Advanced glycation end products (AGEs) are generated via the Maillard reaction, in which non-enzymatic glycation occurs between reducing sugars and amine residues on proteins, lipids, or nucleic acids [1
]. In vivo, the major AGEs appear to be derived from the highly reactive intermediate carbonyl compounds dicarbonyls and oxoaldehydes, including 3-deoxyglucosone, glyoxal (GO), and methylglyoxal (MGO) [3
]. In particular, collagen-linked AGEs initiate oxidative reactions and interact with endothelial cells, thereby leading to vascular dysfunction [4
]. Endothelial dysfunction plays a role in the development of atherosclerosis and diabetic vascular disease [5
]. Thus, AGE accumulation is considered to be associated with the development of diabetic complications.
Phenolic compounds generated as secondary metabolites in plants play protective roles against UV radiation and pathogens [6
]. There is also increasing evidence that plant phenolics have anti-oxidative, anti-aging, anti-diabetic, antimicrobial, and anticancer activities in cellular and animal models [7
]. Dietary antioxidants with low toxicity have been suggested as a remedy for treating diabetic complications, although their therapeutic potential in humans remains to be further investigated. Based on our current scientific understanding, phenolic compounds offer considerable hope for the prevention of chronic human diseases. Plants contain appreciable quantities of bioactive phytochemicals, including phenolic compounds, which are potentially good candidates as AGE inhibitors [8
Peanuts are rich in unsaturated fatty acids that contribute to beneficial health effects with regards to metabolic and cardiovascular disease conditions [11
]. To maintain optimal metabolic control in diabetes, diets rich in high monounsaturated fatty acid (MUFA) have been suggested [12
]. There is increasing evidence that the consumption of phytosterols has diverse beneficial health effects [13
]. Many studies have also reported that peanuts contain several types of phenolic compounds [14
]. However, whereas unsaturated fatty acids and phytosterols are known as significant contributors to the alleviation of diabetes, there has to date been limited study on the properties of the phenolic compounds found in peanuts.
In this study, we aimed to evaluate potential beneficial effects of phenolic compounds in peanuts after removing lipid. Firstly, we developed a simultaneous determination method for 12 phenolic compounds in peanuts. The phenolic contents of peanut extracts were analyzed by ultra-high-performance liquid chromatography (UHPLC) coupled with a triple quadrupole detector, and we also examined the efficacy and validity of the method. Finally, we applied the method for comparison of the phenolic contents of peanut extracted using four different solvents and processed using two different methods. It has been reported that phenolic compounds, including p
-coumaric acid (CMA), resveratrol (RV), catechin (CT), rutin (RT), and others, could have anti-glycation activities [15
]. Therefore, the second aim of this study was to investigate the protective effect of phenolic compounds extracted from peanut against AGE formation and AGE lysis, as well as AGE-induced cellular toxicity via regulation of the apoptotic pathway, including mitogen-activated protein kinases (MAPKs), Bcl-2 family member expression, and reactive oxygen species (ROS) generation. Our results showed that peanut extracts containing various phenolics that can decrease AGEs and alleviate MGO-mediated apoptosis in human umbilical vein endothelial cells (HUVECs) by regulating MAPKs, Bcl-2 family members, and ROS generation.
2. Materials and Methods
The 12 standard phenolic compounds used in this study (trans-cinnamic acid (CNA), (−)-epicatechin (EC), (+)-catechin (CT), caffeic acid (CA), p-coumaric acid (CMA), rutin (RT), isoquercitrin (IQ), trans-ferulic acid (FA), trans-resveratrol (RV), luteolin (LT), quercetin (QT), and chrysoeriol (CE)) were purchased from Extrasynthese (Genay, France). HPLC-grade n-hexane, methanol, acetonitrile, acetone, and acetic acid were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure water of 18.2 Ω was prepared using a water purification system (Barnstead International, Dubuque, IA, USA). Each of the 12 phenolic compounds was prepared by weighing an exact amount and dissolving with methanol in a 50-mL volumetric flask to prepare stock standard solutions. These standard solutions were stored at −20 °C in the dark until use. The concentration of all stock solutions was 0.5 mg/mL. A multi-compound working standard solution at a concentration of 10 mg/L was prepared by mixing together appropriate volumes of each stock solution. This was then diluted serially with methanol to prepare working standards, which were stored in screw-capped amber glass tubes at −20 °C in the dark until use.
P38, phospho-p38, extracellular signal-regulated kinase (ERK), phospho-ERK, Jun N-terminal kinase (JNK) and phospho-JNK antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Bcl-2, Bax, P53, anti-mouse and anti-rabbit antibodies were purchased from Santa Cruz Biotech (Delaware Ave, CA, USA). HUVECs (PCS-100-010) were acquired from the American Type Culture Collection (Rockville, MD, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DCFDA fluorescent dye, MGO, GO, dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). Endothelial growth medium (EGM) was obtained from Lonza (Walkersville, MD, USA), and trinitrobenzenesulfonic acid (TNBS) from Thermo Scientific (Rockford, IL, USA).
2.2. Processing of Peanut Samples
Unprocessed shelled peanuts were purchased from four Korean provinces (Kimcheon, Yecheon, Hongcheon, and Udo Island). Peanuts were processed using two different procedures: roasting and steaming. For the roasting process, raw whole peanuts (kernels with skin) were roasted in a convection oven at 180 °C for 20 min. After roasting, the peanut kernels were cooled in a desiccator at room temperature and maintained in sealed plastic bags at −20 °C until use. For the steaming process, raw whole peanuts were steamed in steamer containing boiling water for 20 min. Raw whole peanuts were used as a control to compare the effect of the two different processing methods.
Sample extraction was performed using the procedure described by Gültekin-Özgüven et al. [16
] with modifications. Peanuts were finely ground using a blender (Food mixer HMF-590; Hanil Electronics, Seoul, Korea). To remove lipid in peanuts, ground peanuts (10 g) were defatted by mixing with hexane (10 g, 100 mL, 10 min 5 times) using a homogenizer (OMNI Macro ES; Omni International, Kennesaw, GA, USA). Lipid-removed peanuts were subjected to extraction using four different solvents: 100% ethanol, 70% ethanol, 80% methanol, and 80% acetone. Fifty milliliters of each solvent was added to 3 g of defatted peanut powder in a screw-capped polyethylene bottle. The phenolic compounds were extracted by ultrasound (Bransonic®
ultrasonic bath; Emerson, Danbury, CT, USA) for 30 min at 40 kHz. Thereafter, the extract was centrifuged (3000 rpm, 10 min, 4 °C) and the pellet was re-extracted with the same solvent. The extraction was repeated three times, and the resultant supernatants were combined. Finally, 20 mL of the combined extracts was evaporated under vacuum at 50 °C in a rotary evaporator. The dried samples were dissolved in 5 mL of methanol and filtered through a 0.2-μm PTFE syringe filter. The prepared extracts were stored in amber glass vials at −20 °C until used.
2.4. UPLC-MS/MS Analysis of Individual Phenolic Compounds
Analytical conditions were optimized according to Flores et al. [17
] with slight modifications. In this study, we used a liquid chromatography–tandem mass spectrometry (LC-MS/MS) system to examine the phenolic compound constituents in peanuts. The application of tandem MS (MS-MS or MS) can be used to characterize individual compounds in a mixture or to identify the structure of compounds by separate ionization and fragmentation steps. Typically, an electrospray ionization (ESI) source is used as an atmospheric pressure ionizer. ESI introduces a parent ion into the mass spectrometer and then collision-induced dissociation (CID) enhances the fragmentation of the parent ion [18
]. The triple quadrupole ion trap is very important because it can serve as an exceptionally high-specificity detector since a mass filter is capable of transmitting only the ion of choice.
Chromatographic analysis of polyphenols was performed using an Acquity UPLC system (Waters, Milford, MA, USA) and separation was achieved using a Cadenza CL-C18 column (100 mm × 2.0 mm, 3 µm particle size; Imtakt Co., Kyoto, Japan). Chromatographic separation was performed using a gradient elution with 0.1% acetic acid (v/v) in 5% acetonitrile as eluent A and 0.1% acetic acid (v/v) in acetonitrile as eluent B. The elution started at 100% of eluent A, which was held for 10 min, prior to being ramped to 30% of B eluent over the course of 15 min. This composition was held for 3 min before being returned to the initial condition in 2 min. The total run time was 20 min. The injection volume was 5 µL, the column temperature was maintained at 40 °C, and the flow rate was set at 0.3 mL/min.
Mass spectrometry analysis was carried out using a Waters Acquity TQD tandem quadrupole mass spectrometer (Waters, Manchester, UK). The ESI source was used in the negative mode. For MS/MS detection, the ionization source parameters were as follows: capillary voltage and the extractor voltage were set at 3.0 kV and 3.0 V, respectively; the source temperature was 150 °C and the desolvation temperature was 400 °C; the cone gas (nitrogen) and desolvation gas were set at flow rates of 50 and 750 L/h, respectively; and the collision-induced dissociation was performed using argon as the collision gas at a pressure of 3.7 × 10−3 mbar in the collision cell. Data acquisition was performed using Masslynx 4.1 software with Quanlynx programs (Waters, Milford, MA, USA).
2.5. Preparation of Glycated Bovine Serum Albumin
MGO and GO-modified BSA (MGO-BSA and GO-BSA) were produced by incubating 20 mg/mL BSA and 20 mM MGO or GO in phosphate-buffered saline (PBS; pH 7.4) in the presence of 0.02% sodium azide (pH 7.4) at 37 °C for 7 days. After incubation, the resultant AGEs were dialyzed with a 7-kDa molecular weight cut-off membrane.
2.6. Cell Culture
HUVECs were cultured in EGM supplemented containing 4% fetal bovine serum and 1% penicillin/streptomycin solution. Cells were cultured under standard cell culture conditions (37 °C in a humidified incubator containing 5% CO2). Cells were used between five and eight passages and at 90% confluency.
2.7. Cell Viability
The MTT assay was used for assessing cell viability. Briefly, HUVECs were seeded at 1.0 × 104 cells/well in 96-well plates and incubated for 24 h at 37 °C. The cells were then pretreated with or without 1, 10, and 100 μg/mL of peanut extracts for 1 h, followed by treatment with MGO (400 µM) for 24 h. Following incubation, MTT solution was added after removing media containing compounds. The cells were incubated for 2 h at 37 °C. The medium was gently removed and the resultant formazan crystals were dissolved in 100 μL DMSO. The absorbance at 570 nm was recorded using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cells were observed using IncuCyte ZOOM™ (Essen BioScience, Ann Arbor, MI, USA).
2.8. Western Blotting Analysis
To confirm alteration in the levels of proteins involved in the MAPK and apoptosis pathways, western blotting experiments were performed. Total proteins were extracted from cultured cells using PRO-PREPTM protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) containing phosphatase. Concentrations of the extracted protein solutions were measured using the Bradford assay. Thereafter, equal amounts of protein were separated on 10% SDS-PAGE gels and then transferred onto nitrocellulose membranes. Membranes were incubated with blocking buffer containing 5% skim milk for 1 h at room temperature and overnight at 4 °C with primary antibodies. After washing, the membranes were incubated with conjugated secondary antibodies for 1 h at room temperature. Chemiluminescence was measured using a ChemiDoc XRS + imaging system (Bio-Rad, Hercules, CA, USA). Protein levels were quantified using Image Lab statistical software (Bio-Rad, Hercules, CA, USA).
2.9. Intracellular ROS Detection
Intracellular ROS were detected using DCFDA as a fluorogenic dye, which interacts with hydroxyl, peroxyl, and other ROS. The intracellular ROS scavenging activity of peanut extracts was measured using DCF-DA. Briefly, 2.0 × 105 cells were dispensed into a 12-well plate and incubated overnight in a 5% CO2 atmosphere at 37 °C. After 24 h, cells were pre-treated with 1, 10, and 100 μg/mL of peanut extracts for 1 h, followed by incubation with MGO for 2 h, and then addition of 10 μM DCF-DA. The cells were then incubated for a further 20 min at 37 °C and washed with PBS. Cells were analyzed by flow cytometry (FACSCalibur flow cytometer; Becton Dickinson, San Jose, CA, USA).
2.10. Inhibitory Effects of Peanut Extracts and Constituent Phenolic Compounds on AGE Formation
The MGO-BSA and GO-BSA assay are exceptional methods for investigating inhibitors of protein glycation and were performed according to the method of Kiho et al. [19
] with slight modifications. BSA (5 mg/mL) was incubated with 2 mM MGO or 2 mM GO in PBS (pH 7.4). A peanut extract or one of the 12 standard phenolic compounds (400 μM) was added shortly before incubation. Sodium azide (0.02%) was then added to the reaction mixture and the cells were incubated for 7 days. Aminoguanidine (AG; 1 mM) was used as a positive control. The formation of AGEs was determined using fluorescence at excitation/emission wavelengths of 355/460 nm with a VICTOR™ ×3 multilabel plate reader (Perkin Elmer, Waltham, MA, USA).
2.11. AGE Breaking Activity of Peanut Extracts and Constituent Phenolic Compounds
A TNBS assay was performed to determine AGE-breaking ability according to Habeeb et al. [20
], with slight modifications. Briefly, 1 mL of MGO-BSA or GO-BSA solution (1 mg/mL) was mixed with peanut extracts (0.1, 0.5, or 1 mg/mL) or one of the 12 standard phenolic compounds (400 μM), and then incubated for 24 h. After incubation, 1 mL of 4% NaHCO3 (pH 8.5) and 1 mL of 0.1% TNBS were added and the solution was incubated at 40 °C for 2 h. After the reaction, 1 mL of 0.1% SDS and 0.5 mL of 1 N HCl were added. AGE-lysis ability was measured using a microplate reader at 340 nm (Molecular Devices, CA, USA).
2.12. Statistical Analyses
The data were expressed as the mean ± SD. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Results were analyzed using one-way ANOVA followed by Bonferroni’s test. A p-value < 0.05 was considered statistically significant.
The phytochemical properties of peanuts, including isoflavones, flavonoids, resveratrol, phenolic acids, and tocopherol, have been intensively studied in recent years [28
]. However, there have been no reports of changes in the contents of the 12 phenolic compounds examined in the present study in peanuts following extraction and processing. In this study, we successfully generated peanut extracts rich in phenolic compounds using hexane, which is the most commonly used and preferred solvent for oilseed extraction [31
We found that the gross contents of phenolic compounds in Udo Island peanuts were the most abundant among the four regional samples we examined. In particular, the resveratrol content of Udo Island peanuts (ND to 3.17 mg/kg) was demonstrably higher than that of the other regional samples (ND to 0.26 mg/kg). However, the seed size of Udo Island peanuts was observed to be only one-third that of the other peanut samples examined, indicating that Udo Island peanut seeds have a larger surface area relative to volume compared with the other samples. Because peanut skin is known to be rich in phenolic content [32
], the smaller seed size seems to contribute to the greater abundance of phenolic compounds in Udo Island peanuts. The contents of phenolic compounds in the peanuts sampled from the four different provinces were found to show a wide variation. Although the reason for the observed differences is not known, the cultivar of samples used and/or the cultural environment might have an effect on phenolic contents [33
Generally, the peanuts consumed by humans have undergone some type of processing. This is because, when peanuts are roasted or steamed, the characteristic unpleasant taste of peanuts is removed and the sweet taste is increased. In addition, studies have shown that thermally processed fruits and vegetables have higher biological activities because of chemical changes that occur during heat treatment [35
Phenolic compounds occur in nature in free or bound forms. According to Xu and Chang, thermal treatment might result in a greater availability of plant phenolic compounds in the matrix [37
]. In the present study, steaming and roasting were conducted at temperatures of 100 °C and 180 °C, respectively. The gross phenolic content was found to be higher following roasting than with steaming. These results indicate that a higher processing temperature contributed to a higher release of phenolic compounds. However, although the total amount of phenolic compounds was increased, the amounts of certain individual phenolic compounds (CT, EC, LT, and CE) were reduced after thermal processing.
Chandrasekara and Shahidi showed that high-temperature (130 °C, 33 min) treatment of cashew nuts resulted in a higher total phenol content (TPC) and antioxidant activity than low-temperature (70 °C, 6 h) treatment [38
]. In addition, syringic acid, gallic acid, p
-coumaric acid, catechin, epicatechin, and epigallocatechin were increased by processing at high temperature. Furthermore, Yu et al. showed that the roasting process (175 °C for 5 min) increased the TPC of peanut skin by 40% compared to the raw peanut skin [39
]. However, in the case of black beans, catechin and epicatechin losses of approximately 49% and 17%, respectively, were detected following thermal processing (boiling, 80 min). In addition, boiling treatment has been shown to cause a greater reduction in TPC and total flavonoid content than steam processing [40
]. Collectively, these previous studies indicate that the phenolic content after thermal processing might differ according to material and experimental conditions.
Polar solvents are generally used for extracting polyphenols with different chemical structures and polarities from plants. Aqueous mixtures of ethanol, methanol, and acetone are frequently used solvents for polyphenol extraction. Ethanol is known to be safe for human consumption. Methanol is considered to be more efficient for the extraction of lower molecular weight polyphenols, whereas aqueous acetone is suitable for extraction of higher molecular weight flavanols [41
]. Accordingly, in the present study, we employed extraction yield as a measure of a solvent’s extraction efficiency. Among the four extraction solvents we examined (100% ethanol, 70% ethanol, 80% methanol, and 80% acetone), 80% methanol was found to be the most effective in terms of extraction yield, whereas absolute alcohol was the least effective. We confirmed that CMA, CT, and EC are the main phenolic compounds in peanut extracts, accounting for over 80% of total phenolics. Very low levels of CNA, CA, FA, and CMA were extracted with absolute ethanol. These results are consistent with the findings of similar studies conducted using macadamia skin waste. Dailey and Vuong showed that the type of extraction solvent used significantly affected the recovery yields of phenolics from macadamia skin [42
]. The combination of organic solvents such as methanol, ethanol, acetonitrile, and acetone with water (50%, v/v
) resulted in the highest recovery yields for TPC and flavonoids, followed by those obtained using absolute methanol, and then water. Absolute ethanol, acetonitrile, and acetone produced the lowest recovery yields.
AGE crosslinking on collagen and elastin leads to increased stiffness of the blood vessel system. AGEs promote an increased accumulation and continued crosslinking of collagen leading to a loss of flexibility [43
]. Furthermore, in diabetes, AGEs have been demonstrated to cause diabetic vascular injury [44
]. In this regard, there has been an increasing interest in the use of natural plant compounds as anti-glycation agents. Many studies have reported that diabetic complications can be ameliorated by using polyphenols via the control of AGEs [45
]. Moreover, inhibition of MGO- or GO-induced AGE formation has been found to play an important role in curing diabetes [46
]. In the present study, the effects of peanut extracts and their constituent polyphenols on diabetes were investigated using AGE-related glucose toxicity experiments. As shown in Figure 1
, treatment with peanut extracts resulted in an increase of free amines and a decrease in fluorescence in a dose-dependent manner. Phenolics in peanut extracts also showed AGE-breaking ability (Figure 4
c,d). The rank order of phenolic constituents in this regard was: LT > FA > IQ > RT > RV > AG > CE > CA > QT > CMA. Among these, LT, FA, IQ, RT, and RV were more effective than the positive control (AG). Phenolic compounds containing a catecholic moiety (such as CA) are the most powerful scavengers of free radicals and they may be used as effective chain-breaking antioxidants [47
]. FA, CNA, and CMA are structurally similar to CA and therefore may also have AGE-breaking activity. Our results show that these phenolic compounds have AGE breaking ability, and that among them, CMA is relatively weaker than the others (Figure 4
c,d). Although CMA had a relatively weak AGE-breaking activity, it comprised the highest proportion (36.3%) of total phenolic compounds in peanut extracts. Thus, we assumed that CMA might play an important role in the AGE-breaking activity of peanut extracts. AGE inhibitory activities have been shown to increase in strength with an increasing number of hydroxyl groups on positions 3,4′ hydroxyl group flavonoids [48
]. Moreover, several studies have reported that positions 6 and 8 of the polyphenol A-ring are the major active sites for trapping MGO [45
]. CT, EC, QT, IQ, CE, RT, and LT have hydroxyl groups at the 3’, 4′ in the B ring and 7 positions, and the 6 and 8 positions in the A-ring as active sites. Based on these characteristics, the anti-glycation activity of phenolic compounds could be predicted and our data showed that these compounds have inhibitory effects on AGE formation and/or an ability to break AGE crosslinks (Figure 4
). Furthermore, RT, LT, and RV have a strong AGE-breaking ability, which is as high as that of AG. Among these, CT and EC constitute more than 45% of the total phenolic content in peanut extracts. Thus, CT and EC might play important roles in the anti-glycation activity of peanut extracts. Previous studies have suggested that AGE formation and breakdown can occur through various mechanisms including antioxidant effects, aldose reductase inhibition, carbonyl trapping, crosslink breaking, and inhibition of non-enzymatic glycation [49
]. The efficacy of phenolic compounds in peanuts to inhibit non-enzymatic glycation and to break AGE crosslinks may be mediated by multiple events, potentially in relation to their antioxidative activities [7
MGO is known to be a highly reactive metabolite of glucose that induces cellular injury and apoptosis in endothelial cells. To evaluate the cytoprotective effects of peanut extracts and the constituent phenolic compounds, we treated HUVECs with MGO (Figure 2
b and Figure 5
). However, with the exception of RT and IQ, the individual components of peanut extracts did not show cytoprotective effects in HUVECs. It can accordingly be assumed that the cytoprotective effects of peanut extracts are attributable to positive interactions such as pharmacodynamic synergy, pharmacokinetic interactions, and complementary mechanisms of action [50
]. Protection of endothelial cells from cytotoxic cell death might be one of the therapeutic implications of peanut-induced anti-diabetic and anti-atherosclerotic effects.
MAPKs play a major role in cell differentiation and cell apoptosis [51
], and it has been reported that MGO induces phosphorylation of ERK, JNK, and P38 in the MAPK pathway [52
]. In this study, we observed that MGO treatment induced phosphorylation of MAPKs, whereas pretreatment with peanut extract decreased MGO-mediated MAPK activation in a dose-dependent manner. These data thus support the supposition that peanut extracts have protective effects against MGO-induced apoptosis in HUVECs. ROS are involved in the Maillard reaction and the formation of free radicals occurs in the early stages of AGE formation [53
]. MGO can increase ROS generation, and ROS generation may play a role in AGE-RAGE interaction [25
]. Figure 2
c shows that MGO treatment increased ROS production in HUVECs, whereas pretreatment with peanut extracts ameliorated ROS production dose dependently. Bcl-2 family member proteins also play critical roles in regulating the process of apoptosis [54
]. Bax and Bcl-2 located in the mitochondrial membrane are transcriptional targets for the tumor suppressor protein, P53, which induces cell apoptosis and regulates mitochondrial function and oxidative stress [55
]. In the present study, treatment of HUVECs with 400 μM MGO decreased the expression of Bcl-2 but increased that of Bax and P53. However, pretreatment with peanut extract attenuated the MGO-induced increase in Bax and P53 expression, and increased Bcl-2 expression. In consideration of these data, we suggest that peanut extracts are likely to protect HUVECs via regulation of the MAPK pathway, Bcl-2 family members, and ROS production. In the present study, we found that the amount and type of phenolics differ according to the pretreatment of peanuts or extraction solvent. In roasted or steamed peanuts, the major phenolic compounds, catechin and epicatechin, are reduced by roasting, whereas the amount of coumaric acid, another major phenolic compound, is significantly increased. In addition, the amounts of rutin, quercetin, and isoquercitrin, which are highly effective among the minor phenolics, were also increased. We accordingly assumed that the efficacy of roasted peanuts extracted with 80% MeOH, in which phenolics change and which yield high amounts of phenolics, will be higher than that of raw or steamed peanuts. Peanuts are a rich source of fatty acids, fiber, phytosterols, and phenolic compounds, which contribute to the health benefits of these nuts [56
]. It has been reported that the regular consumption of peanuts may confer a lower risk of cardiovascular disease and decrease inflammatory markers in adult humans [57
]. Moreover, we have shown that peanut extracts have anti-glycation and cytoprotective effects in HUVECs. When all these facts are considered, we anticipate that use of peanuts as a dietary resource for polyphenols will increase in the future.