Berry anthocyanins: isolation, identification and antioxidant activities

Transcription

1 Journal of the Science of Food and Agriculture J Sci Food Agric 83: (online: 2003) DOI: /jsfa.1511 Berry anthocyanins: isolation, identification and antioxidant activities Marja P Kähkönen, Johanna Heinämäki, Velimatti Ollilainen and Marina Heinonen Department of Applied Chemistry and Microbiology, Division of Food Chemistry, PO Box 27, FIN University of Helsinki, Finland Abstract: Anthocyanins from bilberry, blackcurrant and cowberry were isolated for antioxidant evaluation. Individual compounds were identified and quantified using HPLC and HPLC/ESI MS techniques. Antioxidant and radical-scavenging capacities of the isolates were studied in emulsified methyl linoleate and human low-density lipoprotein (LDL) in vitro and in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test. The total anthocyanin contents in the phenolic extracts of bilberry, blackcurrant and cowberry were 6000, 2360 and 680 mg kg 1 fresh weight respectively. There were four dominant compounds in blackcurrant (glucosides and rutinosides of cyanidin and delphinidin), three in cowberry (monoglycosides of cyanidin) and 15 in bilberry (monoglycosides of cyanidin, delphinidin, malvidin, peonidin and petunidin). Quantification as cyanidin-3-glucoside equivalents gave markedly lower results regarding the total anthocyanin concentration and the content of individual delphinidin and malvidin compounds compared with quantification based on corresponding standard compounds. Berry anthocyanins were highly active radical scavengers in the DPPH test and effective antioxidants in emulsion and human LDL Society of Chemical Industry Keywords: anthocyanins; isolation; identification; quantification; HPLC/ESI MS; antioxidant activity; methyl linoleate; human LDL; DPPH INTRODUCTION Anthocyanins (Fig 1) are flavonoids commonly found in plant tissues, producing blue, red and purple colours. They are the most important water-soluble pigments in plants. 1 Among edible plants, berries (grape, cherry and plum included) with red, blue or purple colours constitute one of the most important sources of anthocyanins. In an earlier study we showed that anthocyanins are especially abundant in bilberry, the wild low-bush blueberry species. Several other berries also have high contents, among them blackcurrant, cowberry, chokeberry, cranberry and cowberry. 2 Other food plants rich in these pigments include red cabbage, red onion and eggplant. 1,3 Along with fresh berries, a variety of berry products such as juice, wine, jam and food colourants (E163 in EC specifications, extracted from grape skin, blackcurrants and other plant materials) contribute significantly to the intake of anthocyanins. There is considerable current interest in the possible health effects of anthocyanins in humans owing to their potential antioxidant effects and their reported positive effects on blood vessel walls More intensive utilisation of berry anthocyanins as food colourants as well as antioxidants is an interesting prospect for the food scientist. A great deal of consideration should be given to the choice of a suitable extraction system and further purification steps for anthocyanins, as they are highly reactive compounds and exceptionally sensitive to ph changes. Anthocyanins are more stable at acidic ph than at neutral or alkaline ph, but exposure to very strong acidic environments for an extensive time in the presence of oxygen may cause acid hydrolysis. 11 Care must be exercised to ensure that acylated anthocyanins possibly present in the sample are not destroyed. Hydrochloric acid or other mineral acids can be replaced with weaker organic acids such as formic or acetic acid. 12,13 In order to produce anthocyanin isolates, the phenolic crude extracts need to be further purified to eliminate other phenolics as well as non-phenolic components, including free sugars and organic acids. Various column chromatographic methods have been widely applied, commonly using C18 stationary phases. 14 Efficient and reliable methods of analysis Correspondence to: Marina Heinonen, Department of Applied Chemistry and Microbiology, Division of Food Chemistry, PO Box 27, FIN University of Helsinki, Finland marina.heinonen@helsinki.fi This paper was partially presented at the COST 916 symposium entitled Bioactive Compounds in Plant Foods held in Tenerife, Spain on April 2001 and at the EUROFOODCHEM XI meeting entitled Biologically-active Phytochemicals in Food held in Norwich, UK on September 2001 Contract/grant sponsor: Finnish Graduate School on Applied Bioscience Contract/grant sponsor: Finnish Ministry of Agriculture and Forestry (Received 4 April 2002; revised version received 27 November 2002; accepted 6 May 2003) 2003 Society of Chemical Industry. J Sci Food Agric /2003/$

2 MP Kähkönen et al HO A OR 4 O + OR 3 R 1 B OH R 2 The aim of this study was to investigate the anthocyanin composition of bilberry (Vaccinium myrtillus), cowberry (Vaccinium vitis-idaea) and blackcurrant (Ribes nigrum), to isolate the anthocyanins from these berries and to determine the antioxidant activity of the isolates using two different lipid models, emulsified methyl linoleate and human LDL. In addition, the radical-scavenging activity of the isolates was investigated using the DPPH radical method. Figure 1. Structural formula of common anthocyanins. Cyanidin: R1 = OH; R2, R3, R4 = H. Delphinidin: R1, R2 = OH; R3, R4 = H. Peonidin: R1 = OCH 3 ;R2,R3,R4= H. Petunidin: R1 = OCH 3 ; R2 = OH; R3, R4 = H. Malvidin: R1, R2 = OCH 3 ;R3,R4= H. Pelargonidin: R1, R2, R3, R4 = H. are also of great importance, as anthocyanins varying in basic skeleton structure and sugar substitution show marked differences in their antioxidant activity. 15 Several recent papers on the bioavailability of anthocyanins suggest that they may be absorbed as intact glycosides into the circulation Therefore it is necessary to perform their analysis as intact glycosides instead of determining the compounds as anthocyanidins after acid hydrolysis. Identification is facilitated by the possibility of applying HPLC combined with a diode array detector and mass spectrometer using a soft ionisation technique such as electrospray ionisation (ESI). Like several other flavonoids, anthocyanins are 15,19 21 powerful free radical scavengers. They also show antioxidant activity in lipid environments such as emulsified methyl linoleate (MeLo), human LDL (low-density lipoprotein) and liposome. 15,22 Moreover, there is some indirect evidence of the antioxidant potential of berry anthocyanins. Fruit extracts (black chokeberry, blackthorn and strawberry) containing high amounts of anthocyanins showed high radical-scavenging activity when using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. 23 A correlation was found between the anthocyanin content and the oxygen radical absorbance capacity (ORAC) in various cultivars of berries belonging to Vaccinium species 24 and in high- and low-bush blueberries, strawberries and raspberries after various storage periods. 25 Several berry extracts rich in phenolics and especially anthocyanins possessed high antioxidant activity in bulk MeLo, but the relationship between anthocyanin content and activity was not statistically significant. 2,26 In fact, anthocyanins and their aglycones were recently shown to be inactive in bulk MeLo when present alone, 15 but synergistic effects with the other phenolic compounds of the extracts cannot be excluded. The activity measurement used affects the results markedly. For example, the anthocyanin concentration of various berries (blackberries, red raspberries, sweet cherries, blueberries and strawberries) correlated with the antioxidant capacity in LDL but not in liposome. 27 EXPERIMENTAL Chemicals Delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-arabinoside, petunidin-3-glucoside, peonidin-3-galactoside and peonidin-3-arabinoside standards were purchased from Polyphenols (Sandnes, Norway). Cyanidin-3-galactoside, cyanidin-3-glucoside, cyanidin-3-rutinoside and malvidin-3-glucoside standards were purchased from Extrasynthése (Genay, France). α-tocopherol, copper sulphate, Na 2 HPO 4 and formic acid were purchased from Merck (Darmstadt, Germany). MeLo was obtained from Nu Check Prep (Elysina, MN, USA), and Emultop (partially hydrolysed soybean lecithin) was a gift from Lucas Meyer GmbH (Hamburg, Germany). Amberlite XAD-7 non-ionic polymeric adsorbent was purchased from Sigma Chemicals Co (St Louis, MO, USA). All solvents were of HPLC grade (Rathburn Chemicals Ltd, Walkerburn, UK), and water was of Milli-Q quality (Millipore Corp, Bedford, MA, USA). Sampling Bilberry (V myrtillus), blackcurrant (R nigrum var Öjebyn) and cowberry (V vitis-idaea) samples(5kg of each) were purchased from a market. The leaves and branches were removed and the samples were packed immediately in vacuo and stored in a freezer at 18 C. Extraction To produce phenolic crude extracts, extraction was carried out according to Norbaek and Kondo. 28 A2g sample of berries was weighed into a centrifuge tube, 20 ml of solvent (CH 3 CN/TFA/H 2 O 49.5:0.5:50 v/v/v) was added and the sample was homogenised for 1 min (Ultra-Turrax T25 mixer, Janke & Kunkel GmbH, Staufen, Germany). The tube was centrifuged (4000 rpm, 15 min) and the clear supernatant was collected. The procedure was repeated twice with another 20 ml of solvent. The supernatants were combined and evaporated to dryness. The solid residues were dissolved in 0.5% TFA (ph 1.5). For identification and quantification, extraction was performed as three replicates. Isolation The crude extracts were further purified, by a method based on the work of Norbaek and Kondo, 28 using Amberlite XAD-7 column chromatography. The 1404 J Sci Food Agric 83: (online: 2003)

3 Berry anthocyanins sample was introduced into the column (diameter 40 mm, length 300 mm), and free sugars and organic and phenolic acids were eluted with 6% CH 3 CN (CH 3 CN/TFA/H 2 O 6.0:0.5:93.5 v/v/v), yielding fraction 1 (X-1). Elution was continued with 50% CH 3 CN (CH 3 CN/TFA/H 2 O 50:0.5:49.5 v/v/v), which yielded the anthocyanin containing fraction (X-2). Finally, the column was washed with CH 3 CN (CH 3 CN/TFA 99.5:0.5 v/v) to elute the remaining phenolics (X-3). Fraction X-2 was further purified using preparative HPLC. The HPLC system (Waters, Milford, MA, USA) consisted of a WISP 712 autosampler, three 501 pumps with a pump control module, a column oven with a temperature control module, a PDA 996 diode array detector and a Millennium 2020C/S software data module. Separation was performed using a Develosil ODS-HG-5 column (250 mm 20 mm, 5 µm; Phenomenex, CA, USA) equipped with a guard column (50 mm 20 mm). The mobile phase consisted of 10% formic acid (solvent A) and 100% CH 3 CN (solvent B). The elution conditions were as follows: isocratic elution 0% B, 0 5 min; linear gradient from 0% B to 15% B, min; to 50% B, 140 min; to 100% B, min. The flow rate was 5.0 ml min 1 and the column oven temperature was 35 C. Detector wavelengths of 280 nm (to monitor all phenolics) and 520 nm (to monitor the anthocyanins) were used. The sample (50 ml) was introduced into the column using an HPLC pump. All anthocyanins were collected together into one fraction. HPLC/DAD analysis Anthocyanin quantification was performed in three stages (for the crude extracts, after XAD-7 purification and for the isolates), using the same equipment and solvents as in preparative HPLC. Analytical separation of anthocyanins was carried out on a Zorbax SB C18 column (150 mm 4.6 mm, 5 µm; Agilent Technologies, CA, USA) equipped with a C18 guard column. The column oven temperature was 40 C. The elution conditions were as follows: isocratic elution 2% B, 0 5 min; linear gradient from 2% B to 9% B, 5 15 min; to 11% B, min; to 30% B, min; to 80% B, min; isocratic elution 80% B, min; linear gradient to 5% B, min; post-time 15 min before next injection; flow rate 1.0 ml min 1 ; injection volumes 5 25 µl. The detection wavelengths were 280 and 520 nm and the spectra from 200 to 600 nm were recorded for all peaks. HPLC/ESI MS analysis An HP 1100 solvent delivery system, autosampler and diode array detector together with HP ChemStation Plus A software Agilent were employed (Agilent Technologies, CA, USA). Separation of anthocyanins was carried out on a narrow-bore Zorbax SB C18 column (150 mm 1.0 mm, 3.5 µm; Agilent Technologies). The mobile phase consisted of 10% formic acid (solvent A) and 100% CH 3 CN (solvent B). The column oven temperature was 40 C. The elution conditions were as follows: isocratic elution 2% B, min; linear gradient from 2% B to 11% B, min; to 40% B, min; to 98% B, min; isocratic elution 98% B, min; linear gradient to 2% B, min; post-time 30 min before next injection; flow rate 0.05 ml min 1 ; injection volumes µl. The UV-vis detection wavelength was 520 nm and the spectra from 200 to 600 nm were recorded for all peaks. The LC/MS instrument was an Esquire-LC ion trap mass spectrometer equipped with an electrospray interface (Bruker Daltonics, Bremen, Germany). The software employed was Esquire- LC NT, version 3.1 (Bruker Daltonics, Bremen, Germany). Electrospray ionisation was performed in positive ion mode under the following conditions: dry temperature 300 C; capillary voltage 2500 V and exit offset 70 V; skimmer 1 potential 20 V; scan range m/z and resolution m/z 0.2; scanning rate m/z s 1 ; trap drive value 41. The nitrogen used as drying gas (5 l min 1 ) and nebuliser gas (50 psi) was produced with a Whatman nitrogen generator (Whatman, Haverhill, MA, USA). Tandem mass spectrometry (MS/MS) was performed using helium (99.996%) as the collision gas. Antioxidant activity in MeLo emulsion The oxidation experiment in 10% oil-in-water emulsion was carried out using 0.4 g of MeLo and 3.6 ml of 1% (w/v) Emultop distilled in Milli-Q water. The tocopherol content in MeLo and Emultop was determined using HPLC. 29 MeLo was found to be free of tocopherols, whereas Emultop contained some γ -and δ-tocopherols (62 and 40 µgg 1 respectively). The anthocyanin isolates were dissolved in methanol at levels of 100 and 500 ppm. The methanolic solutions (100 µl) were pipetted into glass vials (volume 20 ml) and the solvent was evaporated under nitrogen. The MeLo and emulsifier solution were added to the vials, and emulsions were prepared by sonicating the mixture for 3 min in an ice bath (U50 Control Ikasonic sonicator, Janke & Kunkel GmbH). Oxidation was carried out in the dark at 40 C, and formation of hydroperoxides was assessed by measuring the formation of conjugated diene hydroperoxides spectrometrically (Lambda 15 UV-vis spectrophotometer, Perkin-Elmer Norwalk, CT, USA) at 234 nm. 28 The antioxidant activity was expressed as the percentage (%) inhibition of formation of MeLo hydroperoxides after 72 h of oxidation. The ph of the emulsions measured at the beginning and end of the experiment was found to vary between 5.3 and 5.9. Antioxidant activity in human LDL Human LDL was diluted to a protein concentration of 0.2 mg ml 1 using 0.01 M Na 2 HPO 4 (ph adjusted to 7.4) containing 0.15 M NaCl. Samples were incubated at 37 C with 10 µm copper sulphate solution and the anthocyanin isolates (final concentrations 1.4 J Sci Food Agric 83: (online: 2003) 1405

4 MP Kähkönen et al and 4.2 µgml 1 ) in sealed headspace vials. The anthocyanins were added in ethanolic solution (volume 10 µl), and ethanol was removed by nitrogen flushing. The LDL solution (450 µl) was pipetted into the vials, 1340µl of phosphate buffer and 10 µl of copper sulphate were added and the vials were sealed and stirred carefully before placing them in a water bath (37 C). After 2 h of incubation the extent of oxidation was determined by measuring the formation of hexanal using static headspace gas chromatography (Autosystem XL gas chromatograph equipped with HS 40XL headspace sampler; Perkin-Elmer, Shelton, CT, USA) according to Frankel et al. 30 Radical-scavenging activity (RSA) The ability of the anthocyanin isolates to act as free radical scavengers against the DPPH radical was tested spectrophotometrically by measuring the disappearance of the absorbance at 517 nm after adding the antioxidant solution. Anthocyanins absorb at this wavelength as well, but their contribution to the total absorbance was small owing to their low concentration in the cuvette. In a cuvette, 2950 µl of 0.1 mm methanolic DPPH solution was mixed with 50 µl of the antioxidant solution (concentrations 0.1, 0.5 and 1.0 mg ml 1 ). The final concentrations in the cuvette were 1.7, 8.3 and 16.7 µgml 1.The absorption was monitored at intervals of 15 s for 5 min. The result was expressed as the percentage of radicals scavenged after 4 min of reaction time. Statistical analysis A one-way ANOVA test was performed on the antioxidant activity results to investigate significant differences between the isolates. The method used to discriminate among the means was Fischer s least significant difference procedure at 95% confidence level. Simple regression analysis was performed to look for relationships between different tests. P values lower than 0.10 were considered to be significant. The computer program employed was Statgraphics Plus for Windows, version 3.0. RESULTS Isolation The total recovery of anthocyanins after XAD-7 purification, measured using HPLC, was very high for bilberry and blackcurrant and satisfactory for cowberry (Table 1). The purity of the anthocyanin fractions at different stages of isolation was determined by HPLC at a wavelength of 280 nm as the percentage of total anthocyanin peak areas from all peak areas. Above all, this measurement was done to monitor the separation of other phenolics from anthocyanins. The proportion of anthocyanins of all compounds absorbing at 280 nm increased during the isolation process. Compared with the composition of the crude extracts, XAD-7 purification increased the purity by Table 1. Anthocyanin recovery after XAD-7 purification, and distribution of berry anthocyanins between the three XAD fractions, quantified as cyanidin-3-glucoside equivalents Recovery Anthocyanin distribution (%) Berry (%) X-1 X-2 X-3 Blackcurrant Tr Bilberry Cowberry 84 ND 100 Tr ND, not detected; Tr, trace. only 1 4%. Further purification of the X-2 fractions by preparative HPLC raised the purity degree clearly in the blackcurrant and cowberry samples (by 10 and 46% respectively), whereas the increase in the bilberry sample was minimal (2%). Finally, the purity of the blackcurrant and bilberry anthocyanin isolates was 95%, whereas the purity of the cowberry isolate was only 87%. On the basis of the UV vis and mass spectra, the impurities of the cowberry isolate are proanthocyanidins. The absence of ascorbic acid in the isolates could be confirmed by monitoring the HPLC/DAD chromatograms on the basis of retention time and spectral information. Identification The three berries studied differed substantially in their anthocyanin composition. Figure 2 shows the HPLC chromatograms of the anthocyanin isolates at 520 nm. Four main anthocyanins, ie delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside and cyanidin-3-rutinoside, were identified in blackcurrant (Table 2). In addition, two minor anthocyanins, containing the basic skeletons of peonidin and petunidin and the hexose-dioxyhexose sugar moiety, were detected. These may be the 3-rutinosides of peonidin and petunidin, but this tentative identification could not be confirmed, as standard compounds were not available. The anthocyanin composition of bilberry was more complicated than that of cowberry or blackcurrant. A total of 15 different anthocyanins were identified in bilberry, including monoglycosides of delphinidin, cyanidin, petunidin, peonidin and malvidin (Table 2). In cowberry, only three main compounds were identified: cyanidin-3-galactoside, cyanidin-3-glucoside and cyanidin-3-arabinoside (Table 2). Anthocyanin concentrations The anthocyanin (AC) content of the berries was determined from the crude extracts. Bilberry was the richest regarding the total amount of ACs (6000 mg kg 1 fresh weight (fw)). Correspondingly, the total amount of ACs was 2360 mg kg 1 in blackcurrant and 680 mg kg 1 in cowberry. Quantification of single compounds as cyanidin- 3-glucoside equivalents gave lower results compared with quantification that was performed using the 1406 J Sci Food Agric 83: (online: 2003)

5 Berry anthocyanins Cowberry AU Minutes Blackcurrant AU Minutes Bilberry AU Minutes Figure 2. HPLC/DAD chromatograms of cowberry, blackcurrant and bilberry at 520 nm. Refer to peak numbering in Table 2. specific standard compounds available for most anthocyanins (Table 3). The anthocyanin distributions in the isolates corresponded well to the distributions in the crude extracts (Table 4). The peonidin and petunidin rutinosides detected in the crude extract of blackcurrant were not observed in the isolate, as the fraction collection was ended before they eluted in the preparative HPLC system. Antioxidant activity All three AC isolates showed marked inhibition of hydroperoxide formation in MeLo and of hexanal formation in LDL (Table 5). In emulsified MeLo the antioxidant activity of bilberry ACs at the 500 ppm level was higher than that of blackcurrant or cowberry ACs. In LDL all three isolates were highly efficient at the higher concentration used (4.2 µgml 1 ) but inactive at the lower addition level (1.4 µgml 1 ). Blackcurrant ACs were the most efficient DPPH radical scavenger at the highest concentration (16.7 µgml 1 ), followed by bilberry and cowberry ACs (Table 5). At the 1.7 and 8.3 µgml 1 concentrations, blackcurrant and bilberry possessed quite similar activity, cowberry being the least active There were no statistically significant relationships between the radical scavenging activities and the antioxidant activities in emulsion or LDL, nor between the emulsion and LDL test results. DISCUSSION AND CONCLUSIONS The separation of anthocyanins from other phenolics was efficiently accomplished using the combination J Sci Food Agric 83: (online: 2003) 1407

6 MP Kähkönen et al Table 2. Identification of berry anthocyanins using HPLC and ESI MS Peak Retention time [M] + in MS Fragment ion 1 [M] + in MS/MS Fragment ion 2 [M] + in MS/MS a Aglycon Neutral loss in MS/MS Sugar moiety Identification Blackcurrant Dp Hexose Dp-3-glu Dp Hexose + deoxyhexose Dp-3-rut Cn Hexose Cn-3-glu Cn Hexose + deoxyhexose Cn-3-rut Pn Hexose + deoxyhexose Pn-3-rut b Pt Hexose + deoxyhexose Pt-3-rut b Bilberry Dp Hexose Dp-3-gal Dp Hexose Dp-3-glu Cn Hexose Cn-3-gal Dp Pentose Dp-3-ara Cn Hexose Cn-3-glu Pt Hexose Pt-3-gal Cn Pentose Cn-3-ara Pt Hexose Pt-3-glu Pn Hexose Pn-3-gal Pt Pentose Pt-3-ara Pn Hexose Pn-3-glu Mv Hexose Mv-3-gal Pn Pentose Pn-3-ara Mv Hexose Mv-3-glu Mv Pentose Mv-3-ara Cowberry Cn Hexose Cn-3-gal Cn Hexose Cn-3-glu Cn Pentose Cn-3-ara Dp, delphinidin; Cn, cyanidin; Pn, peonidin; Pt, petunidin; Mv, malvidin; glu, glucoside; rut, rutinoside; gal, galactoside; ara, arabinoside. a Further fragmentation of these ions yielded fragment ion 1. b Tentative identification. of XAD-7 column chromatography and preparative HPLC. XAD-7, a non-ionic acrylic polymer adsorbent, was reported to be the most suitable among 16 different solid phase extraction (SPE) phases for anthocyanin purification from black chokeberry juice. 14 Preparative HPLC with C18 columns has been widely used for further purification. 28,31,32 The purity of bilberry isolate appears to be high already after the XAD-7 step, and thus the preparative HPLC step is not necessary. Overall, the current isolation process is relatively simple, but as these molecules are rather labile, consideration has to be given to minimise the effect of light, temperature and oxygen. HPLC combined with DAD and ESI-MS has proved to be a very powerful technique for anthocyanin identification and quantification. Retention times, UV vis spectra, mass/charge ratios as well as the type of anthocyanidin and the nature of sugar substitution can be easily recorded and used together to solve anthocyanin structures. Naturally, if standard compounds are not available, NMR is still necessary to clarify the exact structures of unknown components. The UV vis quantification of all anthocyanins as cyanidin-3-glucoside equivalents was shown to be misleading; in particular, the amount of delphinidin glycosides can be easily underestimated. This is due to differences in the molar absorptivities (ε) of the flavylium cation structures and differences in the distribution of different mesomers in equilibrium. At ph 1.5 the molar absorptivity values (l mol 1 cm 1 ) are for delphinidin-3-glucoside, for cyanidin-3- glucoside, for petunidin-3-glucoside, for peonidin-3-glucoside and for malvidin-3- glucoside. 33 The mesomer distribution at different ph values is affected by methoxylation in ring B, and therefore the absorption of malvidin-3-glucoside was lower than that of cyanidin-3-glucoside at the same molar concentration and ph. The sugar moiety attached to the flavylium cation seems to affect the equilibrium as well. Our results show that cyanidin-3-glucoside had a higher response in the HPLC/DAD analysis than other glycosides of cyanidin. There are several internal and external factors affecting the quantity and quality of anthocyanins in a berry species. These include the genetic (varietal and regional) diversity as well as many environmental variables, ie growing conditions such as light intensity, humidity, temperature, the use of fertilisers, wounding, infections or other stress factors. 34 The large divergence between crops, together with some fundamental 1408 J Sci Food Agric 83: (online: 2003)

7 Berry anthocyanins Table 3. Anthocyanin composition of berry raw extracts quantified as corresponding anthocyanin compounds or as cyanidin-3-glucoside equivalents (mg kg 1 fw; mean ± SD of triplicate assays) Anthocyanin Concentration as corresponding standard %oftotal anthocyanins Concentration as Cn-3-glu equivalents %oftotal anthocyanins Blackcurrant Dp-3-glu 372 ± ± Dp-3-rut 916 ± ± Cn-3-glu 165 ± ± Cn-3-rut 890 ± ± Pn-3-rut a 12 ± ± Pt-3-rut b 1 ± 0 <0.1 2 ± Total 2356 ± ± Bilberry Dp-3-glu 766 ± ± Cn-3-gal 533 ± ± Cn-3-glu 488 ± ± Cn-3-ara 689 ± ± Pt-3-glu 437 ± ± Pn-3-gal 33 ± ± Pn-3-ara 33 ± ± Mv-3-glu 492 ± ± Dp-3-gal c 857 ± ± Dp-3-ara c 859 ± ± Pt-3-gal b 186 ± ± Pt-3-ara b 147 ± ± Pn-3-glu a 160 ± ± Mv-3-gal d 181 ± ± Mv-3-ara d 136 ± ± Total 5997 ± ± Cowberry Cn-3-gal 536 ± ± Cn-3-glu 22 ± ± Cn-3-ara 124 ± ± Total sum 682 ± ± Dp, delphinidin; Cn, cyanidin; Pn, peonidin; Pt, petunidin; Mv, malvidin; glu, glucoside; rut, rutinoside; gal, galactoside; ara, arabinoside. a,b Tentatively identified. a d Corresponding standards not available; quantified as a Pn-3-gal, b Pt-3-glu, c Dp-3-glu, d Mv-3-glu. differences in analytical methods, makes it difficult to compare the results with published data. The total amount and quality of anthocyanins in bilberry, blackcurrant and cowberry are in general agreement with previous papers. 25,35 38 However, data on the amount of individual anthocyanins are scarce, especially for bilberry and cowberry. Iversen 35 determined the anthocyanin content of blackcurrant grown in Denmark (variety Ben Lomond ) by using HPLC with UV detection. The content of delphinidin-3-glucoside was 320 mg (12.7%), delphinidin-3-rutinoside 1690 mg (50.7%), cyanidin-3-glucoside 120 mg (5.0%) and cyanidin-3-rutinoside 1020 mg (31.6%) kg 1 fw, the total concentration being 3150 mg kg 1 fw. Our data for var Öjebyn show a 25% lower total amount and a more even distribution between cyanidin and delphinidin rutinosides. Kalt et al 25 analysed the total anthocyanin content of bilberry (mix of wild clones grown in Europe) spectrophotometrically as malvidin-3-glucoside equivalents. The content was 3700 mg kg 1 fw, which is 38% lower than our result. In their study a total of 24 different anthocyanins were identified and their percentage shares determined as peak area g 1 fw. In addition to compounds detected in the present work, they reported also the presence of acetylated anthocyanins in very small quantities. More recently, two studies have been conducted on the anthocyanin composition of bilberry using HPLC with ESI-MS detection. 36,37 There are some differences between these data. Dugo et al 36 did not detect peonidin-3-arabinoside, while Chandra et al 37 did not detect either the 3-arabinosides of delphinidin, cyanidin and peonidin or peonidin- 3-glucoside in their sample. Andersen 38 determined the total anthocyanin content of cowberry spectrophotometrically as cyanidin-3-glucoside equivalents. The content was 1740 mg kg 1 fw, which is over two times higher than our result. Cyanidin-3- galactoside (88.0%), cyanidin-3-arabinoside (10.6%) and 3-glucoside (1.4%) and delphinidin-3-glucoside (<0.1%) were identified using TLC and HPLC/DAD and quantified as cyanidin-3-glucoside equivalents. The distribution is very similar to our data. Both blackcurrant and bilberry anthocyanins acted effectively in the DPPH test. In our recent J Sci Food Agric 83: (online: 2003) 1409

8 MP Kähkönen et al Table 4. Anthocyanin composition (% of total anthocyanins) of anthocyanin isolates quantified as corresponding anthocyanin compounds Anthocyanin Blackcurrant Bilberry Cowberry Dp-3-glu Dp-3-rut 39.1 Cn-3-gal Cn-3-glu Cn-3-rut 38.7 Cn-3-ara Pt-3-glu 6.0 Pn-3-gal 0.6 Pn-3-ara 1.0 Mv-3-glu 8.4 Dp-3-gal 14.9 Dp-3-ara a 15.3 Pt-3-gal b 2.1 Pt-3-ara b 1.3 Pn-3-glu c 0.1 Mv-3-gal d 3.1 Mv-3-ara d 2.4 Total Dp, delphinidin,cn, cyanidin; Pt, petunidin; Pn, peonidin; Mv, malvidin; gal, galactoside; glu, glucoside; ara, arabinoside; rut, rutinoside. a d Corresponding standards not available; quantified as a Dp-3-glu, b Pt-3-glu, c Pn-3-gal, d Mv-3-glu. study, delphinidin-3-glucoside, cyanidin-3-glucoside and delphinidin-3-rutinoside possessed the highest DPPH radical-scavenging activities among 17 anthocyanins. 15 All three compounds are present in blackcurrant. The anthocyanin composition of bilberry is more complex, but the monoglycosides of delphinidin and cyanidin are dominant. The radical scavenging activity of the cowberry anthocyanin isolate was the lowest even though it consists of cyanidin monoglycosides, all of which in the previous study showed good radical scavenging capacity. The procyanidin impurities may have affected the result. These results demonstrate that berry anthocyanins are powerful antioxidants in emulsified MeLo and LDL. The bilberry isolate prevented the formation of MeLo hydroperoxides most efficiently, but in LDL all three isolates showed high activity. Despite their lower radical scavenging activity, the cowberry anthocyanins are very potent antioxidants in the lipidcontaining models. Currently, as many antioxidant evaluations are conducted merely by means of radical scavenging tests, it must be emphasised that the hydrogen-donating ability of antioxidants in a simple test model does not necessarily indicate their activity in a complex lipid model. Several other factors, including interactions with metal ions, proteins, emulsifiers and other antioxidants as well as distribution between the oil and the water phase, affect the antioxidant action significantly. ACKNOWLEDGEMENTS We gratefully acknowledge Minnamari Edelmann and Satu Vuorela for their skilful technical assistance. This work was supported by the Finnish Graduate School on Applied Bioscience and the Finnish Ministry of Agriculture and Forestry. REFERENCES 1 Bridle P and Timberlake CF, Anthocyanins as natural food colours selected aspects. Food Chem 58: (1997). 2 Kähkönen MP, Hopia AI and Heinonen M, Berry phenolics and their antioxidant activity. J Agric Food Chem 49: (2001). 3 Clifford MN, Anthocyanins nature, occurrence and dietary burden. J Sci Food Agric 80: (2000). 4 Burns J, Gardner PT, O Neil J, Crawford S, Morecroft I, McPhail DB, Lister C, Matthews D, MacLean MR, Lean MEJ, Duthie GG and Crozier A, Relationship among antioxidant activity, vasodilation capacity and phenolic content of red wines. J Agric Food Chem 48: (2000). 5 Youdim KA, Martin A and Joseph JA, Incorporation of the elderberry anthocyanins by endothelial cells increases protection against oxidative stress. Free Rad Biol Med 29:51 60 (2000). 6 Ursini F, Tubaro F, Rong J and Sevanian A, Optimization of nutrition: polyphenols and vascular protection. Nutr Rev 57: (1999). 7 Colatuoni A, Bertuglia S, Magistretti MJ and Donato L, Effects of Vaccinium myrtillus anthocyanosides on arterial vasomotion. Arzneimittel-Forschung 41: (1991). 8 Detre Z, Jellinek H, Miskulin M and Robert AM, Studies on vascular permeability in hypertension: action of anthocyanosides. Clin Physiol Biochem 4: (1986). 9 Kadar A, Robert L, Miskulin M, Tixier JM, Brechemier D and Robert AM, Influence of anthocyanoside treatment on the cholesterol-induced atherosclerosis in the rabbit. Paroi Arterielle. 5: (1979). 10 Mian E, Curri SB, Lietti A and Borbardelli E, Anthocyanosides and the walls of the microvessels: further aspects of the mechanism of action of their protective effect in syndromes due to abnormal capillary fragility. Minerva Med 68: (1977). 11 Revilla E, Ryan JM and Martin-Ortega G, Comparison of several procedures used for the extraction of anthocyanins from red grapes. J Agric Food Chem 46: (1998). Table 5. Antiradical and antioxidant activities of anthocyanin isolates DPPH (µgml 1 ) a Emulsion (ppm) b LDL (µgml 1 ) c Isolate Blackcurrant 12 ± 0a 38 ± 0a 58 ± 1a 60 ± 0a 75 ± 3a 9 ± 1a 96 ± 0a Bilberry 13 ± 0b 38 ± 0a 52 ± 1b 70 ± 5a 90 ± 7b 7 ± 0b 95 ± 1a Cowberry 9 ± 0c 25 ± 0b 36 ± 0c 61 ± 3a 77 ± 7a 4 ± 0c 97 ± 0a Expressed as a radicals scavenged (%) after 4 min reaction time; b inhibition (%) of MeLo hydroperoxide formation after 72 h of oxidation; c inhibition (%) of hexanal formation after 2 h of oxidation. Values in the same column having the same letter are not significantly different at P < J Sci Food Agric 83: (online: 2003)

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