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Czech J. Food Sci., 36, 2018 (3): 233–238 Food Analysis, Food Quality and Nutrition https://doi.org/10.17221/211/2017-CJFS

Determination of and Total Contents in Commercial Apple Juices

Leposava PAVUN 1, Snežana USKOKOVIĆ-MARKOVIĆ 1*, Milena JELIKIĆ-STANKOV 1, Daniela ĐIKANOVIĆ 2 and Predrag ĐURĐEVIĆ 3

1Faculty of Pharmacy and 2Institute for Multidisciplinary Researches, University of Belgrade, Belgrade, Serbia; 3Faculty of Science, University of Kragujevac, Kragujevac, Serbia *Corresponding author: [email protected]

Abstract

Pavun L., Đurđević P., Jelikić-Stankov M., Đikanović D., Uskoković-Marković S. (2018): Determination of flavo- noids and total polyphenol contents in commercial apple juices. Czech J. Food Sci., 36: 233–238.

We propose a sensitive and selective spectrofluorimetric method for the determination of flavonoids as expressed in ‘ equivalent’ in apple juices. The method is based on the strong emission of the aluminium(III)-quercetin complex at 480 nm with excitation at 420 nm, and it is successfully applied for the determination of flavonoids in commercial apple juices and compared with results obtained in reference spectrophotometric determination. The content in commercial apple juices was found to range from 5.53 to 15.55 mg/l quercetin equivalent. The very good agreement between the two methods indicates the suitability of the proposed spectrofluorimetric method for the precise and accurate determination of flavonoids. In addition, the total polyphenol content was determined spectrophotometrically using the Folin-Ciocalteu (FC) method and the antioxidative activity of the tested juices was tested in a DPPH assay and these values were correlated with each other. The obtained profiles of compounds with anti- oxidative ability lead us to conclude that fruit juice labels based only on fruit % might sometimes misinform consumers.

Keywords: aluminium; antioxidant activity; quercetin; spectrofluorimetry; spectrophotometry

Apples and apple juices are traditionally regarded Apples without skin contain less than half the as health foods all over the world. The marked anti- amount of quercetin, the most abundant apple oxidant ability of phenolics from apples against free flavonoid, harboured by whole apples (Persic et al. radicals is thought to account for the positive effects 2017). After cranberries, apples are the fruits with the of apple and apple juices on human health (Hyson second highest level of antioxidant activity. Apples also 2011). Numerous bioactive properties of rank second in total concentration of polyphenolic and flavonoids (anti-, decrease of platelet ag- compounds, and perhaps more importantly, apples gregation, decrease of levels and lowered have the highest proportion of free polyphenols risk of cardiovascular diseases) have been described compared to other fruits (Sun et al. 2002). Because (Procházková et al. 2011; Kay et al. 2012; Russo et the apple peels contain more antioxidant compounds al. 2012; Bubols et al. 2013). Polyphenolic compounds than flesh, especially quercetin, apple peels may have can be a major determinant of the antioxidant poten- higher antioxidant activity and higher bioactivity tial of foods (Parr & Bolwell 2000). Modifying the than the apple flesh. polyphenol content or profile could therefore bolster Moreover, some polyphenolic compounds might be natural sources of antioxidants. lost, and others will increase during juice production,

Supported by the Ministry for Science and Environmental Protection of the Republic of Serbia, Projects No. 172016, 172043 and 173017. 233 Food Analysis, Food Quality and Nutrition Czech J. Food Sci., 36, 2018 (3): 233–238

https://doi.org/10.17221/211/2017-CJFS packaging and storage. Apple polyphenols have been Ciocalteu (FC) reagent, anhydrous sodium carbonate, found to bind to cell wall material, which could , 1,1-diphenly-2-picrylhydrazyl (DPPH), lead to decreased levels of polyphenolic compounds quercetin-dihydrate, aluminium-nitrate and potassium in apple juices (Renard et al. 2001). Processing acetate (all Sigma-Aldrich, USA); , ethanol, of apples has been found to affect the content of and acetic acid (Merck, Germany). bioactive substances in apple juice (Van der Sluis Acetate buffers in 70% (v/v) methanol prepared et al. 2002). Guyot et al. (2003) found that only 42% according to Perrin and Dempsey (1974) were used of total polyphenols were extracted into the juice, for all spectrofluorimetric measurements. leaving over half the total polyphenols in the apple Instruments. The spectrophotometric measure- pomace. However, in modern lifestyles fruit juices ments were performed on a Beckman DU-650 spec- are consumed rather than fresh fruits. Numerous trophotometer, using 1 cm quartz cells. studies have focused on the effects of quercetin as spectra were collected using a a major flavonoid in apples. Fluorolog-3 spectrofluorimeter (Jobin Yvon Horiba, Quercetin is a flavonoid derived from flavone, a France) equipped with a 450 W xenon lamp and a natural polyphenolic antioxidant. Quercetin occurs photomultiplier tube and using 1 cm optical path in nature as easily hydrolysable (quercetin- length quartz cuvettes. The slits on the excitation and 3-galactoside, quercetin-3-, and quercetin- emission beams were both set at 5 nm. The spectra 3-rhamnoside). It is known that the formation of were corrected for the dark counts. In each meas- quercetin aglycon by hydrolysis of quercetin urement, three scans with one-second-integration results in an increase in activity, because the aglycon time were averaged. The emission spectrum of the has approximately two-fold higher antioxidant activity solvent was subtracted. All measurements were per- compared to that of average quercetin glycoside formed at 25°C. (Van der Sluis et al. 2000). Measurements of pH were carried out using a Since quercetin is one of the most common Mettler Toledo MP120 pH meter (± 0.01 pH unit) and one of the most powerful antioxidants, it is equipped with a combination glass electrode. important to have a simple, precise and accurate Spectrophotometric determination of flavonoids. method for the determination of quercetin in different Quercetin was used to construct a calibration curve samples. Several methods have been proposed in the (standard solutions 12.5, 25, 50, 80, and 100 mg/ml literature to determine quercetin in apple and tomato in 80% ethanol (v/v)). The standard solutions or juice, fruits, wines, , serums and pharmaceuticals. samples of juice (0.5 ml) were mixed with 1.5 ml 95%

In spectroscopic methods, the concentration of ethanol (v/v), 0.1 ml 10% Al(NO3)3, 0.1 ml of 1 mol/l flavonoids is expressed as quercetin equivalent (QE) potassium acetate and 2.8 ml deionised water. In the and quercetin is used as a standard for calibration blank, the volume of 10% Al(NO3)3 was substituted by curves. The majority of methods required some pre- the same volume of deionised water. After incubation treatment of samples, such as solid-phase extraction at room temperature for 30 min, the absorbance (Andersen & Markham 2006). of the reaction mixture was measured at 415 nm. In this study, we determined the polyphenolic Flavonoids were expressed as QE. compounds, flavonoids and antioxidative capacities Spectrofluorimetric determination of flavonoids. of commercially available apple juices. We have The stock solution of quercetin was prepared by developed a sensitive spectrofluorimetric method dissolving quercetin-dihydrate in 70% methanol (v/v) based on fluorescence of a complex formed between and was stored in a refrigerator. A working solution aluminium(III)-quercetin (AlQ) with flavonoids (5 × 10–6 mol/l) of the AlQ complex was prepared present in apple juices, and we suggest that this by dilution of the stock solutions of 1 × 10–3 mol/l –4 method is easier and more suitable in comparison Al(NO3)3 and quercetin-dihydrate (1 × 10 mol/l). to the reference spectrophotometric method. An 0.05-ml aliquot of the tested apple juice was pipetted into a 10-ml volumetric flask, followed by –5 0.1 ml of Al(NO3)3, 1 × 10 mol/l, and the flask was MATERIAL AND METHODS filled to the top with acetate buffer pH 3.3 prepared with methanol. The fluorescence intensities of

Reagents. All solutions were prepared in deionised prepared solutions were measured at λex = 420 nm water. The following chemicals were used: Folin- and λem = 480 nm. Calibration curves obtained using 234 Czech J. Food Sci., 36, 2018 (3): 233–238 Food Analysis, Food Quality and Nutrition https://doi.org/10.17221/211/2017-CJFS

Origin 7 software were used to calculate the quercetin Inhibition of free radical by DPPH was calculated content in the tested apple juices. Flavonoid levels from Equation 3: were also expressed as QE.

The obtained data were used to calculate analytical I(%) = [(Ac – As)/Ac] × 100 (3) validation parameters for both spectrofluorimetric where: A – absorbance of the control mixture (containing all and spectrophotometric methods according to c reagents except the test compound); A – absorbance of the Miller and Miller (2005) and ICH Guideline s prepared sample or standard Q2B (1997). The limit of detection (LOD) was calculated by establishing the minimum level at which quercetin can be detected, according to the followingformula: RESULTS AND DISCUSSION

Spectrofluorometric versus spectrophotometric LOD = 3.3 Sb/a (1) determination of flavonoids. A linear dependence where: Sb – standard deviation in intercept; a – slope of cali- of the absorbance of the AlQ complex (A at λ = bration line 415 nm) on the concentration of QE (c) was observed in the interval 5–100 mg/l. For spectrophotometric The limit of quantification (LOQ) was determined determination, the calibration curve equation A = using the following formula: (0.00616 ± 0.0001) c + (0.00137 ± 0.0005) was used. The good linearity of the calibration curve and small LOQ = 10 Sb/a (2) scatter of experimental points resulted in a high coefficient of determination, R2 = 0.99922. The limit Determination of total polyphenol content (TPC) of detection (LOD) was 0.3 mg/l, while the limit of using Folin-Ciocalteu (FC) method. Total polyphenol quantification (LOQ) was 0.9 mg/l. content (TPC) was determined spectrophotometri- The spectrofluorimetric determination of the con- cally according to a slightly modified FC method tent of flavonoids as QE was adapted from the pro- (Singelton & Rossi Jr. 1965; Singh et al. 2007). cedure reported by Pavun et al. (2014) for quercetin One millilitre of the sample was pipetted into a 25-ml determination. Spectrofluorimetric determination volumetric flask containing 9 ml of water, and 1 ml is based on a calibration curve (I = (1.47 ± 0.01) × of FC reagent was added. Ten millilitres of Na CO F 2 3 c + (0.56 ± 0.04)), where the intensity of fluo- solution were added after 5 min and the remainder Querc rescence (%) (λ = 420 nm and λ = 480 nm) is of the volume was filled with deionised water. During ex em linearly dependent on the quercetin concentration oxidation with FC reagent phenolic compounds are (expressed as mg/l). A linear dependence of the reduced to blue-coloured molybdenum and tungsten fluorescence intensity of the complex was observed oxides. After 90 min, the absorbance of the solution in the concentration range 1.5–60.5 mg/l quercetin. was measured at λ = 765 nm against a blank sample The LOD was 0.09 mg/l, while the LOQ was consisting of 10 ml of deionised water instead of the 0.27 mg/l by the spectrofluorimetric method, much diluted sample. The measurements were compared lower values than those obtained using the spectro- to a standard curve of prepared gallic acid solutions photometric method. The high accuracy and repeat- (25–500 mg/l) and expressed as mg of gallic acid ability of the method are demonstrated by the low SD equivalents (GAE) per 1 l ± SD of apple juice. All values. As we have reported in our previous work, measurements were performed in triplicate. C does not interfere with spectrofluorimetric DPPH photometric assay. The free radical- determination of quercetin under the established scavenging activity of the apple juices was evaluated experimental parameters (Pavun et al. 2014). using the stable DPPH radical (Gardner et al. 2000). The proposed spectrofluorimetric method has a The hydrogen atom or electron donation abilities of more than satisfactory sensitivity for the routine the tested juices and pure quercetin were measured determination of flavonoid content in apple juices. from the bleaching of the purple-coloured methanol . solution of DPPH. One millilitre from each tested Flavonoid content in commercial apple juices –4 The results of the spectrofluorimetric and spectro- sample was added to 4 ml of 1 × 10 mol/l methanol photometric determination of flavonoids in six ap- solution of DPPH. After 60 min, the absorbance was ple juices available on the Serbian market are given recorded at 517 nm. 235 Food Analysis, Food Quality and Nutrition Czech J. Food Sci., 36, 2018 (3): 233–238

https://doi.org/10.17221/211/2017-CJFS

Table 1. Determination of favonoid content in commer- 0.00024) was used. The good linearity of the calibra- cial apple juices tion curve and small scatter of experimental points resulted in a high coefficient of determination, R2 = Spectrophotometric Spectrofuorimetric Samples 0.99998. The LOD was 0.2 mg/l, while the LOQ was (mg/l) 0.6 mg/l. The tested apple juices did not harbour 1A 5.53 ± 0.74 5.643 ± 0.004 the declared contents of polyphenolics. The results 2A 8.81 ± 0.72 8.761 ± 0.003 obtained in this work could only be compared with 3A 5.43 ± 0.75 5.589 ± 0.003 already published data (Gliszczynska-Swiglo & 1B 9.84 ± 0.80 9.772 ± 0.003 Tyrakowska 2003) and are comparable to the avail- 2B 15.55 ± 0.85 15.602 ± 0.004 able literature data concerning polyphenol content 3B 11.60 ± 0.62 11.520 ± 0.004 in apple juices. DPPH photometric assay. The antioxidant activi- A – 50% of fruit nectar content; B – 100% of fruit ties of the commercial juices were determined using the DPPH method. All tested juices exhibited strong in Table 1. The samples were chosen from three scavenging activity against DPPH radicals, which different producers, labelled as brands 1, 2, and 3, ranged from 32.6% to 88.26% (Table 2). respectively. According to the fruit juice content, Correlation between antioxidant capacity and samples were marked as A (50% fruit nectar) or B TPC. Numerous examples of the application of the FC (100% of fruit juice). assay to characterise natural products may be found in The flavonoids content in commercial apple juices the literature. In most cases, TPC determined by this was found to range from 5.53 to 15.55 mg/l QE. The method are correlated with the antioxidant capaci- results obtained using both the spectrophotomet- ties, confirming the value of the FC test (Roginsky ric and spectrofluorimetric methods were in good & Lissi 2005). A variety of commercial apple juices agreement. are available on the market, and TPC and antioxidant The amounts of bioactive compounds in fruit, in- activity vary significantly among different brands. cluding flavonoids, depend on many factors, Sometimes juices labelled as consisting of 50% fruit including geographic region, climate, soil proper- nectar content have almost the same quality as those ties, type of cultivar, growing season, harvest date, declared as 100% fruit juice. In this work, a high storage, low-dose irradiation and other conditions correlation was obtained between the TPC and an- (Albach et al. 1981; Patil et al. 2004); therefore, tioxidant capacities. The TPC and the antioxidant the content of flavonoids as well as that of other activity both indicate product quality with respect to compounds, can vary greatly. its biological properties, and both assays should be The tested juices did not have the declared con- applied for the quality control of commercial apple tents of flavonoids. juices. The TPC in commercial apple juices were Determination of TPC by FC method. Polyphe- found to range from 95.37 to 441.91 mg/l GAE. In nolic contents of plant foods depend on a number order to test the correlation between the TPC and of intrinsic (genus, species, cultivars) and extrinsic antioxidant activity of tested samples, correlation (agronomic, environmental, handling, and storage) coefficients were calculated with the SPSS program factors (Tomás-Barberán & Espín 2001). Phenolic content in apple is also influenced by physiological Table 2. Total polyphenol content (TPC) and inhibition disorders in fruits such as watercore and bitter pit activity in commercial apple juices (Zupan et al. 2013, 2016). Phenolic content, which is currently considered Samples TPC (mg/l) DPPH (I%) as a measure of product quality, varied markedly 1A 135.7 37.50 according to juice processing technology and among 2A 191.4 85.02 the different brands of apple juices analysed in this 3A 95.4 32.60 study. A linear dependence of the absorbance (A at 1B 255.9 87.85 λ = 765 nm) on the concentration of GAE (c) was 2B 441.9 88.26 observed in the interval 25–500 mg/l. For the spec- 3B 264.0 86.25 trophotometric determination the standard plot with equation A = (0.00439 ± 0.00001)*c + (0.0036 ± A – 50% of fruit nectar content; B – 100% of fruit

236 Czech J. Food Sci., 36, 2018 (3): 233–238 Food Analysis, Food Quality and Nutrition https://doi.org/10.17221/211/2017-CJFS

(version 18). For all brand A juices, the obtained Andersen O.M., Markham K.R. (2006): Flavonoids: Chem- correlation between the TPC and the antioxidant istry, Biochemistry and Applications. Boca Raton, Taylor activity of tested samples was high and significant & Frencis: 1–36. (R2 = 0.94). For brand B juices, the obtained correla- Awad M.A., de Jager A., van Westing L.M. (2000): Flavonoid tion between the TPC and the antioxidant activity and levels in apple fruit: characteriza- of tested samples was medium-high and significant tion of variation. Scientia Horticulturae, 83: 249–263. (R2 = 0.728). The possible differences could be due Bubols G., Vianna D., Medina-Remón A., Von Poser G., to production process, apple species and the content Lamuela-Raventos R., Eifler-Lima V., Garcia S. (2013): of other compounds such as vitamin C, which may The antioxidant activity of and flavonoids. influence the antioxidant activity. Evaluation of the Mini-Reviews in Medicinal Chemistry, 13: 318–334. A and B brands together indicated a high correla- Gliszczynska-Swiglo A., Tyrakowska B. (2003): Quality of tion between antioxidant activity and fruit content commercial apple juices evaluated on the basis of the (R2 = 0.854). polyphenol content and the TEAC antioxidant activity. The concentration and type of phenolic compounds Journal of Food Science, 68: 1844–1849. varies widely in different parts of the apple fruit, such Guyot S., Marnet N., Sanoner P., Drilleau J. (2003): Variabil- as the skin, core, seeds and the cortex (Awad et al. ity of the polyphenolic composition of cider apple (Malus 2000). As different phenolics are present in differ- domestica) fruits and juices. Journal of Agricultural Food ent parts of the fruit, the concentration of phenolics Chemistry; 51: 6240–6240. in juice, puree and processed apple products may Gardner P., White T., McPhail D., Duthie G. (2000): The vary (Markowski & Płocharski 2006). With this relative contributions of vitamin C, and in mind as well as the many other factors that may phenolics to the antioxidant potential of fruit juices. Food affect the antioxidant activity of commercial fruit Chemistry, 68: 471–474. products, it is clear that sometimes juices with less Hyson D. (2011): A comprehensive review of apples and ap- fruit nectar may have almost the same positive health ple components and their relationship to human health. benefits as juices with higher content of fruits. The Advances in Nutrition, 2: 408–420. antioxidative ability of fruit juices is dependent on Kay C., Hooper L., Kroon P., Rimm E., Cassidy A. (2012): multiple factors, and fruit juice labels stating only the Relative impact of flavonoid composition, dose and struc- fruit % can sometimes misinform the consumers, by ture on vascular function: a systematic review of ran- implying, for example, that a product with a two-fold domised controlled trials of flavonoid-rich food products. higher % of fruit is twice as good for human health. Molecular Nutrition Food Research, 56: 1605–1616. Markowski J., Płocharski W. (2006): Determination of phe- nolic compounds in apples and processed apple products. CONCLUSIONS Journal of Fruit Ornamental Plant Research, 14: 133–142. Miller J.N., Miller J.C. (2005): Statistics and Chemomet- In this work, we developed a simple spectrofluori- rics for Analytical Chemistry. 5th Ed. London, Pearson metric method for determination of quercetin in apple Education. juice after its complexation with aluminium(III) ion. Parr A., Bolwell G. (2000): in the plant and in man. The method provides good accuracy and precision The potential for possible nutritional enhancement of the and may be used for routine analysis. Commercial diet by modifying the phenols content or profile. Journal apple juices were tested for TPC and antioxidative of the Science of Food and Agriculture, 80: 985–1012. activity. The obtained profiles of compounds with an- Patil B., Vanamala J., Hallman G. (2004): Irradiation and tioxidative ability in commercial apple juices resulted storage influence on bioactive components and quality of in the conclusion that fruit juice labels based only early and late season ‘Rio Red’ (Citrus paradisi on fruit % could sometimes misinform consumers. Macf). Postharvest Biology and Technology, 34: 53–64. Pavun L., Đurđević P., Jelikić-Stankov M., Đikanović D., References Ćirić A., Uskoković-Marković S., (2014): Spectrofluori- metric determination of quercetin in pharmaceutical Albach R.F., Redman G.H., Cruse R.R. (1981): Annual and dosage forms. Macedonian Journal of Chemistry and seasonal changes in concentration of ruby red Chemical Engineering, 33: 209–215. grapefruit juice. Journal of Agricultural and Food Chem- Perrin D., Dempsey B. (1974): Buffers for pH and Metal Ion istry, 29: 808–811. Control. London, Chapman and Hall: 77–94. 237 Food Analysis, Food Quality and Nutrition Czech J. Food Sci., 36, 2018 (3): 233–238

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