AGENT FOR INCREASING AMOUNT OF Nrf2 IN NUCLEUS

Abstract

Provided is an agent for increasing the amount of Nrf2 in the nucleus of peripheral blood mononuclear cells, containing papaya fermented food as an active ingredient.

Description

Agent for increasing the amount of Nrf2 in the nucleus

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

[0002] Papaya fermented food (hereinafter also referred to as "FPP") produced by fermenting immature fruits of Carica papaya Linn with edible yeast together with sugar increases maltose and maltotriose when mixed with saliva 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 glucose levels and promote wound healing in type 2 diabetic patients (Patent Document 1).

[0003] Nuclear factor erythroid 2-related factor 2 (Nrf2; Nuclear factor erythroid 2-related factor 2) 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 regulatory system are also said to be involved in neurodegenerative diseases (Non-Patent Documents 8 to 11), but the evaluation of functional foods using plant hormones having an action of activating the Keap1-Nrf2 regulatory 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 cellular level (Non-Patent Document 15). Also, it has been shown that dysfunction of the Keap1-Nrf2 regulatory system and attenuation of the antioxidant response by Nrf2 are related to the aging process itself (Non-Patent Document 16).

[0004] Japanese Patent Application Laid-Open No. 2011-041478

[0005] Baird L, et al., “The Molecular Mechanisms Regulating the KEAP1-NRF2 Pathway.” Mol Cell Biol. 2020 Jun 15;40(13):e00099-20. doi: 10.1128 / MCB.00099-20.Bellezza I, et al., “Nrf2-Keap1 signaling in oxidative and reductive stress.” Biochim Biophys Acta Mol Cell Res. 2018 May;1865(5):721-733. doi: 10.1016 / j.bbamcr.2018.02.010.Yamamoto M, et al., “The KEAP1-NRF2 System: a Thiol-Based Sensor-Effector Apparatus for Maintaining Redox Homeostasis.” Physiol Rev. 2018 Jul 1;98(3):1169-1203. doi: 10.1152 / physrev.00023.2017.Murakami S, et al., “NRF2 signalling in cytoprotection and metabolism.” Br J Pharmacol. 2023 Sep 15. doi: 10.1111 / bph.16246.Zhang Q, et al., “Activation of Nrf2 / HO-1 signaling: An important molecular mechanism of herbal medicine in the treatment of atherosclerosis via the protection of vascular endothelial cells from oxidative stress.” J Adv Res. 2021 Jul 6;34:43-63. doi: 10.1016 / j.jare.2021.06.023.Chen QM, et al., “Nrf2 at the heart of oxidative stress and cardiac protection.Physiol Genomics.” 2018 Feb 1;50(2):77-97. doi: 10.1152 / physiolgenomics.00041.2017.Luo X, et al., “A novel anti-atherosclerotic mechanism of quercetin: Competitive binding to KEAP1 via Arg483 to inhibit macrophage pyroptosis.” Redox Biol. 2022 Nov;57:102511. doi: 10.1016 / j.redox.2022.102511.Chakkittukandiyil A, et al., “The principal molecular mechanisms behind the activation of Keap1 / Nrf2 / ARE pathway leading to neuroprotective action in Parkinson's disease.” Neurochem Int. 2022 Jun;156:105325. doi: 10.1016 / j.neuint.2022.105325Uruno A, et al., “The KEAP1-NRF2 system and neurodegenerative diseases.” Antioxid Redox Signal. 2023 Feb 3. doi: 10.1089 / ars.2023.0005Anandhan A, et al., “α-Syn overexpression, NRF2 suppression, and enhanced ferroptosis create a vicious cycle of neuronal loss in Parkinson's disease.” Free Radic Biol Med. 2022 Nov 1;192:130-140. doi: 10.1016 / j.freeradbiomed.2022.09.015.Kim S, et al., “Nrf2 activator via interference of Nrf2-Keap1 interaction has antioxidant and anti-inflammatory properties in Parkinson's disease animal model.” Neuropharmacology. 2020 May 1;167:107989. doi: 10.1016 / j.neuropharm.2020.107989. Epub 2020 Feb 5. PMID: 32032607.Rathore AS, et al., “Curcumin Modulates p62-Keap1-Nrf2-Mediated Autophagy in Rotenone-Induced Parkinson's Disease Mouse Models.” ACS Chem Neurosci. 2023 Mar 29. doi: 10.1021 / acschemneuro.2c0070Wang Q, et al., “Ellagic acid activates the Keap1-Nrf2-ARE signaling pathway in improving Parkinson's disease: A review.” Biomed Pharmacother. 2022 Dec;156:113848. doi: 10.1016 / j.biopha.2022.113848.Marotta F, et al., “P. Anti-inflammatory and neuroprotective effect of a phytoestrogen compound on rat microglia.” Ann N Y Acad Sci. 2006 Nov;1089:276-81. doi: 10.1196 / annals.1386.033Morgunova, G.V. et al. “Culture medium pH and stationary phase / chronological aging of different cells.” Moscow University Biological Sciences Bulletin.2017, 2, 47-51.Yu C, et al., “The Keap1-Nrf2 System: A Mediator between Oxidative Stress and Aging.” Oxid Med Cell Longev. 2021 Apr 19;2021:6635460. doi: 10.1155 / 2021 / 6635460.

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

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

[0008] This disclosure provides, for example, the inventions described in [1] to

[36] below: [1] A nuclear Nrf2 increasing agent containing fermented papaya food as an active ingredient. [2] The agent according to [1] that promotes the expression of the NQO1 gene and / or the HO-1 gene. [3] The agent according to [1] or [2], wherein the nuclear Nrf2 is nuclear Nrf2 of peripheral blood mononuclear cells. [4] The agent according to any one of [1] to [3] that is 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 dose of 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] that is ingested or administered continuously for one month or more. [7] A composition for increasing the amount of nuclear Nrf2 containing fermented papaya food as an active ingredient. [8] The composition according to [7] that promotes the expression of the NQO1 gene and / or the HO-1 gene. [9] The composition according to [7] or [8], wherein the nuclear Nrf2 is nuclear Nrf2 of peripheral blood mononuclear cells.

[10] The composition according to any one of [7] to [9], which is ingested or administered to a middle-aged or elderly person aged 40 years or older.

[11] The composition according to any one of [7] to

[10] , wherein the dose of the fermented papaya food is 0.5 to 30 g / day for an adult weighing 70 kg.

[12] The composition according to any one of [7] to

[11] , which is ingested or administered continuously for one month or more.

[13] A method for increasing the amount of nuclear Nrf2, comprising administering or having a fermented papaya food ingested as a subject.

[14] The method according to

[13] , which promotes the expression of the NQO1 gene and / or the HO-1 gene.

[15] The method according to

[13] or

[14] , wherein the nuclear Nrf2 is nuclear Nrf2 of peripheral blood mononuclear cells.

[16] The method according to any one of

[13] to

[15] , wherein the fermented papaya food is ingested or administered to a middle-aged or elderly person aged 40 years or older.

[17] The method according to any one of

[13] to

[16] , wherein the dose of the fermented papaya food is 0.5 to 30 g / day for an adult weighing 70 kg.

[18] The method according to any one of

[13] to

[17] , wherein the fermented papaya food is ingested or administered continuously for one month or more.

[19] The use of fermented papaya food in non-therapeutic methods to increase the amount of nuclear Nrf2.

[20] The use according to

[19] , wherein the non-therapeutic method promotes the expression of the NQO1 gene and / or the HO-1 gene.

[21] The use according to

[19] or

[20] , wherein the nuclear Nrf2 is nuclear Nrf2 of peripheral blood mononuclear cells.

[22] The use according to any one of

[19] to

[21] , wherein the fermented papaya food is ingested or administered to a middle-aged or elderly person aged 40 years or older.

[23] The use according to any one of

[19] to

[22] , wherein the dose of the fermented papaya food is 0.5 to 30 g / day for an adult weighing 70 kg.

[24] The use according to any one of

[19] to

[23] , wherein the fermented papaya food is ingested or administered continuously for one month or more.

[25] Fermented papaya food for use in a therapeutic method to increase the amount of nuclear Nrf2.

[26] The fermented papaya food according to

[25] , wherein the therapeutic method promotes the expression of the NQO1 gene and / or the HO-1 gene.

[27] The fermented papaya food according to

[25] or

[26] , wherein the nuclear Nrf2 is nuclear Nrf2 of peripheral blood mononuclear cells.

[28] The fermented papaya food according to any one of

[25] to

[27] , which is ingested or administered to middle-aged or elderly people aged 40 years or older.

[29] The fermented papaya food according to any one of

[25] to

[28] , wherein the dose is 0.5 to 30 g / day for an adult weighing 70 kg.

[30] The fermented papaya food according to any one of

[25] to

[29] , which is ingested or administered continuously for one month or more.

[31] Use of fermented papaya food for the production of a composition for increasing the amount of nuclear Nrf2.

[32] Use according to

[31] , wherein the composition for increasing the amount of nuclear Nrf2 promotes the expression of the NQO1 gene and / or the HO-1 gene.

[33] The use according to

[31] or

[32] , wherein the nuclear Nrf2 is nuclear Nrf2 from peripheral blood mononuclear cells.

[34] The use according to any one of

[31] to

[33] , wherein the composition for increasing the amount of nuclear Nrf2 is ingested or administered to a middle-aged or elderly person aged 40 years or older.

[35] The use according to any one of

[31] to

[34] , wherein the dose of the fermented papaya food is 0.5 to 30 g / day for an adult weighing 70 kg.

[36] The use according to any one of

[31] to

[35] , wherein the composition for increasing the amount of nuclear Nrf2 is ingested or administered continuously for one month or more.

[0009] According to the present invention, it is possible to provide an agent that can increase the amount of Nrf2 in the nucleus.

[0010] This graph shows the amount of intranuclear 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. The vertical axis, "Intranuclear Nrf2 / GAPDH (AU)," represents the ratio of the amount of nuclear Nrf2 to the amount of intracellular GAPDH (obtained by Western blot analysis) for each group, 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. This graph shows the expression levels 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. The vertical axis, "NQO1 / GAPDH (AU)," represents the NQO1 / GAPDH ratio for each group (the ratio of NQO1 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 subject's age, respectively. "*" means that the p-value compared to the vitamin E intake group and baseline was less than 0.01. "ns" means that the p-value compared to baseline was 0.05 or higher, and it was judged that there was no significant difference. 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 the expression level of the HO-1 gene to the expression level of the GAPDH gene, 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 subject's age, 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 no statistically significant difference was determined.

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

[0012] [Intranuclear Nrf2 quantity increasing agent; composition for increasing intranuclear Nrf2 quantity] The intranuclear Nrf2 quantity increasing agent according to this disclosure contains FPP as an active ingredient. The intranuclear Nrf2 quantity increasing agent according to this disclosure can also be called a composition for increasing intranuclear Nrf2 quantity.

[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 "FPP Fermented Papaya Preparation" (registered trademark) or "Immun'Age" (registered trademark). The FPP is certified to ISO 9001:2015, ISO 14001:2015, and ISO 22000:2018, and is produced in a factory that was the first in Japan to obtain FSSC 22000, 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 this lot, the lipid content is 0g per 100g of FPP.

[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 nuclear translocation of Nrf2. 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 conventional method. These fractions can then be subjected to Western blot analysis using anti-Nrf2 antibody and anti-GAPDH antibody, 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 into the nucleus is inhibited by its binding to Keap1 in the cytoplasm, but once this binding is released, Nrf2 translocates into 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 as "+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 intranuclear 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 for improving the balance between the sympathetic and parasympathetic nervous systems (a composition for improving 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 poor balance between the sympathetic and parasympathetic nervous systems. However, by administering the intranuclear Nrf2 increasing agent according to this disclosure for, for example, three months or six 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 therefore can be suitably used as a total antioxidant capacity improving agent (composition for improving total antioxidant capacity). As TAC, for example, the total antioxidant capacity of a plasma sample taken 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 (composition for inhibiting arteriosclerosis), a cardioprotective agent (composition for protecting cardioprotection), a neurodegenerative disease preventive agent (composition for preventing neurodegenerative diseases), an aging retarder (composition for delaying aging), and the like.

[0025] The nuclear Nrf2 increasing agent according to this disclosure may be a food, food additive, pharmaceutical, or quasi-drug. The target subject to which the nuclear Nrf2 increasing agent according to this disclosure may be a mammal, for example, but is 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 people 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 according 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 an 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.

[0031] [Method for increasing the amount of nuclear Nrf2] The method for increasing the amount of nuclear Nrf2 according to this disclosure includes administering or ingesting FPP to a target.

[0032] The specific embodiments of the above method (for example, the administration (intake) of FPP, such as the target of administration (intake), the amount administered, the number of administrations (intakes), and the duration of administration (intake)) can be any of the embodiments described above in [Intranuclear Nrf2 amount increasing agent; composition for increasing intranuclear Nrf2 amount] without limitation.

[0033] FPP may be administered (ingested) to the subject as is, for example, or it may be prepared as an intranuclear Nrf2 increasing agent (composition for increasing intranuclear Nrf2) as described above [Intranuclear Nrf2 increasing agent; composition for increasing intranuclear Nrf2] and administered (ingested) to the subject.

[0034] [Fermented papaya food for use in therapeutic methods to increase nuclear Nrf2 levels] One aspect of this disclosure relates to FPP for use in therapeutic methods to increase nuclear Nrf2 levels.

[0035] The specific embodiments of the above-mentioned FPP (for example, the target of administration (intake), the amount administered (intake), the number of administrations (intakes), the duration of administration (intake), etc.) can be any of the embodiments described above in [Intranuclear Nrf2 amount increasing agent; composition for increasing intranuclear Nrf2 amount] without limitation.

[0036] In this disclosure, "therapeutic method" means a method that includes medical procedures. Medical procedures include, for example, diagnosis, treatment, and prevention performed by a medical professional who possesses the necessary qualifications (such as a medical license).

[0037] [Use of fermented papaya food in non-therapeutic methods to increase nuclear Nrf2 levels] One aspect of this disclosure relates to the use of FPP in non-therapeutic methods to increase nuclear Nrf2 levels.

[0038] The specific embodiments of the above-mentioned use (for example, the administration (intake) of FPP, such as the target of administration (intake), the amount administered (intake), the number of administrations (intakes), and the duration of administration (intake)) can be applied without limitation to the embodiments described above in [Intranuclear Nrf2 amount increasing agent; composition for increasing intranuclear Nrf2 amount].

[0039] In the present disclosure, the term "non-therapeutic method" means a method that does not fall under the category of therapeutic methods. For example, a "non-therapeutic method" means a method that does not include medical acts. That is, a "non-therapeutic method" may be a method that does not include diagnosis, treatment, and prevention and is performed by a medical practitioner having the necessary qualifications (such as a medical license). Also, a "non-therapeutic method" may be a method having purposes such as health purposes and cosmetic purposes.

[0040] [Use of Papaya Fermented Food for the Production of a Composition for Increasing the Amount of Nuclear Nrf2] One aspect of the present disclosure relates to the use of papaya fermented food for the production of a composition for increasing the amount of nuclear Nrf2.

[0041] As specific embodiments of the above use (for example, the embodiment of the composition for increasing the amount of nuclear Nrf2), the embodiments described in the above [Agent for increasing the amount of nuclear Nrf2; Composition for increasing the amount of nuclear Nrf2] can be applied without limitation.

[0042] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to only these examples.

[0043] [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 Helsinki Declaration and its subsequent revisions. This study was designed as a prospective randomized clinical trial comparing FPP (registered trademark) with a commercial antioxidant (i.e., vitamin E) and was conducted using a double-blind method.

[0044] Although the content of this study will be described later, briefly, subjects aged 43 to 75 years were made to ingest FPP or vitamin E daily for 6 months, and the amount of nuclear Nrf2, the expression levels of NQ01 gene and HO-1 gene, TAC, RMSSD, etc. were evaluated before the start of ingestion (hereinafter also referred to as "baseline"), 1 month, 3 months, and 6 months after the start of ingestion, thereby evaluating the effect of FPP on the amount of nuclear Nrf2 and the usefulness of such an effect. The period from the start of ingestion of FPP or vitamin E until 6 months later is also referred to as the "test period".

[0045] <1-1. Selection of Subjects, Physical Examinations, etc.> Persons who agreed to participate by e-mail or phone call and met the following inclusion criteria and the following exclusion criteria in the pre-screening were considered potential participants.

[0046] (Inclusion Criteria) Not in training but leading an active life (whether working or retired). Having a social attitude. Having normal blood pressure. Not smoking or having quit smoking more than 6 months ago. Having a stable diet and sleep pattern. Not taking food supplements or having stopped taking them 3 months before the study participation. Having no musculoskeletal problems. Being a non-drinker or drinking lightly occasionally and not drinking distilled spirits.

[0047] (Exclusion Criteria) Having had or currently having cancer, stroke, myocardial infarction, diabetes, thromboembolism, hypertension, obesity, digestive diseases (ulcer, ulcerative colitis, Crohn's disease), malabsorption syndrome, blood diseases, endocrine diseases, chronic fatigue syndrome, post-COVID complications, suspected post-COVID vaccine side effects, fibromyalgia, rheumatic diseases, autoimmune diseases, depression or other major mental disorders. Having undergone surgery (excluding minor skin problems) within the past 6 months. Having used food supplements within the past 3 months. Smoking or having quit smoking within 6 months. Engaging in strenuous exercise once a week. Drinking a large amount of alcohol. Using illegal drugs. Having an unstable diet style. Having a sleep disorder. Regularly using high-caffeine beverages.

[0048] Among the potential participants, all subjects provided written informed consent and underwent a medical history interview, physical examination, clinical examination, measurement of HRV, body measurement, and resting blood pressure measurement. In addition, all subjects had their resting electrocardiogram (ECG) evaluated. All subjects were within 20% of their ideal body weight. Also, the body mass index (BMI) of all subjects was calculated. The age of the subjects was 43 to 75 years.

[0049] (HRV Measurement) For the above HRV measurement, a Body Health Analyzer Pro (manufactured by Binacor) was used. 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 measurement was 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, respiration, etc.

[0050] <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 a group that took a sachet containing 3g of FPP and a white cellulose film tablet twice a day (hereinafter also referred to as the "FPP intake group"), or a group that took a sachet containing 3g of sugar and a white film tablet containing 400 IU of vitamin E twice a day (hereinafter also referred to as the "vitamin E intake group"). The FPP intake group and the vitamin E intake group took the prescribed sachets and film tablets twice a day every day during the study period.

[0051] <1-3. Subjects' Diet 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 revealed 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 the subjects' diet reports and interviews with their families.

[0052] <1-4. Gradual Exercise Walking Test (GEWT)> During the six weeks of the above-mentioned study period, subjects were subjected to the GEWT on the first and third days of each week. The GEWT was designed not to improve performance, but rather to provide subjects with exercise stressors that increased oxidative stress (McGinnis, Graham, et al. “Acute hypoxia and exercise-induced blood oxidative stress.” International journal of sport nutrition and exercise metabolism 24.6 (2014): 684-693.). This design was made to make the GEWT more closely resemble real-life situations and to mix aerobic and anaerobic elements. Specifically, the GEWT proceeded as follows (1) to (3). The intensity of the GEWT was increased every two minutes. (1) First 6 minutes: Warm-up (increase intensity from 4.5 km / h to 6.5 km / h during this time) (2) 7-12 minutes: Intensive aerobic test (increase intensity from 7 km / h to 8 km / h during this time) (3) 12-22 minutes: Anaerobic phase (increase intensity from 8 km / h until fatigue or up to 11 km / h during this time)

[0053] 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 on the morning of the day after the GEWT.

[0054] <1-5. Collection of Whole Blood and Plasma Samples> Blood samples from 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. A portion of the plasma sample was then transferred to a tube and stored at -80°C until testing.

[0055] <1-6. Evaluation of TAC> The 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, the TAC was calculated from the following formula (I): TAC = [Amount of antioxidant (nmol) × Dilution ratio (%) × Trolox molar concentration (ng / nmol)] / Pipette volume (μL) … (Formula (I))

[0056] <1-7. Analysis of Nrf2 gene genotype> The genotypes of the Nrf2 gene (-653A / G, -651G / A, and -617C / A) were analyzed. Specifically, DNA was isolated from the whole blood sample 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 fragment was then directly subjected to 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, and then a final extension at 72°C for 8 minutes. Sequencing was performed using a Perkin Elmer ABI 3100 DNA sequencer (Applied Biosystems).

[0057] <1-8. Recovery of mRNA and protein samples (whole cell fraction and nuclear fraction) from whole blood> The whole blood sample was diluted 1:1 by volume with phosphate-buffered saline (PBS), and then stratified by centrifugation at 800 x g for 25 minutes at 20°C using a Ficol-Histopaque gradient centrifuge (Sigma-Aldrich). 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 x 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). After that, 2 x 10 ６ PBMC cells from cells were added to 100 μL of mRNA cell lysate (Ambion Biotech), thereby lysing the PBMC cells and obtaining an mRNA sample.

[0058] 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." Pesticide biochemistry and physiology 164 (2020): 47-57.

[0059] First, regarding the procedure for extracting the core fraction, the specific steps are as follows: 3 × 10 ６Cells' PBMC 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.

[0060] 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 three 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.

[0061] <1-9. Evaluation of Nuclear Nrf2 Amount> The amount of nuclear Nrf2 was evaluated by performing Western blot analysis on the above nuclear fraction. Specifically, the above nuclear fraction was separated by electrophoresis using SDS-PAGE and transferred to a nitrocellulose membrane. Then, rabbit polyclonal primary antibody and anti-rabbit secondary antibody against Nrf2 (both from ThermoFisher, PA-kit) were added and co-incubated. Subsequently, the intensity of the bands was analyzed using Image Version 1.56 (National Institutes of Health), and evaluated by optical density via a digital image analyzer. In addition, the optical density of the GAPDH band was measured by performing Western blot analysis on the above cytoplasmic fraction using an anti-GAPDH antibody. The amount of nuclear Nrf2 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).

[0062] <1-10. Evaluation of NQO1 and HO1 gene expression levels> 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 described 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).

[0063]

[0064] <1-11. Measurement of RMSSD> 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 RMSSD (milliseconds) was calculated based on that.

[0065] <1-12. Statistical Analysis> In some cases, data from the FPP intake group and the vitamin E intake group were analyzed by arranging the subjects in each group by age and dividing them equally into four groups (referred to as the "first quantile" to the "fourth quantile," in order from the youngest to the oldest). The age ranges for the first, second, third, and fourth quantiles 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 quantiles of the vitamin E intake group were 43-50 years, 51-58 years, 59-66 years, and 67-75 years, respectively.

[0066] The Hardy-Weinberg equilibrium, and the comparison of allele frequencies between different databases, as well as the comparison 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 performed 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.

[0067] [2. Results] The procedures described in [1. Materials and Methods] were well followed in both the FPP intake group and the vitamin E intake group. Minor violations of the above procedures were observed in some subjects, but these violations were not considered to affect this study, and the subjects were not excluded. Specifically, these violations included missing one dose of medication (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.

[0068] <2-1. BMI, etc.> No significant changes were observed in blood pressure and oxygen saturation, measured the day before the GEWT, within two hours after the GEWT, and the day after the GEWT, as well as in the subjects' BMI, from the start to the end of the study period. However, a non-significant decreasing trend was observed in the subjects' BMI and total cholesterol from the start to the end of the study period.

[0069] <2-2. Genotype of the Nrf2 gene> Based on the results of <1-7. Analysis of the genotype of the Nrf2 gene> above, 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%).

[0070] <2-3. Nuclear Nrf2 Amount> The results of <1-9. Evaluation of Nuclear Nrf2 Amount> 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 amounts 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 amount 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 amount.

[0071] 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.

[0072] <2-4. Expression Level of the NQO1 Gene> Figure 2 shows the results for the NQO1 gene from <1-10. Evaluation of Expression Levels of the NQO1 and HO1 Genes> above. 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.

[0073] <2-5. Expression Level of the HO-1 Gene> The results for HO-1 from <1-10. Evaluation of Expression Levels of the NQO1 and HO-1 Genes> above are shown in Figure 3. As shown in Figure 3, in the FPP intake group, the mRNA expression level of the HO-1 gene was significantly higher than 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.

[0074] The increased expression levels of the NQO1 gene and the HO-1 gene, as described above, are thought to be due to increased nuclear Nrf2 levels, specifically because Nrf2 promoted the transcription of antioxidant stress enzyme genes downstream of antioxidant response elements (AREs).

[0075] <2-6. TAC> The results of <1-6. Evaluation of TAC> above are shown in Table 2. 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 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 due to an increase in 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.

[0076]

[0077] <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 nervous systems based on HRV. A high RMSSD means that the parasympathetic nervous system is active, and a low RMSSD means that the parasympathetic nervous system is inactive.

[0078] 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 in the "third and fourth quantiles" was significantly lower than in the "first and second quantiles." From this, it was considered 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 nerve activity and improve the balance between the sympathetic and parasympathetic nervous systems.

[0079]

Claims

1. A peripheral blood mononuclear cell Nrf2 increasing agent containing fermented papaya 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 or older.

4. 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.

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

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