![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Pharmacology, University of Bologna, 40126 Bologna, Italy
2To whom correspondence should be addressed. E-mail: phrelia{at}biocfarm.unibo.it.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • apples • antioxidant properties • cultivation methods • cold storage
There is growing interest in the health benefits of phytochemicals contained in fruits and vegetables. A considerable body of evidence indicates that phytochemicals such as phenolic compounds play a critical role in the prevention of oxidative damage to biomolecules and associated pathologies in humans, including heart diseases and cancer (1,2).
Epidemiological studies have shown only associations between total intake of fruits and vegetables and beneficial effects, with limited evidence linked to individual phytochemicals. This conforms with the observation that brussels sprouts, onions, and tomatoes (3–6) but not single antioxidants such as ß-carotene, vitamin E, vitamin C, and coenzyme Q10 reduce the levels of biomarkers for free radical DNA damage in urine or lymphocytes in humans (7–11). This lack of evidence for the health-protecting activities of individual phytochemicals could be because the real health benefits of fruits and vegetables come from the concerted action of a cocktail of antioxidants present in whole plant foods (12). A recent study showed that fresh apples provide greater levels of antioxidant and anticancer activity than are provided by dietary supplementation with vitamin C (13).
Variability in the levels of protective components in foods arising from factors in the complete food production chain can confound the identification of associations between food intake and reduced risk for diseases (14). In particular, a realistic evaluation should also consider the various factors such as cultivation methods, industrial processing, and storage that may affect the final concentration of phenolics in food and their eventual bioactivity. The lack of knowledge regarding the effects of these factors has implications for the reliability of the intake data used in epidemiological studies.
At present, there is a general tendency to assay the total antioxidant capacity (TAC)3 of whole food plants by various analytical methods and to evaluate the relation between dietary intake, in terms of TAC, and pathologic processes (15,16). Experimental methods that use normal or cancerous epithelial cells should be integrated into the analytical methods, because they allow evaluation at the cellular level of the antioxidant and anticancer activity of phytochemicals present in food plants.
The present study investigated the influence of commercial cold-storage periods on the antioxidant content and health-related properties of apples grown either by organic or integrated systems. Apples are an important product in the plant food market, and they provide a major source of phytochemicals in the human diet (17). Recent studies highlighting the health-related properties of apples have reported correlations between apple consumption and reductions in the risk of stroke (18), heart disease (19), and lung cancer (20). Apples are usually picked in autumn (September to October), then retailed throughout the following year. Therefore, the cold-storage time of shop-bought apples varies considerably. This raises the question of the effect of various cold-storage times on the health-related properties of apples.
Another potential variable is the use of organic cultivation, which is increasing both in North America and Europe. Recent studies indicate that organic fruits and vegetables may contain higher levels of phytochemicals than their conventionally produced counterparts (21–23). This raises the question of whether organically grown produce provides greater health benefits. Although organic produce is often compared with produce cultivated by methods based on the use of synthetic fertilizers and pesticides, in the European Community these so-called conventional methods have been officially replaced by integrated systems that reduce the use of chemicals by combining organic and conventional techniques (24). Comparisons of the health-related effects of integrated and organically produced fruit are not yet available.
We previously reported that cold storage affects the bioactivity of fruit (25). We then investigated the effects of commercial storage practices on the health-related properties of apples grown by either strictly organic or integrated systems. After analyzing the effects of up to 6 mo of cold storage on levels of total phenolics, vitamin C, and total antioxidant activity, we examined bioactivity in terms of intracellular antioxidant, cytoprotective, and antiproliferative activity in human colon carcinoma (Caco-2) cells in vitro. In particular, we evaluated intracellular antioxidant and cytoprotective effects in Caco-2 cells differentiated to normal intestinal epithelia (26), which provided a suitable model for assessment of the physiological response of the intestinal epithelia to oxidative injury (27,28).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Selection and preparation of apples. Golden Delicious apples from integrated and strictly organic cultivars grown in Pergine Valsugana (Italy) were harvested in October 2001. The apples were selected in the field and examined immediately after picking, were all of similar size, and did not present any physical defects, signs of phatogen or degradation processes, or other characteristics unsuitable for marketing purposes. The apples were collected in crates and either analyzed immediately (after 7 d) or kept under normal cold-storage conditions at 0°C for 3 or 6 mo. To simulate commercial conditions, all the apples were kept at 20°C for the final 7 d before analysis.
Extraction of phenolics. The phenolics were extracted as described by Sun et al. (29). Briefly, 100 g of edible apple with or without skin was mixed with 250 mL of 80:20 methanol:water and homogenized (Ultra-Turrax) for 1 h. The mixture was centrifuged for 10 min at 500 x g, and the clear supernatant was collected. The procedure was repeated with another 250 mL of solvent, and the supernatants were combined and dried completely. The phenolic extracts were stored at –80°C until use.
Analysis of total phenolics. Total phenolic concentration was assayed by spectrophotometric analysis, using the Folin-Ciocalteu colorimetric method (30). Extract dissolved in 0.2 mL of methanol was added to 1 mL of Folin-Ciocalteau’s reagent and 0.8 mL of 7.5% sodium carbonate. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. The absorbance was measured at 750 nm with a spectrophometer (Tecan Spectra Classic). The phenolic concentration was expressed as the gallic acid equivalent (GAE) in mg/100 g edible apple.
Analysis of vitamin C. Vitamin C concentration was assayed by HPLC after extraction in 5% metaphosphoric acid as described by Carbonaro et al. (22). The HPLC system consisted of a Waters 510 pump, a Waters 996 detector, and a Hypersil ODS C18 column (4.6 x 250 mm; Waters). After separation, the ascorbic acid content was measured by UV absorption at 254 and 220 nm, respectively. The ascorbic acid concentration was calculated on the basis of a calibration curve and expressed as mg/100 g edible apple.
Analysis of total antioxidant activity. The total antioxidant activity level was assayed using the stable DPPH· radical (31). In a 96-well plate, 50 µL of each sample was added to 250 µL of 90 µmol/L DPPH· dissolved in methanol. The plate was covered with aluminum foil and left to stand at room temperature for 30 min. The reduction in absorbance was measured at 525 nm with a spectrophometer (Tecan Spectra Classic). The amount of reduction in absorbance was compared to that of vitamin C as a standard, and the result was expressed as the vitamin C equivalent antioxidant capacity (VCEAC) in mg/100 g edible apple (32).
Cell cultures. Human Caco-2 cells (a gift from Fabio Dall’Olio, Department of Experimental Pathology, University of Bologna, Bologna, Italy) were routinely grown at 37°C in a humidified incubator with 5% CO2 in DMEM supplemented with 20% fetal calf serum (FCS), 2 mmol/L glutamine, 50 kU/L penicillin, and 50 mg/L streptomycin. To evaluate the intracellular antioxidant and cytoprotective activity of the apple extracts, Caco-2 cells were seeded at a density of 8 x 104 cells/cm2 in multiwell dishes; once the cells were confluent, the medium was changed every 48 h, using DMEM with 20% FCS. The experiments were performed using completely differentiated cultures at 12 to 14 d postseeding.
Analysis of intracellular antioxidant and cytoprotective activity. The intracellular antioxidant and cytoprotective activity of the apple extracts was evaluated against both formation of intracellular reactive oxygen species (ROS) and cytotoxicity in differentiated Caco-2 cells after treatment with t-BuOOH, a compound used to induce oxidative stress.
The formation of intracellular ROS was assayed using a fluorescent probe, DCFH-DA, as described by Wang et al. (33). Briefly, differentiated Caco-2 cells were incubated for 24 h with extracts equivalent to 50 mg/L of apple. The cells were washed with PBS and then incubated with 5 µmol/L DCFH-DA in PBS in 5% CO2 at 37°C for 30 min. After removal of the DCFH-DA and further washing, the cells were incubated with 3 mmol/L t-BuOOH in PBS for 1 h. After incubation, the fluorescence of the cells from each well was measured at 485 and 535 nm with a spectrofluorometer (Spectra Max Gemini, Molecular Devices). Intracellular antioxidant activity was expressed as the percentage of inhibition of intracellular ROS caused by exposure to t-BuOOH. At least 3 independent experiments were performed for each sample.
Cytotoxicity was monitored by trypan blue uptake as previously described (34). Briefly, differentiated Caco-2 cells were incubated for 24 h with extracts equivalent to 50 mg/L of apple, washed with PBS, and then incubated with 3 mmol/L t-BuOOH in 5% CO2 at 37°C. After 3 h of incubation, the cells were collected by gentle scraping in PBS and dispersed by repeated gentle pipetting. An aliquot of the cell suspension was then diluted 1:1 with 0.5% trypan blue in 10 mmol/L sodium phosphate buffer (pH 7.2) and placed on a Neubauer hemocytometer with a cover slip. The percentages of stained cells were recorded in at least 3 separate counts. Cytoprotective activity was expressed as the percentage of inhibition of cytotoxicity caused by exposure to t-BuOOH. At least 3 independent experiments were performed for each sample.
Analysis of cell proliferation. The antiproliferative activity of the apple extracts was assayed in human cancer cells in vitro as described by Eberhardt et al. (13). Briefly, Caco-2 cells were seeded in 96-well microtiter plates at a density of 1.5 x 104 cells/cm2. After 24 h of incubation at 37°C in 5% CO2, the growth medium was removed, and medium containing apple extract equivalent to 50 mg/L of edible apple was added to the cells. After 96 h of incubation, cell proliferation was measured with the colorimetric MTT assay (35), a colorimetric method utilizing a tetrazolium reagent. Antiproliferative activity was expressed as the percentage of inhibition of proliferation of Caco-2 cells. At least 3 replications of the experiment were performed for each sample.
Statistical analysis. Data are presented as means ± SD. The statistical analysis used Student’s t test for comparison of means and Pearson’s correlation coefficient for relations among variables. Differences were considered significant at P < 0.05. Analyses were performed using STATISTICA 4.5 software (Statsoft) on a Windows platform.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
|
These data indicate that the first 3 mo of cold storage decreases the phenolic concentration of apple skin but not the vitamin C concentration, irrespective of organic or integrated cultivation. Previous studies reported a small decrease in the total catechin concentration of unpeeled Golden Delicious after about 5 mo of cold storage (39) and a slow decline of total phenolic concentration in Granny Smith peel during the first 4 mo of storage (40). The results of the present study suggest that in Golden Delicious peel, such reductions in phenolics mainly occur in the first 3 mo of storage.
The total antioxidant activity of apples, expressed as VCEAC in mg/100 g edible apple, was 3- to 4-fold higher in apples with skin than in those without skin (integrated, P < 0.001; organic, P < 0.001; Table 1). In apples with skin, the first 3 mo of cold storage strongly reduced the VCEAC values (integrated, 334.3 ± 44.2, P = 0.01; organic, 402.4 ± 57.6, P = 0.003) to values similar to those of apples without skin at baseline. Subsequently, however, there was no further drop in VCEAC levels in apples with skin (at 6 mo: integrated, 320.3 ± 50.0; organic, 340.4 ± 69.5; both P > 0.05 compared to 3 mo). However, cold storage did not affect the VCEAC levels of apples without skin (integrated, 251.6 ± 48.8 and 240.8 ± 39.2; organic, 303.5 ± 50.5 and 280.0 ± 45.9, at 3 and 6 mo, respectively; all P > 0.1 compared to baseline). These data indicate that the first 3 mo of cold storage strongly reduces the total antioxidant activity of apple skin, irrespective of the cultivation method. It is noteworthy that a moderate reduction in total phenolics in apple skin translates into a much stronger reduction in total antioxidant activity.
To investigate bioactivity, we first examined the intracellular antioxidant and cytoprotective effects of the fruit extracts in Caco-2 colon cancer cells differentiated to normal intestinal epithelia. We then assessed the antiproliferative activity of the apple extracts in undifferentiated Caco-2 cells.
Freshly picked apples with skin showed higher intracellular antioxidant activity than those without skin (integrated, P = 0.04; organic, P = 0.04; Fig. 1). Moreover, apples with skin also showed higher cytoprotective activity (integrated, P = 0.02; organic, P = 0.04) and antiproliferative activity (integrated, P = 0.02; organic, P = 0.02). Remarkably, the type of cultivation did not affect the intracellular antioxidant, cytoprotective, and antiproliferative activity of freshly picked apples with or without skin (all P > 0.1). These data highlight the health-related benefits of unpeeled whole fresh apples.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
The present study clearly indicates that factors such as cold storage may affect the health-related properties of apples. An individual would probably need to consume at least 2 apples stored for 6 mo or more to obtain the health benefits provided by 1 freshly picked apple. Epidemiological studies on the cancer-preventive benefits of fruits and vegetables should take into account the cold-storage bias for apples, and possibly for other products. More primary research is needed to ascertain whether cold-storage time negatively affects other varieties of fruits and vegetables. Studies to determine which phytochemical compounds are most affected by cold storage are also needed. In the meantime, as previously noted (25), the public should be made aware that freshly picked apples are likely to provide the strongest health benefits. Consumers should also be provided with ready access to information on the harvest date of retail produce.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: Caco-2, human colon carcinoma cells; DCFH-DA, 7'dichlorodihydrofluorescein diacetate; DPPH·, 1,1-diphenyl-2-picrylhydrazyl radical; FCS, fetal calf serum; GAE, gallic acid equivalent; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; TAC, total antioxidant capacity; t-BuOOH, tert-butyl hydroperoxide; VCEAC, vitamin C equivalent antioxidant capacity.
Manuscript received 5 December 2003. Initial review completed 7 January 2004. Revision accepted 15 February 2004.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Ness, A. R. & Powles, J. W. (1997) Fruit and vegetables, and cardiovascular disease: a review. Int. J. Epidemiol. 26:1-13.
2. Key, T. J., Allen, N. E., Spencer, E. A. & Travis, R. C. (2002) The effect of diet on risk of cancer. Lancet 360:861-868.[Medline]
3. Verhagen, H., de Vries, A., Nijhoff, W. A., Schouten, A., van Poppel, G., Peters, W. H. & van den Berg, H. (1997) Effect of brussels sprouts on oxidative DNA-damage in man. Cancer Lett. 114:127-130.[Medline]
4. Verhagen, H., Poulsen, H. E., Loft, S., van Poppel, G., Willems, M. I. & van Bladeren, P. J. (1995) Reduction of oxidative DNA-damage in humans by brussels sprouts. Carcinogenesis 16:969-970.
5. Lean, M. E., Noroozi, M., Kelly, I., Burns, J., Talwar, D., Sattar, N. & Crozier, A. (1999) Dietary flavonols protect diabetic human lymphocytes against oxidative damage to DNA. Diabetes 48:176-181.[Abstract]
6. Boyle, S. P., Dobson, V. L., Duthie, S. J., Kyle, J. A. & Collins, A. R. (2000) Absorption and DNA protective effects of flavonoid glycosides from an onion meal. Eur. J. Nutr. 39:213-223.[Medline]
7. Carr, A. & Frei, B. (1999) Does vitamin C act as a pro-oxidant under physiological conditions?. FASEB J. 13:1007-1024.
8. Halliwell, B. (2000) The antioxidant paradox. Lancet 355:1179-1180.[Medline]
9. McCall, M. R. & Frei, B. (1999) Can antioxidant vitamins materially reduce oxidative damage in humans?. Free Radic. Biol. Med. 26:1034-1053.[Medline]
10. van Poppel, G., Poulsen, H., Loft, S. & Verhagen, H. (1995) No influence of beta carotene on oxidative DNA damage in male smokers. J. Natl. Cancer Inst. 87:310-311.
11. Halliwell, B. (1999) Vitamin C: poison, prophylactic or panacea?. Trends Biochem. Sci. 24:255-259.[Medline]
12. Liu, R. H. (2003) Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. Am. J. Clin. Nutr. 78:517S-520S.
13. Eberhardt, M. V., Lee, C. Y. & Liu, R. H. (2000) Antioxidant activity of fresh apples. Nature 405:903-904.[Medline]
14. Dekker, M. & Verkerk, R. (2003) Dealing with variability in food production chains: a tool to enhance the sensitivity of epidemiological studies on phytochemicals. Eur. J. Nutr. 42:67-72.[Medline]
15. Halvorsen, B. L., Holte, K., Myhrstad, M. C., Barikmo, I., Hvattum, E., Remberg, S. F., Wold, A. B., Haffner, K. & Baugerod, H. et al. (2002) A systematic screening of total antioxidants in dietary plants. J. Nutr. 132:461-471.
16. Pellegrini, N., Serafini, M., Colombi, B., Del Rio, D., Salvatore, S., Bianchi, M. & Brighenti, F. (2003) Total antioxidant capacity of plant foods, beverages and oils consumed in Italy assessed by three different in vitro assays. J. Nutr. 133:2812-2819.
17. Hertog, M. G., Hollman, P. C., Katan, M. B. & Kromhout, D. (1993) Intake of potentially anticarcinogenic flavonoids and their determinants in adults in the Netherlands. Nutr. Cancer 20:21-29.[Medline]
18. Knekt, P., Isotupa, S., Rissanen, H., Heliovaara, M., Jarvinen, R., Hakkinen, S., Aromaa, A. & Reunanen, A. (2000) Quercetin intake and the incidence of cerebrovascular disease. Eur. J. Clin. Nutr. 54:415-417.[Medline]
19. Knekt, P., Jarvinen, R., Reunanen, A. & Maatela, J. (1996) Flavonoid intake and coronary mortality in Finland: a cohort study. Br. Med. J. 312:478-481.
20. Le Marchand, L., Murphy, S. P., Hankin, J. H., Wilkens, L. R. & Kolonel, L. N. (2000) Intake of flavonoids and lung cancer. J. Natl. Cancer Inst. 92:154-160.
21. Weibel, F. P., Bickel, R., Leuthold, S. & Alfoldi, T. (2000) Are organically grown apples tastier and healthier? A comparative field study using conventional and alternative methods to measure fruit quality. Acta Hort. 517:417-426.
22. Carbonaro, M., Mattera, M., Nicoli, S., Bergamo, P. & Cappelloni, M. (2002) Modulation of antioxidant compounds in organic vs conventional fruit (peach, Prunus persica L., and pear, Pyrus communis L.). J. Agric. Food Chem. 50:5458-5462.[Medline]
23. Asami, D. K., Hong, Y. J., Barrett, D. M. & Mitchell, A. E. (2003) Comparison of the total phenolic and ascorbic content of freeze-dried and air-dried marionberry, strawberry, and corn grown using conventional, organic and sustainable agricultural practices. J. Agric. Food Chem. 51:1237-1241.[Medline]
24. Sansavini, S. (1997) Integrated fruit production in Europe: research and strategies for a sustainable industry. Sci. Hort. 68:25-36.
25. Hrelia, P., Tarozzi, A. & Cantelli Forti, G. (2003) Diet and risk of cancer. Lancet 361:258.
26. Hidalgo, I. J., Raub, T. J. & Borchardt, R. T. (1989) Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96:736-749.[Medline]
27. Manna, C., Galletti, P., Cucciolla, V., Moltedo, O., Leone, A. & Zappia, V. (1997) The protective effect of the olive oil polyphenol (3,4-dihydroxyphenyl)-ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in Caco-2 cells. J. Nutr. 127:286-292.
28. Manna, C., D’Angelo, S., Migliardi, V., Loffredi, E., Mazzoni, O., Morrica, P., Galletti, P. & Zappia, V. (2002) Protective effect of the phenolic fraction from virgin olive oils against oxidative stress in human cells. J. Agric. Food Chem. 50:6521-6526.[Medline]
29. Sun, J., Chu, Y. F., Wu, X. & Liu, R. H. (2002) Antioxidant and antiproliferative activities of common fruits. J. Agric. Food Chem. 50:7449-7454.[Medline]
30. Kahkonen, M. P., Hopia, A. I., Vuorela, H. J., Rauha, J. P., Pihlaja, K., Kujala, T. S. & Heinonen, M. (1999) Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem. 47:3954-3962.[Medline]
31. du Toit, R., Volsteedt, Y. & Apostolides, Z. (2001) Comparison of the antioxidant content of fruits, vegetables and teas measured as vitamin C equivalents. Toxicology 166:63-69.[Medline]
32. Kim, D. O., Lee, K. W., Lee, H. J. & Lee, C. Y. (2002) Vitamin C equivalent antioxidant capacity (VCEAC) of phenolic phytochemicals. J. Agric. Food Chem. 50:3713-3717.[Medline]
33. Wang, H. & Joseph, J. A. (1999) Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic. Biol. Med. 27:612-616.[Medline]
34. Baker, S. S. & Baker, R. D., Jr (1993) Caco-2 cell metabolism of oxygen-derived radicals. Dig. Dis. Sci. 38:2273-2280.[Medline]
35. Carmichael, J., DeGraff, W. G., Gazdar, A. F., Minna, J. D. & Mitchell, J. B. (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47:936-942.
36. Burda, S., Oleszek, W. & Lee, C. Y. (1990) Phenolic compounds and their changes in apples during maturation and cold storage. J. Agric. Food Chem. 38:945-948.
37. Escarpa, A. & Gonzalez, M. C. (1998) High-performance liquid chromatography with diode-array detection for the determination of phenolic compounds in peel and pulp from different apple varieties. J. Chromatogr. 823:331-337.
38. Arts, I. C., van de Putte, B. & Hollman, P. C. (2000) Catechin contents of foods commonly consumed in the Netherlands. 1. Fruits, vegetables, staple foods, and processed foods. J. Agric. Food Chem. 48:1746-1751.[Medline]
39. van der Sluis, A. A., Dekker, M., de Jager, A. & Jongen, W. M. (2001) Activity and concentration of polyphenolic antioxidants in apple: effect of cultivar, harvest year, and storage conditions. J. Agric. Food Chem. 49:3606-3613.[Medline]
40. Golding, J. B., McGlasson, W. B., Wyllie, S. G. & Leach, D. N. (2001) Fate of apple peel phenolics during cool storage. J. Agric. Food Chem. 49:2283-2289.[Medline]
41. Wolfe, K., Wu, X. & Liu, R. H. (2003) Antioxidant activity of apple peels. J. Agric. Food Chem. 51:609-614.[Medline]
42. Trewavas, A. (2001) Urban myths of organic farming. Nature 410:409-410.[Medline]
43. Bourn, D. & Prescott, J. A. (2002) Comparison of the nutritional value, sensory qualities, and food safety of organically and conventionally produced foods. Crit. Rev. Food Sci. Nutr. 42:1-34.[Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
