Identification and Quantification of Phenolic Compounds in Grapes

Transcription

1 Identification and Quantification of Phenolic Compounds in Grapes M. Rivera-Dominguez Centro de Investigación en Alimentación y Desarrollo, Hermosillo, Sonora and Facultad de Ciencias Naturales Universidad Autónoma de Querétaro Juriquilla, 76230, Querétaro Mexico E.M. Yahia Facultad de Ciencias Naturales Universidad Autónoma de Querétaro Juriquilla, 76230, Querétaro Mexico N. Wlodarchak and M. Kushad Department of Natural Resources and Environmental Sciences University of Illinois Urbana, IL USA Keywords: grapes, antioxidants, phytochemicals, phenolic compounds Abstract Grapes are a major source of phenolic compounds. Phenolic compounds play an important role in the quality of grapes and wines, and the antioxidant activity of grapes has been positively correlated with their phenolic composition. This work was focused on the identification of phenolic compounds in the fruit extracts of Norton, also known as Cynthiana grapes (Vitis aestivalis). Identification and quantification were performed using high performance liquid chromatography-mass spectrometry (HPLC-MS TOF) with an electrospray ionization interface in negative mode ion. Detection was carried out at and 320 nm. Cactechin, epicatechin, isoremnetin, siringic acid, caffeic acid, gallic acid, protocatechuic acid, miricetyn, hidroxybenzoic acid, quercetyn, stilbenes, such as resveratrol, pteroestibene, transpiceid, piceatannol and E-viniferin were identified in the extract, and caffeic acid, gallic acid, protocatechuic acid, miricetyn, hidroxybenzoic acid and quercetyn were also quantified. The major concentrations found were of epicatechin, caffeic acid and catechin, followed by protocatechuic acid, hidroxybenzoic acid, gallic miricetyn and quercetyn corresponding to , , , , , , , ug/g dry weight, respectively. INTRODUCTION Phenolic compounds are secondary metabolites, ubiquitous in the plant kingdom, and have shown to exert beneficial influence on human health, including being potent antioxidants (Manach et al., 2004). The antioxidant activity of grapes has been positively correlated with phenolics such as anthocyanins, flavonols, flavan-3-ols, and hydroxybenzoates, among others (Landbo and Meyer, 2001). Grapes are considered one of the major sources of phenolic compounds among different fruits (Maxcheix et al., 1990). The phenolic compounds in Vitis vinifera include phenolic acids, stilbenes, and flavonoids, which include flavonols, flavanols, and anthocyanins, and play an important role in the quality of grapes and wines (Downey et al., 2006). Anthocyanins are directly responsible for the color in grapes and young wines, whereas astringency and structure of wines are related to catechins, and proanthocyanidins, while flavonols contribute to bitterness (Hufnagel et al., 2008). Health is of prime importance in one s life. Consumption of adequate amounts of fruits and vegetables is considered essential for a healthy life. The new food pyramid recommends the consumption of 2 to 3 fruits daily (Willet and Stampfer, 2003). A higher intake of fruits and vegetables has been correlated with a lower incidence of major human illnesses like cardiovascular disease (CVDs) and cancer (Block et al., 1992; Surh, 2003; Proc. 6 th International Postharvest Symposium Eds.: M. Erkan and U. Aksoy Acta Hort. 877, ISHS

2 Brandt et al., 2004). Grapes and other dietary constituents derived from it like grape juice and wine have attracted a great deal of attention in recent years. The composition and properties of grapes have been extensively investigated. Some reports indicate that grapes contain large amounts of phenolic compounds (Somers and Ziemelis, 1985; Macheix, 1990; Ricardo-da-Silva, 1990), which play an important role in human health, such as lowering of human low-density lipoprotein (Frankel et al., 1993; Tussedre et al., 1996). It has been demonstrated that wine and other products derived from grapes have high antioxidant capabilities (Alonso et al., 2002). A large number of polyphenols and flavonoids such as p-coumaric acid, cinnamic acid, caffeic acid, ferulic acid, vannilic acid, catechin, epicatechin, quercetin, proanthocyanidins, besides trihydroxy stilbenes such as resveratrol and polydatin have been reported from grapes. In addition to these, viniferin, a potent antifungal agent, and anthocyanins, which are strong antioxidants that inhibit platelet aggregation, are also present in grapes (Escarpa and Gonzalez, 2001). The objective of the present study was to identify the composition of phenolic compounds in grape extracts using HPLC:MS-TOF. MATERIALS AND METHODS Chemicals and Standards HPLC grade solvents and analytical reagent grade formic acid were purchased from J.T. Baker (USA). Gallic acid, protocatechuic acid, caffeic acid, miricetyn, hidroxybenzoic acid, quercetin, siringic acid, ferullic acid, isoramnetin, cinnamic acid and chlorogenic acid were purchased from Sigma-Aldrich Co. (St. Louis Mo, USA). Samples Freeze-dried powder from the fruit of Norton grapes was used. Ripe berries were harvested from three six-year-old vines grown on silty-loam soil in central Illinois. Vines were trained to a two wire double curtain. Fruits were harvested at optimum maturity based on commercial standards developed for wine grapes. Berries were cleaned with double distilled water, dried with paper towels, and frozen in liquid nitrogen. Frozen berries were lyophilized and ground into powder using a coffee mill. Extraction of Phenolic Compounds Phenolic extractions were made according to Carreiri et al. (2003) with some modification. 0.5 g of freeze-dried whole grapes samples were homogenized in 25 ml methanol/ethanol solution (8:2) for 16h. Extract was centrifuged at 27,000 g for 15 min and the pellet was re-extracted with 5 ml for one h and centrifuged again at the same speed and time. Supernatants were combined, and then evaporated to dryness in a rotary evaporator at 30 C, the residue was reconstituted in 1.5 ml of 100% methanol, and filtered with a 0.2 µm nylon filter. Phenolic separation was done with a Hitachi L-6200A HLPC System with Intelligent solvent delivery unit, autosampler, a column oven, a solvent degasser, a UV- VIS detector, and a Shimadzu CR501 integrator. The column used was RP-C18 (LiChrospher, 250 mm 4.6, 5 µm) and a LiChrocart, guard column (25 4 mm) (Merck, Darmstadt, Germany). Chromatographic Conditions Column temperature was 30 C, flow rate was 0.5 ml/min, and the mobile phase was a 70:22:8 solution of 1% formic acid (v/v):methyl cyanide (acetonitrile):2-propanol in a isocratic elution. Phenolics were detected at 306 nm absorbance. Fractions of the two peaks detected at around 14 and 18 mins (Fig. 1) were collected and were labeled as samples 1 and 2 as follows: GWEXTR (grape whole extract); Sample 1, peak time min (S1P 14.13), and Sample 2, peak time min (S2P 17.87). Samples dissolved in methanol were filtered through a 0.45 µm nylon filter (Millipore Corporation USA), and injected into an HPLC system and ESI-Mass 1234

3 spectrophotometer in the negative mode ion. High-Perfomance Liquid Chromatography (HPLC) HPLC used was an Agilent HPLC series 1100 (Waldbronn, Germany) equipped with ChemStation software, a G1322A degasser, a G1311quaternary pump, a G1316A column oven, and a G1315A diode array detector (DAD). Analysis was performed using a XTerra RP18 column with a particle size of 5µm and mm length (Waters, Ireland), operating at a temperature of 25 C. The phenolic compounds were analyzed using water (eluent A), and acetonitrile (eluent B) at a flow rate of 0.5 ml/min. Phenols were separated with a linear gradient from 2% B to 100% B in 60 min, maintaining this condition for 70 min, and finally the gradient was decreased from 100% B to 2% B in 5 min. The injection volume for all samples was 50 µl. Detection was carried out at and 320 nm. The identification of the different chromatographic peaks was confirmed with standards and retention times. High-Perfomance Liquid Chromatography-Electrospray Mass Spectrometry (HPLC-MS) Analysis HPLC-DAD was coupled to a time of flight (TOF) mass spectrometer (Agilent, Palo Alto, CA) equipped with an electrospray ionization (ESI) source and Mass Hunter manager software (A.02.01) operating in a negative mode ionization. Nitrogen was used as dry gas at a flow rate of 9 ml/min and 350 C, with nebulizing (45 psi). The spectra were taken in the presence of formic acid to promote (M+H) + ion production (electrospray voltage 4.0 kv). Phenols were separated with a linear gradient from 2% B to 100% in 60 min, maintaining this condition up to 70 min, and finally decreased the gradient from 100% B to 2% B in 5 min, at a flow rate of 0.5 ml/min. The injection volume for all extract samples was 50 µl. RESULTS AND DISCUSSION Presence of Phenolic Compounds in Grape Extract The identification of the peaks detected under full-scan conditions was obtained by analyzing each peak of the spectrum scan, and throughout the extracted-ion chromatograms of the ion current at m/z values corresponding to the ions of the individual investigated compounds. In this way, some phenolic compounds were identified in grape extract (Table 1) including stilbenes, flavonols, flavanols, anthocyanins and hydroxycinnamic acids and derivatives. Stilbenes, such as resveratrol, pteroestibene, trans-piceid, piceatannol, astringin, and ε-viniferin were also identified in the grape extract. Figure 2 shows the spectrum of grape extract indicating the retention time for cactechin, epicatechin, isoramnetin, siringic acid, caffeic acid, gallic acid, protocatechuic acid, miricetyn, hidroxybenzoic acid and quercetyn identified. Figures 3 and 4 show the MS-TOF spectrum of sample peaks from grape, and Table 2 shows the phenolic compounds detected in sample peaks. Resveratrol (227.22), quercetin-3-galactoside+na (486.24), caftaric acid (311.22), caffeic acid (179.13), kaempferol hexose malonate (489.37), pteroestibene (255.28), querecetin-3- glucoside/quercetin-3-glucuronide (477.47), flavonol diglycoside (599.41) were detected in both sample peaks S1P and S2P However, ferulic acid (193.18) and hidroxybenzoic acid derivative (189.15) were detected only in S1P14.13 peak. Also, catechin/epicatechin (289.12), hexahydroxydiphenoyl (HHDP) galloylglucopiranoside (633.50), quercentin diglycoside (595.51) and chlorogenic acid (353.26) were detected only in S2P peak. Quercetyn, hidroxybenzoic acid, caffeic acid, protocatechuic acid, gallic acid, siringic acid, miricetyn, catechin and epicatechin were quantified from grape extract (Table 3). The major concentrations found were of epicatechin, caffeic acid and catechin, followed by protocatechuic acid, hidroxybenzoic acid, siringic acid, gallic acid, miricetyn and quercetyn corresponding to , 1.486, , , , , , 1235

4 0.0297, mg/g dry weight, respectively. There are several reports including the phenolic compounds in wines and grape seeds and skin. Grape seeds and skin are a good source of phenolic compounds and provide the astringent taste to wine. We observed that catechin, epicatechin were the major flavonols present in grape extract. In agreement to our results, the major flavonoids present in grape skin and seeds are gallic acid and monomers such as catechin and epicatechin and in addition to various anthocyanins (Yilmaz and Toledo, 2004). In this way, Cantos et al. (2000) reported that the phenolic content such as caffeoyl tartaric, procyanidin B1, cyanidin-3- glucoside, trans-piceid, peonidin-3-glucoside, mailvidin-3- glucoside, quercetin-3-glucuronide, quercetin-3-glucoside, trans-resveratrol, cisresveratrol, malvidin-3-acetylglucoside and malvidin-3-p-coumaroylglucoside were found in skin of red grape Napoleon. Also, Cantos et al. (2002) reported the induction of stilbenes in grape skin influenced by UV-C irradiation. Monagas et al. (2003) reported in wine, grape seeds and skin, the monomeric and polymeric flavan-3-ols composition of three grape cultivars, and concluded that all of the monomeric flavan-3-ols and oligomeric proantocyanidins found in wines were also present in the seeds and that the skin polymeric anthocyanin were also found in wine. The content of flavan-3-ols (catechin and epicatechin) was higher in wine than in the seeds and skin, and was higher in the seeds than in the skin. Other authors mentioned that biotic and abiotic stress, some manipulation and postharvest treatments influence the composition of phenolic compounds in grapes. In this context, Dugo et al. (2004) studied the composition of stilbenes and flavonoids content in wine obtained from grapes previously treated with pesticides. Others used ozone (González-Barrio et al., 2006), UV irradiation (Jiménez-Sánches et al., 2007; Cantos et al., 2000, 2002) and maceration temperatura (Koyama et al., 2007), with the goal of increasing the composition of some phenolic compounds. However, few reports mentioned the phenolic content in whole berries grape (Nicolleti et al., 2008; Jensen et al., 2008). Jensen et al. (2008) analyzed the phenolics profiles in order to predict the color of wine and found considerable amounts of gallic acid, catechin, epicatechin, hydroxycinnamatos in wine and in grape extract from several cultivars. Our results showed catechin and epicatechin as the principal components. However, in 10 grape varieties of Vitis vinifera analized by Nicolleti et al. (2008), 15 phenolic compounds comprising phenolic acids, flavonols, catechines, stilbenes and anthocyanins were simultaneously separated and identified in whole berries. These phenolic compounds included gallic acid, protocatchuic acid, caftaric acid, catechin, epicatechin, delphinidin 3-O-glicoside, cyaniding 3-O-glucoside, petunidin 3-Oglucoside, peonidin 3-O-glucoside, trans-piceid, rutin, quercetin 3-O-glucoside, kaemphero 3-O-glucoside, and trans-resveratrol. The majority of the compounds reported by Nicolleti et al. were also reported in our work, with the exception of delphinidin 3-Oglicoside, cyaniding 3-O-glucoside, petunidin 3-O-glucoside, peonidin 3-O-glucoside and malvidin 3-O-glucoside. Also, in addition to resveratrol and trans piceid, we have detected other stilbenes such as pteroestibene, piceatannol, astringin, and ε-viniferin in the grape extract. In conclusion, the phenolic compounds identified in grape extract included stilbenes, flavonols, flavanols, anthocyanins and hydroxycinnamic acids and derivatives. Stibenes, such as resveratrol, pteroestibene, trans-piceid, piceatannol, astringin, and ε-viniferin were also identified in the grape extract. Literature Cited Alonso, A.M., Guillen, D.A., Barroso, C.G., Puertas, B. and Gracia, A Determination of antioxidant activity of wine byproducts and its correlation with polyphenolic content. J. Agric. Food. Chem. 50: Block, G., Patterson, B. and Subar, A Fruits, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr. Cancer 18:1-29. Brandt, K., Christesen, L.P., Hansen-Moller, J., Hansen, S.L., Haraldsdottir, J., Jespersen, 1236

5 L., Purup, S., Kharazmi, A., Barkholt, V., Frokiaer, H. and Kobaek-Larsen, M Health promoting compounds in vegetables and fruits: a systematic approach for identifying plant components with impact on human health. Trends Food Sci. Technol. 15: Cantos, E., Espín, J.C. and Tomás-Barberán, F.A Postharvest stilbene-enrichment of red and white table grape varieties using UV-C irradiation pulses. J. Agric. Food Chem. 50: Cantos, E., García-Viguera, V., de Pascual-Teresa, S. and Tomás-Barberán, F.A Effect of postharvest ultraviolet irradiation on resveratrol and other phenolics of cv. Napoleon table grapes. J. Agric. Food Chem. 48: Careri, M., Corradini, C., Elveiri, L., Nicoletti, I. and Zagnoni, I Direct HPLC analysis of quercetin and trans-resveratrol in red wine, grape, and wine making byproducts. J. Agric. Food Chem. 51: Downey, M.O., Dokoozlian, N.K. and Krstic, M.P Cultural practice and environmental impacts on the flavonoid composition of grapes and wine: A review of recent research. Am. J. Enol. Vitic. 57: Dugo, G., Saitta, M., Giuffrida, D., Vilasi, F. and La Torre, G.L Determination of resveratrol and other phenolic compounds in experimental wines from grape subjected to different pesticide treatment. Ital. J. Food Sci. 16: Escarpa, A. and Gonzalez, M.C An overview of analytical chemistry of phenolic compounds in foods. Crit. Rev. Anal. Chem. 31: Frankel, E.N., Waterhouse, A.L. and Kinsella, J.E Inhibition of human LDL oxidation by resveratrol. Lancet 341: González-Barrio, R., Beltrán, D., Cantos, E., Gil, M.I., Espín, J.C. and Tomás-Barberán, F.A Comparison of zone and UV-C treatments on the postharvest stilbenoid monomer, dimer, and trimer induction in var. Superior white table grapes. J. Agric. Food Chem. 54: Hufnagel, J.C. and Hofmann, T Orosensory-directed identification of astringent mouth feel and bitter-testing compounds in red wine. J. Agric. Food Chem. 56: Jensen, J.S., Demiray, S., Egebo, M. and Meyer, A.S Prediction of wine color attributes from the phenolic profiles of red grapes (Vitis vinifera). J. Agric. Food Chem. 56: Jiménez-Sánchez, J.B., Crespo, E., Corral, J.M., Orea, M.J., Santos Delgado and González Ureña, A Elicitation of trans-resveratrol by laser resonant irradiation of table grapes. Appl. Phys. B. 87: Koyama K., Goto-Yamamoto, N. and Hashizume, K Influence of maceration temperature in red wine vinification on extraction of phenolics from berry skin and seeds of grape (Vitis vinifera). Biosci. Biotechnol. Biochem. 71(4): Landbo, A.K. and Meyer, A.S Ascorbic acid improves the antioxidant activity of European grape juices by improving the juices ability to inhibit lipid peroxidation of human LDL in vitro. Int. J. Food Sci. Technol. 36: Macheix, J.J., Fleuriet, A. and Billot, J Fruit phenolics. Boca Raton FL: CRC Press. Manach, C., Scalbert, A., Morand, C. and Jimenez, C Bioavailability and bioefficacy of polyphenols in humans. Am. J. Clin. Nutr. 79: Maxcheix, J.J., Fleuriet, A. and Billot, J The main phenolics of fruits. p In: Fruit Phenolics, CRC Press: Boca Raton, FL. Monagas, M., Gómez-Cordovéas, C., Bartolomea, B., Laureano, O. and Ricar Da Silva, J. M Monomeric, oligomeric, and olymeric flavan-3-ol composition of wines and grapes from Vitis vinifera L. cv. Graciano, Tempranillo, and Cabernet Sauvignon. J. Agric. Food Chem. 51: Nicoletti, I., Bello, C., De Rossi, A. and Corradini, D Identification and quantification of phenolic compounds in grapes by HPLC-PDA-ESI-MS on a semimicro separation scale. J. Agric. Food Chem. 56(19):

6 Ricardo-da-Silva, J.M., Rosec, J.P., Bourzeix, M. and Heredia, N Separation and quantitative determination of grape and wine procyanidins by HPLC. J. Sci. Food Agric. 53: Somers, T.C. and Ziemelis, G Spectral evaluation of total phenolic components in Vitis vinifera: grapes and wines. J. Sci. Food Agric. 36: Surh, Y.J Cancer chemoprevention with dietary phytochemicals. Nature Rev. Cancer 3: Tussedre, P.L., Frankel, E.N., Waterhouse, A.L., Peleg, H. and German, J.B Inhibition of in vitro human LDL oxidation by phenolic antioxidants from grapes and wines. J. Sci. Food Agric. 70: Willet, W.C. and Stampfer, M.J Rebuilding the food pyramid. Sci. Am Yilmaz, Y. and Toledo, R.T Major flavonoids in grape seeds and skins: Antioxidant capacity of catechin, epicatechin, and gallic acid. J. Agric. Food Chem. 52: Tables Table 1. Identification of phenoloc compounds in grape extract (GWEXTR). Phenolic group Stilbenes Flavonols Hidroxycinamic acids and derivatives Flavonols Phenolic Std TOF Retention time (min) DAD TOF , , 45.0, , , , 21.0, , 32.2, , , , Identification Trans piceid Resveratrol Piceatanol ε-viniferin Pteroestilbene Astringin Catechin Epicatechin Epicatechin dimer Gallic acid Protocatechuic acid Siringic acid Caffeic acid Ferulic acid Sinapic acid Hidroxybenzoic acid p-coumaric acid Cinnamic acid Chlorogenic acid 2-Hidroxycinnamic acid Vanillic acid Cafeoil hexosa Kaempferol Miricetyn Quercetin Quercetin-3- glucoronide Rutin Negative ion (m/z) Isoramnetin Anthocyanins 20.0, 23.5 Malvidin DAD: diode array detector (HPLC); TOF: time of fligth mass spectrometry 1238

7 Table 2. Phenolic compounds detected in MS-TOF analysis of sample peaks from grape extracts obtained by HPLC separation. Sample Peak S1P S2P m/z HHDP: Hexahydroxydiphenoyl Compound detected Resveratrol Quercetin-3-galactoside +Na Caftaric acid Ferulic acid Caffeic acid Hidroxybenzoic acid derivative Kaempferol hexose malonato Pteroestilbene Quercetin-3-glucoside/Quercetin-3- glucoronide Flavonol diglycoside Resveratrol Quercetin galactosido +Na Ceftaric acid Kaempferol hexose malonate Pteroestilbene Quercetin-3-glucoside/Quercetin-3- glucuronide Caffeic acid Flavonol diglycoside Catechin/Epicatechin HHDP galloyl-glucopyranoside Quercetin diglycoside Chlorogenic acid MS-TOF time (min) 40.33, , 40.44, , , , , 44, , , Table 3. Quantification of phenolic compounds in grape extract (GWEXTR). Phenolic compound λ (nm) mg/g dry weight Epicatechin Cafeic acid Catechin Protocatechuic acid Hidroxybenzoic acid Siringic acid Gallic acid Miricetyn Quercetin

8 Figures Fig. 1. Phenolic separation of grape extract using UV-HPLC Fig. 2. MS-TOF spectrum from grape extract (GWEXTR). A: MS-TOF scan and B:DAD scan. 1:Gallic acid, 2:Protocatechuic acid, 3:Caffeic acid, 4:Catechin, 5:Epicatechin, 6:Hidroxybenzoic acid, 7:Siringic acid, 8:Miricetyn, 9:Quercetin, 10:Isoramnetin. Fig. 3. MS-TOF spectrum from grape extract S1P A:MS-TOF scan B:DAD scan. Fig. 4. MS-TOF spectrum from grape extract S2P A:MS-TOF scan B: DAD scan. 1240