Monterrey, Monterrey, Nuevo León, Mexico

Transcription

1 Phytochemicals and antioxidant activity of comminuted orange (Citrus sinensis L.) Zamantha Escobedo-Avellaneda a, Vinicio Serment-Moreno a, Aurora Valdez-Fragoso a, Hugo Mujica-Paz a, and Jorge Welti-Chanes a a Department of Biotechnology and Food Engineering, Instituto Tecnológico y de Estudios Superiores de Monterrey, Monterrey, Nuevo León, Mexico (zamantha.avella@gmail.com, vsermentm@gmail.com, a.valdez@itesm.mx, hmujicap@yahoo.com, jwelti@itesm.mx) ABSTRACT Orange (Citrus sinensis L.) is a prominent source of phytochemical, secondary metabolites of plants that provide health benefits due to a risk reduction of chronic illness acquisition such as cancer and cardiovascular disease. Phenolic compounds, vitamin C, total carotenoids, and antioxidant activity of comminuted orange, a natural basis used to enhance sensory properties of soft drinks, were analyzed spectrophotometrically. Free and total phenolics were analyzed by the Folin-Ciocalteau colorimetric method. Vitamin C was measured as reduced and total ascorbate. The oxygen radical antioxidant activity (ORAC) assay was used to determine the antioxidant capacity. Total phenolics compounds (285.5±9.0 mg gallic acid equivalents/100 g sample) were 86.5 times higher than total carotenoids (3.3±0.2 mg -carotene equivalents/100 g sample). The ratio between free and bound phenolics was found of 1.7 (181.1±7.4 and mg gallic acid equivalents/100 g sample for free and bound phenolics respectively). Most vitamin C was found in the reduced form (44.9±1.1 for L-ascorbic acid versus 3.9 mg L-ascorbic acid equivalents/100 g sample for dehydroascorbic acid). The antioxidant activity, mainly attributed to hydrophilic phytochemicals such as vitamin C and phenolics was of ±154.9 mol trolox equivalernts/100 g sample. Knowledge of phytochemicals and the antioxidant activity of comminuted orange is fundamental for evaluating the level in which this product could improve soft drinks quality, and for designing the most adequate method to preserve it without reducing bioavailability and functionality of these compounds. Keywords: comminuted orange; phenolics compounds; vitamin C; total carotenoids; and antioxidant activity. INTRODUCTION Orange (Citrus sinensis L.), a hesperidium belonging to the Rutaceae family, is the most widely grown and commercialized citrus specie. Orange is composed by an external layer (peel) formed by flavedo (epicarp or exocarp) and albedo (mesocarp), and an inner material called endocarp that contains vesicles with juice. Juice, flavedo and albedo account for about 50, 10 and 25% (w/w) respectively of the whole fruit [1]. Besides sugars, acids, and polysaccharides, oranges are an important source of phytochemicals such as phenolics, vitamin C and carotenoids. These compounds also known as nutraceuticals provides health benefits due to a risk reduction of chronic illness such as cancer and cardiovascular disease [2-3]. One of the mechanisms by which these phytochemicals exerts their beneficial effects in human health has been related to their antioxidant activity. Phenolics in fruits and vegetables, as well as vitamin C, are said to be effective antioxidants. It was shown that vitamin C contributes in 100% to the total antioxidant activity of Florida orange juice [4]. Vitamin C scavenges free radicals such as O2 -, OH., peroxyl radicals and singlet oxygen, protecting the intracellular and extracellular structures [4-5]. Carotenoids prevent potentially damaging radical production due to their polyene structure [3]. Some authors have found a direct relationship between phenolic compounds and their antioxidant activity [4, 6] whereas others are not in agreement [7]. These differences may be attributed to the extraction procedure and antioxidant activity determination method. Phenolics can be found free or bound; therefore the extraction method must be highlighted to avoid underestimation. It has also been shown a direct correlation between vitamin C and its potential activity against oxidant species, but this was not the case for carotenoids [4]. Both juice and peel contain nutraceuticals, nevertheless it has been demonstrated that they are more abundant in citrus peel [8-10]. One of the products derived from citrus is comminuted orange, that is obtained by grinding defined proportions of peel and juice, and that is mainly used as a natural basis to enhance sensory properties of soft drinks. In addition to augmenting beverages quality, production costs and wastes are reduced because orange peel is used in comminuted elaboration. Nutraceuticals and antioxidant activity of comminuted orange must be evaluated in order to determine the level in which this product is a source of these compounds, and it can provide antioxidant activity.

2 Thus, the aim of this work was to quantify phenolic compounds, vitamin C, total carotenoids, and antioxidant activity of comminuted orange. MATERIALS & METHODS Reagents 2 N Folin-Ciocalteau phenol reagent, trichloroacetic acid (TCA), DL-dithiothreitol 99% (DTT), N- ethylmaleimida 98% HPLC (NEM), gallic acid, L-ascorbic acid, -Bipyridyl, 2,2 -Azobis(2- methylpropionamidine) dihydrochloride (AAPH), fluorescein sodium salt, and trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid) were purchased from Sigma-Aldrich (USA). HCl, potassium phosphate monobasic, potassium phosphate dibasic, ethanol, and sodium phosphate monobasic were acquired from Fermont (Mexico). Phosphoric acid (85% HPLC grade), ferric chloride, tetrahydrofuran (THF) HPLC grade were obtained from Fisher Scientific (Mexico). Methanol was purchased from DEQ (Mexico). Sodium carbonate and sodium phosphate dibasic were acquired from CTR Scientific (Mexico). Unless otherwise noted, all solvents and reagents were of analytical grade. Comminuted orange preparation Juice, flavedo and albedo (71.6, 16.0 and 12.4% w/w respectively) of commercially mature Valencia oranges acquired in June 2010 in Monterrey, Nuevo León, Mexico, were grinded in a domestic blender (Oster, BRLY-Z00-013, Mexico) to obtain a homogenous paste. Phenolic compounds 250 mg of comminuted were mixed with either 5 ml of methanol: water (1:1 v/v) (for free phenolics) or 1.2 M HCl in (1:1 v/v) methanol/water (for total phenolics). The mixture was vortexed at 3000 rpm/1 min (VWR, model , USA) and then heated at 90 C (Fisher Scientific, model 102, USA) for 3 h with vortexing every 30 min. Sample was cooled to reach room temperature, diluted to 10 ml with methanol and centrifuged at 5000 rpm/10 min/4 C (IEC, Centra MP4R, USA) [11]. 50 L free or total phenolic extracts were placed in a 2 ml microcentrifuge tubes followed by 650 L of water. Then 50 L of the Folin- Ciocalteau reagent were added. The resulting mixture was vortexed and incubated 5 min at room temperature. Finally, 250 μl of 1 N sodium carbonate solution were added and the test tubes were incubated at 37 C/2 h. 200 l sample from each assay tube were transfer to a clear 96- flat bottom well microplate (Costar, USA), and the absorbance was measured at 765 nm in a microplate reader (Synergy HT; Bio-Tek, USA) [12]. Concentration was expressed as mg gallic acid equivalents/100 g comminuted orange in wet basis (mg GAE/100 g). Phenolic concentration represents the mean of 18 lectures of absorbance. Bound phenolics were determined by subtracting free phenolics from total phenolics. Vitamin C 0.25 g of comminuted orange were mixed with 1 ml of 6% TCA and then centrifuged at 13,000 g/5 min/ 4 C (VWR, galaxy 16DH, USA). Supernatants were transferred to new tubes [13]. 25 L of 75 mm potassium phosphate buffer and 50 L of sample were added to microcentrifuge tubes for both reduced and total ascorbate content. 25 l of 10 mm DTT were added to the total ascorbate tubes and then incubated at room temperature/10 min. Then 25 l of 0.5% NEM were added to the same tubes and 50 l of water to the reduced ascorbate tubes. 125 l 10% TCA, 100 l 43% H 3 PO 4, 100 l 4% -bipyridyl and 50 l 3% FeCl 3 were added to all assay tubes. Tubes were vortexed and incubated at 37 C for 1 h. 200 l sample from each assay tube were transfer to a clear 96- flat bottom well microplate (Costar, USA) and the absorbance was read at 525 nm in a microplate reader. Concentration was expressed as mg L-ascorbic acid equivalents/100 g comminuted orange in wet basis (mg L-AAE/100 g). For each comminuted orange sample, vitamin C concentration represents the mean of 6 lectures of absorbance. Oxidized ascorbate was determined by subtracting reduced vitamin C from total vitamin C. Total carotenoids 75 mg of comminuted orange were weighed in microcentrifuge tubes under subdued light. Then 1.5 ml of ethanol: THF (1:1 v/v) were added and an ultrasound treatment at 45 W/10 min (VWR, model 50 T, USA) was applied. Tubes were centrifuged at 5000 g/5 min/4 C. Adequate dilution of supernatants was measured at 450 nm (Genesys 10 UV thermo electron, USA) [14-15]. Concentration was expressed as mg -carotene equivalents/100 g comminuted orange in wet basis (mg -CE/100 g). For each extract, concentration represents the mean of 6 lectures of absorbance. Antioxidant activity 25 l of adequate dilution (in sodium phosphate buffer solution, PBS, 75 mm, ph 7.4) of comminuted orange was placed in polystyrene black plates with 96 round bottom wells (Costar, USA). A microplate reader

3 (Synergy HT; Bio-Tek, USA) was used to automatically dispense 150 l of 1 M fluorescein. After 30 min of incubation at 37 C, 25 l of 153 M AAPH were dispensed. Fluorescence (485/20 nm excitation wavelength and 528/20 nm emission wavelength) was measured during 1 h/37 C [16]. ORAC values were expressed as mol trolox equivalents/100 g comminuted orange in wet basis ( mol TE/100 g), and they represent the mean of 9 lectures. Statistical analysis Results were statistically evaluated by ANOVA (Minitab 14) with a confidence level of RESULTS & DISCUSSION Concentration of free and total phenolics of fresh comminuted orange is shown in Figure 1. The Folin- Ciocalteau colorimetric method used for phenolic determination is based in a single electron transfer reaction in which phenolic compounds are oxidized at high ph, yielding a colored product with max at 765 nm. It has been shown that other reducing agents such as vitamin C can interfere in the analysis [17]. Nevertheless, it is thought that vitamin C does not interfere in the analysis of comminuted orange due to its sensitivity to the high temperature used for free and total phenolics extraction. The amount of total and free phenolics was found as 285.5±9.0 and 181.1±7.4 mg GAE/100 g respectively. Free phenolics were subtracted from total phenolics, and 36.6% of conjugated phenolics were calculated, indicating that most phenolics are in free form. This percentage is similar to that obtained in orange juice by Sun et al. [18] (30% of bound phenolics). Concentration of total phenolics found in comminuted orange is similar to that reported by the U.S. Department of Agriculture [19] for navel orange (337 mg GAE/100 g wb). Compared with other foodstuffs widely recognized by their high phenolics content, such as blackberry, blueberry and red grape with total phenolics of 447, 311, and 170 mg GAE/100 g wb respectively [19], comminuted orange could be considered as an important source of these compounds. Figure 1. Phenolic compounds of comminuted orange. L-ascorbic acid (reduced form) and dehydroascorbic acid (oxidized form) account for total vitamin C. 48.8±1.5 and 44.9±1.1 mg L-AAE/100 g for total and reduced vitamin C were found respectively in comminuted orange (Figure 2). Despite of the O 2 incorporated to the comminuted orange by the milling process, contributing to the oxidation of vitamin C, only 8 % was found as dehydroascorbic acid. The USDA [19] reported 50 and 136 mg L-AAE/100 g wb of total vitamin C for juice and peel orange respectively. Total vitamin C of orange components was analyzed, but oranges were different from the ones used for the comminuted orange of the present research. Juice, flavedo and albedo accounted for 27.4±1.1, 120.1±0.8, and

4 7.0±1.4 mg L-AAE/100 g respectively. According to these results and considering the percentage of each component used for comminuted orange formulation, the total amount of vitamin C must be 39.2 mg L- AAE/100 g, while experimentally for that lot of comminuted orange a total vitamin C content of 41.3±1.29 mg L-AAE/100 g was obtained, which is very similar to the calculated value. Figure 2. Vitamin C content of comminuted orange. Total carotenoid content in comminuted orange was 3.3±0.2 mg -CE/100 g wb, while in dry basis this value corresponds to 21.0±1.1 mg -CE/100 g db. 8 mg -CE/100 ml freeze dried orange juice, and 44.5 mg - CE/100 g freeze dried peel were reported by Yuan-Chuen et al. [9] and Yuan-Chuen et al. [10] respectively. Using data from these authors, and considering the orange juice and flavedo plus albedo (peel) proportions used in comminuted orange formulation, the total carotenoid content calculated is 18.4 mg -CE/100 g db, value similar to 21.0±1.1 mg -CE/100 g db. Because phenolics are 86.5 times higher than total carotenoids, they are the main phytochemicals in the product. Antioxidant capacity of comminuted orange, mainly attributed to hydrophilic compounds such as phenolics and vitamin C, was ±154.9 mol TE/100 g. This value is much higher than that reported by the USDA [20] for orange juice (726 mol TE/100 g). The antioxidant activity in the product was improved by the flavedo and albedo with higher concentration of nutraceuticals than orange juice. Compared with other foodstuffs widely recognized as sources of antioxidants, such as blackberry, blueberry and red grape with antioxidant activity of 5802, 3463, and 1837 mol TE/100 g respectively [20], the antioxidant capacity of comminuted could be considered as high. Due to the content of carotenoids and other lipophilic compounds in peel orange such as limonene, in further research it will be necessary to determine the L-ORAC (lipophilic antioxidant activity) of comminuted orange to know its total antioxidant capacity. Many research studies have pointed out that vitamin C greatly contributes with the total antioxidant activity of orange juice. In order to determine its contribution in comminuted orange, it is necessary to quantify the antioxidant capacity of L- ascorbic acid standard and to relate it with the concentration of vitamin C in the product. CONCLUSION Analysis of total and free phenolics, total and reduced vitamin C, total carotenoids and antioxidant activity of comminuted orange was performed. Knowledge of the concentration of these nutraceuticals is fundamental for evaluating the level in which comminuted orange is a source of these compounds, and can provide antioxidant activity. This is also important for designing the most adequate method to preserve it without reducing nutraceuticals availability and functionality. The concentration of nutraceuticals and antioxidant activity of comminuted orange was improved by the addition of peel as compared with orange juice. Results show that orange comminuted can be recognized as an important source of nutraceuticals like foodstuffs such as blackberry, blueberry, and red grape ±9.0 and 181.1±7.4 mg GAE/100 g comminuted orange wb were found for total and free phenolics respectively. Total vitamin C was 48.8±1.5 mg L-ascorbic acid

5 equivalents/100 g. Total carotenoids were 3.3±0.2 mg -CE/100 g. The antioxidant activity, mainly attributed to hydrophilic compounds such as vitamin C and phenolics, was ±154.9 mol TE/100 g. REFERENCES [1] Izquierdo L. & Sendra J.M Citrus fruits: Composition and Characterization. In: Caballero B., Trugo L. & Finglas P. (Eds.). Encyclopedia of Food Sciences and Nutrition (Vol. 2). Academic Press, Oxford, UK. [2] Diplock, A.T Antioxidants and Disease Prevention. Molecular Aspects of Medicine, 15, [3] Faulks M. & Southon S Carotenoids, Metabolism and Disease. In: Wildman R.E.C. (Ed.). Handbook of Nutraceuticals and Functional Foods. CRC Press, Florida, USA. [4] Gardner P.T., White T.A.C., McPhail D.B. & Duthie, G.G The Relative Contributions of Vitamin C, Carotenoids and Phenolics to the Antioxidant Potential of Fruit Juices. Food Chemistry, 68, [5] Francis, F.J. (2000). Wiley encyclopedia of food science and technology (Vol. 4, pp ). Wiley- Interscience, USA. [6] Anagnostopoulou M.A., Kelafas P., Papageorgiou V.P., Assimopoulou A.N. & Boskou D Radical Scavenging Activity of Various Extracts and Fractions of Sweet Orange Peel (Citrus sinensis). Food Chemistry, 94, [7] Wang H., Cao G.H., & Prior R.L Total Antioxidant Capacity of Fruits. Journal of Agruculture and Food Chemistry, 44, [8] Ersus S. & Cam M Determination of Organic Acids, Total Phenolic Content, and Antioxidant Capacity of Sour Citrus Aurantium Fruits. Chemistry of Natural Compounds, 43 (5), [9] Yuan-Chuen W., Yueh-Chueh C. & Yu-Hua K Quantitation of Bioactive Compounds in Citrus Fruits Cultivated in Taiwan. Food Chemistry, 102, [10] Yuan-Chuen W., Yueh-Chueh C. & Hsing-Wen H The Flavonoid, Carotenoid and Pectin Content in Peels of Citrus Cultivated in Taiwan. Food Chemistry, 106, [11] Vinson J.A., Su X., Zubik L. & Bose P Phenol Antioxidant Quantity and Quality in Foods: Fruits. Journal of Agricultural and Food Chemistry, 49, [12] Waterhouse L. (2002). Determination of Total Phenolics by Folin-Ciocalteau Colorimetry. In Wrolstad R. E., Acree T. E., An H., Decker E. A., Penner M. H., Reid D.S., Schwartz S.J., Shoemaker C. F. & Sporns P. (Eds.). Current Protocols in Food Analytical Chemistry. John Wiley & Sons, Inc. [13] Gillespie K.M. & Ainsworth E.A Measurement of Reduced, Oxidized and Total Ascorbate Content in Plants. Nature Protocols, 2 (4), [14] Bechoff A., Westby A., Owori C., Menya G., Dhuique-Mayer C., Dufourc D.& Tomlinsa K Effect of Drying and Storage on the Degradation of Total Carotenoids in Orange-Fleshed Sweetpotato Cultivars. Journal of the Science of Food and Agriculture, 90, [15] Rodriguez-Amaya D.B. & Kimura M HarvestPlus Handbook for Carotenoid Analysis (pp ). HarvestPlus, USA. [16] Tabart J., Kevers C., Pinc J., Defraigne J.O. & Dommes J Comparative Antioxidant Capacities of Phenolic Compounds Measured by Various Test. Food Chemistry, 113, [17] Jimenez-Alvarez D., Giufrida A., Vanrobaeys F., Golay P.A., Cotting C., Lardeau A. & Keely B.J High- Throughput Methods to Asses Lipophilic and Hydrophilic Antioxidant Capacity of Food Extracts in Vitro. Journal of Agricultural and Food Chemistry, 56, [18] Sun J., Chu Y.F., Wu X., & Liu R.H Antioxidant and Antiproliferative Activities of Common Fruits. Journal of Agricultural and Food Chemistry, 50, [19] USDA National Nutrient Database for Standard Reference. [20] U.S. Department of Agriculture, Agricultural Research Service Oxygen Radical Absorbance Capacity (ORAC) of Selected Foods, Release 2. Nutrient Data Laboratory Home Page:

6