CN118615334A - Immune cell activator, anti-aging agent, method for activating immune cells, and method for preventing or preventing aging - Google Patents

Abstract

The present invention addresses the problem of providing an immune cell activator, an anti-aging agent, a method for activating immune cells, and a method for preventing or preventing aging. The solution is that the present invention provides an immune cell activator comprising Vaccinium uliginosum as an active ingredient. In addition, the present invention provides an anti-aging agent characterized by comprising Vaccinium uliginosum as an active ingredient, and by activating immune cells to exclude senescent cells. Furthermore, the present invention provides a method of activating immune cells in a subject in need thereof, the method comprising: the subject was administered with Vaccinium uliginosum to activate the immune cells. Furthermore, the present invention provides a method of preventing or preventing aging in a subject in need thereof, the method comprising: vaccinium uliginosum was applied to the above subjects to exclude senescent cells by activating immune cells.

Description

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

Technical Field

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 prophylactic aging.

Background Art

It is known that senescent cells increase in vivo with aging (Non-patent Document 1). It is known that senescent cells are eliminated by immune cells (e.g., NK cells, monocytes/macrophages, T cells, etc.) (e.g., Non-patent Document 2), and it is also reported that immune cells that eliminate senescent cells decrease with aging (Non-patent Document 3), so the activation of immune cells is considered to be important in the removal of senescent cells caused by aging.

It is known that the expression of NKp30 and NKp46, activation markers of NK cells, which are one of the immune cells that remove senescent cells, decreases 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), suggesting that the expression of NKp46 is very important for the activation of NK cells.

Hibiscus or fermented products of its extracts are known to activate immune cells such as NK cells (Patent Document 1). There is a demand for the search for other substances having a high immune cell activating effect comparable to that of hibiscus or fermented products of its extracts.

Prior art literature

Patent Literature

Patent Document 1: International Publication No. 2022/176174

Patent Document 2: Japanese Patent Application Publication No. 2008-526956

Non-patent literature

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. 2011Apr; 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

Problems to be solved by the invention

The present invention aims to provide a novel immune cell activator for activating immune cells and a method for activating immune cells using the immune cell activator. In addition, the present invention aims to provide a novel anti-aging agent for eliminating senescent cells by activating immune cells and a method for preventing or preventing aging using the anti-aging agent.

Methods for solving problems

The present inventors have conducted intensive studies and have found for the first time that bilberry has an effect of activating immune cells, thereby completing the present invention. That is, the present invention includes the following inventions.

[1] An immune cell activator comprising Vaccinium vitis-idaea as an active ingredient.

[2] The immune cell activator according to claim 1, wherein the immune cell is a NK cell.

[3] The immune cell activator according to claim 1 or 2, characterized in that senescent cells are eliminated by activating immune cells.

[4] The immune cell activator according to any one of [1] to [3], wherein the vaccinium is obtained by squeezing the juice of vaccinium vaccinium fruit.

[5] An anti-aging agent, characterized in that it contains bilberry as an active ingredient and eliminates senescent cells by activating immune cells.

[6] The anti-aging agent according to [5], wherein the immune cells are NK cells.

[7] The anti-aging agent according to [5] or [6], wherein the vaccinium is obtained by squeezing the juice of vaccinium vaccinium fruit.

[8] A method for activating immune cells in a subject in need thereof, the method comprising:

Applying lingonberry to the above-mentioned subject activates the above-mentioned immune cells.

[9] The method according to [8], wherein the immune cells are NK cells.

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

[11] The method according to any one of [8] to [10], wherein the bilberry is obtained by squeezing the juice of bilberry fruits.

[12] A method for preventing or prophylaxis of aging in a subject in need thereof, the method comprising:

Applying lingonberry to the above subjects can eliminate senescent cells by activating immune cells.

[13] The method according to [12], wherein the immune cells are NK cells.

[14] The method according to [12] or [13], wherein the bilberry is obtained by squeezing the juice of bilberry fruits.

Effects of the Invention

According to the present invention, immune cells can be activated, thereby eliminating senescent cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression level of NKp46 mRNA in NK cells when Vaccinium vitis-idaea fruit juice and Hibiscus sylvestris flower fermented extract were added as relative values, with the expression level of negative control sterilized water being 100.

FIG. 2 shows the expression level of NKp46 mRNA in NK cells when Vaccinium vitis-idaea fruit juice and Hibiscus sabdariffa flower fermented extract were added at various concentrations as relative values, with the expression level of negative control sterilized water being 100.

DETAILED DESCRIPTION

Hereinafter, the mode for implementing the present invention will be described, but the technical scope of the present invention is not limited to the following mode. In addition, the following contents described for implementing the present invention can be mutually applied in the description of the immune cell activator, anti-aging agent, immune cell activation method, and method for preventing or preventing aging as long as there is no special limitation. In addition, the prior art documents cited in this specification are also cited in this specification, and are integrated into this specification as a whole.

It is known that bilberry removes/inhibits active oxygen caused by ultraviolet rays, and a skin condition improving composition containing bilberry for skin whitening and wrinkle improvement is known (Patent Document 2). However, there has been no report on the effect of bilberry on activating immune cells. The present invention is based on the discovery of the inventors that bilberry activates immune cells.

In one embodiment, the present invention provides an immune cell activator comprising bilberry as an active ingredient. In another embodiment, the present invention provides an anti-aging agent comprising bilberry as an active ingredient and activating immune cells to eliminate senescent cells. Hereinafter, the immune cell activator and anti-aging agent of the present invention are sometimes collectively referred to as the preparation of the present invention.

In the present specification, "blueberry" refers to a plant of the genus Vaccinium of Ericaceae known as a broad blueberry including alpine blueberry (Vaccinium uliginosum var.alpinum) and blueberry (Vaccinium uliginosum var.japonicum) in a narrow sense. Blueberry juice in the present invention is sometimes expressed as "Wild blueberry juice concentrate (Vaccinium uliginosum)" in English, and sometimes expressed as "concentrated blueberry juice" in Chinese.

The "Vaccinium vitis-idaea" used in the present invention can be used as the whole plant, leaves, flowers, stems, roots, fruits, etc. When using fruits, the fruits including the peel can be used. In addition to fruits, other parts such as flowers, roots, etc., or the whole plant body can also be included.

The "Vaccinium vitis-idaeae" that can be used in the present invention can be in any form such as a dried plant, a fresh plant, a powder, a small fragment, a paste, an extract, etc. For example, it can be prepared by a known method. For example, it can be in any form such as the collected plant itself, a dried plant, a material obtained by subdividing the plant by crushing, cutting, crushing, grinding, homogenizing, mixing, etc., a fresh plant or dried material, or an extract obtained by extraction by any method.

The "Vaccinium vitis-idaea" that can be used in the present invention can be a substance obtained by squeezing the fruit of Vaccinium vitis-idaea. The squeezed juice of Vaccinium vitis-idaea can be further concentrated. For example, the juice obtained by washing the fruit of Vaccinium vitis-idaea with water in advance as needed to remove foreign matter, crushing it, and squeezing it is concentrated by heating, vacuuming, freezing, drying, etc. In the case of a concentrate, any concentration ratio can be adopted, for example, 1 to 20 times, 2 to 15 times, 5 to 10 times, 6 to 8 times, for example, 7 times.

If bilberry juice is commercially available, the bilberry juice may be used, for example, products of companies such as those described in the following examples may be used. Alternatively, an extract as described in Patent Document 1 may be used.

By applying the present invention, immune cells can be activated. In this specification, so-called immune cells are cells that play the role of "immune system" in the body, and examples thereof include NK cells, T cells, B cells, monocytes/macrophages, dendritic cells, etc. The immune cells that can be activated by applying the present invention are preferably NK cells.

NK cells are short for natural killer cells, and are a type of cytotoxic lymphocytes that function as a major factor in innate immunity. NK cells do not express T cell antigen receptors (TCR), CD3, and surface immunoglobulin (Ig) B cell receptors, but are generally CD16 (FcγRIII)-positive and CD56-positive cells in humans. NK cells are cytotoxic, and the small granules in their cytoplasm contain special proteins such as perforin and proteases known as granzymes. If perforin is released near a cell that is targeted for killing, it forms a hole in the cell membrane of the target cell, and granzymes and related molecules can enter from here, inducing apoptosis.

NK cells are usually activated in response to interferon or cytokines derived from macrophages. The cytotoxic activity of NK cells is highly regulated by two surface receptors called "activation receptors" and "inhibitory receptors." NK cells are known to play an important role in tumor resistance to host, and also play a role in killing virus-infected cells.

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 activation markers of NK cells, decrease with age (Almeida-Oliveira A., et al., Hum Immunol. 2011 Apr; 72 (4): 319-329.). Based on the above knowledge, the present inventors searched for substances that increase the expression level of NK cell activation markers, and found that the above-mentioned lingonberry exerts this effect, thereby completing the present invention.

In this specification, the term "senescent cells" refers to cells that show an increased expression of senescence markers compared to normal cells. Examples of senescence markers include senescence-related acidic β-galactosidase, p53, p16 ^INK4a , p21 ^CIP1 , etc. It is known that senescent cells are characterized by irreversible proliferation arrest in the G1 phase and are formed by the inhibition of genes that promote cell cycle operation and the increased expression of p53, p16 ^INK4a , and p21 ^CIP1 that inhibit the cell cycle.

Senescent cells may be senescent cells caused by replicative cell senescence, premature cell senescence, or cell senescence induced by treatment. Senescent cells caused by replicative cell senescence may be senescent cells that have undergone multiple cell divisions, such as 40 or more, 50 or more, 60 or more, 70 or more, or 80 or more cell divisions. Senescent cells caused by premature cell senescence are not limited, but may be senescent cells induced by ultraviolet rays, reactive oxygen species, environmental toxins, smoking, ionizing radiation, deformation of chromatin structure, excess mitogen signals, carcinogenic mutations, and the like. In addition, in other embodiments, premature cell senescence is cell senescence that can be induced by ionizing radiation. In other specific embodiments, premature cell senescence can be induced by ectopic transfection using Ras protein. Senescent cells caused by cell senescence induced by treatment are senescent cells that can be induced by radiotherapy, chemotherapy, DNA damage therapy, and the like.

Senescent cells that can be eliminated by using the present invention can generally be eukaryotic cells. Examples of senescent cells include, for example, mammary epithelial cells, keratinocytes, cardiomyocytes, chondrocytes, endothelial cells (large blood vessels), endothelial cells (capillaries), epithelial cells, fibroblasts, hair papilla cells, hepatocytes, melanocytes, osteoblasts, fat precursor cells, cells of the immune system, skeletal muscle cells, smooth muscle cells, adipocytes, neurons, glial cells, contractile cells, exocrine epithelial cells, extracellular matrix cells, hormone secreting cells, keratinized epithelial cells, islet cells, crystalline cells, mesenchymal stem cells, adenocarcinoma cells of the pancreas, Paneth cells of the small intestine, cells of the hematopoietic system, cells of the nervous system, cells that support sensory organs and peripheral nerve cells, and moist stratified barrier epithelial cells, but are not limited thereto.

The number of senescent cells in various organs and tissues generally increases with age. The accumulation of senescent cells can further aggravate the 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 diseases. In one embodiment, aged tissues in which senescent cells accumulate lack the ability to respond to stresses that require proliferation, thereby causing the reduction in health observed with aging. Therefore, in one embodiment, by applying the present invention, senescent cells are eliminated by immune cells, and aging can be prevented or prevented.

In one embodiment, the present invention can be provided as a composition comprising bilberry or the above-mentioned immune cell activator or anti-aging agent. The form of the composition is arbitrary, as long as it is appropriately selected according to the conditions such as the ingredients and the purpose, and examples thereof include compositions such as cosmetics, medicines, quasi-drugs, and food and beverage products. In addition, the composition of the present invention can be in various forms such as powder, liquid, solid, tablet, capsule, granule, paste, gel, emulsion, cream, sheet, spray, and foam. There is no particular limitation on the content of bilberry or immune cell activator or anti-aging agent in the composition of the present invention, as long as it contains an effective amount for immune cell activation.

The preparation or composition of the present invention preferably contains bilberry in an amount that can fully exert the effect of the present invention, and its blending amount can be appropriately determined according to their type, purpose, form, utilization method, etc. For example, bilberry as an active ingredient can be 0.0001-100 mg/ml, 0.0001-90 mg/ml, 0.001-50 mg/ml, 0.01-5 mg/ml, 0.01-1 mg/ml, 0.05-0.5 mg/ml, 0.1-0.2 mg/ml (w/v), etc. per total weight, as long as the effect of the present invention is exerted, there is no limitation. In a certain embodiment, the preparation of the present invention is composed of bilberry.

Furthermore, the preparation or composition of the present invention can be used in combination with any selected additives as needed. The additives may include excipients and the like.

As the excipient, any substance can be used as long as it is a substance commonly used when making a desired form, and for example, starches such as wheat starch, rice starch, corn starch, potato starch, dextrin, and cyclodextrin, crystalline cellulose, lactose, glucose, granulated sugar, reduced maltose, starch syrup, oligofructose, oligolactose and other sugars, sorbitol, erythritol, xylitol, lactitol, mannitol and other sugar alcohols can be mentioned. These excipients can be used alone or in combination of two or more.

In addition, the preparation or composition of the present invention may appropriately select and use other ingredients as needed, such as colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH adjusters, oils, powders, colorants, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, humectants, fragrances, various medicinal ingredients, preservatives, pH adjusters, neutralizers and other known substances.

In one embodiment, the present invention provides a method for activating immune cells in a subject in need thereof, the method comprising:

Applying lingonberry to the above-mentioned subject activates the above-mentioned immune cells.

Furthermore, in other embodiments, the present invention provides a method of preventing or prophylaxis of aging in a subject in need thereof, the method comprising:

Applying lingonberry to the above subjects can eliminate senescent cells by activating immune cells.

In this specification, the so-called "subject" is a mammal including a human, preferably a human subject. Regarding the method of applying lingonberry, for example, topical administration (on the skin, inhalation, enema, eye drops, ear drops, nasal, vaginal, etc.), enteral administration (oral, tube, injection, etc.), parenteral administration (intravenous, arterial, percutaneous, intramuscular injection, etc.) can be mentioned. In addition, it can be taken as a food or drink.

The method of the present invention is a method for beauty treatment and may not be performed by a doctor or medical practitioner.

In other embodiments, the present invention also provides use of Vaccinium myrtillus in the manufacture of a medicament for immune cell activation, or a medicament for preventing or prophylaxis of senescence by eliminating senescent cells through activation of immune cells.

In other embodiments, the present invention also provides Vaccinium myrtillus for use in immune cell activation, or in the prevention or prophylaxis of senescence by eliminating senescent cells through the activation of immune cells.

Example

Hereinafter, the present invention will be described in further detail based on Examples, but these examples do not limit the present invention at all.

Material

The following materials were used in this example.

Primary cultured human CD56+ NK cells (ATTC, PCS-800-019(TM))

Culture medium: 20% fetal bovine serum RPMI (1X) + GlutaMAX-1 RPMI Medium 1640 [+] 25 mM HEPES (gibco, Ref; 72400-047, Lot: 2193150)

Weigh 200 mg of 7-fold concentrated lingonberry juice (Lot; 210209-1, Heilongjiang Shangzhi Green Berries Co., Ltd. (China)) and dissolve in 800 μL of dH ₂ O.

Hibiscus flower fermented extract (Hibiscus (registered trademark), catalog number: 05694, Technoble Co., Ltd. (Japan))

Experiment 1

Commercially available (ATCC) primary cultured human NK cells were cultured in 20% fetal bovine serum RPMI (1X) + GlutaMAX-1 RPMI Medium 1640 [+] 25 mM HEPES medium (Gibco) (37°C, 100% humidity, 5% CO2).

NK cells were seeded in a 24-well plate at a concentration of 2×10 ⁵ cells/well in 1 ml of culture medium. To the culture medium, 0.2 mg/ml (w/v) of concentrated bilberry juice and 0.1% (v/v) of hibiscus flower fermented extract were added, and culture was performed. As a negative control, the same amount of dH ₂ O was used. After 48 hours of culture, RNA was extracted from the recovered cells using RNeasy (registered trademark) Mini Kit (Qiagen). Using the extracted RNA, semi-quantitative PCR (Light Cycler 480; Roche) of the NKp46 gene and the ribosomal S9 gene, which is a housekeeping factor, was performed using TaqMan RNA-to CT 1-step Kit (Thermo Fisher SCIENTIFIC). The primers used were the ribosomal S9 gene (Hs00183118_m1; Thermo Fisher SCIENTIFIC, Lot: 2038445) and the NKp46 gene (Hs02339424_g1; Thermo Fisher SCIENTIFIC, Lot: 2079026), which are housekeeping factors, and the expression level of the NKp46 gene was divided by the expression level of the ribosomal S9 gene for normalization. The results were tested for significance using the multiple comparison test (Dunnett) on the statistical software Excel.

The results are shown in Figure 1. Vaccinium myrtillus significantly promoted the expression of activation marker (NKp46) in primary cultured human NK cells (N=4, ^* p<0.05, ^** P<0.001). In addition, the expression level was comparable to that of hibiscus flower fermented extract (Patent Document 2) which is known to have immune cell activation effects.

Experiment 2

A, B, and C, for which no effect was observed in Experiment 1, were excluded. For Vaccinium japonicum, for which an effect was observed, Vaccinium japonicum was added to the culture medium at a final concentration of 0.1 mg/ml (w/v) or 0.2 mg/ml (w/v) in order to confirm the concentration dependence. In addition, the cells were cultured in the same manner as in Experiment 1, and the expression levels of the ribosomal S9 gene and NKp46 gene were measured, and normalization and significance difference tests were performed.

The results are shown in FIG2 (N=4, ^* P<0.05, ^** P<0.001). It was found that the effect of promoting the expression of the NKp46 gene by Vaccinium vitis-idaea was observed in a concentration-dependent manner.

Claims (7)

1. An immune cell activator comprises Vaccinium uliginosum as an active ingredient.

2. The immune cell activator of claim 1, wherein the immune cell is an NK cell.

3. The immune cell activator according to claim 1 or 2, wherein senescent cells are excluded by activation of immune cells.

4. Immune cell activator according to claim 1 or 2, wherein the vaccinium uliginosum is obtained by squeezing the fruit of the vaccinium uliginosum.

5. An anti-aging agent, characterized by comprising Vaccinium uliginosum as an active ingredient, and by activating immune cells to exclude senescent cells.

6. The anti-aging agent of claim 5, wherein the immune cells are NK cells.

7. The anti-aging agent according to claim 5 or 6, wherein said vaccinium uliginosum is obtained by squeezing the fruit of vaccinium uliginosum.