JP2022006259A - Composition for improving immunosenescence - Google Patents

Abstract

To provide a novel composition for improving immunosenescence.SOLUTION: A composition for improving and/or inhibiting immunosenescence contains Agrimonia pilosa extract as an active ingredient.SELECTED DRAWING: Figure 1

Description

The present invention relates to a composition for improving immunosenescence.

It has been pointed out that the aging of young people leads to the maturation and stabilization of the living body, but the aging (aging) after adulthood causes various deteriorations in physiological functions. Among such declines in physiological function, the phenomenon of decline in immune function is notable. Specific changes in immunity with aging include a decrease in acquired immune response ability, an increase in inflammatory predisposition, and an increase in autoimmune risk due to a decrease in thymic function. Such changes in immunity associated with aging are called immunosenescence.
Immunosenescence is a term that comprehensively describes the characteristics of age-related changes in the immune system, and is generally based on three signs: decreased adaptive immune response, increased inflammatory predisposition, and increased autoimmunity risk. It is characterized. That is, it is characterized by having the dual nature of protecting the body from infection sources and reducing the normal immune response that eliminates them, while increasing chronic inflammation and autoimmune response. All immune cells undergo qualitative and functional changes with aging, but T cells are said to have the greatest changes with aging because the function of the thymus that produces T cells declines at an early stage.
The composition of T cell subsets changes with age. It is known that naive T cells decrease and memory T cells increase, especially in the state of immunosenescence. In addition, the ability of aged T cells to produce cytokines is also attenuated, and the expression of inhibitory receptors that suppress the activation of T cells such as PD-1 is increased. The number of cells expressing PD-1, which is one of the so-called checkpoint molecules, increases. As a result, self-activation of T cells is suppressed, and PD-L1 and PD-L2, which are ligands that bind to PD-1, are expressed on the surface of antigen-presenting cells, vascular endothelium, etc., and accompany aging. Increases and suppresses T cells. In this way, the indicators of immunosenescence have become clear.
From the findings so far, it is considered that in order to improve immunosenescence, improvement is possible by normalizing a subset of T cells and reducing PD-1-expressing cells.

Furthermore, some proposals have been made to improve immunosenescence from this point of view. Patent Document 1 proposes a method of inhibiting the expression of PD-1 in an immune cell by contacting the CD11b modulator that binds to the CD11b of the immune cell with the immune cell.
Further, Patent Document 2 combines autologous plasma enriched with stem cells by granulocyte colony stimulating factor (G-CSF) and stem cell-rich plasma obtained from cord blood of a donor compatible with ABO / Rh type. Proposals have been made to enhance transplant immunity to reinforce aged T cells by administering stem cell-rich plasma. However, such a method has not yet been put into practical use.

Currently, drugs and methods for improving immunosenescence are being sought, and drugs and methods for improving immunosenescence are being sought.

On the other hand, plant-derived extracts are used for various medical purposes. Among them, Agrimony japonica extract has been attracting attention in recent years.
Agrimonia japonica (Scientific name: Agrimonia pyrosa) is a perennial plant belonging to the genus Agrimonia in the Rosaceae family and is used as a Chinese herbal medicine. The crude drug name is Senzuru-Kusa. It is known that the above-ground foliage of Agrimony japonica contains essential oil and tannin, and tannin, which is the main component of the essential oil, has an astringent action to tighten the cell tissue. In addition, the aqueous extract boiled in water is said to have a biliary effect that helps the gallbladder work. In addition to tannin, roots are known to contain phenolic glycosides, agrimonolides, phytosterols, vanyl acid, taxifolin and the like.

As a pharmacological effect of Agrimony japonica extract, Patent Document 3 describes a nerve activating composition containing Agrimony japonica extract as an active ingredient.
Patent Document 4 describes a whitening agent, an anti-aging agent, and a skin cosmetic containing an Agrimony japonica extract.
Patent Document 5 describes an α-glucosidase inhibitor containing Agrimony japonica extract.
Patent Document 6 describes an external skin preparation agent and an allergen activator containing Agrimony japonica extract as an active ingredient.
However, it is not known that Agrimony japonica extract can be used for suppressing or improving the above-mentioned immunosenescence.

Special Table 2018-524300 Japanese Unexamined Patent Publication No. 2019-112463 Japanese Unexamined Patent Publication No. 2018-8888 JP-A-2010-65009A Japanese Unexamined Patent Publication No. 2006-16388 Japanese Unexamined Patent Publication No. 2008-174529

In the process of studying T cell differentiation, the present inventors screened various plant extracts and found an extract that controls T cell differentiation. Then, it was found that this extract improves the immunosenescence associated with aging and restores the response of cell-mediated immunity, and made the present invention.
That is, it is an object of the present invention to provide a composition for improving and / or suppressing immunosenescence associated with aging.

The configuration of the present invention is as follows.
(1) A composition for improving and / or suppressing immunosenescence containing Agrimony japonica extract as an active ingredient.
(2) The composition for improving and / or suppressing immunosenescence according to (1), wherein the immunosenescence is due to aging.
(3) The composition for improving and / or suppressing immunosenescence according to (1) or (2), wherein immunosenescence is a decrease in naive T cells and / or a decrease in immune response due to an increase in memory T cells.
(4) The immunosenescence according to any one of (1) to (3), wherein immunosenescence is a decrease in immune response due to an increase in PD-1 expressing T cells among CD4 positive and / or CD8 positive T cells. A composition for improving and / or suppressing aging.
(5) A composition for improving, alleviating, or suppressing biological abnormalities due to immunosenescence, which contains Agrimony japonica extract as an active ingredient.

The present invention provides a composition for improving and / or suppressing immunosenescence.
The present invention also provides a composition containing Agrimony japonica extract as an active ingredient for improving or alleviating various biological abnormalities caused by immunosenescence or suppressing biological abnormalities.

Among the CD4 + T cells, the counter plot developed with CD4 and PD-1 is shown. The data shows one representative result out of n = 5. It is a graph of the data obtained by averaging the ratio (%) of PD-1 + CD4 + T cells shown in FIG. Among the CD8 + T cells, the counter plot developed with CD8 and PD-1 is shown. The data shows one representative result out of n = 5. It is a graph of the data obtained by averaging the ratio (%) of PD-1 + CD8 + T cells shown in FIG. It is a dot plot developed with CD44 and CD62L among CD4 + T cells. In FIG. 5, the ratio (%) of naive T cells (CD44lowCD62High cells) is graphed. In FIG. 5, the ratio (%) of memory T cells (CD44highCD62Low cells) is graphed.

As used herein, "immunosenescence" is a decrease in thymic function or a change (decrease) in the function of immunocompetent cells, such as a decrease in the number of naive T cells in T cells and / or a decrease in the number of memory T cells. It refers to an increase in PD-1 expressing T cells among CD4 + and / or CD8 positive T cells that are in an increased state.
Further, in the specification of the present application, "improvement of immunosenescence" means
1. 1. Regressing the naive / memory T cell ratio that changed with aging to a young age.
2. 2. To reduce PD-1 expressing T cells among CD4 + and / or CD8 positive T cells.
It means to realize either or both of.
Furthermore, "suppression of immunosenescence" means
1. 1. Suppressing the decrease in the number of naive T cells in T cells and / or suppressing the increase in memory T cells with aging,
2. 2. Suppressing the increase of PD-1 expressing T cells among CD4 + and / or CD8 positive T cells.
It means to realize either or both of.
The various biological abnormalities caused by immunosenescence in the present invention include "decreased acquired immune response ability, increased inflammatory predisposition, increased autoimmune risk" and "decreased acquired immune response ability, inflammatory predisposition". "Disease associated with increased and increased autoimmune risk".

Agrimonia japonica (scientific name: Agrimonia pyrosa) is a perennial plant belonging to the genus Agrimonia of the Rosaceae family, and inhabits forest edges, wilderness, and roadsides in Honshu, Shikoku, and Kyushu, and is easily available. Also known as Ryugaso. In the present invention, Agrimony japonica can use commercially available dried products as herbal medicines, folk remedies, and raw materials for health foods (herbal teas). The dried whole plant is marketed under the crude drug name of Senzuru grass. You can also use this.
In addition, it is possible to collect native or cultivated whole plants and naturally dry or heat-dry them. This is chopped and soaked in about 10 times the amount of water or ethanol having a water content of 0 to 99.5% (v / v) for 3 to 5 days to extract at room temperature, or with a reflux condenser. Extraction is performed at 50 to 80 ° C. for 5 to 24 hours, and the mixture is filtered to collect the Kinmizuhiki extract. Water and ethanol are removed from this extract by a vacuum vacuum drying device such as a rotary evaporator or a freeze-drying device to obtain Agrimony japonica extract.

The composition for improving and / or suppressing age-related immunosenescence containing Agrimony japonica extract as an active ingredient is administered to animals and humans as a pharmaceutical composition as it is or together with a conventional pharmaceutical pharmaceutical carrier. be able to. The dosage form of the pharmaceutical composition is not particularly limited, and may be appropriately selected as necessary. Examples thereof include oral preparations such as tablets, capsules, granules, fine granules and powders, and parenteral preparations such as injections and suppositories. In the case of oral preparations, it is usually appropriate for adults to take 1 to 1000 mg of the dry mass of the extract in several divided doses a day.

In the present invention, oral preparations such as tablets, capsules, granules, fine granules and powders are produced according to a conventional method using excipients such as starch, lactose, sucrose, mannitt, carboxymethyl cellulose, cornstarch and inorganic salts. Will be done. In addition to the composition of the present invention, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a colorant, a fragrance and the like can be appropriately used for this type of oral preparation.

The compounding ingredients other than the active ingredient are illustrated below. Examples of the binder include starch, dextrin, arabic gum, gelatin, hydroxypropyl starch, sodium methylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol and the like. Examples of the disintegrant include starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose. Examples of the surfactant include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polyoxyethylene sorbitan fatty acid ester and the like. Examples of the lubricant include talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like. Examples of the fluidity accelerator include light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like.

Further, the content of the composition containing the Agrimony japonica extract as an active ingredient for improving or alleviating biological abnormalities due to immunosenescence or suppressing the occurrence of biological abnormalities is not particularly limited. Usually, the Agrimony japonica extract is blended so as to contain 0.01 to 50% by mass, preferably 0.1 to 30% by mass, per composition.
Hereinafter, the present invention will be described in more detail with reference to an example of an effect test using animals as an example.

<Effect test of Agrimony japonica extract on immunocompetent cells>
1. 1. Test method (1) Preparation of test sample-Preparation of Agrimony japonica extract 400 to 500 g of Agrimony japonica crude drug (Sentsurukusa, Tochimoto Tenkaido Co., Ltd.) was pulverized with a mixer. Then, 10 times the amount of the pulverized substance, 99.5% ethyl alcohol was added, and the mixture was sufficiently immersed. This was stirred at room temperature for 4 days and then solid-liquid separated to obtain an extract. This step was repeated 3 times, and the obtained extract was evaporated and removed with ethyl alcohol and water by a rotary evaporator by a conventional method to obtain a dry product. This dry matter was used in the following tests as the Agrimony japonica extract of the present invention.

(2) Test animals / old animals: C57BL / 6J mice 78 weeks old at the time of arrival (male, 78 weeks old, Charles River Laboratories, Japan).
-Young animals: C57BL / 6J mice 7 weeks old at the time of arrival (male, 7 weeks old, Japan Charles River Co., Ltd.).
Aged animals were acclimated and bred with a purified feed (AIN-93M Oriental Yeast Co., Ltd.) for one week after arrival, and then 5 animals per group were used. It was divided and used for the test.
In addition, 5 young animals (Young) were acclimated and bred with purified feed (AIN-93M Oriental Yeast Co., Ltd.) for 1 week after arrival, and then blood was collected.

(3) Method of administering Agrimony japonica extract to test animals The above-mentioned Agrimony japonica extract is mixed with purified feed AIN-93M, and the mixture is sufficiently stirred and mixed so that the Agrimony japonica extract is 0.5% by mass, and this is used as a test feed. As a result, they were allowed to take freely for 4 months from the start of the test.

(4) Method for confirming the effect In order to confirm the immunosenescence of the test animal and its improvement and / or inhibitory effect, the ratio of the T cell subset was confirmed.
After the breeding period of old animals, whole blood was collected by cardiac blood sampling under isoflurane suction anesthesia, and the whole blood was separated into plasma and blood cells. Blood cells were centrifuged at Ficoll (GE Healthcare Life Science). This leukocyte fraction was used to analyze a subset of T cells.
Similarly, young animals collected whole blood by cardiac blood sampling under isoflurane suction anesthesia and separated it into plasma and blood cells.
Blood was collected from young animals and leukocyte fractions were collected in the same manner as in old animals.
Subset analysis of T cells was performed by label staining using each antibody for subset identification and cell sorting by a cell sorter "FACSAria II ™" (BD Biosciences).

The antibodies used for T cell subset analysis are:
APC / cy7-anti-CD4 antibody (Biolegend): CD4 + T cell marker APC-anti-PD-1 antibody (eBioscience): PD-1 expressing cell marker FITC-anti-CD44 antibody (Biolegend) and PE / Cy7-anti-CD62L antibody (Biolegend): Naive T cell and memory T cell marker PerCP / Cy5.5-anti-CD8 antibody (Biolegend): CD8 + cell marker Staining was performed using each antibody, and the cell sorter "FACSAria II (trademark)" (BD Biosciences). The T cell subset was analyzed by.
The ratio of expressed cells analyzed based on the sorting results was statistically analyzed by the Tukey-Kramer method and a significant difference test was performed. When the P value was 0.05 or less, it was determined that there was a significant difference.

2. 2. Analysis result by flow cytometer "+" shown in the result of this experiment indicates that it is expressed in the cell. For example, in the case of PD-1 + CD4 + T cells, T cells expressing both PD-1 and CD4 are shown.
(1) Analysis using PD-1 and CD4 as markers FIG. 1 shows one representative result of the counter plots in which CD4 + T cells were developed on CD4 and PD-1. The numerical value in the figure is the ratio (%) of PD-1 + CD4 + T cells. Further, FIG. 2 shows an averaged and graphed ratio (%) of PD-1 + CD4 + T cells shown in FIG. From FIGS. 1 and 2, it can be seen that the proportion of PD-1 + CD4 + T cells increased due to aging, and the proportion of PD-1 + CD4 + T cells decreased due to the administration of Agrimony japonica extract. That is, PD-1 + CD4 + T cells increased with aging, but decreased by administration of Agrimony japonica extract.
PD-1 + CD4 + T cells are known to suppress cell-mediated immunity, and the fact that the ratio of these cells per leukocyte is reduced is that the kinmizuhiki extract has the cell-mediated immunity associated with aging. It was a result supporting the decrease, that is, the improvement of immunosenescence.

(2) Analysis using PD-1 and CD8 as markers FIG. 3 shows one representative result of the counter plots developed by CD8 + T cells on CD8 and PD-1. The numerical value in the figure is the ratio (%) of PD-1 + CD8 + T cells as in FIG. 1. Further, FIG. 4 shows an averaged and graphed ratio (%) of PD-1 + CD8 + T cells shown in FIG. From these results, it can be seen that PD-1 + CD8 + T cells increase with aging of mice and decrease with administration of Agrimony japonica extract.
That is, it was clarified that the group to which Agrimony japonica extract was administered markedly suppressed PD-1 + CD8 + T cells, which increased with aging.
From the above analyzes (1) and (2), Agrimony japonica extract has an effect of suppressing PD-1 expressed on CD4 and / or CD8-positive T cells caused by immunosenescence and improving immunosenescence. It became clear.

(3) Analysis using CD44 and CD62L as markers Naive T cells are cells with low CD44 expression and high CD62L expression (CD44low CD62High), and memory T cells are cells with high CD44 expression and low CD62L expression (CD44highCD62Llow). Is.
FIG. 5 shows a dot plot of CD4 + T cells developed on CD44 and CD62L.
The sorting two-dimensional map arranged by the expression level of CD44 and the expression level of CD62L shows different distribution patterns in each of the three groups of young animals (Young), Vehickle, and Agrimony japonica extract (AP) administration group, but young. The aged animals and the Agrimony japonica extract-administered group showed similar patterns. That is, in the young animals and the Agrimony japonica-administered group, the density of memory T cells is low, the expression of CD44 is low, and the density of naive T cells is high.
Vehicle, on the other hand, shows the opposite pattern. That is, it was found that the density of memory T cells was high and the density of naive T cells was low.

There are two types of T cells: a naive phenotype (naive T cell) that has not been sensitized with an antigen and a memory phenotype (memory T cell) that has been sensitized with an antigen.
The above results show that the composition of the T cell subset of the aged animals in the Agrimony japonica-administered group has changed to the same T cell subset composition as the young animals with less memory T cells and more naive T cells. There is.
That is, it was clarified that the Agrimony japonica extract has an action of making the composition of the T cell subset by aging the same as that of the young animal.

(4) Reanalysis with CD4 The cell group analyzed in (3) above was reanalyzed using CD4 as a marker. By this reanalysis, the ratio of CD4 + T cells, ie naive T cells or memory T cells, per T cell expressing CD44 and CD62L can be known.
1) Ratio of naive T cells FIG. 6 shows the ratio (%) of naive T cells obtained by reanalyzing FIG. 5.
That is, in the case of young animals, the ratio of naive T cells was 80.1%. On the other hand, Vechcle decreased to 35.6%. In contrast, the Agrimony japonica extract-administered group recovered to 59.4%.

2) Ratio of memory T cells FIG. 7 shows the ratio of memory T cells obtained by reanalyzing FIG. 5.
In the case of young animals, the proportion of memory T cells was 14.7%. On the other hand, Vechcle increased to 56.3%. In contrast, the Agrimony japonica extract-administered group decreased to 33.5%.

That is, the ratio of naive T cells to memory T cells in CD4 + T cells is shown in Table 1 below.

As is clear from FIGS. 6, 7 and 1, Agrimony japonica extracts bring the proportion of CD4 + T cell subsets of aged animals closer to the ratio of naive T cells to memory T cells, similar to that of young animals. It was revealed that the subset ratio was improved. That is, it was revealed that the Agrimony japonica extract acts to bring the ratio of naive T cells to memory T cells of the subset of CD4 + T cells closer to the ratio of the subset to young animals.

3. 3. Summary of effect test of Agrimony japonica extract The following is a summary of the action and effect clarified from the above analysis results.
(1) Agrimony japonica extract improves and / or suppresses immunosenescence.
(2) The main point of action of Agrimony japonica extract is T cells.
(3) Kinmizuhiki extract suppresses an increase in PD-1 expressed in CD4 positive and / or CD8 positive T cells, which is a cause of immunosenescence, which is an immunosenescent aging-related T cell.
(4) Agrimony japonica extract acts to normalize the ratio of naive T cells to memory T cells of immunosenescent CD4 + T cells.
(5) Agrimony japonica extract improves immunosenescence as described in (1) to (4) above. Therefore, diseases associated with "decreased acquired immune response, increased inflammatory predisposition, increased autoimmune risk" and "decreased acquired immune response, increased inflammatory predisposition, increased autoimmune risk" caused by immunosenescence. It acts to improve, alleviate, or suppress the occurrence of abnormal biological diseases.

Claims (5)

A composition for improving and / or suppressing immunosenescence containing Agrimony japonica extract as an active ingredient. The composition for improving and / or suppressing immunosenescence according to claim 1, wherein the immunosenescence is due to aging. The composition for improving and / or suppressing immunosenescence according to claim 1 or 2, wherein the immunosenescence is a decrease in the immune response due to a decrease in naive T cells and / or an increase in memory T cells. The improvement of immunosenescence and / or the improvement of immunosenescence according to any one of claims 1 to 3, wherein the immunosenescence is a decrease in the immune response due to an increase in PD-1 expressing T cells among CD4-positive and / or CD8-positive T cells. Or an inhibitory composition. A composition for improving, alleviating, or suppressing biological abnormalities due to immunosenescence, which contains Agrimony japonica extract as an active ingredient.

Images