Food Sci. Biotechnol. 22(1): 225-231 (2013) DOI 10.1007/s10068-013-0031-y
RESEARCH ARTICLE
Reactive Oxygen Species Scavenging Activities of Naturally Occurring Colorants Hye Ryung Kang, Hyo Jung Kim, Hwa-Jin Suh, Oh Oun Kwon, Kyung Sun Kim, and Jong-Sang Kim
Received: 25 July 2012 / Revised: 1 September 2012 / Accepted: 4 September 2012 / Published Online: 28 February 2013 © KoSFoST and Springer 2013
Abstract Twenty-five kinds of plant extract used as natural dyeing agents were tested for their reactive oxygen species (ROS) scavenging activities using several assay systems. Among plant extracts examined in this study the hot water extracts from logwood, wattle, pomegranate, dragon’s blood, and quebracho showed relatively strong antioxidant activity. In particular, the pigments extracted from pomegranate and wattle effectively suppressed intracellular generation of ROS induced by hydrogen peroxide in mouse hepatoma hepa1c1c7 cells as well as showed a strong oxygen radical scavenging activity in in vitro assay systems, suggesting their potential usefulness as health functional food ingredients and natural food colorants. Keywords: reactive oxygen species, plant pigment, natural colorant
Introduction Plants produce more than 200,000 different types of compounds (1), including many pigments. Naturally occurring pigments have been used as not only colorants since prehistoric times but also contain health benefits as evidenced for some flavonoids, carotenoids, phytoestrogens, and others. For instance, plant carotenoids are red, orange, Hye Ryung Kang, Hyo Jung Kim, Jong-Sang Kim () School of Applied Biosciences and Food Science and Biotechnology, Kyungpook National University, Daegu 702-701, Korea Tel: +82-53-950-5752; Fax: +82-53-950-6750 E-mail:
[email protected] Hwa-Jin Suh, Oh Oun Kwon Gyeongbuk Natural Color Industry Institute, Youngcheon, Gyeongbuk 770-060, Korea Kyung Sun Kim Whasoomok Company, Yeongcheon, Gyeongbuk 770-831, Korea
and yellow lipid-soluble pigments found embedded in the membranes of chloroplasts and chromoplasts. Their color is masked by chlorophyll in photosynthetic tissues, but in late stages of plant development these pigments contribute to the bright colors of many flowers and fruits and the carrot root. Carotenoids protect photosynthetic organisms against potentially harmful photo-oxidative processes and are essential structural components of the photosynthetic antenna and reaction center complexes (2). The many flavonoids in plants have extensive biologic properties including the promotion of human health and reduction of the risk of disease. Most flavonoids augment the activity of antioxidant vitamins, protect lipid oxidation, inhibit platelet aggregation, and have anti-inflammatory, and antitumor action (3). Some plants including fruits and vegetables contain a variety of isoprenoid compounds that exhibit several health benefits including anticancer activities. These compounds derived from mevalonate pathway, include the tocotrienols and monoterpenes such as limonene, geraniol, menthol, carvone, β-ionone, and perillyl alcohol (4). The terpenoids and tocotrienols increase tumor latency and decrease multiplicity. The same compounds elicit the reduction of total and LDL-cholesterol, thereby reducing the risk of cardiovascular disease (3). Currently, 26 natural colorants including anthocyanins, curcumin, carminic acid, lycopene, betanin, paprika, and saffron are permitted for use as colorants exempt from cerification in the United States (5). More than 40 colorants have been authorized as food additives by the EU and have been assigned an E number. Sixteen of these are of plant origin. Juices or extracts from some fruits and vegetables are also used for coloring purposes. The stability and efficacy of natural colors are usually influenced by several factors including the presence of temperature, light, oxidizing and reducing agents, acids, or
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time of storage (5). There is still a lack of natural colors to completely replace synthetic colorants, and hence, efforts to search more options are needed. We learned that many naturally occurring colorants have been utilized for dyeing textile and paper, and conducted preliminary investigational study including reactive oxygen species (ROS) scavenging activity of those colorants prior to confirming their potential as health functional food ingredients. In this study, it is demonstrated that some water extracts enriched with plant pigments have strong antioxidant activities and therby potenital to be utilized as health functional food ingredients as well as food colorants.
Materials and Methods Plant colorants and reagents Twenty-five kinds of plant color extract were obtained from Hwasoomok Co. (Youngcheon, Korea). The samples were prepared by extracting dry raw materials with 10-fold water at 55oC for 1 h, followed by concentration and spray drying. The scientific and common names of plants used in this study and the part used for sample preparation were listed in Table 1. All chemicals used were of reagent grade. Determination of total phenolics Total phenolics were determined using Folin-Ciocalteu reagent (8). Briefly, 100 µL of extract was mixed with 50 µL of sodium carbonate (10%, w/v) solution, followed by addition of 15 µL of Folin-Ciocalteu reagent (previously diluted 5-fold with distilled water). After 5 min at room temperature, the sample mixture was transferred to 96-well microplate and then absorbance at 655 nm was measured using microplate reader. Results are expressed as gallic acid equivalents (GAE). DPPH radical scavenging assay The DPPH radical scavenging activities of the samples were evaluated as previously described (10,11). Briefly, 50 µL of sample solution was added to 200 µL of 200 µM DPPH radical solution, which was freshly made. After 30 min of incubation at 37oC, the absorbance at 515 nm was measured using microplate reader. Synthetic antioxidant reagent, Trolox was used as a positive control, and all tests were carried out in triplicate. ABTS+ radical scavenging assay ABTS working solution was prepared by mixing 100 µL of 70 mM ABTS, 900 µL deionized water, and 17.5 µL of 140 mM potassium persulfate, and the reaction solution was shaken in the dark at room temperature for 16 h before use (12). It was diluted with ethanol so that its absorbance was adjusted to 0.7± 0.02 at 750 nm. ABTS (900 µL) was added to 96-well microplate containing 10 µL of samples. Absorbance was
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measured at 750 nm after 5 min using microplate reader (Infinite 200; Tecan). The percentage of radical scavenging activity was calculated by comparing the absorbance values of control without sample. All determinations were performed 3 times. Oxygen radical absorbance capacity (ORAC) assay Antioxidant activities of the sample extracts in different concentrations (between 0.125 and 1 µg/mL) were investigated for their peroxyl radical scavenging capacity using ORAC assay system. The ORAC assay was carried out using a Tecan fluorescence microplate reader (Infinite 200; Tecan, Grodig, Austria) with fluorescent filters (excitation wavelength 485 nm, emission filter 535 nm). In the final assay mixture, fluorescein (91.4 nM) was used as a target of free radical attack with either AAPH (final conc. 11.4 mM) as a peroxyl radical generator in peroxyl radical scavenging capacity (ORACROO·) assay (6,7). Trolox (1 µM) was used as a control standard and prepared fresh on a daily basis. The analyzer was programmed to record the fluorescence of fluorescein every 2 min after AAPH was added. All fluorescence measurements were expressed relative to the initial reading. Final results were calculated based on the difference in the area under the fluorescence decay curve between the blank and each sample. All data were expressed as µmol of Trolox equivalents (TE). One ORAC unit is equivalent to the net protection area provided by 1 µM of Trolox. Ferric reducing antioxidant power (FRAP) assay The FRAP of the samples was determined as described previously (9). Briefly, 170 µL of H2O and 7 µL samples were mixed with 30 µL of FRAP reagent [containing 300 mmol/L acetate buffer (pH 3.6), 10 mmol/L TPTZ solution, 20 mmol/L FeCl3 solution, and distilled water], and the absorbance at 593 nm was recorded after 4 min using microplate reader. FRAP values of unknowns were calculated on the basis of standard curves established using ferrous sulfate at 0.1-1.0 mmol/L. Lipid peroxidation inhibition assay Assay of lipid peroxidation was performed according to the method described previously (13). Mice were sacrificed by cervical dislocation. The brain, perfused liver, and kidney were isolated and homogenized with 40 parts (20 parts for kidney) of isotonic phosphate buffer saline using a homogenizer on ice. The homogenate was centrifuged at 10,000×g for 5 min, and the supernatant was used for the in vitro lipid peroxidation assay. Briefly, various concentrations of each sample were mixed with 0.5 mL of 0.15 M potassium chloride and 0.5 mL of mouse tissue homogenate. Peroxidation was initiated by adding 100 µL of 20 mM ferric chloride. After incubation at 37oC for 30
ROS Scavenging Activity of Natural Colorants
min, the reaction was stopped by adding 0.5 mL of ice-cold 0.25 M HCl containing 15% tricarboxylic acid (TCA), 0.38% thiobarbituric acid (TBA), and 0.5% butylated hydroxytolune (BHT). The reaction mixture was heated at 90oC for 60 min, cooled, and centrifuged at 3,000×g for 3 min, and the supernatant was read at 532 nm using microplate reader. A negative control without added sample was also run simultaneously while BHT was used as a positive control. The lipid peroxidation rate was calculated as follows; lipid peroxidation rate=(sample OD/ blank OD)×100. Measurement of intracellular ROS Oxidative stress was quantified in cells by 2,7-dichlorofluorescein (DCF) assay according to Wang and Joseph (14), with slight modifications. A mouse hepatoma cell line (hepa1c1c7) was obtained from ATCC (Manassas, VA, USA). For routine maintenance, cells were grown in α-minimal essential medium (α-MEM, BD, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) at 37oC in an atmosphere of 5% CO2/95% air under saturating humidity and passaged every other day (1:6 split ratio) by trypsinization with 0.25% trypsin/0.02% EDTA sodium salt solution (Thermo Fisher Scientific Inc., Waltham, MA, USA). The cells (1×105 cells/well) were seeded into a blackbottom 96-well plate and cultivated for 24 h using α-MEM containing 10% FBS. Cells were further incubated in the absence and presence of sample (12.5, 25, and 50 µg/mL) for another 24 h. Then cells were incubated with dichlorfluorescein diacetate (DCFDA) dissolved in dimethyl sulfoxide (DMSO, final concentration 10 µM) for 30 min, followed by the addition of H2O2 (final conc. 500 µM) for 1 h. Fluorescence was measured at 485 and 535 nm of excitation and emission, in a fluorescence microplate reader (Infinite 200; Tecan). Most of the steps including incubation of the reaction mixture containing dye and oxidant, washing, and fluorometric determination were performed in the dark. The fluorescence intensity of sample was expressed as relative percentage to the negative control in which neither sample nor H2O2 was added. Statistical analysis Values of the results expressed as means±standard deviation (SD). Significant difference of means was analyzed using analysis of variance (ANOVA), followed by Duncan’s multiple range test to determine statistical difference among groups. Statistical significance was defined as p
