The Effect of Freezing and Extraction Solvent on the Total Polyphenol Content Measurement in Whole ‘Royal Gala’ Apple Fruit D. Stefanellia, S. Brady, T. Plozza, C. Trenerry and R. Jones Future Farming Systems Research Department of Primary Industries Victoria, Knoxfield Centre Private Bag 15, Ferntree Gully DC Victoria 3156 Australia Keywords: antioxidants, phenolics, methanol, acetone, storage, UHPLC-PDA-MSn Abstract The Folin-Cioccalteau (FC) procedure is commonly used to measure the total polyphenol content (TPP) of fruit. There is at present no agreement on the most effective solvent for this assay, however, the most widely reported extractants are acetone or methanol based. There is also limited knowledge on the effect of freezing the fruit on the extraction efficiency of the solvent used for the assay. The aim of this study was to examine the effects of freezing on the extraction efficiencies of a variety of solvents by measuring the TPP values for ‘Royal Gala’ apples stored at -20°C for up to six months. The polyphenol composition was determined concurrently on each of the extracts by ultra-high pressure liquid chromatography-photodiode array-ion trap mass spectrometry (UHPLC-PDA-MSn). Four different extractants were evaluated: acetone-water 50-50% (A50); acetone 70%, water 29.5% and acetic acid 0.5% (AWA); methanol-water 90-10% (M90); and methanol-water 50-50% (pH 2.0), followed by acetone-water 70-30% (M50A70). TPP values were determined in fruit frozen for 1, 15, 30, 90 or 180 days and were compared with values for fresh apples. A50 provided the highest TPP values (120 mg GAE/100 g FW) in fresh apples, followed by AWA (110 mg GAE/100 g FW), M90 (84 mg GAE/100 g FW) and M50A70 (81 mg GAE/100 g FW). There was a significant interaction between extractant type and freezing time. TPP values decreased over time with A50 (1020%), remained constant with AWA and M90 and increased with M50A70 (1025%). AWA provided the most consistent TPP values throughout the experiment. If differences were detected, AWA gave the highest values while M90 the lowest when the polyphenol levels were determined by UHPLC-PDA-MS. This experiment suggests that AWA is the optimal extractant for measuring TPP values in frozen apple fruit after storage for up to 6 months and is also suitable for identifying selected polyphenols in fruit by UHPLC-PDA-MSn. INTRODUCTION Phenolics in foods have gained much attention in recent times, due to their antioxidant activities and possible beneficial implications for human health. Apples are one of the most important dietary sources of these phytochemicals and a major effort has been put into producing fruit with increased phytochemical concentrations and subsequently a higher antioxidant capacity (Carbone et al, 2011). The health properties of these natural products depend on the contents of bioactive compounds, mainly phenolics and vitamin C (Gorinstein et al., 2009) as major contributors to the total antioxidant capacity of fruits (Heo et al., 2007), which is commonly determined by the FolinCiocalteau (FC) method to measure the total polyphenol content (TPP). According to Robards (2003) the data on phenolic contents in fruits and vegetables are incomplete and not totally reliable due to the wide diversity of extraction and quantification procedures. In addition, Luthria et al. (2006, 2007) observed significant differences in the extraction a
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Proc. 7th International Postharvest Symposium Eds.: H. Abdullah and M.N. Latifah Acta Hort. 1012, ISHS 2013
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efficiency of phenolic compounds, but not in the number of components extracted. The most widely reported extractants used in the FC assay are acetone or methanol based. Duportet et al. (2012) demonstrated that different solvent-based extraction methods can result in significantly different metabolite profiles, which impact substantially in the interpretation of data, while Arranz et al. (2009) suggested that total plant polyphenols have been underestimated due to processing methods which could leave compounds behind (non-extracted phenolics). In addition to the effect of solvent and method of extraction, sample preparation could play a role in the efficiency of identification and quantification of phenolics (Luthria et al, 2006), especially in fruit that are rich in sugars and therefore highly hygroscopic. Some researchers conduct their analysis on fresh samples and others routinely freeze the samples before processing or analysis. This could potentially influence the results, however, to date the effect of freezing on apple polyphenolics has not been studied. There is also no direct comparison of the various methods being used, making it impossible to determine the optimal method. Moreover, most of the sample preparations listed in the literature involve some kind of immediate processing of the produce, severely limiting the number of samples that can be processed at once and therefore the complexity of an experiment. In this study we proposed to test (a) the effect of freezing whole fruit (-20°C) and subsequent storage for up to six months and (b) the efficiency of a range of solvents on extracting and identifying phenolics, to measure the antioxidant content (TPP) of apples and the polyphenolic profile by UHPLC-PDA-MSn. MATERIALS AND METHODS ‘Royal Gala’ apple fruit (n=240) were collected from Montague Fresh Ltd. (Narre Warren, VIC, Australia). Four replicates of 10 fruit were utilized in each extraction for water soluble total plant phenols analysis. Fruit for extraction were randomly selected from the boxes. Immediately after random selection of fruit to be extracted as fresh (control) the remaining fruit were placed whole in a -20°C walk in freezer. Frozen fruit were extracted after 1, 15, 30, 90 or 180 days of storage. From each sample of 10 fruit 30 g of material was sub-sampled to be extracted with each of the four extractants. Each 10 fruit sample constituted one block, four blocks were utilized at each storage time. Anova analysis was performed and means separated by LSD (>0.05) with Genstat version 12 (VSN International, UK). Extraction Protocol and Total Plant Phenols Analysis A subsample consisting of a slice 0.5 cm wide was cut from the central part of each of the 10 frozen fruit with a bench saw. The central core was removed from the slices, which were then chopped with a knife and mixed for homogeneity. Four 30 g subsamples were then weighed into 80 ml beakers and completely submerged in 40 ml of each extractant to avoid thawing while exposed to air. The material was blended for 2 minutes while still frozen, and the blender rinsed with an additional 20 ml of extractant. All material was poured into 250 ml conical flasks and placed on a shaker at 4°C. Shaking time and extraction procedure varied with the extractant. Four extractants were tested in the experiment; 50:50 (v/v) acetone-water (A50); 90:10 (v/v) methanol-water (M90); 70:0.5:29.5 (v/v) acetone-acetic acid-water (AWA); and 50:50 (v/v) methanol-water adjusted to pH 2 with HCl followed by 70:30 (v/v) acetone-water (50M70A) following Arranz et al. (2009) without sample digestion. AWA and M90 were shaken for 1 hour at 4°C, and poured into two 50 ml plastic tubes equally distributed and centrifuged (Model 5810 R, Eppendorf, Hamburg) at 4000 rpm for 10 minutes at 4°C. After centrifugation the supernatant was decanted into 250 ml volumetric flasks and the remaining tissue pellet mixed with 35 ml of extractant and centrifuged as before. Supernatants from each extraction were combined in the volumetric flasks and brought to volume. 50A was shaken for 2 hours at 4°C then centrifuged as previously described. The 1460
supernatant was decanted into 250 ml volumetric flasks and the remaining tissue pellet extracted twice more with 30 ml of extractant. The supernatants from each extraction were combined in the volumetric flasks and made to volume. The 50M70A combination comprised two separate extractants. First the 50:50 (v/v) methanol-water extract was shaken for 1 hour at 4°C, then centrifuged as previously described and decanted into 250 ml volumetric flasks. The remaining tissue pellet was mixed with 25 ml of 50:50 (v/v) methanol-water, centrifuged, and the supernatant decanted. The tissue pellet was then mixed with 25 ml of 70:30 (v/v) acetone-water and shaken for one hour at 4°C. After centrifugation the supernatant was decanted and the tissue pellet mixed with an additional 20 ml of 70:30 (v/v) acetone-water, centrifuged and the supernatant decanted. The supernatants from each extraction were combined in the volumetric flasks and made to 250 ml volume. Subsamples from the different extractions were placed in 1.5 ml tubes and mixed in a 1:5 dilution with water. Tubes were micro centrifuged at 10,000 rpm for 10 minutes (Model Sarvall MC 12V, Dupont Inc. Newtown, UK). Water soluble total plant phenols were measured with a microplate spectrometer (Varioskan Flash, Thermoscientific Corp., Melbourne, Australia) following the Folin-Ciocalteau (FC) method (Singleton et al., 1999). UHPLC-PDA-MSn The analyses were performed with an Agilent 1290 series Ultra High Pressure Liquid Chromatography system consisting of a binary pump, cooled autosampler (5°C), column heater (40°C), and UV/Vis detector (Agilent, Waldron Germany) connected to a Thermo Fisher LTQ ion trap mass spectrometer operating in the negative ion Electrospray Ionization (ESI) mode (Thermo Fisher, San Jose, USA). The compounds were separated with a 2.7 µm Ascentis Express 2.1×50 mm C8 column (Supelco, Bellefonte, PA, USA) fitted with a C8 guard column. The mobile phase consisted of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid at a flow rate of 0.3 ml/min. The elution profile was: 0 min, 100% A; 20 min, 50% A (linear gradient); 26 min, 20% A; 27 min, 100% A, and the column equilibrated for 3 min before the next injection. UV/Vis chromatograms were acquired at 280, 360 and 520 nm, and spectral data from 190600 nm. The mass spectrometer was tuned by infusing a 10 µg/ml solution of rutin at a rate of 10 µl/min, mixed with 0.2 ml/min UHPLC mobile phase via a T-piece before entering the mass spectrometer. The ESI source voltage was set to 2.5 kV and the capillary voltage set at -42 V. The heated capillary was maintained at 300°C and the sheath, auxiliary and sweep gases were at 30, 5 and 2 units respectively. Full scan ms1 data was acquired in Turbo mode with a mass range of 150-1000. Data dependent ms2, ms3 and ms4 data were also collected in Turbo mode, with a normalized collision energy of 35. Excalibur V2.1 software was used to process the data. Phenolic compounds were identified by their characteristic UV spectra and comparison of mass spectral data with published data (Parveen et al., 2011; Mari et al., 2010). RESULTS AND DISCUSSION In fresh fruit, A50 extracted significantly more TPP followed by AWA and M90 and 50M70A (Table 1). After 24 h frozen storage AWA extracted the highest TPP (Table 1). After 15 days frozen storage AWA extracted a significantly higher amount of TPP followed by the other three extractants which did not differ statistically (Table 1). After 30 days frozen storage AWA extracted a significantly higher amount of TPP followed by 50M70A and A50, and M90 extracted the least (Table 1), while after 90 days AWA again extracted a significantly higher amount of TPP followed by the other three extractants which did not differ statistically (Table 1). At 180 days frozen storage AWA and A50 extracted significantly higher amount of TPP followed by 50M70A and M90 which extracted the same (Table 1). For all the extractants apart from AWA, after 24 h storage there was a significant reduction in TPP content when compared with fresh and with all other storage times. This drop was probably due to the fact that the fruit were not 1461
completely frozen to the core, therefore reducing the possible extractability (Arranz et al., 2009). A50 as an extractant showed a dramatic statistical drop in TPP values during frozen storage when compared with fresh fruit, with a 40% decline after 24 h frozen storage. Despite values increasing at the subsequent storage dates (-23, -18, -20% at 15, 30 and 90 days respectively), TPP never reached fresh values even after 180 days storage (-10%) (Table 1). AWA did not show the 24 h freezing drop, however TPP values were the lowest when compared with the subsequent frozen storage dates. Storing fruit frozen for 15, 30, 90 and 180 days showed similar TPP values. However 15 and 90 days had slightly higher values than fresh fruit (7 and 6% respectively), while all other storage dates were comparable with fresh values (Table 1). It is not clear why A50 did not extract comparable values with fresh tissue during frozen storage while AWA did. According to Downey and Hanlin (2010) there was no difference between 50 and 70% acetone in extracting tannins from grape skin. Even if some of the phenols could have destabilised due to the lack of an acidic environment (Arranz et al., 2009; Downey et al., 2007), it still does not explain the difference with fresh values. 50M70A, apart from the 24 h drop (-12%), extracted statistically higher TPP values than fresh at every other frozen date (23, 20, 24% at 15, 30, 90 days respectively), even if at 180 days increased values were slightly lower than the other frozen times (11%) (Table 1). It is not clear why 50M70A would overestimate TPP during freezing when compared with fresh values. Despite the statistical drop after 24 h freezing in M90 (-10%) all of the other frozen storage dates were comparable to fresh fruit values, apart for 90 days which showed the highest values (22%) (Table 1), at which most of the other extractants also showed higher values. UHPLC-PDA-MSn The majority of compounds present in the UHPLC-PDA-MSn chromatogram were identified by comparison of the UV, mass spectral (ms) and retention time (rt) data reported for similar fruit extracts in the literature (Parveen et al., 2011; Mari et al., 2010) – see Figures 1 and 2 and Table 2. The 22 compounds were grouped into (1) hydroxycinnamic acids, (2) flavanols, (3) procyanidins (4) anthocyanins, (5) flavonols and (6) dihydrochalcones. Two “unidentified” compounds were classified as hydroxycinnamic acids (A and B) and two other “unidentified” compounds classified as anthocyanins (A and B) through comparison with the UV, ms and rt data of other compounds in the chromatogram. Quantitative data were not available as pure standards could not be readily sourced for the majority of compounds. However, comparisons could be made between the levels of each compound in the different extraction solvents from the corresponding peak areas, e.g., 4-p-coumaroyl quinic acid: A50, peak area 18.41 units, AWA, peak area 19.97 units, 50M70A, peak area 18.26 units and M90, 16.29 units. Anova analysis was performed on the area data and means separated by LSD (>0.05). The various groups of compounds behaved differently with the tested extractants. For hydroxycinnamic acids, the two unidentified compounds showed the greatest differences between extractants; epicatechin and the proocyanidins displayed the most significant difference, with M90 extracting the least amount in comparison with the other three solvents. This is in agreement with data reported in the literature (Khanal et al., 2009). For flavonols, the only significant difference was for avicularin, with M90 extracting again the least amount and there was no significant difference for dihydrochalcones. In general, if differences were detected, AWA gave the highest values while M90 the lowest when the polyphenol levels were determined by UHPLC-PDAMSn. No further conclusions for the four unidentified compounds can be made until the structures are confirmed. CONCLUSION This experiment suggests that it is possible to freeze and store whole apple fruit and accurately measure TPP at a later date. The extractant that provided the most consistent and highest values for TPP analysis was AWA. Our results also indicate that 1462
acetone based solvents are a more efficient extractant than methanol based solvents for the determination of most phenolic compounds, in particular epicatechin and procyanidins in ‘Royal Gala’ apples. ACKNOWLEDGEMENTS This is a publication from Premium Fruit, a Victorian Department of Primary Industries project. Also thanks to Janine Jaeger, Janet Tregenza, and Christine Frisina for their technical assistance. Literature Cited Arranz, S., Saura-Calixto, F., Shaha, S. and Kroon, P.A. 2009. High contents of nonetractable polyphenols in fruits suggest that polyphenol content of plant foods have been underestimated. J. Agri. Food Chem. 57:7298-7303. Carbone, K., Giannini, B., Picchi, V., Lo Scalzo, R. and Cecchini, F. 2011. Phenolic composition and free radical scavenging activity of different apple varieties in relation to the cultivar, tissue type and storage. Food Chem. 127:493-500. Diportet, X., Aggio, R.B.M., Carneiro, S. and Villas-Bôas, S. 2012. The biological interpretation of metabolomic data can be misled by the extraction method used. Metabolomics 8:410-421. Downey, M.O., Mazza, M. and Krstic, M.P. 2007. Development of a stable extract for anthocyanins and flavonols from grape skin. Amer. J. Enol. Vitic. 58:358-364. Downey, M.O. and Hanlin, R.L. 2010. Comparison of ethanol and acetone mixtures for extraction of condensed tannin from grape skin. S. Afr. J. Enol. Vitic. 31:154-159. Gorinstein, S., Park, Y.-S., Heo, B.-G., Namiesnik, J., Leontowicz, H., Leontowicz, M., Ham, K.-S., Cho, J.-Y. and Kang, S.-G. 2009. A comparative study of phenolic compounds and antioxidant and antiproliferative activities in frequently consumed raw vegetables. Eur. Food Res. Technol. 228(6):903-911. Heo, H.J., Kim, Y.J., Chung, D. and Kim, D.-O. 2007. Antioxidant capacities of individual and combined phenolics in a model system. Food Chem. 104:87-92. Khanal, R.C., Howard, L.R. and Prior, R. 2009. Procyanidin composition of selected fruits and fruit byproducts is affected by extraction method and variety. J. Agri. Food Chem. 57:8839-8843. Luthria, D.L. 2006. Significance of sample preparation in developing analytical methodologies for accurate estimation of bioactive compounds in functional foods. J. Sci. Food Agri. 86:2266-2272. Luthria, D.L., Biswas, R. and Natarajan, S. 2007. Comparison of extraction solvents and techniques used for the assay of isoflavones from soybeans. Food Chem. 105:325-333. Mari, A., Tedesco, I., Nappo, A., Russo, G., Malorni, A. and Carbone, V. 2010. Phenolic compound characterisation and antiproliferative activity of ‘Annurca’ apple, a southern Italian cultivar. Food Chemistry 123:157-164. Parveen, I., Threadgill, M.D., Hauck, B., Donnison, I. and Winters, A. 2011. Isolation, identification and quantitation of hydroxycinnamic acid conjugates, potential platform chemicals, in the leaves and stems of Miscanthus × giganteus using LC-ESI-MSn. Phytochemistry 72:2376-2384. Robards, K. 2003. Strategies for the determination of bioactive phenols in plants, fruit and vegetables. Journal of Chromatography A 1000:657-691. Singleton, V.L., Orthofer, R. and Lamuela-Raventós, R.M. 1999. Analysis of total phenolics and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. p.152-178. In: L. Packer (ed.), Methods in Enzymology: Oxidants and Antioxidants, Part A, 299. Elsevier, San Diego, USA.
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Tables Table 1. Water soluble total plant phenols (TPP) in ‘Royal Gala’ apples at different storage times depending on extractant used. Extractant1 50:50 A-W 90:10 M-W 70:0.5:29.5 A-Ac-W 50:50 M-W at pH 2 followed by 70:30 A-W 1 2 3
Fresh
1 119.8a2A3 74.2bD 83.7cB 75.5bC 109.9bBC 103.5aC 81.4cC
71.6bD
Frozen storage days 15 30 90 92.5cC 98.8bC 96.5bC 83.0dB 82.8cB 102.7bA 118.5aA 115.7aAB 117.0aA 100.3bA
98.0bA
101.1bA
180 107.6aB 83.3cB 112.6aAB 90.6bB
All concentrations are in volume/volume. A = acetone, W = water, M = methanol, Ac = acetic acid. Small letters, to be read vertically, refer to significant differences between extractants within the same storage time (fresh or frozen storage days) at p
