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Keywords:

  • apple;
  • banana;
  • neurotoxicity;
  • orange;
  • oxidative stress

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

ABSTRACT:  Banana, orange, and apple are the major fruits in Western and Asian diets. In order to find the effects of these fruits, neuron like PC12 cells were exposed to the extracts of these fruits before H2O2 treatment. We found a significant viability of PC12 cells by the MTT reduction test, which indicated that the phenolics of banana, orange, and apple fruits prevented oxidative stress-induced neurotoxicity. Additional tests by lactate dehydrogenase and trypan blue exclusion assays showed that the extracts reduced oxidative stress-induced neuronal cell membrane damage. These results suggest that fresh apples, banana, and orange in our daily diet along with other fruits may protect neuron cells against oxidative stress-induced neurotoxicity and may play an important role in reducing the risk of neurodegenerative disorders such as Alzheimer's disease.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Alzheimer's disease (AD) is a progressive and neurodegenerative disorder characterized by loss of memory and cognition. Many studies indicate that the brain of an AD patient is subjected to increased oxidative stress resulting from free radical damage, and the resulting cellular dysfunctions are widely believed to be responsible for neuronal degeneration in AD (Runyons and others 2005). Oxidative neuronal cell damage has been implicated in neurodegenerative disorders such as AD (Behl 1999; Lambeth 2007). It is mediated by reactive oxygen species (ROS), including hydrogen peroxide (H2O2), superoxide anion, and hydroxyl radicals, which are generated as byproducts of normal and irregular metabolic processes that utilize molecular oxygen. ROS can attack cellular biomolecules. For example, increased oxidation of protein and DNA is reported in AD patients, and decreased levels of polyunsaturated fatty acids coupled with increased lipid peroxidation are also found in the AD brain. These oxidative stress-induced damages disrupt cellular function and membrane integrity, thereby leading to apoptosis (Fiers and others 1999; Zana and others 2007). It has been demonstrated that ROS generate cell death through apoptosis (Deng and others 1999). There are many types of physiological inducers of oxidative stress, which are able to cause apoptotic cell death. For instance, H2O2 induces apoptosis in many different cell types (Deng and others 1999), and this effect will be inhibited by addition of antioxidants such as vitamin C (Jang and Surh 2003). As the major component of ROS, H2O2 has been extensively used as an inducer of oxidative stress in many in vitro models (Satoh and others 1996).

Natural antioxidants have been reported to play a major role in blocking oxidative stress induced by free radicals. Recently, natural foods and food-derived components, such as antioxidative vitamins and phenolic phytochemicals, have received a great deal of attention because they are safe and not concerned as “medicine”; some of these are known to function as chemopreventive agents against oxidative damage. Vitamin C has been considered to be one of the most potent antioxidative agents of fruits and vegetables and shows essential chemopreventive effects without toxicity at a relatively high level (Lee and others 2002). However, the contribution of vitamin C to the total antioxidant activity of fruits was determined to be generally less than 15% (Wang and others 1996), while polyphenolic phytochemicals contribute significantly to the total antioxidant capacity of fruits (Eberhardt and others 2000).

Fruits and vegetables contain many different antioxidant substances. These naturally occurring substances present in the human diet have been identified as potential chemopreventive agents (Grundman and Delaney 2002). We previously reported that the antioxidative and antiproliferative activities of apples are the effect of synergistic activities of phenolics rather than vitamin C, and the total antioxidant capacity of apples is due to the contributions of phenolics (Eberhardt and others 2000; Lee and others 2002). However, the effects of phytochemicals of fruits and vegetables against oxidative stress-induced neurotoxicity are relatively unknown. In the present study, we have investigated the possible protective effect of apple, banana, and orange phenolics, 3 major fresh fruits consumed by North Americans and Koreans (Ministry of Agriculture & Forestry, Republic of Korea 2004; USDA/Economic Research Service 2007), against oxidative stress-induced apoptosis in neuronal PC12 cell.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Materials

Roswell Park Memorial Inst. (RPMI) 1640 medium, fetal bovine serum, horse serum, penicillin, and streptomycin were obtained from Gibco BRL (Grand Island, N.Y., U.S.A.). All other chemicals were the products of Sigma (St. Louis, Mo., U.S.A.). Fresh Fuji apples were picked at commercial maturity during the 2002 harvest season at the New York State Agricultural Experiment Station orchard in Geneva, N.Y., U.S.A. Fresh bananas and oranges were obtained from a local grocery store. Immediately upon arrival in the laboratory after harvest, apples were stored in a 2 to 5 °C cold room. They were carefully cut into slices, the pits were removed, and the freeze-dried samples were ground to powder using a laboratory mill (Thomas Scientific, Swedesboro, N.J., U.S.A.) and then stored at −20 °C until analyzed.

Extraction of phenolics

The phenolics in fruits were extracted from 10 g of dried samples (apple with skin, banana, and orange) using 80% aqueous methanol by the ultrasound-assisted method (Lee and others 2003). The mixture was sonicated for 20 min with a continual stream of nitrogen gas purging to prevent possible oxidative degradation of phenolics. The mixture was filtered through Whatman nr 2 filter paper (Whatman Intl. Ltd., Brentford, U.K.) using a chilled Bűchner funnel and rinsing with 50 mL of absolute methanol. Extraction of the residue was repeated using the same conditions. The 2 filtrates were combined and transferred into a 1 L evaporating flask with an additional 50 mL of 80% aqueous methanol. The solvent was removed using a rotary evaporator at 40 °C. The remaining phenolic concentrate was first dissolved in 50 mL of absolute methanol and diluted to a final volume of 100 mL using deionized distilled water (ddH2O). The mixture was centrifuged at refrigerated temperatures at 12000×g for 20 min and stored at −20 °C until analyses.

Cell culture

PC12 cells were propagated in RPMI 1640 medium containing 10% heat-inactivated horse serum, 5% fetal bovine serum, 50 units/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 5% CO2 (Heo and others 2002). The PC12 cell line was derived from a transplantable rat pheochromocytoma. The cells respond reversibly to nerve growth factor (NGF) by induction of the neuronal phenotype.

Determination of cell viability

PC12 cells were plated at a density of 104 cells/well on 96-well plates in 100 μL RPMI, and the cell viability was determined by the conventional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) reduction assay (Heo and others 2001). The cells were incubated with 0.25 mg MTT/mL (final concentration) for 2 h at 37 °C, and the reaction was stopped by adding solution containing 50% dimethylformide and 20% sodium dodecyl sulfate (pH 4.8). The amount of MTT formazan product was determined by measuring absorbance using a microplate reader (Bio-Rad, Hercules, Calif., U.S.A.) at a test wavelength of 570 nm and a reference wavelength of 630 nm.

Measurement of cell membrane toxicity

PC12 cells were precipitated by centrifugation at 2000×g for 2 min at room temperature, 50 μL of the supernatants were transferred into new wells, and LDH was determined using the in vitro toxicology assay kit (Sigma). Total cellular LDH activity was determined by solubilizing the cell with 0.2% Triton X-100 (Heo and others 2001). Damage of the plasma membrane was evaluated by measuring the amount of the intracellular enzyme LDH released into the medium.

The trypan blue exclusion assay was based on the capability of viable cells to exclude the dye. Since viable PC12 cells maintained membrane integrity, the cells did not allow trypan blue dye to pass through the cell membrane. Cells with damaged membrane appeared blue due to their accumulation of dye, and were counted as dead. The dye of 0.4% trypan blue was added to PC12 cells, and after 5 min cells were loaded into a hematocytometer and counted for the dye uptake. The number of viable cells was calculated as percent of the total cell population (Heo and others 2001).

Statistical analysis

All data were expressed as mean ± SD. Statistical analysis was performed by Student's t-test. Statistical comparisons within the same group were performed for paired observations. A P value of < 0.05 was considered significant.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

The oxidative stress-induced neurotoxicity was examined by determining the percentage of MTT reduction after incubation of PC12 cells for 2 h with H2O2. Hydrogen peroxide (400 μM) caused a significant decrease in cell viability (45 ± 3.2%), but pretreatment of PC12 cell with fruit phenolics blocked oxidative stress-induced cytoxicity in a dose-dependent manner. Quercetin (60 μM) was used as positive control. Especially, apple with the highest cell viability among 3 samples at 2000 μg/mL showed 83% of quercetin which is a major antioxidative phenolic in apples (Figure 1). The protective effects of banana and orange phenolics were lower than that of apple, especially at the highest concentration (2000 μg/mL), but the protective effects of banana and orange, compared to the control, showed 118% and 103%, respectively. These data showed the same pattern of the results on their antioxidant capacity. Previous reports represented that the relative total antioxidant capacities of the samples evaluated by using the ABTS radical chromogens compared to vitamin C were as follows in decreasing order: apple (2.3) > banana (1.8) > orange (1.6) (Heo and others 2004; Heo and Lee 2005).

image

Figure 1—. Cell viability effect of fruit phenolics on H2O2-induced cytotoxicity in PC12 cell system. PC12 cells were pretreated for 10 min with various concentrations. The cells were then treated with 400 μM H2O2 for 2 h. Levels of cell viability were measured using the MTT assay as described in Materials and Methods. Quercetin (60 μM) was applied as positive control. Results shown are means ± SD (n= 3). Significant difference (P < 0.05) was observed on the H2O2-induced apoptosis.

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This result clearly demonstrated that PC12 cell death by oxidative stress was suppressed by pretreatment with phenolics, of which fruit extracts showed the highest protective effect at 2000 μg/mL (> 100% of control). MTT dye reduction assay is based on the catalytic activity of some metabolic enzymes in intact mitochondria (Jang and Surh 2003). Mitochondria may be one of the most sensitive primary targets of oxidative injury in neuronal cells (Wallace 1992). This may be due to the fact that mitochondrial DNA (mtDNA) does not encode for any repair enzymes and, unlike nuclear DNA, it is not covered by protective histones. In addition, mtDNA is close to the site where free radicals are generated during oxidative phosphorylation (Shoffner and others 1993). Indeed, an increased frequency of mutations in mtDNA has been found in autopsy samples of the AD brains (Saraiva and others 1985), and many studies have suggested mitochondrial defects in the pathogenesis of the AD patient (Shoffner and others 1993). Furthermore, apoptosis is the process of animal cell suicide. It is one of the main types of programmed cell death, and includes complicated biochemical events leading to a characteristic cell death. The process of programmed cell death is controlled by a diverse range of cell signals that may originate either extrinsic or intrinsic inducers. Extrinsic or extracellular signals may involve nitric oxide (NO), hormones, growth factors, and cytokines (Wilson 2000). There is a growing body of evidence which shows that NO is able to induce programmed cell death by helping to diminish the membrane potential of mitochondria and then make it more permeable. The NO is also a free radical, which is relevant to oxidative stress-induced neurotoxicity. Therefore, fruit phenolics with the highest concentration (2000 μg/mL) may protect PC12 cells from cellular oxidative stress-induced mitochondrial defect and NO-induced apoptosis. Consequently, these results suggest that PC12 cell protection (> 100%) by these fruit extracts is partially due to the mitochondrial protection and anti-apoptotic mechanisms.

It has been proposed that amyloid β (Aβ) proteins produced from amyloid precursor protein (APP) play an important role in AD (Selkoe 1994). Aβ proteins are generated by 2 successive cleavage events: β-secretase cleaves the N-terminus of APP, while γ-secretase cleaves the C-terminus. Most APP will be cut at Val40 (Aβ40) or Ala42 (Aβ42) (Selkoe 1999). This Aβ protein has been identified as a possible source of oxidative stress in the AD brain because it can acquire a free radical state contributing to its toxic effects (Pappolla and others 1998). In another study, experiments on a transgenic mouse model of AD support that Aβ-induced neurotoxicity is mediated by oxidative stress. For example, it has been reported (Pappolla and others 1998) that Cu/Zn superoxide dismutase (SOD) and hemoxygenase-1 (HO-1), biomarkers of oxidative stress, were promoted in aged transgenic mice. This study was further verified in the PC12 cell system. Both SOD and HO-1 levels were increased in PC12 cells following treatment with Aβ or H2O2. Hence, these studies strongly suggest that free radicals are involved in Aβ-induced neurotoxicity. Brain is considered abnormally sensitive to oxidative damage (Floyd and Carney 1992), and in fact early studies on the peroxidation of brain membranes supported this conception (Zaleska and Floyd 1985). Since brain is enriched in the more easily peroxidizable fatty acids (arachidonic acid and docosahexaenoic acid [DHA]), the cell membrane of brain is considered abnormally sensitive to oxidative damage (Markesbery and Carney 1999). To examine the probability of oxidative stress-induced membrane damage, we have assessed the protective effect of these fruits phenolics on H2O2-induced cytotoxicity using the LDH assay: measuring the activity of this stable enzyme released into the medium from apoptotic PC12 cells. A quantitative analysis of LDH activity can determine what percentage of cells is dead. Figure 2 shows that treatment with H2O2 caused an increase in LDH release into the medium and a decrease in the number of viable cells (81 ± 2.3%). Pretreatment with the apple extracts exhibited more efficient inhibitory activity of LDH release in PC12 cell system, while banana and orange extracts were less effective on LDH release. The cell viability of apple extract with 2000 μg/mL concentration was the 88% of quercetin. To confirm if apple extracts block the H2O2-induced membrane damage, the trypan blue exclusion assay, which directly measures the viable cells maintaining the capability of excluding the dye and may reflect more precisely the integrity of viable cell membrane, was also used. H2O2-induced oxidative stress increased plasma membrane damage and the fruit phenolics protected the PC12 cells from neurotoxicity in a dose-dependent manner (Figure 3). These data showed the same pattern as LDH assay: the protective activity of apple extracts was more effective than those of banana and orange. These results indicate the double protective effects of the fruit phenolics with antioxidative activity on mitochondrial disruption and oxidative stress-induced membrane damage.

image

Figure 2—. Inhibition effect of LDH release of fruit phenolics on H2O2-induced membrane damage in PC12 cells. PC12 cells were pretreated for 10 min with various concentrations. The cells were treated with H2O2 (400 μM) for 2 h. LDH activity in culture supernatants was measured with a colorimetric LDH assay kit. Basal and total LDH activities were determined in intact cells and cell solubilized with 0.2% Triton X-100, respectively, and LDH release was calculated as [(sample LDH − basal LDH) / (total LDH − basal LDH)]× 100 (%). Quercetin (60 μM) was applied as positive control. All data are presented as the means ± SD (n= 3) and values obtained from 3 separate cultures. Statistical analysis indicated that the influence of the compounds used had significant effect on H2O2-induced membrane toxicity (LDH release) (P < 0.05).

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image

Figure 3—. Preventive effects of fruit phenolics on H2O2-induced membrane damage in PC12 cells. PC12 cells were plated at low density in 24-well plate. Cells were incubated with the phenolics for 10 min before the addition of 400 μM H2O2. Cultures were observed after additional 2 h, and trypan blue exclusion staining was performed. Quercetin (60 μM) was applied as positive control. Data are presented as mean ± SD for 1 representative triplicate determination and are expressed as the percent survival compared to the corresponding controls (P < 0.05).

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AD is subjected to increased oxidative stress resulting from Aβ-induced free radicals, and the resulting neurotoxicity is widely believed to be responsible for neuronal degeneration in AD (Runyons and others 2005). Oxidative neurotoxicity has also been implicated in neurodegenerative disorders such as AD (Behl 1999; Lambeth 2007). In addition, natural antioxidants from fruits and vegetables have been reported to play a key role in inhibiting oxidative stress induced by free radicals. Consequently, it is believed that antioxidative agents or free-radical scavengers might have some beneficial activities for the prevention of oxidative stress-induced neurotoxicity. We previously reported the concentrations and composition of the major phenolics of 6 apple cultivars (Golden Delicious, Cortland, Monroe, Rhode Island Greening, Empire, and NY674) grown in New York State in 2001 (Lee and others 2003; Heo and Lee 2004). The relative total antioxidant capacity of phenolics evaluated by using the ABTS radical chromogens compared to vitamin C was as follows: quercetin (3.06) > epicatechin (2.67) > procyanidin B2 (2.36) > phloretin (1.63) > vitamin C (1.00) > chlorogenic acid (0.97). Especially, quercetin showed the highest relative contribution on total antioxidant activity (34.7%). Therefore quercetin has the most powerful antioxidant capacity (lowest EC50 value) among the major phenolics in 6 apple cultivars. In this point, our results with the cell viability of apple extract were mainly due to the quercetin including other antioxidant phenolics.

The inflammatory reaction hypothesis on AD has been interesting when it was demonstrated that the products of inflammatory reaction, such as cytokines (Shalit and others 1994) and free radicals (Harman 1996), were neurotoxic in experimental neuron models. These products of inflammatory reactions may represent extracellular signals, which initiate and promote neuronal degeneration in AD. Mitogen-activated protein (MAP) kinase (for example, p38 protein families) initiates the induction of genes such as inducible nitric oxide synthase (iNOS) and cycloxygenase-II (COX II) as well as several cytokines, and these genes make cytotoxic inflammatory reaction in neuronal cells (Robinson and others 1999). Protein MAP kinases mediate phosphorylation of sequential proteins, and in many cases the subsequent phosphorylated protein then becomes a protein kinase itself. It has been reported that p38 was activated by interleukin-1β (IL-1β) and H2O2 (Robinson and others 1999). Activation of p38 includes the double phosphorylation of threonine and tyrosine amino acids on a specific domain (Thr180-Gly181-Try182) of the inactivated protein, which resides in the cytosolic part. Upon activation, the phosphorylated protein then moves into the nucleus where it catalyzes phosphorylation and the activation of specific target transcription factors, including cyclic AMP-responsive element binding protein (CREB) and DNA damage-inducible gene (CHOPP/GADD153) and monocyte enhancement factor 2C (MEF2C). Therefore, anti-inflammatory agents may play an important role in neurodegenerative disorders such as AD. Several studies indicated that phenolics inhibited the production of inflammatory reaction-induced nitric oxide (NO: inflammatory mediator) through the blocking of the expression level of iNOS, COX-II gene in a concentration-dependent manner (Youdim and Joseph 2001; Jang and Surh 2003). In addition, our previous studies showed that apple had many antioxidative phenolics, and quercetin, which is one of the major antioxidative phenolics, exerted as a much stronger antioxidant than vitamin C (Lee and others 2003).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Chemoprevention has been suggested as a useful strategy for the management of AD and chronic disease. Many natural substances present in the human diet have been known as potential chemopreventive agents (Youdim and Joseph 2001). Our study demonstrated that antioxidants in the major fresh fruits consumed in the United States and Korea protected neuronal cells from oxidative stress. In addition, it has been reported that apple juice with antioxidative phytochemicals protected brain tissue against oxidative damage, and improved cognitive performance in genetically induced AD mice (Rogers and others 2003). Therefore, additional consumption of fresh fruits such as apple, banana, and orange may be beneficial to ameliorate chemopreventive effects in neurodegenerative disease such as AD.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

This study was supported by the fund of Research Promotion Program (RPP-2007-036), Gyeongsang Natl. Univ., and Technology Development Program for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References