Nuclear Nrf2 level increasing agent

Abstract

To provide an agent that can increase the amount of Nrf2 in the nucleus. [Solution] A nuclear Nrf2 increaser containing fermented papaya food as an active ingredient.

Description

Technical Field

[0001] The present invention relates to an agent for increasing the amount of Nrf2 in the nucleus.

Background Art

[0002] A papaya fermented food (hereinafter also referred to as "FPP") produced by fermenting immature fruits of Carica papaya Linn with sugar and edible yeast increases maltose and maltotriose when mixed with saliva compared to when mixed with water. Oral ingestion of FPP is expected to increase oligosaccharides and regulate the intestinal environment, and is also expected to suppress an increase in blood glucose levels and promote wound healing in type 2 diabetes patients (Patent Document 1).

[0003] Nuclear factor erythroid 2-related factor 2 (Nrf2) is widely present in vivo, and its association with cell protection, phase II detoxifying enzyme arrays, and chaperone proteins has been reported (Non-Patent Documents 1 to 4). Also, recent studies have suggested the possibility that Nrf2 exhibits anti-atherosclerotic and cardioprotective effects through experiments using Nrf2 knockout mice (Non-Patent Documents 5 to 7). In addition, redox abnormalities associated with disorders of the Keap1-Nrf2 control system are also thought to be involved in neurodegenerative diseases (Non-Patent Documents 8 to 11). However, the evaluation of functional foods using plant hormones having an action of activating the Keap1-Nrf2 control system against neurodegenerative diseases is limited to in vitro or in vivo experiments (Non-Patent Documents 12 to 14), or remains at the level of complex considerations regarding longevity at the cell level (Non-Patent Document 15). Also, it has been shown that dysfunction of the Keap1-Nrf2 control system and attenuation of the antioxidant response by Nrf2 are associated with the aging process itself (Non-Patent Document 16).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-licensed literature

[0005] [Non-licensed document 1] Baird L, et al., "TheMolecular Mechanisms Regulating the KEAP1-NRF2 Pathway." Mol Cell Biol. 2020Jun 15;40(13):e00099-20. doi: 10.1128 / MCB.00099-20.

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Outdoor Configuration 6

Direct Environment 7

Outdoor Tools 8

Non-licensed literature 9

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Non-licensed Document 13

[0006] The present invention aims to provide an agent that can increase the amount of Nrf2 in the nucleus. [Means for solving the problem]

[0007] The present inventors have found that FPP can increase the amount of nuclear Nrf2.

[0008] The present disclosure provides, for example, the inventions described in the following [1] to [6]. [1] An agent for increasing the amount of nuclear Nrf2, containing a papaya fermented food as an active ingredient. [2] The agent according to [1], which promotes the expression of NQO1 gene and / or HO-1 gene. [3] The agent according to [1] or [2], wherein the nuclear Nrf2 is nuclear Nrf2 in peripheral blood mononuclear cells. [4] The agent according to any one of [1] to [3], which is ingested or administered to middle-aged and elderly humans aged 40 or above. [5] The agent according to any one of [1] to [4], wherein the dosage of the papaya fermented food is 0.5 to 30 g / day for an adult weighing 70 kg. [6] The agent according to any one of [1] to [5], which is continuously ingested or administered for 1 month or more.

Effects of the Invention

[0009] According to the present invention, an agent capable of increasing the amount of nuclear Nrf2 can be provided.

Brief Description of the Drawings

[0010] [Figure 1]A graph showing the amount of nuclear Nrf2 in peripheral blood mononuclear cells 1, 3, and 6 months after the start of FPP or vitamin E intake in the FPP intake group and the vitamin E intake group. "Nuclear Nrf2 / GAPDH (AU)" on the vertical axis means the ratio of nuclear Nrf2 / GAPDH (the ratio of the amount of nuclear Nrf2 obtained by Western blot analysis to the amount of intracellular GAPDH) in each group when the baseline (before FPP or vitamin E intake) is set to 1. "I", "II", "III", and "IV" respectively represent the 1st quartile, 2nd quartile, 3rd quartile, and 4th quartile based on the age of the subjects. "*" means that the p-value for the vitamin E intake group and the baseline was less than 0.01. "**" means that the p-value for the 1st and 2nd quartiles was less than 0.05. "ns" means that the p-value was 0.05 or more compared to the baseline and no significant difference was determined. [Figure 2] A graph showing the expression level of the NQO1 gene in peripheral blood mononuclear cells 1, 3, and 6 months after the start of FPP or vitamin E intake in the FPP intake group and the vitamin E intake group. "NQO1 / GAPDH (AU)" on the vertical axis means the ratio of NQO1 / GAPDH (the ratio of the expression level of the NQO1 gene obtained by reverse transcription quantitative polymerase chain reaction (RT-qPCR) to the expression level of the GAPDH gene) in each group when the baseline (before FPP or vitamin E intake) is set to 1. "I", "II", "III", and "IV" respectively represent the 1st quartile, 2nd quartile, 3rd quartile, and 4th quartile based on the age of the subjects. "*" means that the p-value for the vitamin E intake group and the baseline was less than 0.01. "ns" means that the p-value was 0.05 or more compared to the baseline and no significant difference was determined. [Figure 3]This graph shows the expression levels of the HO-1 gene in peripheral blood mononuclear cells 1, 3, and 6 months after the start of FPP or vitamin E intake in the FPP intake group and the vitamin E intake group. The vertical axis, "HO-1 / GAPDH(AU)", represents the HO-1 / GAPDH ratio for each group (the ratio of HO-1 gene expression to GAPDH gene expression obtained by reverse transcription quantitative polymerase chain reaction (RT-qPCR)) with baseline (before FPP or vitamin E intake) set to 1. "I", "II", "III", and "IV" represent the first, second, third, and fourth quantiles based on the age of the subjects, respectively. "*" means that the p-value compared to the vitamin E intake group and baseline was less than 0.01. "**" means that the p-value compared to the first and second quantiles was less than 0.05. "ns" means that the p-value compared to baseline was 0.05 or greater, and it was judged that there was no significant difference. [Modes for carrying out the invention]

[0011] The embodiments of this disclosure will be described in detail below.

[0012] The nuclear Nrf2 increasing agent relating to this disclosure contains FPP as an active ingredient.

[0013] As mentioned above, FPP is a fermented product derived from papaya, produced by fermenting unripe Carica papaya (Carica papaya Linn) fruit with sugar and edible yeast.

[0014] It is preferable that FPP is manufactured by Osato Laboratory Co., Ltd. and sold by Osato International Co., Ltd. (Patent Document 1, Non-Patent Document 1). The FPP is available as "FPPFermented Papaya Preparation" (registered trademark) or "Immun'Age" (registered trademark). The FPP is certified to ISO9001:2015, ISO14001:2015, and ISO22000:2018, and is produced in a factory that was the first in Japan to obtain FSSC22000, which is said to be the strictest food safety standard in Europe and the United States, thus guaranteeing quality, environmental friendliness, and safety.

[0015] According to analysis by the Japan Food Research Laboratories, 100g of FPP contains 91.2g of carbohydrates, along with small amounts of protein (0.3g), potassium (14.9mg), and water (8.5g) (Lot No. 091; Analysis test report dated May 27, 2014). In the same lot, the lipid content per 100g of FPP was 0g.

[0016] As mentioned above, FPP can be produced by fermenting unripe Carica papaya fruit with sugar and edible yeast.

[0017] The nuclear Nrf2 increasing agent according to this disclosure can increase the amount of nuclear Nrf2. The increase in the amount of nuclear Nrf2 by the nuclear Nrf2 increasing agent according to this disclosure may be due to an increase in Nrf2 expression or to the promotion of Nrf2 translocation into the nucleus. The nuclear Nrf2 may be nuclear Nrf2 from peripheral blood mononuclear cells (hereinafter also referred to as "PBMCs").

[0018] The amount of nuclear Nrf2 can be evaluated by Western blot analysis. For example, nuclear and cytoplasmic fractions can be extracted from target cells, such as peripheral blood mononuclear cells, using a standard method. These fractions can then be subjected to Western blot analysis using anti-Nrf2 antibodies and anti-GAPDH antibodies, respectively. The amount of nuclear Nrf2 in these cells can then be evaluated by dividing the resulting Nrf2 band intensity by the GAPDH band intensity.

[0019] Nrf2's translocation to the nucleus is inhibited in the cytoplasm by its binding to Keap1, but once this binding is released, Nrf2 translocates to the nucleus and exhibits its physiological activity. For example, nuclear Nrf2 can promote the expression of genes whose expression is regulated by antioxidant response elements (AREs) by binding to them. Therefore, the nuclear Nrf2 increasing agent according to this disclosure can increase the amount of Nrf2 in the nucleus, thereby increasing the binding of Nrf2 to AREs and promoting the expression of genes whose expression is regulated by AREs. Genes whose expression is regulated by ARE include genes for antioxidant enzymes such as heme oxygenase-1 (HO-1), quinone oxidoreductase-1 (NQO1), superoxide dismutase, glutathione synthase, thioredoxin reductase, thioredoxin, glutathione peroxidase, and glutamate cysteine ​​ligase; genes for xenobiotic metabolic phase II detoxification enzymes such as glutathione-S-transferase, epoxydohydrase, and aldokereductase; and genes for drug transporters such as multidrug resistance-related protein 1 and multidrug resistance-related protein 2, with the NQO1 gene and / or the HO-1 gene being preferred.

[0020] The Nrf2 gene has single nucleotide polymorphisms (SNPs) such as -653A / G, -651G / A, and -617C / A, but the SNPs of the Nrf2 gene are not particularly limited. "-653A / G" refers to a SNP where, with the Nrf2 gene transcription start site being "+1", subtracting 1 from "+1" for each nucleotide upstream (promoter side) results in "-653", and either adenine (A) or guanine (G) is present at that nucleotide. The -617C / A SNP may be, for example, C / C type, C / A type, or A / A type, and C / C type and / or C / A type is preferred. "C / C type" means that at the base position -617, both alleles are cytosine (C), and "C / A type" means that at the base position -617, one allele is cytosine (C) and the other allele is adenine (A).

[0021] The nuclear Nrf2 increasing agent according to this disclosure can improve the balance between the sympathetic and parasympathetic nervous systems by activating the activity of the parasympathetic nervous system, and therefore can be suitably used as an agent to improve the balance between the sympathetic and parasympathetic nervous systems. For example, middle-aged and elderly people (especially elderly people) have decreased parasympathetic nervous system activity and an imbalance between the sympathetic and parasympathetic nervous systems. However, by administering the nuclear Nrf2 increasing agent according to this disclosure for, for example, 3 months or 6 months or more, parasympathetic nervous system activity can be activated and the balance between the sympathetic and parasympathetic nervous systems can be improved.

[0022] The balance between the sympathetic and parasympathetic nervous systems can be indicated by the root mean square (RMSSD) of the time difference between consecutive heartbeats. That is, a high RMSSD indicates high parasympathetic activity, while a low RMSSD indicates decreased parasympathetic activity. RMSSD can be calculated based on heart rate variability (HRV) measured by conventional methods.

[0023] The nuclear Nrf2 increasing agent according to this disclosure can improve total antioxidant capacity (TAC), and is therefore suitably used as a total antioxidant capacity enhancer. For example, the total antioxidant capacity of a plasma sample collected from a subject can be determined by a conventional method.

[0024] Furthermore, the intranuclear Nrf2 increasing agent according to this disclosure can be suitably used as an arteriosclerosis inhibitor, cardioprotective agent, neurodegenerative disease preventive agent, aging retarder, and the like.

[0025] The nuclear Nrf2 increasing agent relating to this disclosure may be a food, food additive, pharmaceutical, or quasi-drug. The target organism to which the nuclear Nrf2 increasing agent relating to this disclosure may be a mammal, for example, but preferably a human. The above human is not particularly limited, but from the viewpoint of significantly increasing the amount of nuclear Nrf2, middle-aged and elderly people aged 40 years or older are preferred, and such humans include, for example, people aged 40 to 64 years, people aged 65 to 75 years, people aged 65 years or older, or people aged 75 years or older. The amount of nuclear Nrf2 may decrease with age, but the nuclear Nrf2 increasing agent relating to this disclosure can restore (increase) the amount of nuclear Nrf2 in middle-aged and elderly people aged 40 years or older.

[0026] Furthermore, it is preferable that the person described above is a healthy person. A healthy person may be a person who meets the participation and exclusion criteria of the examples described later.

[0027] FPP can be prepared in various forms such as granules, powders, and fine granules to suit oral administration, and additives such as excipients, binders, and lubricants can be added as appropriate during preparation.

[0028] The amount of FPP used as the active ingredient may be 0.5 to 30 g / day for an adult weighing 70 kg, preferably 1 to 20 g / day, more preferably 3 to 15 g / day, and most preferably 3 to 9 g / day.

[0029] The intake or administration of FPP may be 1 to 5 times per day, preferably 1 to 3 times per day, and more preferably 2 times per day.

[0030] FPP may be used for continuous intake or administration for one month or more, for three months or more, or for six months or more. [Examples]

[0031] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0032] [1. Materials and Methods] The design of this study was approved by the ReGenera Research Association Committee on October 12, 2023, in accordance with the Declaration of Helsinki and its subsequent revisions. This study was designed as a prospective, randomized clinical trial comparing FPP® with a commercially available antioxidant (i.e., vitamin E) and was conducted in a double-blind manner.

[0033] The details of this study will be described later, but in summary, subjects aged 43 to 75 years will be given FPP or vitamin E daily for 6 months, and nuclear Nrf2 levels, NQ01 and HO-1 gene expression levels, TAC, RMSSD, etc. will be evaluated before the start of intake (hereinafter also referred to as "baseline"), and at 1 month, 3 months, and 6 months after the start of intake. Based on this, the effect of FPP on nuclear Nrf2 levels and the usefulness of this effect will be evaluated. The period from the start of FPP or vitamin E intake to 6 months after is also referred to as the "test period".

[0034] <1-1. Selection of subjects, physical examination, etc.> Those who agreed to participate via email or telephone and who met the following participation and exclusion criteria for pre-screening were considered potential participants.

[0035] (Participation criteria) They do not train but lead an active lifestyle (whether employed or retired). They are sociable. They have normal blood pressure. They do not smoke or have quit smoking more than 6 months ago. They have a stable eating and sleeping pattern. They do not take food supplements or have stopped taking them 3 months prior to joining the study. They have no musculoskeletal problems. They are abstaining from alcohol or drink only occasionally and lightly, and do not consume distilled spirits.

[0036] (exclusion criteria) You have a history of or currently suffer from any of the following: cancer, stroke, myocardial infarction, diabetes, thromboembolism, hypertension, obesity, gastrointestinal disorders (ulcers, ulcerative colitis, Crohn's disease), malabsorption syndrome, blood disorders, endocrine disorders, chronic fatigue syndrome, post-COVID complications, suspected post-COVID vaccine side effects, fibromyalgia, rheumatic diseases, autoimmune diseases, depression, or other major mental disorders. You have had surgery (excluding minor skin problems) within the last 6 months. You have used any food supplements within the last 3 months. You smoke or have quit smoking within the last 6 months. You engage in strenuous exercise once a week. You drink large amounts of alcohol. You use illegal drugs. You have an unstable eating pattern. You have sleep disorders. You regularly consume high-caffeine beverages.

[0037] All potential participants were provided with written informed consent and underwent medical history taking, physical examination, clinical tests, HRV measurement, anthropometric measurements, and resting blood pressure measurement. Resting electrocardiogram (EKG) was also evaluated for all participants. All participants were within 20% of their ideal weight. Body mass index (BMI) was calculated for all participants. Participants' ages ranged from 43 to 75 years.

[0038] (HRV measurement) The Body Health Analyzer Pro (Binacor) was used to measure HRV as described above. This device connects a Bluetooth-enabled finger device to specific software to analyze the variability and characteristics of heart rhythm and estimate the relevant variables of the sympathetic and parasympathetic nervous systems. HRV measurements were performed after the subjects rested in a quiet room for 10 minutes prior to the measurement. Visual analysis of the obtained raw data was also performed to check for artificial noise caused by movement, ectopic pulses, and respiration.

[0039] <1-2. Intake of FPP and Vitamin E> All subjects were randomized using a random number generator to be assigned in a 1:1 ratio to either the group that received a sachet containing 3g of FPP and a white cellulose film tablet twice daily (hereinafter also referred to as the "FPP intake group"), or the group that received a sachet containing 3g of sugar and a white film tablet containing 400 IU of vitamin E twice daily (hereinafter also referred to as the "vitamin E intake group"). Both the FPP intake group and the vitamin E intake group received the aforementioned prescribed sachets and film tablets twice daily throughout the study period.

[0040] <1-3. Subjects' meals during the study period> This study was conducted in accordance with the Good Clinical Practice (GCP) standards for human medicinal products of the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). Analysis of the subjects' diets during the six months prior to the study period confirmed that their diets were Mediterranean diets (15%, 40-45%, and 35-38% of energy (calories) consisted of protein, carbohydrates, and fat, respectively. Of the lipids, 22-24% were monounsaturated fatty acids (MUFAs), 8-12% were extra virgin olive oil, 8-10% were saturated fatty acids (SFAs), and 4% were polyunsaturated fatty acids (PUFAs)). The details of the subjects' diets were obtained from their dietary reports and interviews with their families.

[0041] <1-4. Gradual Exercise Walking Test (GEWT)> During the six-week trial period described above, subjects underwent GEWT on the first and third days of each week. The GEWT was designed not to improve performance, but rather to provide subjects with an exercise stressor that increased oxidative stress (McGinnis, Graham, et al. “Acute hypoxia and exercise-induced blood oxidative stress.” Internationaljournal of sport nutrition and exercise metabolism 24.6 (2014): 684-693.). This design was intended to make the GEWT more closely resemble real-life situations and to mix aerobic and anaerobic elements. Specifically, the GEWT proceeded as follows: The intensity of the GEWT was increased every two minutes. (1) First 6 minutes: Warm-up (during this time, the intensity was increased from 4.5 km / h to 6.5 km / h) (2) 7-12 minutes: Intensive aerobic test (intensity increased from 7 km / h to 8 km / h during this time) (3) 12-22 minutes: Anaerobic phase (during this time, the intensity was increased from 8 km / h until fatigue occurred or up to 11 km / h)

[0042] Furthermore, during the above-mentioned trial period, the subjects' blood pressure and oxygen saturation levels were measured as needed on the day before the GEWT, within two hours after the GEWT, and the morning after the GEWT.

[0043] <1-5. Collection of whole blood and plasma samples> Blood samples from the subjects were collected from the FPP intake group and the vitamin E intake group before the start of FPP or vitamin E intake, and at 1 month, 3 months, and 6 months after the start of the study period. First, 10 mL of blood collected by venipuncture was placed in an anticoagulant EDTA tube, and the anticoagulant was dispersed. This obtained a whole blood sample. The whole blood sample was centrifuged at 25°C for 15 minutes at 2500 rpm to obtain a plasma sample. Subsequently, a portion of the plasma sample was transferred to a tube and stored at -80°C until testing.

[0044] <1-6. Evaluation of TAC> TAC was evaluated using a conventional method. Specifically, 200 μL of the above plasma sample was mixed with 400 μL of ethanol. Then, 800 μL of hexane was added, the resulting mixture was shaken briefly, and centrifuged at 1000 × g for 5 minutes. Subsequently, 200 μL of the lipid phase was collected as a lipid extract, dried under a nitrogen stream, and stored at -80°C. The dried lipid extract was then dissolved in 200 μL of methanol and centrifuged at 5000 × g for 1 minute. The supernatant (120 μL) was subjected to photochemical analysis, Trolox auxiliary calibration, and analysis using a photosensitizer. Based on these analytical results, TAC was calculated from the following formula (I). TAC = [Amount of antioxidant (nmol) × Dilution ratio (%) × Trolox molar concentration (ng / nmol)] / Pipette volume (μL) ... (Equation (I))

[0045] <1-7. Analysis of the Nrf2 gene genotype> We analyzed the genotypes of the Nrf2 gene (-653A / G, -651G / A, and -617C / A). Specifically, DNA was isolated from the whole blood samples using the modified phenol-chloroform extraction method, and the region from -738 to -461 within the NFE2L2 promoter was amplified by PCR. The resulting DNA fragments were then directly sequenced. The PCR was performed under the following conditions: initial denaturation at 95°C for 4 minutes, followed by 35 cycles of 95°C for 1 minute, 56°C for 1 minute, and 72°C for 1 minute, and then a final extension at 72°C for 8 minutes. Sequencing was performed using a Perkin Elmer ABI 3100 DNA sequencer (Applied Biosystems).

[0046] <1-8. Recovery of mRNA and protein samples (total cell fraction and nuclear fraction) from whole blood> The whole blood sample described above was diluted 1:1 by volume with phosphate-buffered saline (PBS), and then stratified using a Ficoll-Histopaque gradient centrifuge (Sigma-Aldrich) at 800×g for 25 minutes at 20°C. The layer rich in peripheral blood mononuclear cells (PBMC cells), such as lymphocytes, was collected. This layer was washed twice with PBS (ThermoFisher) containing a phosphorylation enzyme inhibitor. The centrifugation conditions for this washing were 400×g for 10 minutes at 4°C. After discarding the supernatant, the PBMC cell pellet was washed with a buffer solution. The obtained PBMC cells were counted using an automated system (BiosRad CD375 Hercules). Subsequently, 2×10⁶ cells were counted. 6 PBMC cells from cells were added to 100 μL of mRNA cell lysate (Ambion Biotech), and the PBMC cells were lysed to obtain an mRNA sample.

[0047] The whole cell fraction and nuclear fraction for Western blot analysis, described later, were obtained using an efficient and practical method described in Chen, Lu, et al. "The Nrf2-Keap1 pathway: A secret weapon against pesticide persecution in Drosophila Kc cells." Pesticidebiochemistry and physiology 164 (2020): 47-57.

[0048] First, regarding the procedure for extracting the core fraction, the specific steps are as follows: 3 × 10 6PBMC cells from cells were added to a PBS solution containing a phosphorylation enzyme, a protease inhibitor, and 0.1 v / v% Triton. The cells were lysed, gently mixed, and microcentrifuged to separate them into a supernatant (for the cytoplasmic fraction) and a pellet (for the nuclear fraction). The supernatant was collected and mixed with 4×Laemmli sample buffer in a 3:1 volume ratio to obtain the cytoplasmic fraction. The pellet (approximately 20 μL) was resuspended in 1000 μL of PBS solution containing 0.1 v / v% Triton and centrifuged for 10 seconds. The supernatant was then discarded, and Laemmli buffer was added to obtain the nuclear fraction.

[0049] Next, regarding the procedure for extracting the whole cell fraction, the specific steps are briefly described as follows: 1 × 10 6 PBMC cells from cells were transferred to a microcentrifuge tube and lysed with a PBS solution containing 0.1 v / v% Triton and a protease-phosphorylation enzyme inhibitor mixture. These were then mixed with 4×Laemmli sample buffer. The resulting PBMC cell lysate was sonicated on ice at level 2 for 5 seconds × 3 times. The lysate was then heated at 100°C for 5 seconds to obtain the whole cell fraction, which was stored at -80°C after heating.

[0050] <1-9. Evaluation of intranuclear Nrf2 levels> The amount of Nrf2 in the nucleus was evaluated by Western blotting analysis of the nuclear fraction described above. Specifically, the nuclear fraction was separated by electrophoresis using SDS-PAGE and transferred to a nitrocellulose membrane. Subsequently, rabbit polyclonal primary antibody and anti-rabbit secondary antibody against Nrf2 (both from ThermoFisher, PA-kit) were added and co-incubated. The band intensity was then analyzed using Image Version 1.56 (National Institutes of Health), and evaluated by optical density via a digital image analyzer. The cytoplasmic fraction was also subjected to Western blotting analysis using an anti-GAPDH antibody, and the optical density of the GAPDH band was measured. The amount of Nrf2 in the nucleus was evaluated by dividing the value of (Nrf2 band intensity) / (GAPDH band intensity) for each sample by the baseline value of (Nrf2 band intensity) / (GAPDH band intensity).

[0051] <1-10. Evaluation of the expression levels of the NQO1 and HO1 genes> The expression levels of the NQO1 and HO1 genes were evaluated by subjecting the above mRNA samples to reverse transcription quantitative polymerase chain reaction (RT-qPCR). mRNA purification was performed using the Qiagen RNeasy Plus Mini Kit (Qiagen) according to the kit's protocol. Subsequently, the amount and purity of the mRNA were confirmed using Thermo Nanodrop (ThermoFisher). RNA quality was also confirmed by agarose gel electrophoresis. The mRNA samples were denatured at 65°C for 5 minutes, and then RNA-cDNA conversion was performed using reverse transcription reagent (BioRad). After that, residual genomic DNA was removed using DNase. For RT-qPCR, the primer sets used were the NQO1 primer set (forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 2) and the HO1 primer set (forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 and reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 4), as listed in Table 2 below, and the GAPDH primer set (forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 5 and reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 6) as an internal control gene. Real-time PCR was performed three times for each sample using a CFX96-Touch (Bio-Rad). The amount of mRNA in each sample was evaluated by dividing the value of (expression level of NQO1 gene or HO1 gene) / (expression level of GAPDH) for each sample by the baseline value of (expression level of NQO1 gene or HO1 gene) / (expression level of GAPDH).

[0052] [Table 1]

[0053] <1-11. Measuring RMSSD> The RMSSD was measured before the start of FPP or vitamin E intake, and at 1 month, 3 months, and 6 months after the start of the study period. More specifically, the subjects' HRV was measured in the same manner as described above (measurement of HRV), and the RMSSD (milliseconds) was calculated based on that.

[0054] <1-12.Statistical analysis> The data for the FPP intake group and the vitamin E intake group were also analyzed by arranging the subjects in each group by age and dividing them equally into four groups (referred to as the "first quartile" to the "fourth quartile," from youngest to oldest). The age ranges for the first, second, third, and fourth quartiles of the FPP intake group were 43-50 years, 51-58 years, 59-66 years, and 67-75 years, respectively. The age ranges for the first, second, third, and fourth quartiles of the vitamin E intake group were 43-50 years, 51-58 years, 59-66 years, and 67-75 years, respectively.

[0055] Hardy-Weinberg equilibrium, and comparisons of allele frequencies between different databases, and comparisons of exclusion rates by sex and age are χ². 2 The analysis was performed using statistical tests. Comparisons of means between different genotype groups were analyzed using the Kruskal-Wallis test with post-hoc analysis using the Holm method. Statistical significance was defined as p-value < 0.05. All statistical analyses were performed using R software version 3.6.2.

[0056] [2. Results] In both the FPP intake group and the vitamin E intake group, the procedures described in [1. Materials and Methods] were well followed. Although some subjects exhibited minor violations of the above procedures, these violations were not considered to affect the study and were not excluded. Specifically, these violations included one missed dose (one in the FPP group and two in the vitamin E group), one subject in the FPP group engaging in three hours of intensive cycling per week followed by three days of complete rest, and one subject in the vitamin E group taking nonsteroidal anti-inflammatory drugs for two days after prolonged driving. Furthermore, no gastrointestinal or allergic symptoms were reported during the study period.

[0057] <2-1.BMI etc.> From the start to the end of the study period, no significant changes were observed in blood pressure, oxygen saturation, or the subjects' BMI, measured the day before the GEWT, within two hours after the GEWT, and the day after the GEWT. However, a non-significant downward trend was observed in the subjects' BMI and total cholesterol from the start to the end of the study period.

[0058] <2-2. Genotype of the Nrf2 gene> As a result of the above <1-7. Analysis of Nrf2 gene genotype>, the single nucleotide polymorphism (-617C / A) of the Nrf2 gene in the subjects was C / C type in 38 subjects (55.8%), C / A type in 28 subjects (41.2%), and A / A type in 2 subjects (2.9%).

[0059] <2-3.Nuclear Nrf2 amount> The results of the above <1-9. Evaluation of Nuclear Nrf2 Levels> are shown in Figure 1. As shown in Figure 1, in the FPP intake group, at 1, 3, and 6 months after the start of FPP intake, nuclear Nrf2 levels were significantly higher in all four quartiles based on the subject's age, compared to baseline and the vitamin E intake group. On the other hand, in the vitamin E intake group, no significant increase in nuclear Nrf2 compared to baseline was observed at 1, 3, and 6 months after the start of vitamin E intake. Furthermore, in the FPP intake group, the nuclear Nrf2 level in the fourth quartile was significantly lower than that in the first and second quartiles, at 1, 3, and 6 months after the start of FPP intake. No influence of gender was observed on these results. From the above, it was shown that FPP has the effect of increasing nuclear Nrf2 levels.

[0060] Furthermore, these findings were the same whether we extracted results from subjects with the Nrf2 gene type C / C or from subjects with the Nrf2 gene type C / A, based on the results from <1-9. Evaluation of Nuclear Nrf2 Amount> above. Such analysis could not be performed for subjects with the Nrf2 gene type A / A, as there were only two such subjects.

[0061] <2-4. Expression level of the NQO1 gene> Of the results for the NQO1 gene from the above <1-10. Evaluation of the expression levels of the NQO1 and HO1 genes>, Figure 2 shows the results for the NQO1 gene. As shown in Figure 2, in the FPP intake group, at 1, 3, and 6 months after the start of FPP intake, the mRNA expression level of the NQO1 gene was significantly higher than at baseline and in the vitamin E intake group in all quartiles from the 1st to the 4th based on the subject's age. On the other hand, in the vitamin E intake group, no significant increase in the mRNA expression level of the NQO1 gene compared to baseline was observed at 1, 3, and 6 months after the start of vitamin E intake. No influence of sex was observed on these results. From the above, it was shown that FPP has the effect of increasing the expression level of the NQO1 gene.

[0062] <2-5. Expression level of the HO-1 gene> Of the results for HO-1 from the above <1-10. Evaluation of Expression Levels of NQO1 and HO1 Genes>, Figure 3 shows the results. As shown in Figure 3, in the FPP intake group, the mRNA expression level of the HO-1 gene was significantly higher than at baseline and in the vitamin E intake group in the first and second quantiles based on the subject's age one month after the start of FPP intake, and in the first to fourth quantiles three and six months after the start of FPP intake. On the other hand, in the third and fourth quantiles one month after the start of FPP intake, the mRNA expression level of the HO-1 gene was significantly lower than in the first and second quantiles one month after the start of FPP intake. Furthermore, in the vitamin E intake group, no significant increase in HO-1 gene mRNA expression level compared to baseline was observed at any of the following times: one, three, and six months after the start of vitamin E intake. No influence of sex was observed on these results. From the above, it was shown that FPP has the effect of increasing the expression level of the HO-1 gene.

[0063] The increased expression levels of the NQO1 and HO-1 genes mentioned above, given the increased amount of nuclear Nrf2, are thought to be due to Nrf2 promoting the transcription of antioxidant stress enzyme genes downstream of antioxidant response elements (AREs).

[0064] <2-6.TAC> Table 2 shows the results of the above <1-6. Evaluation of TAC>. In Table 2, "*" means that the p-value relative to baseline was less than 0.01. As shown in Table 2, in both the FPP intake group and the vitamin E intake group, an increasing trend in TAC was observed at 3 and 6 months after the start of FPP or vitamin E intake. This increasing trend was significant at 6 months in the FPP intake group and at 3 and 6 months in the vitamin E intake group. From the above, it was shown that FPP and vitamin E have the effect of increasing TAC. It is thought that FPP increases TAC by promoting the transcription of antioxidant stress enzyme genes by increasing the amount of nuclear Nrf2. Since vitamin E did not increase the amount of nuclear Nrf2, it is thought that vitamin E increases TAC by a different mechanism than FPP and vitamin E.

[0065] [Table 2]

[0066] <2-7.RMSSD> The results of the above <1-11. Measurement of RMSSD> are shown in Table 3. In Table 3, "*" means that the p-value for the third and fourth quantiles was less than 0.01. Here, RMSSD indicates the balance between the sympathetic and parasympathetic nervous systems based on HRV; a high RMSSD means that parasympathetic activity is active, and a low RMSSD means that parasympathetic activity is decreased.

[0067] In all four groups, the baseline RMSSD values ​​shown in Table 3 were insufficient, indicating decreased parasympathetic nervous system activity and a poor balance between sympathetic and parasympathetic nervous systems based on HRV. Furthermore, as shown in Table 3, in both the FPP intake group and the vitamin E intake group, the RMSSD values ​​for the "third and fourth quantiles" were significantly lower compared to the "first and second quantiles." This suggests that the "third and fourth quantiles" had decreased parasympathetic nervous system activity and a worse balance between sympathetic and parasympathetic nervous systems based on HRV than the "first and second quantiles." As shown in Table 3, in all groups, including the "first and second quantiles" and "third and fourth quantiles" in the FPP intake group, and the "first and second quantiles" and "third and fourth quantiles" in the vitamin E intake group, there was a tendency for RMSSD to improve with continued intake of FPP or vitamin E. Therefore, it was shown that FPP intake can activate parasympathetic nervous system activity and improve the balance between the sympathetic and parasympathetic nervous systems.

[0068] [Table 3]

Claims

1. A nuclear Nrf2 increasing agent containing fermented papaya food as an active ingredient.

2. The agent according to claim 1, which promotes the expression of the NQO1 gene and / or the HO-1 gene.

3. The agent according to claim 1 or 2, wherein the nuclear Nrf2 is nuclear Nrf2 from peripheral blood mononuclear cells.

4. The agent according to claim 1 or 2, which is to be ingested or administered to middle-aged or elderly people aged 40 or older.

5. The agent according to claim 1 or 2, wherein the dose of fermented papaya food is 0.5 to 30 g / day for an adult weighing 70 kg.

6. The agent according to claim 1 or 2, which is ingested or administered continuously for one month or more.

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