium.7 The vertical fashion made lutein and β-carotene
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favorable to react with ferric di -TPTZ complex at the membrane surface. Unexpectedly, although lycopene
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located in bilayer core, its FRAP activity showed highly concentration-dependent increase. It has been
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demonstrated that the parallel fashion of lycopene could decrease the penetration barrier for small molecules
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to the liposomal membrane.31 Thus, we speculated that the loosened membrane structure created more
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opportunities of lycopene to react with ferricyanide. For canthaxanthin, undoubtedly, the slight increase of ACS Paragon Plus Environment
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FRAP activities and lower DPPH scavenging ability was the consequence of their poor incorporation ability.
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Lipid Peroxidation Assay. Another mechanism of the antioxidant defense system is the lipid
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peroxidation inhibition capacity (LPIC). EYPC may undergo peroxidation due to the presence of
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polyunsaturated acyl chains in lipid molecules, leading to high membrane permeability.32 Thus, the
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protection from lipid peroxidation owing to the carotenoids antioxidation may in turn retain the incorporated
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carotenoids more effectively. The product of peroxidation can be determined spectrophotometrically by the
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thiobarbituric acid reactive substances (TBARS) and the inhibition rate of TBARS after initial preparation of
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liposomes is shown in Figure 5. Within a certain concentration range, the lipid peroxidation during liposome
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preparation was inhibited by lycopene (0.25%-0.75%), β-carotene (0.25%-1.25%), lutein (0.25%-1.50%)
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and canthaxanthin (0.25%). Especially, the more lutein molecules got incorporated (0.25% to 1.25%), the
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more significant the increase of TBARS inhibition rate was (P
