APTEFF, 38, UDC: : : DOI: /APT C BIBLID: (2007) 38, Original scientific paper

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1 APTEFF, 38, UDC: : : DI: /APT C BIBLID: (2007) 38, riginal scientific paper SLID-PHASE EXTRACTIN F ANTIXIDANT CMPUNDS FRM CMMERCIAL CRANBERRY EXTRACT AND ITS ANTIRADICAL ACTIVITY Vesna T. Tumbas, Sonja M. ilas, Jasna M. anadanovi -Brunet, Gordana S. etkovi and Sla ana M. Savatovi This study is concerned with the fractionation and determination of major antioxidant compounds (phenols, flavonoids, anthocyanins and vitamin C) in commercial cranberry extract. The total content of phenolics, flavonoids and total and monomers of anthocyanins, determined spectrophotometrically, was 1.67 mg/g, 0.41 mg/g, 5.12 mg/g and 3.32 mg/g. The content of vitamin C, determined volumetrically, was mg/g. Commercial cranberry extract was dissolved in 80 % acetone and the solution was fractionated using solid phase extraction (SPE) in order to abstract vitamin C, neutral and acidic phenols. The free radical scavenging activity of the cranberry extract and its fractions was investigated on stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) and reactive hydroxyl radicals employing electron spin resonance (ESR) spectroscopy. The most effective fractions were those containing vitamin C (AA DPPH = AA H =100%), neutral (AA DPPH =89.50% and AA H =43.11%) and acidic (AA DPPH =83.98% and AA H =38.58%) phenols. The presence of vitamin C, abstracted from cranberry extract, was determined by Fe(III)-mediated ascorbate oxidation which yields characteristic ESR doublet spectrum of ascorbyl radical. KEYWRDS: Cranberry extract, phenols, vitamin C, anthocyanins, SPE, free radical scavenging activity, ESR INTRDUCTIN The reactive oxygen species and free radicals are associated with certain diseases such as cancer, atherosclerosis, and coronary heart diseases, which are so-called lifestylerelated diseases. Hence, intake of constituents from different natural sources that have radical scavenging activities (antioxidants) is considered to be important in preventing such diseases. The addition of antioxidants has become popular as means of increasing the shelf life of food, cosmetic and pharmaceutical products and to reduce wastage and quality losses by inhibiting and delaying oxidation. Vesna T. Tumbas, M.Sc., Assist., Dr. Sonja M. ilas, Prof., Dr. Jasna M. anadanovi -Brunet, Assoc. Prof., Dr. Gordana S. etkovi, Assoc. Prof., Sla ana M. Savatovi, M.Sc.University of Novi Sad, Faculty of Technology, Novi Sad, Bulevar Cara Lazara 1, Serbia 157

2 Many antioxidant compounds, both synthetic and natural, have been examined in attempts to control oxidation in different products. However, concern about the safety of the commonly used synthetic antioxidants had led to increasing interest in natural alternatives which occur in plants as secondary metabolites (1, 2). Natural antioxidants are primarily plant polyphenolic compounds which are multifunctional and can act as reducing agents (free radical terminators), metal chelators, and singlet oxygen quenchers. The most common plant phenolic antioxidants include flavonoids, benzoic and cinnamic acid derivatives, coumarins, and tocopherols (3). Because of the high content of vitamin C and phenolic compounds, many cultivars and native species of berries are good source of natural antioxidants (4). Cranberry (Vaccinium macrocarpon, Ericaceae) is recognized as a good source of dietary phenolic acids and flavonoids, including anthocyanins, flavonol glycosides and proanthocyanidins (5,6), as well as vitamin C (7). The protective properties of cranberry and its products include antioxidant, anticancer and cardiovascular-protective activities (8). In this study, the content of phenols, flavonoids and anthocyanins was determined spectrophotometrically and vitamin C volumetrically in commercial powdered cranberry extract. A solid-phase extraction (SPE) with CHRMABND PS-H and CHRMA- BND C 18 cartridges was used to separate vitamin C and fractionate phenolic compounds from the cranberry extract. The antioxidant activity of cranberry extract and the obtained fractions on stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical and reactive hydroxyl radical formed in the Fenton reaction was evaluated using electron spin resonance (ESR) spectroscopy. EXPERIMENTAL Methanol, hydrogen peroxide and acetic acid were obtained from Zorka Šabac (Serbia). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 5,5-dimethyl-1-pyroline-N-oxide (DMP), Folin-Ciocalteu reagent, chlorogenic acid and rutin were purchased from Sigma Chemicals Co. (USA). These chemicals were of analytical reagent grade. ther chemicals and solvents used were of the highest analytical grade. Commercial powdered cranberry extract was purchased from herbal tea and dietetic production factory Fructus, Ba ka Palanka, Serbia. Total phenolics. Total phenolics in extract were determined spectrophotometrically using the Folin-Ciocalteu reagent and the results are expressed as mg chlorogenic acid per g dry weight (9). Total flavonoids. Total flavonoids in extract (expressed as mg rutin per g dry weight) were estimated spectrophotometrically according to Markham (10). Total anthocyanins. The total anthocyanins content in extract was estimated spectrophotometrically using the ph differential method (11). Anthocyanins were quantified as cyanidin-3-glucoside using an extinction coefficient of and resultant values were expressed in terms of mg anthocyanins per g dry weight. Vitamin C. The vitamin C content in extract was analyzed volumetrically using Tillman s method (12) and the results are expressed as mg per g dry weight. Extraction and fractionation of antioxidant compounds. Cranberry extract (5 g) was dissolved in 20 ml of 80% acetone, filtered and passed through two solid-phase extraction 158

3 cartridges, first through a CHRMABND PS-H to remove vitamin C, and then through a CHRMABND C 18 to fractionate neutral and acidic phenolic compounds. CHRMABND PS-H cartridge was preconditioned by sequentially passing 3 ml of methanol and 3 ml of distilled water. The sample was adjusted to ph 11 with diluted NaH solution, loaded onto column (Fr1) and washed first with 3 ml of 10% NH 4 H (Fr2), then with 3 ml methanol/water (20:80, v/v) (Fr3). The adsorbed vitamin C was eluted with 2 x 2 ml methanol/acetone (1:1, v/v) with 5% acetic acid (Fr4). The CHRMABND C 18 column was preconditioned for isolation of neutral flavonoids and phenolics, by passing 8 ml of methanol and 4 ml of ph 7 distilled water. The effluent portion (Fr1) was adjusted to ph 7, and passed through the preconditioned cartridge. The sample was loaded onto the preconditioned column (Fr5), washed with 10 ml of ph 7 distilled water (Fr6), and eluted with 12 ml of methanol (Fr7). For the isolation of acidic phenolics the CHRMABND C 18 cartridge was preconditioned by passing 8 ml of methanol and 4 ml of 0.01 M HCl. The effluent portion (Fr5) was adjusted to ph 2 with 2 M HCl, passed through the preconditioned CHRMABND C 18 cartridge (Fr8), washed with 5 ml of 0.1 M HCl (Fr9), and eluted with 12 ml of methanol (Fr10). The cranberry extract and its fractions (Fr2, Fr3, Fr4, Fr6, Fr7, Fr8, Fr9, and Fr10) were evaporated to dryness under reduced pressure. The obtained weights of fractions were: m Fr2 = g; m Fr3 = g; m Fr4 = g; m Fr6 = g; m Fr7 = g; m Fr8 = g; m Fr9 = g; m Fr10 = g. Fractions were dissolved in 80% acetone in order to obtain the final concentration of 100 mg/ml. ESR measurements. All ESR spectra were recorded on an ESR spectrometer Bruker 300E (Rheinstetten, Germany). DPPH radical assay. Blank probe was obtained by mixing 600 l 0.4 mm methanolic solution of DPPH and 200 l of 80% acetone. A volume of 10 l of 100 mg/ml of cranberry extract or its fractions dissolved in 80% acetone was added to a mixture of 190 l of 80% acetone and 600 l of 0.4 mm methanolic solution of DPPH radical (probe). The final concentration of investigated cranberry extract and its fractions was 1.25 mg/ml. After that the mixture was stirred for 2 min and transferred to a quartz flat cell ER-160FT. ESR measurements were performed under the following conditions: field modulation 100 khz, modulation amplitude G, receiver gain , time constant ms, conversion time ms, center field G, sweep width G, x- band frequency 9.45 GHz, power 7.96 mw, temperature 23 C. The antioxidative activity on DPPH radicals (AA DPPH ) of the extract was defined as: AA DPPH =100 (h o -h x )/h o [%] where h o and h x are the height of the second peak in the ESR spectrum of DPPH radicals of the blank and the probe, respectively. Hydroxyl radical assay. The Fenton reaction was conducted by mixing 0.2 ml 80% acetone, 0.2 ml 10mM H 2 2, 0.2 ml 10 mm FeCl 2 x4h 2 and 0.2 ml 0.3 M DMP as a spin trap (blank). The influence of cranberry extract and its fractions on the formation and transformation of hydroxyl radicals was investigated by adding the extract to the Fenton reaction system in the final concentrations 1.25 mg/ml. ESR spectra were recorded after 5 min, with the following spectrometer settings: field modulation 100 khz, 159

4 modulation amplitude G, receiver gain , x-band frequency 9.64 GHz, power 20 mw, temperature 23 C. The antioxidative activity on hydroxyl radicals (AA H ) of the extract was defined as: AA H =100 (h o -h x )/h o [%] where h o and h x are the height of the second peak in the ESR spectrum of DMP-H spin adduct of the blank and the probe, respectively. Ascorbyl radical detection. xidation of ascorbate was initiated by the addition of 250 l of 10 M Fe(III)-EDTA in 20 mm HEPES buffer (ph 7,2) at the 250 l of Fr4. ESR measurements of ascorbyl radical were performed after 3.5 min of iron addition to Fr4, at room temperature with x-band frequency 9.42 GHz, field modulation 100 khz, time constant ms, conversion time ms, center field G, sweep width G, power 10 mw, modulation amplitude 0.25 G and receiver gain RESULTS AND DISCUSSIN The total content of phenolics, flavonoids, and anthocyanins, as well as the content of vitamin C in cranberry extract are shown in Table 1. Table 1. The total content of phenolics, flavonoids, anthocyanins, and vitamin C in cranberry extract ANTIXIDANT CMPUNDS TTAL CNTENT (mg/g) Phenolics 1.67 ± 0.07 Flavonoids 0.41 ± 0.02 Anthocyanins 5.12 ± 0.25 Monomers of anthocyanins 3.32 ± 0.15 Vitamin C ± 4.32 The results in Table 1 indicate that cranberry extract contains very high amount of vitamin C ( mg/g). n the other hand, flavonoids are present at the lowest level (0.41 mg/g). Also, significant contents of anthocyanins and their monomers are present in cranberry extract (5.12 mg/g and 3.32 mg/g). Solid-phase extraction was employed to separate and fractionate vitamin C and phenolic compounds from the cranberry extract. Free radical scavenging activities of cranberry extract and its fractions were tested in DPPH and hydroxyl free radical system (Fig. 1) 160

5 H radicals DPPH radicals AA (%) Extract Fr 2 Fr 3 Fr 4 Fr 6 Fr 7 Fr 8 Fr 9 Fr 10 Fractions Fig. 1. Free radical scavenging activity of cranberry extract and its fractions As can be seen from the results presented in Fig. 1, the most effective cranberry extract fraction in both assays was Fr4. The concentration of 1.25 mg/ml caused total reduction of DPPH and H radicals (AA DPPH =AA H =100%). Fractions Fr7, Fr10 and Fr8 also showed marked antioxidant activity in both free radical systems. Generally, these activities are higher on DPPH free radicals (89.50%, 83.98% and 71.82%, respectively) than on hydroxyl radicals (43.11%, 38.58% and 29.53%, respectively). The probable causes of these differences are: (I) the extracts are very complex mixtures of a number of different compounds with distinct polarity, sometimes showing synergistic actions, (II) the kinetic constants of reactions between hydroxyl radical and polyphenols are generally higher than for the reaction between DPPH radicals and polyphenols, (III) the different system and spin traps are used to measure AA DPPH and AA H (13). Higher rates of free radical scavenging activity of the fractions Fr4, Fr7, Fr8 and Fr10 are probably due to the higher levels of all the major contributors to the antioxidant activity. Fr4 represents vitamin C abstracted from cranberry extract. Vitamin C (ascorbate, AscH ) is a water soluble donor, chain-breaking antioxidant and reacts with radicals very rapidly by the following mechanism: AscH + R Asc + RH forming a very stable, resonance stabilized, ascorbyl radical (Asc - ): H H 161

6 Vitamin C reduces almost two DPPH molecules (14): CH 2 H CH 2 H CH 2 H HHC DPPH HHC DPPH HHC H H H DPPH-H DPPH-H H H H L-ascorbic acid Semi-dehydroascorbic acid Dehydroascorbic acid This can explain the dominant antioxidative activity of Fr4. The presence of vitamin C in Fr4 was confirmed by Fe(III)-EDTA-mediated ascorbate oxidation, the formation of ascorbyl radical being detected by electron spin resonance (ESR) spectroscopy: Fe(III)-EDTA + ascorbate Fe(II)-EDTA + ascorbyl radical The representative ESR doublet spectrum characteristic for ascorbyl radical (a H = =1.8 G), obtained in iron-mediated ascorbate oxidation in Fr4, is shown in Fig Fig. 3. ESR spectrum of ascorbyl radical obtained in iron-mediated ascorbate oxidation in Fr4 Fr7 contains neutral and Fr10 acidic phenolics extracted from cranberry extract. Chen et al. reported that neutral fraction of cranberry juice has (+)-catechin, myricetin and quercetin and the acidic fraction chlorogenic acid, p-coumaric acid, benzoic acid and 3,4,5-trimethoxycinnamic acid (6). Natural phytochemicals such as phenolic compounds commonly involve an aromatic ring as a part of the molecular structure, with one or more hydroxyl groups. The antioxidative activity can therefore be attributed to the hydrogendonating ability and direct scavenging activity of the investigated compounds (15). According to the literature, one DPPH molecule forms complex with one aryl radical formed from phenolic compounds (16). Fr8 contains other antioxidant compounds which do not include vitamin C, neutral and acidic phenolics. Further investigations should include determination of antioxidant composition in all fractions in order to provide more exact conclusions about the antioxidant mechanisms and interactions between the constituents.

7 CNCLUSIN The analysis of the composition of powdered commercial cranberry extract showed the presence of phenolic compounds (1.669 mg/g), flavonoids ( mg/g), total anthocyanins (5.12 mg/g), anthocyanin monomers (3.32 mg/g) and vitamin C ( mg/g). The fraction containing vitamin C (Fr4) showed highest antioxidant activity (AA DPPH = AA H =100%) in both investigated free radical systems. The presence of vitamin C in Fr4 was confirmed by iron-mediated oxidation of ascorbate and ESR detection of the ascorbyl radical. Significant antioxidant activity was also observed in the presence of neutral (Fr7, AA DPPH = 89.50% and AA H = 43.11%), acidic (Fr10, AA DPPH = 83.98% and AA H = 38.58%) phenols, and the remaining fraction (Fr8, AA DPPH = 71.82% and AA H = 29.53%). This study suggests that cranberry is a very rich source of major antioxidant compounds and could play an important role in improving antioxidant intake in the human diet. REFERENCES 1. Harborne, J.B.: Plant Phenolics, in Encyclopaedia of Plant Physiology, Vol 8, Secondary Plant Products, Eds. E.A. Bell and B.V. Charlwood, Springer, Berlin (1980) pp Vinson, J.A., Hao, Y., Su, X. and L. Zubik: Phenol antioxidant quantity and quality in foods. J. Agric. Food Chem. 46 (1998) Shahidi, F. and P.K.J.P.D. Wanasundara: Phenolic antioxidants. Critical Rev Food Sci Nutr 32 (1992) Moyer, R.A., K.E. Hummer, C.E. Finn, B. Frei and R.E. Wrolstad: Anthocyanins, Phenols, and Antioxidant Capacity in Diverse Small Fruits: Vaccinium, Rubus and Ribes. J. Agric. Food Chem. 50 (2002) Hakkinen, S.H., M. Heinonen, S. Karenlampi, H. Mykkanen, J. Ruuskanen and R. Torronen: Screening of Selected Flavonoids and Phenolic Acids in 19 Berries. Food Res. Int. 32 (1999) Chen, H., Y. Zuo and Y. Deng: Separation and Determination of Flavonoids and Phenolic Compounds in Cranberry Juice by High-performance Liquid Chromatography. J. Chromatogr. A 913 (2001) Hakkinen, S.H., S. Karenlampi, M. Heinonen, H. Mykkanen, J. Ruuskanen and R. Torronen: Content of Flavonols Quercetin, Myricetin, and Kaempferol in 25 Edible Berries. J. Agric. Food Chem. 47 (1999) Tumbas, V.T., Savatovi, S.M., ilas, S.M., anadanovi -Brunet, J.M., G.S. etkovi : Cranberry a Good Source of Natural Antioxidants. Acta Periodica Tehnologica 37 (2006) Singleton, V.L., rthofer, R. and R.M. Lamuela-Raventos, R.M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods in Enzymology, 299 (1999)

8 10. Markham, K.R.: Methods in Plant Biochemistry, Academic Press, London, UK (1989) pp Cheng, G,W. and P.J. Breen: Activity of Phenylalanine Ammonialyase (PAL) and Concentrations of Anthocyanins and Phenolics in Developing Strawberry Fruits. J. Am. Soc. Hortic. Sci. 116 (1991) JAAC. fficial methods of analysis. Vitamin C (ascorbic acid) in vitamin preparations and juices: 2,6-Dichloroindophenol titrimetric method, Association of fficial Analytical Chemists, Washington, DC (1984) p etkovi, G.S., Djilas, S.M., anadanovi -Brunet, J.M., V.T. Tumbas: Antioxidant properties of marigold extracts, Food Res. Int. 37 (2004) Brand-Williams, W., Cuvelier, M.E., C. Berset: Use of a Free Radical Method to Evaluate Antioxidant Activity, Lebensm. Wiss. U. Technol. 28 (1995) anadanovi -Brunet, J.M., Ðilas, S.M., etkovi, G.S., V.T. Tumbas: Free-Radical Scavenging Activity of Wormwood (Artemisia absinthium L.) Extracts. J. Sci. Food Agric. 85 (2005) etkovi, G.S., Djilas, S.M., anadanovi -Brunet, J.M., V.T. Tumbas: TLC Analysis and Scavenging Activity of Marigold (Calendula officinalis L.) Extracts. Acta Periodica Technologica 34 (2003) ,.,. -,. e - (, 1,67 / ;, 0,41 / ;, 5,12 /, 3,32 / )., -, 121,74 /. 80% (SPE),. 2, (DPPH) (ESR). - (AA DPPH = AA H = 100%), (AA DPPH = 89.50% and AA H = 43.11%) (AA DPPH = 83.98% and AA H = 38.58%). - Fe(III) - ESR. Received 4 September 2007 Accepted 27 September