2021 年 27 巻 5 号 p. 747-757
Maillard reaction products (MRPs) produced during thermal processing and storage of foods have received considerable attention because of their potential antioxidant activity. We investigated whether heat-dried tomato (Solanum lycopersicum L.) extracts and their MRPs confer longevity and neuroprotective effects in Caenorhabditis elegans. Heat-drying of mature green tomato resulted in increased absorbance at 294 nm and 420 nm, which indicated the presence of MRPs. Heat-dried green tomato extract and its MRP fraction exhibited DPPH and ABTS radical scavenging activities and significantly extended the lifespan of C. elegans. The MRP fraction protected against β-amyloid-, 1-methyl-4-phenylpyridinium (MPP+)-, and H2O2-induced neurotoxicity in C. elegans. These results suggest that the dry processing of unripe green tomato induces MRP formation that might protect against neurodegeneration.
Aging is characterized by a general physiological decline over time and may involve disruptions in oxidative stress response, aberrant cell signaling, and mitochondrial dysfunction. It is now suspected that this progressive deterioration of cellular functions during aging may significantly increase the risk for several cardio-metabolic and neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD) (Filippopoulou et al., 2017). Therefore, elucidating molecular mechanisms relevant to aging and age-associated pathologies to facilitate discovery of therapeutic targets and clinical or dietary interventions that can promote healthy aging has emerged as a vibrant area of rigorous scientific study.
The roundworm Caenorhabditis elegans, a small (∼1 mm total length), free-living, and soil-dwelling nematode, is among the most widely used models for aging-related research. Their rapid life cycle coupled with high reproductive capacity makes C. elegans a suitable tool for mutagenesis and compound screening. In AD research, specific strains, such as transgenic C. elegans strains that express high levels of human β-amyloid (Aβ) under the control of a muscle-specific promoter (CL2006), are often used (Wu and Luo, 2005). This transgenic AD model relates Aβ expression and cellular toxicity to an easily visible progressive paralytic phenotype, and thus greatly facilitates the evaluation of pharmacological intervention strategies. CL2006 is an ideal model to investigate the therapeutic potential of AD treatment drugs, because this strain constitutively expresses Aβ in the body wall muscles (Ng et al., 2014). C. elegans also has a short adult lifespan and is an established model for biological aging (Braungart et al., 2004; Brenner, 2003; Larsen, 1993). Recently, a 1-methyl-4-phenylpyridinium (MPP+) toxicity-induced PD model in C. elegans that can recapitulate many key features of PD, including dopaminergic neuron degeneration, was established. This C. elegans model is increasingly used to screen neuroprotective agents in vivo (Lu et al., 2010).
During thermal processing and food storage, some biochemical reactions, such as the Maillard reaction, occur and form components that could be beneficial for health. The Maillard reaction occurs between amino acids or proteins and reducing sugars in heat-treated foods such as bakery products and coffee (Delgado-Andrade et al., 2006; Oh et al., 2016; Tamanna and Mahmood, 2015). This results in the formation of Maillard reaction products (MRPs) that have a positive effect on flavor, color, and texture (Oh et al., 2016). Some MRPs have beneficial antioxidative and antibacterial effects (Ganesan et al., 2014; Hiramoto et al., 2004).
Fruits and vegetables contain various healthy compounds. Many studies suggest that there are high contents of tomatidine and specific amino acids, including Asn, Ser, Pro, Tyr, and Val, in unripe green tomatoes, although their amounts decrease as the fruit ripens (Choi et al., 2010; Sorrequieta et al., 2013). Thus, we hypothesized that dry heat treatment of green tomatoes can produce MRPs with high antioxidant activity that might confer longevity and neuroprotective effects. In this study, we investigated the effect of dried tomato extracts and their MRPs on wild-type C. elegans as a model organism. Furthermore, the neuroprotective effects of MRPs against β-amyloid, MPP+, and H2O2 neurotoxins in C. elegans were evaluated.
Materials Tomato (Solanum lycopersicum L. ‘Reika’) was obtained from the Education and Research Center of Alpine Field Science, Shinshu University (Nagano, Japan). In this study, green tomato (mature unripe tomato) and red tomato (mature ripe tomato) were harvested on 46 and 59 d after plant flowering, respectively. All reagents were of analytical grade. We obtained 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and MPP+ from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were obtained from Wako Pure Chemical Industries (Osaka, Japan).
Preparation of dried tomato and its extract The green and red tomato fruits were washed, sliced to 3 mm thickness, and dried in a constant temperature oven (DKN812, Yamato, Tokyo, Japan) at 40 °C for 7 d or freeze-dried using a freeze-dryer (FDU-1100, EYELA, Tokyo, Japan). All samples were ground into a powder using an A10 basic analytical grinder mill (IKA, Staufen, Germany), passed through a 0.5 mm particle size sieve, and stored at −25 °C until use. Tomato extract was prepared by adding 30 mg ground tomato to 30 mL distilled water. The solution was then mixed at 12 000 rpm for 2 min using a Polytron PT 3100 (Kinematica AG, Lucerne, Switzerland) and sonicated for 10 min. The extract was finally filtered through a 0.22 µm membrane filter and stored at −25 °C until use.
MRP preparation using gel filtration chromatography Dried tomato samples (40 mg/mL) were dissolved in 50 mM ammonium bicarbonate buffer (pH 7.5). The sample solution was vortexed, sonicated for 10 min, and centrifuged at 7 000 g for 15 min. The supernatant was filtered through a 0.45 µm membrane filter and loaded onto a HiPrep 26/60 Sephacryl S-200 HR gel filtration column (GE Healthcare, Chicago, IL, USA) on an ÄKTA Pure 25 chromatography system (GE Healthcare). Elution was carried out with 50 mM ammonium bicarbonate buffer (pH 7.5) at a flow rate of 0.5 mL/min, and the absorbance was measured at 280 nm. The column bed void fraction was determined from the peak retention volume of blue dextran, and each fraction (F1, F2, and F3) of the applied samples was collected and freeze-dried.
Browning intensity The absorbance of tomato samples at 294 nm and 420 nm was determined using a UV-1700 spectrophotometer (Shimadzu, Kyoto, Japan) according to the method described by Ganesan et al. (2014) with slight modifications. Briefly, 10 mg/mL freeze-dried or heat-dried tomato samples were prepared in distilled water. The solution was then homogenized at 12 000 rpm for 2 min using a Polytron PT 3100 (Kinematica AG) and sonicated for 10 min. The extract was finally filtered through a 0.45 µm membrane filter, and the absorbance was measured at 294 nm and 420 nm.
Reducing sugar content Reducing sugars in the dried tomato extract were measured by the dinitrosalicylic acid method (Deshavath et al., 2020). Standard glucose solutions were used to develop a calibration curve. The reducing sugar content was calculated as per g dry weight of the dried tomato sample.
Free radical scavenging activity of tomato samples The capacity of the dried tomato extract and collected fraction (F1, F2, and F3) to scavenge free radicals was assessed using ABTS and DPPH assays (Blois, 1958). The results were expressed as µg 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalent/mg sample.
C. elegans maintenance All C. elegans strains and their food, E. coli OP50, were obtained from the Caenorhabditis Genetics Center (CGC) (Minneapolis, MN, USA). Wild-type N2 and AD model CL2006 worms were used for lifespan assays. The worms were maintained according to the standard protocols described by Stiernagle (2006) at 20 °C on nematode growth medium (NGM) agar plates containing E. coli OP50. S-complete solution was prepared according to the literature (Solis and Petrascheck, 2011).
Lifespan assay using wild-type C. elegans Wild-type C. elegans lifespan was determined according to the method described by Amrit et al. (2014). Briefly, 12 larval stage 4 (L4) of the C. elegans N2 strain from a synchronized population were transferred to eight NGM plates and allowed to become adults in the presence or absence of tomato extract (1 mg/mL) or the collected fraction (F1, F2, and F3, 500 µg/mL) at 20 °C. Worms were scored as dead or alive every other day. Missing worms were censored based on their lifespan count. Significance was determined by the log-rank test, and the mean was calculated by averaging the days of C. elegans death for each condition. The results were obtained from three independent experiments (50–120 worms/treatment in each experiment).
Lifespan assay using CL2006 strains CL2006 strains were used to assess paralysis according to the method described by Diomede et al. (2013) with some modifications. Briefly, 12 larval stage 4 (L4) nematodes from a synchronized population were transferred to eight NGM plates and allowed to become adults in the presence or absence of F1 (500 µg/mL) at 16 °C. Worms were transferred every day until all nematodes were dead. The number of worms paralyzed was scored starting from L4 larval stage (day 0), and for each consecutive day until all worms were dead. Significance was determined by the log-rank test, and the mean was calculated by averaging the days of C. elegans death for each condition. The results were obtained from three independent experiments (50–120 worms/treatment in each experiment).
Neurotoxicity assay MPP+-induced neurotoxicity in wild-type C. elegans was performed using the method described by Lu et al. (2010) with some modifications. L1 larvae were added to 96-well plates at an average of 12 worms per well in a 40 µL solution containing E. coli OP50. A 952 µM MPP+ solution and the tested tomato sample dissolved in S-complete solution were added to achieve a final volume of 50 µL per well. L1 larvae were incubated with MPP+ alone or in the presence of various concentrations of F1. Forty-eight hours after exposure to MPP+, worm viability was visually inspected under a stereomicroscope. The results of the MPP+-treated groups were normalized and expressed as a percentage of the normal controls. The results were obtained from three independent experiments (80–130 worms/treatment in each experiment).
Oxidative stress assay Oxidative stress was induced by H2O2 in wild-type C. elegans. L1 larvae were added to 96-well plates at an average of 12 nematodes per well in a 40 µL solution containing E. coli OP50. A 2 mM H2O2 solution and the tested tomato sample dissolved in S-complete solution were added to achieve a final volume of 50 µL per well. L1 larvae were incubated with H2O2 alone or in the presence of various concentrations of F1. Forty-eight hours after exposure to H2O2, worm viability was visually inspected under a stereomicroscope. The results from the H2O2-treated groups were normalized and expressed as a percentage of normal controls. The results were obtained from three independent experiments (80–130 worms/treatment in each experiment).
Statistical analysis Data are expressed as mean ± standard deviation (SD) for each group. Significant differences between groups were assessed using one-way ANOVA, followed by Tukey's post hoc comparison test. Statistical significance was set at p < 0.05, p < 0.01, or p < 0.001. For lifespan assays, C. elegans survival was plotted using Kaplan-Meier survival curves and analyzed by log-rank tests using Graph Pad Prism software (version 9.01).
Browning intensity and radical-scavenging activity of dried tomato extracts To confirm that the Maillard reaction occurs during the tomato drying process, the browning intensity of the dry samples was determined by measuring the UV absorbance at 294 nm and 420 nm (Table 1). The results showed that heat-dried samples had higher absorbance values than freeze-dried samples. No marked differences were observed between the green and red tomato samples. On the other hand, the heat-dried samples had a lower amount of reducing sugars than the freeze-dried samples. These results suggest that during heat-treatment, MRPs were produced via the Maillard reaction between reducing sugars and proteins or amino acids. The free radical-scavenging activity in the tomato samples was determined using the DPPH and ABTS methods. The results showed that green tomato samples possessed higher free radical-scavenging activity than the red tomato samples using both assays. Furthermore, the heat-dried green tomato (HGT) extract exhibited the highest antioxidant properties among the tested samples.
| Absorbance | Reducing sugar | DPPH | ABTS | ||
|---|---|---|---|---|---|
| 294 nm | 420 nm | (g of glucose/g dry weight) | (µg Trolox eq/mg) | (µg Trolox eq/mg) | |
| Freeze-dried green tomato | 0.863 ± 0.000 a | 0.666 ± 0.001 b | 0.137 ± 0.001 c | 23.501 ± 0.130 c | 95.723 ± 1.765 c |
| Heat-dried green tomato | 1.290 ± 0.002 b | 0.744 ± 0.001 d | 0.071 ± 0.000 a | 37.133 ± 0.252 d | 99.821 ± 1.013 d |
| Freeze-dried red tomato | 0.822 ± 0.010 a | 0.413 ± 0.009 a | 0.153 ± 0.001 d | 16.750 ± 0.231 a | 89.860 ± 1.708 a |
| Heat-dried red tomato | 1.105 ± 0.010 b | 0.708 ± 0.000 c | 0.121 ± 0.002 b | 17.373 ± 0.522 b | 92.244 ± 0.282 b |
Different letters show significant differences at p < 0.05.
Effect of crude dried tomato extracts on C. elegans lifespan Extracts of dried tomato fruit (500 µg/mL) were added to agar plates containing the standard C. elegans laboratory diet. The median C. elegans lifespan increased significantly when treated with the HGT extract (Fig. 1). Treatment with the freeze-dried green tomato extract resulted in a slight decrease in the median lifespan for unknown reasons. There was no significant difference between treatment with control and extract samples from freeze-dried and heat-dried red tomatoes. These results suggest that the extract obtained from HGT possesses lifespan-extending effects in C. elegans.
Effect of crude dried tomato extracts on the C. elegans lifespan.
N2 worms were treated with 1 mg/mL crude dried tomato extracts. (A) Lifespan curve from a single experiment, representative of n = 3 independent experiments. (B) Median lifespan. The results are presented as mean ± SD. * p < 0.05, ** p < 0.01 compared to control.
MRP isolation from HGT extract To isolate MRPs from the HGT extract, the extract sample was subjected to gel filtration chromatography using Sephacryl S-200. Three fractions (F1, F2, and F3) were obtained (Fig. 2). F1 was eluted in the column void volume (MW > 250 kDa) and showed the highest UV absorbance at 294 nm and 420 nm among the fractions (Table 2). The free radical-scavenging activity of F1 was also higher than that of F2 and F3.
Gel filtration chromatography of heat-dried tomato extract.
The sample was dissolved to 40 mg/mL in ammonium bicarbonate buffer (pH 7.5) and applied to a HiPrep 26/60 Sephacryl S-200 HR gel filtration column. Elution was carried out at a flow rate of 0.5 mL/min and the absorbance at 280 nm was measured.
| Fraction | Absorbance | DPPH (mg Trolox eq/mg) | ABTS (mg Trolox eq/mg) | |
|---|---|---|---|---|
| 294 nm | 420 nm | |||
| F1 | 0.721 ± 0.027 c | 0.884 ± 0.000 b | 746.355 ± 29.461 | 195.426 ± 6.923 b |
| F2 | 0.175 ± 0.000 b | 0.050 ± 0.000 a | ND | 9.691 ± 1.761 a |
| F3 | 0.038 ± 0.000 a | ND | ND | ND |
Different letters show significant differences at p < 0.05. ND, Not detected
Effect of chromatographic fractions isolated from HGT extract on C. elegans lifespan We next investigated the effect of chromatographic fractions of HGT extract on C. elegans lifespan. Compared with the control, F1 extended the median and maximum lifespan (Fig. 3A), but there was no significant difference among F2, F3, and the control (Fig. 3B). These results suggest that MRPs contained in F1 may extend the lifespan of wild-type C. elegans.
Effect of heat-dried green tomato (HGT) extract fractions on the C. elegans lifespan.
N2 worms were treated with 500 mg/mL collected fractions (F1, F2, and F3) of heat-dried green tomato extract. (A) Lifespan curve from a single experiment, representative of n = 3 independent experiments. (B) Median lifespan. The results are presented as mean ± SD. *** p < 0.001 compared to control.
Neuroprotective effects of HGT-derived MRPs in C. elegans To investigate the effect against Aβ-induced toxicity, we used transgenic CL2006 C. elegans expressing human Aβ specifically in the body wall muscle. F1 significantly extended the median and maximum CL2006 lifespan compared to the control (Fig. 4A). The median lifespan was increased by 32% after treatment with F1 (Fig. 4B).
Effects of Maillard reaction products isolated from HGT extract on the lifespan of CL2006 transgenic C. elegans.
CL2006 worms were treated with 500 mg/mL F1. (A) Lifespan curve from a single experiment, representative of n = 3 independent experiments. (B) Median lifespan. The results are presented as mean ± SD. *** p < 0.001 compared to control.
Next, we investigated the protective effect against MPP+-induced neuronal injury. The survival rates indicated a protective effect of F1 (Fig. 5). MPP+ treatment resulted in a remarkable decrease in survival rate. However, F1 treatment with MPP+ increased the survival rate in a dose-dependent manner. Additionally, the survival rate was significantly decreased by H2O2 treatment. Similar to previous experiments, F1 treatment increased survival in a dose-dependent manner (Fig. 6).
Effects of Maillard reaction products isolated from HGT extract on MPP+-induced neurotoxicity in C. elegans.
N2 worms were treated with 50, 100, 200, or 300 mg/mL F1 in the presence of MPP+. Each experiment was repeated independently at least three times. The results are presented as mean ± SD. ** p < 0.01, *** p < 0.001 compared to control.
Effects of Maillard reaction products isolated from HGT extract on H2O2-induced neurotoxicity in C. elegans.
N2 worms were treated with 50, 100, 200, or 300 mg/mL F1 in the presence of H2O2. Each experiment was repeated independently at least three times. The results are presented as mean ± SD. *** p < 0.001 compared to control.
Many studies have focused on the antioxidant activity of MRPs in processed foods such as roasted coffee beans and bakery products (Delgado-Andrade et al., 2006; Rufián-Henares and de la Cueva, 2009). In this study, we isolated MRPs with high antioxidant activity from HGT fruit. In the Maillard reaction, reducing sugars react with amino acids or proteins to form complex MRPs in three major stages: early, intermediate, and final stage (Boggio et al., 2000; Martins et al., 2000). UV absorbance at 294 nm is often used to indicate the intermediate MRPs, while the final stage is monitored by measuring the absorbance at 420 nm (browning intensity). The most significant increases in absorbance at 294 nm and 420 nm were observed in HGT. An increment in the antioxidant activity of MRP is closely related to browning (Vhangani and Van Wyk, 2013), suggesting that the Maillard reaction proceeded to the final stage. On the other hand, freeze-dried samples also showed high absorbance at 294 nm and 420 nm, indicating MRP production. The Maillard reaction begins in the initial stages of ripening and progresses during fruit storage (Obulesu and Bhattacharya, 2011). Since the antioxidant activities of freeze-dried tomato samples were lower than those of dry-heated tomato samples, the antioxidant activities might be attributed to brown polymers formed at the final stages of the Maillard reaction.
During tomato fruit ripening, Asn, Ser, Pro, Tyr, and Val content declines, whereas Asp and Glu content increases (Boggio et al., 2000). Echavarría et al. (2013) reported that MRPs derived from Trp and Lys show the highest absorbance at 420 nm, and MRPs from Tyr showed the highest free radical-scavenging activity among all tested MRPs. The major sugars of tomato are glucose and fructose (Davies and Kempton, 1975), and the amount of reducing sugars increases during fruit ripening (Gautier et al., 2008). Since the dried green tomato samples exhibited higher antioxidant activity than the dried red tomato samples, amino acid metabolism during tomato fruit ripening might mainly affect the antioxidant activity of MRPs produced from each tomato sample.
In regards to the lifespan experiments, worms treated with the HGT extract had a longer median lifespan than the control C. elegans. To further understand the reason for this lifespan extension, the HGT extract was analyzed by gel filtration chromatography. Among the three collected fractions, only F1 showed a high absorbance at 420 nm and strong free radical scavenging activity. These results suggest that F1 contains high molecular weight MRPs derived from the HGT extract.
During the past decade, the C. elegans AD model has been extensively used to study the protective effects of natural products against AD. Interestingly, supplementation with F1 extended the lifespan of CL2006, a transgenic C. elegans strain that constitutively expresses Aβ in muscle cells. Coffee extracts also protect against Aβ toxicity in a transgenic C. elegans AD model, possibly by the activation of skn-1 (Nrf2 transcription factor in mammals) (Dostal et al., 2010). Nrf2 activation can induce an increased antioxidant response, thereby increasing stress resistance and lifespan. Oxidative stress is associated with aging and age-related diseases (Liguori et al., 2018). Accumulating evidence indicates that aging is mainly caused by damage from free radical reactions (Harman, 1981). Our study demonstrates that MRPs obtained from HGT increased the lifespan and attenuated H2O2-induced neurotoxicity, which results from mitochondrial dysfunction causing cell death. In contrast, HGT-derived MRPs showed a potential neuroprotective effect in MPP+-treated C. elegans. MPP+ is a dopaminergic neuronal toxin that induces oxidative stress and apoptosis. Therefore, further research is necessary to better understand the underlying mechanisms responsible for neuroprotection against Aβ accumulation and oxidative stress.
In conclusion, we demonstrate that unripe green tomato-derived MRPs have antioxidant activities that increase longevity and extend neuroprotective effects against various neurotoxins, such as Aβ, MPP+, and H2O2 in C. elegans. These findings provide useful information for developing beneficial compounds that promote longevity and healthy aging. The potential extension of lifespan and neuroprotective effects of HGT extract might be the result of antioxidant capacities of the MRPs formed during the drying process. Further investigation is necessary to clarify the detailed mechanism of action.
Conflict of interest There are no conflicts of interest to declare.
