Immune cell activator, anti-aging agent, method for activating immune cells, and method for preventing or preventing aging

Abstract

The present invention provides an immune cell activator containing hibiscus or a fermented extract thereof as an active ingredient. The present invention also provides an anti-aging agent characterized in that said anti-aging agent contains hibiscus or a fermented extract thereof as an active ingredient and eliminates senescent cells by activating immune cells. The present invention additionally provides a method for activating immune cells in a subject in need thereof, wherein the method includes administering hibiscus or a fermented extract thereof to the subject and activating the immune cells thereof. In addition, the present invention provides a method for preventing or protecting against aging in a subject in need thereof, wherein the method includes administering hibiscus or a fermented extract thereof to the subject and eliminating senescent cells by activating immune cells.

Description

[Technical field]

[0001] The present invention relates to an immune cell activator, an anti-aging agent, a method for activating immune cells, and a method for preventing or inhibiting aging. [Background technology]

[0002] It is known that senescent cells increase in the body with age (Non-Patent Document 1). Senescent cells are known to be eliminated by immune cells (e.g., NK cells, monocytes / macrophages, T cells, etc.) (e.g., Non-Patent Document 2), and it has also been reported that immune cells that eliminate senescent cells decrease with age (Non-Patent Document 3). Therefore, activation of immune cells is considered to be important for eliminating senescent cells due to age.

[0003] It is known that the expression levels of NKp30 and NKp46, which are activation markers of NK cells, which are one of the immune cells that remove senescent cells, decrease with age (Non-Patent Document 3). It is known that NKp46 is involved in the formation of immune synapses between NK cells and target cells (Non-Patent Document 4), and it has been suggested that the expression of NKp46 is very important for the activation of NK cells.

[0004] It is known that hibiscus or a fermented product of its extract promotes the synthesis of hyaluronic acid in epidermal cells, and a skin topical composition containing the same for improving wrinkles and sagging skin is known (Patent Document 1). However, as described below, the effect of hibiscus or a fermented product of its extract in activating immune cells was revealed for the first time by the present inventors, and has not been reported before. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2019-34899 A [Non-patent literature]

[0006] [Non-Patent Document 1] van Deursen JM., Nature., 2014, 509:439-446 [Non-Patent Document 2] Sagiv A., and Krizhanovsky V., Biogerontology. 2013 Dec;14(6):617-628. [Non-Patent Document 3] Almeida-Oliveira A., et al., Hum Immunol. 2011 Apr;72(4):319-329. [Non-Patent Document 4] Hadad U., et al., J Immunol May 1, 2014, 192 (1 Supplement) 121.13 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a novel immune cell activator that activates immune cells and a method for activating immune cells using the same. Another object of the present invention is to provide a novel anti-aging agent that eliminates aging cells by activating immune cells, and a method for preventing or preventing aging using the same. [Means for solving the problem]

[0008] As a result of intensive research, the present inventors have found for the first time that hibiscus or a fermented product of its extract has the effect of activating immune cells, and have completed the present invention. That is, the present invention includes the following inventions.

[0009] [1] An immune cell activator containing a fermented product of hibiscus or its extract as an active ingredient. [2] The immune cell activator described in Item 1, wherein the immune cell is a NK cell. [3] The immune cell activator according to item 1 or 2, characterized in that senescent cells are eliminated by activating immune cells. [4] The immune cell activator according to any one of items 1 to 3, wherein the fermentation product of hibiscus or an extract thereof is obtained by lactic acid fermentation of hibiscus or an extract thereof.

[0010] [5] An anti-aging agent comprising a fermented product of hibiscus or its extract as an active ingredient, and characterized by eliminating senescent cells by activating immune cells. [6] The anti-aging agent according to Item 5, wherein the immune cells are NK cells. [7] The anti-aging agent according to item 5 or 6, wherein the fermentation product of hibiscus or an extract thereof is obtained by lactic acid fermentation of hibiscus or an extract thereof.

[0011] [8] A method for activating immune cells in a subject in need thereof, comprising: administering to said subject a fermented product of hibiscus or an extract thereof, thereby activating said immune cells. A method comprising: [9] The method of item 8, wherein the immune cells are NK cells.

[10] The method according to item 8 or 9, wherein senescent cells are eliminated by activating the immune cells.

[11] The method according to any one of items 8 to 10, wherein the fermentation product of hibiscus or an extract thereof is obtained by lactic acid fermentation of hibiscus or an extract thereof.

[0012]

[12] A method for preventing or preventing aging in a subject in need thereof, comprising: administering to said subject a fermented product of hibiscus or an extract thereof, thereby activating immune cells and eliminating senescent cells. A method comprising:

[13] The method of claim 12, wherein the immune cells are NK cells.

[14] The method according to item 12 or 13, wherein the fermented product of hibiscus or an extract thereof is obtained by lactic acid fermentation of a hibiscus flower extract. Effect of the Invention

[0013] According to the present invention, it is possible to activate immune cells, thereby making it possible to eliminate senescent cells. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram showing the screening method used to select the immune cell activator or anti-aging agent of the present invention. [Diagram 2] 2 is a graph showing the activation of NK cells by the immune cell activator or anti-aging agent of the present invention. The expression level of NKp46 mRNA in NK cells when various concentrations of hibiscus fermentation product (v / v%) were added is shown, normalized with the expression level of RPS9 mRNA as an endogenous control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, the mode for carrying out the present invention will be described, but the technical scope of the present invention is not limited to the following mode. Furthermore, unless otherwise specified, the contents described in the following mode for carrying out the present invention can be mutually applied in the description of immune cell activators, anti-aging agents, immune cell activation methods, and methods for preventing or preventing aging. Note that the prior art documents cited in this specification are also incorporated herein by reference, and are incorporated in their entirety into this specification.

[0016] <Immune cell activator and anti-aging agent> In one embodiment, the present invention provides an immune cell activator comprising a fermented product of hibiscus or an extract thereof as an active ingredient. In another aspect, the present invention provides an anti-aging agent comprising a fermented product of hibiscus or an extract thereof as an active ingredient, and characterized in that it activates immune cells, thereby eliminating senescent cells.

[0017] In this specification, "hibiscus" refers to a plant of the genus Hibiscus in the family Malvaceae, such as roselle (Hibiscus sabdariffa L.), rose of Sharon (Hibiscus syriacus), Hibiscus mutabills, Hibiscus coccineus, Hibiscus tiliaceus, and Hibiscus schizopetalus. The "hibiscus or an extract thereof" used in the present invention may be used in the form of the whole plant, leaves, flowers, stems, roots, seeds, etc. When flowers are used, the flowering time and size are not particularly limited, and petals, calyx, or all of them may be used.

[0018] The method for preparing the "hibiscus extract" that can be used in the present invention can be a known method, for example, it can be prepared with reference to JP 2019-34899 A. As an example, the procedure for preparing the "hibiscus extract" is described below.

[0019] The hibiscus extracting part is washed with water if necessary to remove foreign matter, and then either left as is or dried, and then chopped or crushed as necessary, and then contacted with an extracting solvent to carry out extraction. Extraction can be carried out by contacting with the extracting solvent according to a conventional method such as the immersion method, but supercritical extraction or steam distillation may also be used.

[0020] Examples of extraction solvents include water; lower alcohols such as methanol, ethanol, and propanol; higher alcohols such as oleyl alcohol, stearyl alcohol, and octyldodecanol; polyhydric alcohols such as ethylene glycol, 1,3-propanediol, 1,3-butylene glycol, and glycerin; esters such as ethyl acetate, butyl acetate, methyl propionate, and glyceryl trioctanoate; ketones such as acetone and methyl ethyl ketone; ethers such as ethyl ether, isopropyl ether, and the like; and hydrocarbon solvents such as n-hexane, toluene, and chloroform, and these can be used alone or in combination of two or more kinds.

[0021] When a mixed solvent is used, the mixing ratio is preferably in the range of 1:1 to 25:1 in terms of volume ratio (hereinafter the same) for a mixed solvent of water and ethyl alcohol; in the range of 1:1 to 15:1 for a mixed solvent of water and glycerin; or in the range of 1:1 to 15:1 for a mixed solvent of water and 1,3-propanediol or 1,3-butylene glycol.

[0022] When preparing an extract, the pH is not particularly limited, but is generally preferably in the range of pH 3 to 9. If necessary, the extraction solvent may be blended with an alkalinity regulator such as sodium hydroxide, sodium carbonate, or potassium hydroxide, or an acidity regulator such as citric acid, hydrochloric acid, phosphoric acid, or sulfuric acid to adjust the pH to a desired level.

[0023] The extraction conditions, such as the extraction temperature and extraction time, vary depending on the type and pH of the solvent used and are not limited. For example, when water or 1,3-butylene glycol, or a mixture of water and 1,3-butylene glycol is used as the solvent, the extraction temperature may be in the range of 0°C to 90°C, and the extraction time may be 0.5 hours to 7 days.

[0024] If necessary, the extraction site may be subjected to a hydrolysis treatment prior to or in parallel with the extraction treatment, which may improve the skin irritation, efficacy, storage stability, etc. of the extract, thereby enabling the extract to be used more effectively.

[0025] In this specification, the term "fermented product of hibiscus or an extract thereof" refers to a product obtained by a known fermentation process using a microorganism (e.g., lactic acid bacteria, yeast, koji mold, or Bacillus subtilis) from "hibiscus or an extract thereof," and can be prepared, for example, with reference to JP 2019-34899 A. The "fermented product of hibiscus or an extract thereof" that can be used in the present invention is preferably obtained by lactic acid bacteria fermentation, and for example, "Hibiscus (registered trademark)" (also called "hibiscus flower fermented extract") (Technoble Co., Ltd. (Japan)), which is commercially available as a product prepared by fermenting hibiscus extract using lactic acid bacteria, can be applied to the present invention.

[0026] By applying the present invention, immune cells can be activated. In this specification, immune cells are cells that play a role in the "immune system" in the living body, and examples of such cells include NK cells, T cells, B cells, monocytes / macrophages, and dendritic cells. Immune cells that can be activated by applying the present invention are preferably NK cells.

[0027] NK cells, short for natural killer cells, are a type of cytotoxic lymphocyte that acts as a key factor in innate immunity. NK cells do not express T cell antigen receptors (TCR), CD3, or surface immunoglobulin (Ig) B cell receptors, but are usually CD16 (FcγRIII)- and CD56-positive in humans. NK cells are cytotoxic, and their small granules in the cytoplasm contain specialized proteins such as perforin and proteases known as granzymes. When released in close proximity to a cell targeted for killing, perforin creates pores in the plasma membrane of the target cell, allowing the entry of granzymes and related molecules, inducing apoptosis.

[0028] NK cells are usually activated in response to interferon or macrophage-derived cytokines. The cytotoxic activity of NK cells is highly regulated by two types of surface receptors called "activating receptors" and "inhibitory receptors." NK cells are known to play an important role in host rejection of tumors and also play a role in killing virus-infected cells.

[0029] It is also known that activated immune cells, such as NK cells and T cells, play a role in eliminating senescent cells (Sagiv A., and Krizhanovsky V., Biogerontology. 2013 Dec;14(6):617-628.). Among them, it is known that the expression levels of NKp30 and NKp46, which are known as NK cell activation markers, decrease with age (Almeida-Oliveira A., et al., Hum Immunol. 2011 Apr;72(4):319-329.). Based on the above findings, the present inventors searched for a substance that increases the expression level of NK cell activation markers, and found that the above-mentioned hibiscus or a fermented product of its extract exhibits this effect, leading to the completion of the present invention.

[0030] As used herein, the term "senescent cells" refers to cells that exhibit increased expression of senescence markers compared to normal cells. Senescence markers include senescence-associated acid β-galactosidase, P53, P16, and INK4a , P21 CIP1 Senescent cells are characterized by irreversible proliferation arrest in the G1 phase, suppression of genes that promote cell cycle progression, and the downregulation of p53 and p16 INK4a , p21 CIP1 It is known that their formation is related to increased expression of

[0031] Senescent cells may be caused by replicative cellular senescence, premature cellular senescence, or therapy-induced cellular senescence. Senescent cells caused by replicative cellular senescence may have undergone multiple cell divisions, for example, 40 or more, 50 or more, 60 or more, 70 or more, or 80 or more cell divisions. Senescent cells caused by premature cellular senescence may be induced by, but are not limited to, ultraviolet light, reactive oxygen species, environmental toxins, smoking, ionizing radiation, distortion of chromatin structure, excessive mitogenic signaling, oncogenic mutations, and the like. In another embodiment, premature cellular senescence may be induced by ionizing radiation. In another particular embodiment, premature cellular senescence may be induced by ectopic transfection with Ras protein. Senescent cells caused by therapy-induced cellular senescence may be induced by radiation therapy, chemotherapy, DNA damage therapy, and the like.

[0032] The senescent cells that can be eliminated by using the present invention can generally be eukaryotic cells. Examples of senescent cells include, but are not limited to, mammary epithelial cells, keratinocytes, cardiac muscle cells, chondrocytes, endothelial cells (large blood vessels), endothelial cells (micro blood vessels), epithelial cells, fibroblasts, hair papilla cells, hepatocytes, melanocytes, osteoblasts, adipose precursor cells, immune system cells, skeletal muscle cells, smooth muscle cells, adipocytes, neurons, glial cells, contractile cells, exocrine epithelial cells, extracellular matrix cells, hormone-secreting cells, keratinized epithelial cells, pancreatic islet cells, lens cells, mesenchymal stem cells, pancreatic adenocarcinoma cells, intestinal Paneth cells, hematopoietic cells, nervous system cells, cells supporting sensory organs and peripheral nerve cells, and moist stratified barrier epithelial cells.

[0033] The number of senescent cells in various organs and tissues usually increases with age. The accumulation of senescent cells can lead to more deterioration under aging and aging-related diseases. For example, the accumulation of senescent cells in aged tissues can contribute to age-related tissue dysfunction, reduced regenerative capacity, and disease. In one embodiment, aged tissues with accumulated senescent cells lack the ability to respond to stresses that require proliferation, resulting in the reduced healthiness seen with aging. Thus, in one embodiment, by applying the present invention, senescent cells can be eliminated by immune cells, preventing or preventing aging.

[0034] In one embodiment, the present invention may be provided as a composition comprising the immune cell activator or anti-aging agent described above. The composition of the present invention may be in various forms such as powder, liquid, solid, granular, paste, gel, milky lotion, cream, sheet, spray, foam, etc.

[0035] The content of the immune cell activator or anti-aging agent in the composition of the present invention is not particularly limited as long as it contains an effective amount for activating immune cells.

[0036] Furthermore, the composition of the present invention may contain additives, which may be selected arbitrarily and used in combination, if necessary. The additives may include excipients and the like.

[0037] The excipient may be any one that is normally used when the desired form is obtained, and examples thereof include starches such as wheat starch, rice starch, corn starch, potato starch, dextrin, and cyclodextrin, crystalline celluloses, sugars such as lactose, glucose, sugar, reduced maltose, starch syrup, fructooligosaccharides, and emulsified oligosaccharides, and sugar alcohols such as sorbitol, erythritol, xylitol, lactitol, and mannitol. These excipients may be used alone or in combination of two or more.

[0038] Furthermore, the composition of the present invention may contain other components, as necessary, which may be appropriately selected from known components such as colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH adjusters, oils, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants, UV absorbers, moisturizers, fragrances, various medicinal ingredients, preservatives, pH adjusters, and neutralizing agents.

[0039] <Method for activating immune cells and method for preventing or preventing aging> In one embodiment, the present invention provides 1. A method for activating immune cells in a subject in need thereof, comprising: administering to said subject a fermented product of hibiscus or an extract thereof to activate said immune cells.

[0040] In another embodiment, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: 1. A method for preventing or preventing aging in a subject in need thereof, comprising: administering to the subject a fermented product of hibiscus or an extract thereof, thereby eliminating senescent cells by activating immune cells.

[0041] In the present specification, the term "subject" refers to a mammal, including humans, and preferably to a human subject. Methods for administering hibiscus or a fermented product of its extract include, for example, topical administration (on the skin, inhalation, enema, eye drops, ear drops, nasal, vaginal, etc.), enteral administration (oral, tube, injection, etc.), and parenteral administration (intravenous, intraarterial, transdermal, intramuscular, etc.). EXAMPLES

[0042] The present invention will be described in more detail below with reference to examples, but these are not intended to limit the present invention in any way.

[0043] 1.Material The following materials were used in this example: Primary cultured human NK cells (Promocell) Culture medium: RPMI1640 medium supplemented with 20% fetal bovine serum and essential / non-essential amino acids (Nacalai Tesque) Fermented Hibiscus Flower Extract (Hibiscus®, Catalog Number: 05694, Technoble Co., Ltd. (Japan))

[0044] 2. Experimental Method Commercially available (Promocell) primary cultured human NK cells were cultured in RPMI1640 medium (Nacalai Tesque) supplemented with 20% fetal bovine serum and essential / non-essential amino acids (37°C, 100% humidity, 5% CO 2 ).

[0045] 4 x 10^ suspended in 1 ml of medium 5 NK cells were seeded in a 24-well plate, and hibiscus flower fermented extract (05694) diluted with PBS was added at the concentrations shown in Figure 2. After 24 hours of culture, RNA was extracted from the collected cells using RNeasy plus (Qiagen), and cDNA was synthesized using GoScript transcriptase (Promega). Based on the cDNA, semi-quantitative PCR of the NKp46 gene and ribosomal S9 gene was performed using GoTaq qPCR reagent (Promega) (Light Cycler 480; Roche). The primers used were ribosomal S9 gene 5'-TGCTGACGCTTGATGAGAAG-3', 5'-CGCAGAGAAGTCGATGTG-3' as a housekeeping factor, and NKp46 gene; 5'-CCACCGAGGGACATACCGAT-3', 5'-GTGCAAGGCTGGTGTTCTCA-3'. The expression level of the NKp46 gene was divided by the expression level of the ribosomal S9 gene to standardize it. The results were tested for significance using multiple comparison tests (Tukey) on the statistical software SPSS.

[0046] 3.Results The fermented hibiscus flower extract promoted the expression of the activation marker (NKp46) in primary cultured human NK cells at concentrations of 0.01 (v / v)% or more (N=4, *p<0.001) (Figure 2).

Claims

1. An NK cell activator comprising, as an active ingredient, a fermented product of hibiscus or an extract thereof.

2. The NK cell activator according to claim 1 , wherein the fermentation product of hibiscus or an extract thereof is obtained by fermenting hibiscus or an extract thereof with lactic acid bacteria.

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