Agents that increase the amount of Nrf2 in the nucleus

Abstract

To provide an agent capable of increasing the amount of intranuclear Nrf2. [Solution] An agent for increasing the amount of Nrf2 in the nucleus, which contains fermented papaya food as an active ingredient.

Description

[Technical field]

[0001] The present invention relates to an agent for increasing the amount of intranuclear Nrf2. [Background technology]

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

[0003] Nuclear factor erythroid 2-related factor 2 (Nrf2) is widely present in the body, and its association with cytoprotection, phase II detoxification enzyme arrays, and chaperone proteins has been reported (Non-Patent Documents 1-4). In addition, recent studies have suggested that Nrf2 may exert anti-atherosclerotic and cardioprotective effects through experiments using Nrf2 knockout mice (Non-Patent Documents 5-7). In addition, redox abnormalities associated with disorders of the Keap1-Nrf2 control system are also said to be involved in neurodegenerative diseases (Non-Patent Documents 8-11), but the evaluation of functional foods using plant hormones that have the effect of activating the Keap1-Nrf2 control system for neurodegenerative diseases has been limited to in vitro or in vivo experiments (Non-Patent Documents 12-14), or has been limited to consideration of complex longevity at the cellular level (Non-Patent Document 15). In addition, it has been shown that dysfunction of the Keap1-Nrf2 control system and blunting 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. [Non-licensed document 2] Bellezza I, et al., "Nrf2-Keap1 signaling in oxidative and reductive stress." Biochim Biophys ActaMol Cell Res. 2018 May;1865(5):721-733. doi: 10.1016 / j.bbamcr.2018.02.010. [Non-licensed document 3] Yamamoto M, et al., "TheKEAP1-NRF2 System: a Thiol-Based Sensor-Effector Apparatus for MaintainingRedox Homeostasis." Physiol Rev. 2018 Jul 1;98(3):1169-1203. doi:10.1152 / physrev.00023.2017.

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[0006] An object of the present invention is to provide an agent capable of increasing the amount of intranuclear Nrf2. [Means for solving the problem]

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

[0008] The present disclosure provides, for example, the inventions described in the following [1] to [6]. [1] An agent that increases the amount of Nrf2 in the nucleus, containing fermented papaya as an active ingredient. [2] The agent described in [1], which promotes expression of the NQO1 gene and / or HO-1 gene. [3] The agent described in [1] or [2], wherein the intranuclear Nrf2 is intranuclear Nrf2 in peripheral blood mononuclear cells. [4] The agent according to any one of [1] to [3], which is to be ingested or administered to middle-aged or elderly people aged 40 years or older. [5] The agent according to any one of [1] to [4], wherein the dosage of the fermented papaya 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 to be taken or administered continuously for one month or more. Effect of the Invention

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

[0010] [Figure 1]This is a graph showing the amount of nuclear Nrf2 in peripheral blood mononuclear cells in the FPP intake group and the vitamin E intake group 1, 3, and 6 months after starting FPP or vitamin E intake. The vertical axis "Nuclear Nrf2 / GAPDH (AU)" means the nuclear Nrf2 / GAPDH (ratio of the amount of nuclear Nrf2 to the amount of intracellular GAPDH obtained by Western blot analysis) of each group, with the baseline (before intake of FPP or vitamin E) being set to 1. "I", "II", "III", and "IV" respectively mean the first, second, third, and fourth quantiles 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 first and second quantiles was less than 0.05. "ns" means that the p value was 0.05 or more against the baseline, and it was determined that there was no significant difference. [Diagram 2] 1 is a graph showing the expression level of NQO1 gene in peripheral blood mononuclear cells in the FPP intake group and the vitamin E intake group 1, 3, and 6 months after starting the intake of FPP or vitamin E. The vertical axis "NQO1 / GAPDH (AU)" means the NQO1 / GAPDH (the ratio of the expression level of NQO1 gene to the expression level of GAPDH gene obtained by reverse transcription quantitative polymerase chain reaction (RT-qPCR)) of each group, with the baseline (before the intake of FPP or vitamin E) being set at 1. "I", "II", "III", and "IV" respectively mean the 1st, 2nd, 3rd, and 4th quantiles 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 against the baseline, and it was determined that there was no significant difference. [Diagram 3]1 is a graph showing the expression level of HO-1 gene in peripheral blood mononuclear cells in the FPP intake group and the vitamin E intake group 1, 3, and 6 months after starting FPP or vitamin E intake. "HO-1 / GAPDH (AU)" on the vertical axis means HO-1 / GAPDH (the ratio of the expression level of HO-1 gene to the expression level of GAPDH gene obtained by reverse transcription quantitative polymerase chain reaction (RT-qPCR)) of each group, with the baseline (before intake of FPP or vitamin E) being set at 1. "I", "II", "III", and "IV" mean the 1st, 2nd, 3rd, and 4th quantiles based on the age of the subjects, respectively. "*" 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 quantiles was less than 0.05. "ns" means that the p value was 0.05 or more against the baseline, and it was determined that there was no significant difference. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, embodiments of the present disclosure will be described in detail.

[0012] The agent for increasing the amount of intranuclear Nrf2 according to the present disclosure contains FPP as an active ingredient.

[0013] As mentioned above, FPP is a papaya-derived fermented product produced by fermenting unripe Carica papaya Linn fruits together with sugar using edible yeast.

[0014] The FPP is preferably 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 produced in a factory that is certified with ISO9001:2015, ISO14001:2015, and ISO22000:2018, and is the first in Japan to obtain FSSC22000, which is said to be the strictest food safety standard in Europe and the United States, and is guaranteed in terms of quality, environment, and safety.

[0015] According to an analysis by the Japan Food Research Laboratories, 91.2g of FPP per 100g is carbohydrate, with small amounts of protein (0.3g), potassium (14.9mg) and water (8.5g) included (Lot No. 091; Analysis Test Report dated May 27, 2014). In addition, the lipid content of 100g of FPP in this same lot is 0g.

[0016] FPP can be produced by fermenting unripe Carica papaya fruits with sugar using edible yeast as described above.

[0017] The agent for increasing the amount of nuclear Nrf2 according to the present disclosure can increase the amount of nuclear Nrf2. The increase in the amount of nuclear Nrf2 by the agent for increasing the amount of nuclear Nrf2 according to the present disclosure may be due to an increase in the expression level of Nrf2, or may be due to promotion of translocation of Nrf2 into the nucleus. The above-mentioned nuclear Nrf2 may be the nuclear Nrf2 of peripheral blood mononuclear cells (hereinafter also referred to as "PBMC").

[0018] The amount of intranuclear Nrf2 can be evaluated by Western blot analysis. For example, nuclear and cytoplasmic fractions are extracted from cells such as peripheral blood mononuclear cells of a subject by a conventional method, and the nuclear and cytoplasmic fractions are subjected to Western blot analysis using an anti-Nrf2 antibody and an anti-GAPDH antibody, respectively, and the amount of intranuclear Nrf2 in the cells can be evaluated based on the value obtained by dividing the band intensity of Nrf2 by the band intensity of GAPDH.

[0019] In the cytoplasm, Nrf2 is inhibited from translocating into the nucleus by binding with Keap1, but when the binding with Keap1 is released, Nrf2 translocates into the nucleus and exhibits its physiological activity. For example, Nrf2 in the nucleus can promote the expression of genes whose expression is controlled by ARE by binding to an antioxidant response element (ARE). Therefore, the agent for increasing the amount of Nrf2 in the nucleus according to the present disclosure can increase the amount of Nrf2 in the nucleus, thereby increasing the binding of Nrf2 to ARE and promoting the expression of genes whose expression is controlled by ARE. Examples of genes whose expression is controlled by ARE include genes for antioxidant enzymes such as heme oxygenase-1 (HO-1), quinone oxidoreductase-1 (NQO1), superoxide dismutase, glutathione synthetase, thioredoxin reductase, thioredoxin, glutathione peroxidase, and glutamate cysteine ​​ligase; genes for phase II detoxification enzymes of the xenobiotic metabolism system such as glutathione-S-transferase, epoxide hydrolase, and aldo-ketoreductase; and genes for drug transporters such as multidrug resistance-associated protein 1 and multidrug resistance-associated protein 2, with the NQO1 gene and / or the HO-1 gene being preferred.

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

[0021] The agent for increasing the amount of intranuclear Nrf2 according to the present disclosure can improve the balance between the sympathetic and parasympathetic nerves by activating the activity of the parasympathetic nerves, and can therefore be suitably used as an agent for improving the balance between the sympathetic and parasympathetic nerves. For example, middle-aged and elderly people (especially elderly people) have reduced parasympathetic nerve activity and a poor balance between the sympathetic and parasympathetic nerves, but by ingesting the agent for increasing the amount of intranuclear Nrf2 according to the present disclosure for, for example, 3 months or 6 months or more, the activity of the parasympathetic nerves can be activated and the balance between the sympathetic and parasympathetic nerves can be improved.

[0022] The balance between the sympathetic and parasympathetic nerves can be indexed by the root mean square of the successive differences (RMSSD) of the time difference between successive heartbeats. In other words, a high RMSSD means that the parasympathetic nerves are more active, and a low RMSSD means that the parasympathetic nerves are less active. The RMSSD can be calculated based on heart rate variability (HRV) measured by a conventional method.

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

[0024] Furthermore, the agent for increasing the amount of intranuclear Nrf2 according to the present disclosure can be suitably used as an arteriosclerosis inhibitor, a cardioprotective agent, an agent for preventing neurodegenerative diseases, an agent for retarding aging, and the like.

[0025] The agent for increasing the amount of intranuclear Nrf2 according to the present disclosure may be a food, a food additive, a medicine, or a quasi-drug. The subject to which the agent for increasing the amount of intranuclear Nrf2 according to the present disclosure can be applied may be, for example, a mammal, and is preferably a human. The above-mentioned human is not particularly limited, but from the viewpoint of significantly increasing the amount of intranuclear Nrf2, a middle-aged human aged 40 years or older is preferable, and examples of the human include a human aged 40 to 64 years, a human aged 65 to 75 years, a human aged 65 years or older, or a human aged 75 years or older. The amount of intranuclear Nrf2 may decrease with age, but the agent for increasing the amount of intranuclear Nrf2 according to the present disclosure can restore (increase) the amount of intranuclear Nrf2 in a middle-aged human aged 40 years or older.

[0026] The human is preferably a healthy human. A healthy human may be a human who satisfies the inclusion criteria and exclusion criteria in the examples described below.

[0027] FPP can be formulated into various forms, such as granules, powders, and fine granules, for oral ingestion. During formulation, additives, such as excipients, binders, and lubricants, can be added as appropriate.

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

[0029] FPP may be taken or administered 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 to be taken or administered continuously 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 in accordance with the Declaration of Helsinki and its subsequent amendments and was approved by the ReGenera Research Association Committee on October 12, 2023. The study was designed as a prospective, randomized clinical trial comparing FPP® with a commercial antioxidant (i.e., vitamin E) and was conducted in a double-blind manner.

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

[0034] <1-1. Selection of subjects, physical examination, etc.> Potential participants were those who agreed to participate by email or telephone and met the pre-screening inclusion and exclusion criteria listed below.

[0035] (Participation criteria) Not training but leading an active lifestyle (whether working or retired); Have a sociable attitude; Have normal blood pressure; Do not smoke or have quit smoking more than 6 months ago; Have stable dietary and sleep patterns; Do not take food supplements or have stopped taking them 3 months before participating in the study; Do not have any musculoskeletal problems; Are abstainers or occasional light drinkers, not drinking distilled alcohol.

[0036] (exclusion criteria) Past or current cancer, stroke, myocardial infarction, diabetes, thromboembolism, hypertension, obesity, digestive disease (ulcer, ulcerative colitis, Crohn's disease), malabsorption syndrome, blood disease, endocrine disease, chronic fatigue syndrome, post-COVID complications, suspected post-COVID vaccine side effects, fibromyalgia, rheumatic disease, autoimmune disease, depression or other major psychiatric disorder. Has undergone surgery within the past 6 months (except minor skin problems). Has used food supplements within the past 3 months. Smokers or has quit smoking within the past 6 months. Exercises intensely once a week. Drinks large amounts of alcohol. Uses illegal drugs. Has an unstable eating style. Has a sleep disorder. Regularly uses highly caffeinated beverages.

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

[0038] (HRV measurement) The Body Health Analyzer Pro (Binacor) was used to measure the HRV. This device connects a Bluetooth-enabled finger device with specific software, analyzes the variability and characteristics of the heart rate rhythm, and estimates the sympathetic and parasympathetic related variables. The HRV measurement was performed after the subjects had rested in a quiet room for 10 minutes in advance. In addition, a visual analysis of the obtained raw data was performed to check for artificial noise due to movement, ectopic beats, breathing, etc.

[0039] <1-2. Intake of FPP and Vitamin E> All subjects were randomized using a random number generator and assigned in a 1:1 ratio to either one of two groups: one taking a sachet containing 3 g of FPP and a white cellulose film tablet twice a day (hereinafter referred to as the "FPP intake group"), or one taking a sachet containing 3 g of sugar and a white film tablet containing 400 IU of vitamin E twice a day (hereinafter referred to as the "vitamin E intake group"). The FPP intake group and vitamin E intake group took the aforementioned prescribed sachet and film tablet twice a day every day during the study period.

[0040] <1-3. Subject's diet during the study period> This study was conducted in accordance with the International Conference on Harmonization of Technical Requirements for Human Use (ICH) Good Clinical Practice (GCP) for human pharmaceuticals. Analysis of the subjects' diet during the 6 months prior to the study period confirmed that the diet was Mediterranean (15%, 40-45%, and 35-38% of energy (calories) were protein, carbohydrate, and fat, respectively. Of the fats, 22-24% were monounsaturated fatty acids (MUFA), 8-12% were extra virgin olive oil, 8-10% were saturated fatty acids (SFA), and 4% were polyunsaturated fatty acids (PUFA). The contents of the diet consumed by the subjects were obtained from the subjects' dietary reports and interviews with their families.

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

[0042] During the above-mentioned test period, blood pressure and oxygen saturation of the subjects were examined as necessary on the day before GEWT, within 2 hours after GEWT, and on the morning of the day after GEWT.

[0043] <1-5. Collection of whole blood and plasma samples> Blood samples were collected from the subjects in the FPP and vitamin E intake groups before the start of FPP or vitamin E intake, and 1, 3, 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 to disperse the anticoagulant. This resulted in a whole blood sample. The whole blood sample was centrifuged at 2500 rpm at 25°C for 15 minutes to obtain a plasma sample. A portion of the plasma sample was then transferred to a tube and stored at -80°C until testing.

[0044] <1-6.TAC Evaluation> The TAC was evaluated by 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, and the resulting mixture was shaken briefly and centrifuged at 1000×g for 5 min. Then, 200 μL of the lipid phase was collected as lipid extract, dried under a nitrogen stream, and stored at −80° C. Then, the dried lipid extract was dissolved in 200 μL of methanol and centrifuged at 5000×g for 1 min. The supernatant (120 μL) was subjected to photochemical analysis, Trolox-assisted calibration, and analysis using a photosensitizer. Based on these analytical results, the TAC was calculated from the following formula (I). TAC = [antioxidant amount (nmol) x dilution ratio (%) x Trolox molar concentration (ng / nmol)] / pipette volume (μL) ... (Formula (I))

[0045] <1-7. Genotype analysis of the Nrf2 gene> The genotypes of the Nrf2 gene (-653A / G, -651G / A, and -617C / A) were analyzed. Specifically, DNA was isolated from the whole blood samples by a modified phenol-chloroform extraction method, and the region from -738 to -461 in the NFE2L2 promoter was amplified by PCR, and the resulting DNA fragments were subjected to direct sequencing. 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, followed by a final extension at 72°C / 8 minutes. Sequencing was performed using a Perkin Elmer ABI 3100 DNA sequencer (Applied Biosystems).

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

[0047] The whole cell fraction and nuclear fraction for the Western blot analysis described below were obtained by 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, the specific procedure for extracting the nuclear fraction is briefly described below. 6PBMC cells of 1000 cells were added to a PBS solution containing kinase, protease inhibitors and 0.1v / v% Triton, the cells were lysed, gently mixed, and microcentrifuged to separate into a supernatant (for cytoplasmic fraction) and a pellet (for nuclear fraction). The supernatant was collected and mixed with 4x Laemmli sample buffer in a volume ratio of 3:1 to prepare the cytoplasmic fraction. The pellet (approximately 20μL) was resuspended in 1000μL of PBS solution containing 0.1v / v% Triton and centrifuged for 10 seconds. The supernatant was then discarded and Laemmli buffer was added to prepare the nuclear fraction.

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

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

[0051] <1-10. Evaluation of the expression levels of the NQO1 gene and the HO1 gene> The expression levels of the NQO1 gene and the HO1 gene were evaluated by subjecting the above mRNA samples to reverse transcription quantitative polymerase chain reaction (RT-qPCR). The purification of mRNA was performed using a Qiagen RNeasy Plus Mini Kit (Qiagen) according to the kit's protocol. The amount and purity of the mRNA were then confirmed using Thermo Nanodrop (ThermoFisher). The quality of the RNA 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 a reverse transcription reagent (BioRad). Residual genomic DNA was then removed using DNase. As the primer set for RT-qPCR, the primer set for NQO1 (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 primer set for HO1 (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) shown in Table 2 below, and the primer set for GAPDH (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 were used. Real-time PCR was performed three times each using a CFX96-Touch (manufactured by Bio-Rad). The amount of mRNA in the sample was evaluated by dividing the value of (expression amount of NQO1 gene or HO1 gene) / (expression amount of GAPDH) of each sample by the value of (expression amount of NQO1 gene or HO1 gene) / (expression amount of GAPDH) of the baseline.

[0052] [Table 1]

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

[0054] <1-12.Statistical analysis> Data from the FPP and vitamin E intake groups were sometimes analyzed by arranging the subjects in each group in order of age and dividing them into four groups (referred to as the "1st" to "4th" quantiles, starting from the youngest). The age ranges for the 1st, 2nd, 3rd, and 4th quantiles in the FPP intake group were 43-50 years, 51-58 years, 59-66 years, and 67-75 years, respectively. The age ranges for the 1st, 2nd, 3rd, and 4th quantiles in the vitamin E intake group were 43-50 years, 51-58 years, 59-66 years, and 67-75 years, respectively.

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

[0056] [2. Results] The procedures described in [1. Materials and Methods] were well followed in both the FPP and vitamin E intake groups. Some subjects made minor violations of the procedures, but these were not considered to affect the study and were not excluded. The violations were one missed dose (one in the FPP group and two in the vitamin E group), one subject in the FPP group did 3 hours of intensive cycling per week but then took 3 days of complete rest, and one subject in the vitamin E group took nonsteroidal anti-inflammatory drugs for 2 days after driving for a long period of time. No gastrointestinal or allergic symptoms were reported during the study.

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

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

[0059] <2-3.Nuclear Nrf2 amount> The results of <1-9. Evaluation of intranuclear Nrf2 levels> are shown in Figure 1. As shown in Figure 1, in the FPP intake group, the intranuclear Nrf2 levels were significantly higher in all 1st to 4th quartiles based on the subjects' age at 1, 3, and 6 months after the start of FPP intake than at baseline and in the vitamin E intake group. On the other hand, in the vitamin E intake group, no significant increase in intranuclear Nrf2 was observed compared to baseline at 1, 3, and 6 months after the start of vitamin E intake. In addition, in the FPP intake group, the intranuclear Nrf2 levels in the 4th quartile were significantly smaller than those in the 1st and 2nd quartiles at 1, 3, and 6 months after the start of FPP intake. There was no effect of gender on these results. From the above, it was shown that FPP has the effect of increasing intranuclear Nrf2 levels.

[0060] These findings were the same when extracting the results of subjects whose Nrf2 gene is C / C type from the results of <1-9. Evaluation of intranuclear Nrf2 amount> above, as well as when extracting the results of subjects whose Nrf2 gene is C / A type. As there were only two subjects whose Nrf2 gene is A / A type, such an analysis could not be performed.

[0061] <2-4. Expression level of NQO1 gene> Among the results of <1-10. Evaluation of the expression levels of NQO1 gene and HO1 gene> above, the results for the NQO1 gene are shown in Figure 2. As shown in Figure 2, in the FPP intake group, the mRNA expression levels of the NQO1 gene were significantly higher in all of the first to fourth quartiles based on the subjects' ages at 1, 3, and 6 months after the start of FPP intake, compared with baseline and the vitamin E intake group. On the other hand, in the vitamin E intake group, no significant increase in the mRNA expression level of the NQO1 gene was observed compared with baseline at 1, 3, and 6 months after the start of vitamin E intake. There was no effect of gender 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 of HO-1 gene> Among the results of <1-10. Evaluation of expression levels of NQO1 gene and HO1 gene> above, the results for HO-1 are shown in Figure 3. As shown in Figure 3, in the FPP intake group, the mRNA expression levels of HO-1 gene were significantly higher in the first and second quartiles based on the age of the subjects 1 month after the start of FPP intake, and in the first to fourth quartiles 3 and 6 months after the start of FPP intake, compared with baseline and the vitamin E intake group. On the other hand, in the third and fourth quartiles 1 month after the start of FPP intake, the mRNA expression levels of HO-1 gene were significantly lower than the first and second quartiles 1 month after the start of FPP intake. In addition, in the vitamin E intake group, no significant increase in the mRNA expression levels of HO-1 gene was observed compared with baseline at 1, 3, and 6 months after the start of vitamin E intake. There was no effect of gender on these results. From the above, it was shown that FPP has the effect of increasing the expression level of HO-1 gene.

[0063] Considering that the amount of Nrf2 in the nucleus is increased, the above-mentioned increased expression of the NQO1 gene and the HO-1 gene is thought to be due to Nrf2 promoting the transcription of antioxidant stress enzyme genes downstream of the antioxidant response element (ARE).

[0064] <2-6.TAC> The results of <1-6. Evaluation of TAC> are shown in Table 2. In Table 2, "*" means that the p-value against the baseline was less than 0.01. As shown in Table 2, in both the FPP intake group and the vitamin E intake group, a tendency for TAC to increase was observed 3 and 6 months after the start of FPP or vitamin E intake. The above tendency for increase 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 considered that FPP increases TAC by promoting the transcription of antioxidant stress enzyme genes by increasing the amount of Nrf2 in the nucleus. Since vitamin E did not increase the amount of Nrf2 in the nucleus, it is considered that it increases TAC by a mechanism different from that of FPP and vitamin E.

[0065] [Table 2]

[0066] <2-7.RMSSD> The results of <1-11. Measurement of RMSSD> above 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 nerves based on HRV, and a high RMSSD means that the parasympathetic nerve activity is vigorous, and a low RMSSD means that the parasympathetic nerve activity is reduced.

[0067] In all four groups, the RMSSD at baseline shown in Table 3 was insufficient, and parasympathetic activity was reduced, suggesting poor balance between sympathetic and parasympathetic nerves based on HRV. As shown in Table 3, the RMSSD in the "third and fourth quartiles" was significantly lower than that in the "first and second quartiles" in both the FPP intake group and the vitamin E intake group. This suggests that the "third and fourth quartiles" have lower parasympathetic activity than the "first and second quartiles," and that the balance between sympathetic and parasympathetic nerves based on HRV is poorer. As shown in Table 3, the RMSSD tended to improve with continued intake of FPP or vitamin E in both the "first and second quartiles" and "third and fourth quartiles" in the FPP intake group and the "first and second quartiles" and "third and fourth quartiles" in the vitamin E intake group. Therefore, it was shown that ingestion of FPP can activate the parasympathetic nervous system and improve the balance between the sympathetic and parasympathetic nervous systems.

[0068] [Table 3]

Claims

1. An agent for increasing the amount of Nrf2 in the nuclei of peripheral blood mononuclear cells, comprising 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, which is ingested or administered to middle-aged or elderly people aged 40 years or older.

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

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

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