Flavonoid-containing composition

Abstract

The inventors' primary objective was to provide a technology that suppresses inflammation, particularly in aging tissues. [Solution] The above problem can be solved by a composition containing at least one selected from the group consisting of genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin, and glycosides thereof.

Description

[Technical Field]

[0001] This disclosure relates to compositions containing at least one selected from the group consisting of genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin, and glycosides thereof. [Background technology]

[0002] As we age, the structure and function of blood vessels deteriorate, such as arteriosclerosis and decreased endothelial function, which is known to increase the risk of various diseases. For example, stroke, vascular dementia, myocardial infarction, hypertension, diabetes, and renal failure have been reported to be closely related to vascular aging (Non-Patent Literature 1). Since these diseases are factors that cause a decline in quality of life and an increase in mortality, preventing and / or improving vascular aging is important from the perspective of extending healthy life expectancy.

[0003] In recent years, research has focused particularly on vascular endothelial cells within blood vessels. For example, Non-Patent Literature 2 reports that in transgenic mice (E-DNIκB mice) in which functional NF-κB signaling in vascular endothelial cells was inhibited, inflammation and oxidative stress in vascular endothelial cells were reduced, the progression of vascular aging was suppressed, and lifespan was extended. Furthermore, Non-Patent Literature 3 shows that in control mice fed a high-calorie diet, p53 expression in vascular endothelial cells was significantly increased, leading to metabolic abnormalities, while in vascular endothelial cell-specific p53-deficient mice (EC-p53KO), insulin sensitivity was improved and fat accumulation decreased. It should be noted that p53 is a factor known to be involved in aging. These studies suggest that inhibiting vascular aging may contribute to reducing disease risk and extending healthy life expectancy. Approaches to prevent or delay vascular aging include the use of antioxidants and anti-inflammatory drugs, and lifestyle improvements (moderate exercise, balanced diet, smoking cessation, etc.). In recent years, it has also been reported that certain flavonoids and polyphenols may have anti-vascular aging effects. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Han Y, Kim SY. Endothelial senescence in vascular diseases: current understanding and future opportunities in senotherapeutics. Exp Mol Med. 2023;55(1):1-12. doi:10.1038 / s12276-022-00906-w [Non-Patent Document 2] Hasegawa Y, Saito T, Ogihara T, et al. Blockade of the nuclear factor-κB pathway in the endothelium prevents insulin resistance and prolongs life spans. Circulation. 2012;125(9):1122-1133. doi:10.1161 / CIRCULATIONAHA.111.054346 [Non-Patent Document 3] Yokoyama M, Okada S, Nakagomi A, et al. Inhibition of endothelial p53 improves metabolic abnormalities related to dietary obesity. Cell Rep. 2014;7(5):1691-1703. doi:10.1016 / j.celrep.2014.04.046 [Overview of the project] [Problems that the invention aims to solve]

[0005] In recent years, there has been a growing demand for personalized medicine and personalized supplements, which are health management tailored to each individual's age, gender, lifestyle, health condition, genetic predisposition, etc. In light of these circumstances, the inventors of this invention primarily aimed to provide a technology that suppresses inflammation, especially in aging tissues. [Means for solving the problem]

[0006] The inventors have found that at least one selected from the group consisting of genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin, and their glycosides, can exert anti-inflammatory effects, particularly in aging tissues. Further improvements have led to this disclosure.

[0007] This disclosure includes, for example, the following subjects: Section 1. An anti-inflammatory composition for aging tissue, comprising at least one selected from the group consisting of genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin, and glycosides thereof. Section 2. The composition according to item 1, wherein the aging tissue is vascular endothelial tissue. Section 3. A composition according to item 1 or 2 for use against inflammatory responses induced by endotoxins. Section 4. It contains at least one selected from the group consisting of nobiletin, apigenin, and naringenin, and their glycosides. A composition used to suppress the expression of at least one inflammation-related gene selected from the group consisting of CCL2, PAI-1, and MMP-1 in aging tissue. Section 5. It contains at least one selected from the group consisting of genistein, nobiletin, apigenin, and naringenin, and their glycosides. A composition used to suppress the expression of at least one inflammation-related gene selected from the group consisting of PAI-1 and MMP-1 in aging tissue. Item 6. The composition according to item 4 or 5, wherein the aged tissue is vascular endothelial tissue. Item 7. The composition according to item 4 or 5, which is used to suppress the expression of the inflammation-related gene induced by endotoxin. Item 8. The composition according to any one of items 1 to 7, wherein the aged tissue is the tissue of a subject aged 40 or older.

Advantages of the Invention

[0008] According to the present disclosure, a technique for suppressing inflammation, particularly in aged tissues, can be provided.

Brief Description of the Drawings

[0009] [Figure 1] Test 1: Fluorescence microscope images (left in the figure) and fluorescence intensities (relative values with the fluorescence intensity at p.4 as 1, right in the figure) when detecting SA-β-gal positive cells using the β-galactosidase detection fluorescent reagent SPiDER-βGal are shown. In the figure, the upper end of the bar graph indicates the average value, and the error bar indicates the standard deviation (the same applies hereinafter). **p < 0.01, n = 6. [Figure 2] Test 1: Results of analyzing SA-β-gal positive cells by flow cytometry are shown. **p < 0.01, n = 5. [Figure 3] Test 1: Results of analyzing cells highly expressing SA-β-gal by flow cytometry are shown. **p < 0.01, n = 4. [[ID=^{33}]] [Figure 4] Test 1: Results of analyzing the cell cycles of cells at p.4 and p.21 by flow cytometry are shown. The vertical axis of the graph indicates the percentage in all cells (% cell). n = 2. [Figure 5] Test 1: Gene expression levels of IL-8, CXCL1, ICAM-1, p16, and p21 are shown. **p < 0.01, n = 6. [[ID=3^{9}]] [Figure 6] Test 1: Gene expression levels of SIRT1, NRF1, and TFAM are shown. **p < 0.01, *p < 0.05, n = 6. [Figure 7] Test 1: The results of analyzing the protein expression levels of p16, p21, and SIRT1 by Western blotting are shown. Above the figure, the quantitative results of band intensity performed using Image J are shown. *p < 0.05, **p < 0.01, n = 5 - 6 for each group. [Figure 8] Test 2: The results of analyzing the gene expression levels of IL-8, CXCL1, PAI-1, and MMP-1 in p.4 and p.21 cells with (labeled as "E.coli LPS" in the figure) or without (labeled as "control" in the figure) the addition of E.coli LPS are shown. The results represent the relative values when the gene expression level when culturing p.4 without the addition of E.coli LPS is set as 1. *p < 0.05, **p < 0.01, n = 3. [Figure 9] (Continued from Figure 8) Test 2: The results of analyzing the gene expression levels of E-selectin and CCL2 in p.4 and p.21 cells with (labeled as "E.coli LPS" in the figure) or without (labeled as "control" in the figure) the addition of E.coli LPS are shown. The results represent the relative values when the gene expression level when culturing p.4 without the addition of E.coli LPS is set as 1. *p < 0.05, **p < 0.01, n = 3.

Modes for Carrying Out the Invention

[0010] Hereinafter, each embodiment included in the present disclosure will be described in more detail. The present disclosure preferably includes compositions for anti-inflammation in aging tissues and the like, but is not limited thereto, and the present disclosure includes all that are disclosed herein and can be recognized by those skilled in the art.

[0011] I. Compositions of the Disclosure The compositions included in this disclosure contain at least one selected from the group consisting of genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin, and their glycosides. Hereinafter, the compositions included in this disclosure may be referred to as "the compositions of this disclosure." Furthermore, genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin, and their glycosides, may be collectively referred to as "the flavonoids of this disclosure." The compositions of this disclosure may contain one of the flavonoids of this disclosure alone or in combination of two or more.

[0012] Genistein is a type of isoflavone that can be found in soybeans and other leguminous plants. The genistein used in the art of this disclosure may be of plant origin or may be chemically synthesized. The molar mass of genistein is 270.24 g / mol. The structure of genistein is shown below. [ka]

[0013] Quercetin is a type of flavonoid and a polyphenol compound that can be found mainly in plants such as fruits, vegetables, and tea leaves. The quercetin used in the technology of this disclosure may be of plant origin or chemically synthesized. The molar mass of quercetin is 302.236 g / mol. The structure of quercetin is shown below. [ka]

[0014] Fisetin is a type of flavonoid and a polyphenol compound that can be found in strawberries, apples, kiwis, grapes, etc. The fisetin used in the technology of this disclosure may be of plant origin or chemically synthesized. The molar mass of fisetin is 286.24 g / mol. The structure of fisetin is shown below. [ka]

[0015] Nobiletin is a type of polymethoxyflavonoid that can be found mainly in the peels of citrus fruits. The nobiletin used in the art of this disclosure may be of plant origin or chemically synthesized. The molar mass of nobiletin is 402.39 g / mol. The structure of nobiletin is shown below. [ka]

[0016] Kaempferol is a type of flavonoid that can be found in fruits, vegetables, tea leaves, wine, etc. The kaempferol used in the technology of this disclosure may be of plant origin or chemically synthesized. The molar mass of kaempferol is 286.24 g / mol. The structure of kaempferol is shown below. [ka]

[0017] Apigenin is a flavonoid that can be found in celery, parsley, chamomile, and other plants. The apigenin used in the technology of this disclosure may be of plant origin or may be chemically synthesized. The molar mass of apigenin is 270.05 g / mol. The structure of apigenin is shown below. [ka]

[0018] Naringenin is a type of flavanone that can be found mainly in citrus fruits. The naringenin used in the art of this disclosure may be of plant origin or chemically synthesized. The molar mass of naringenin is 272.26 g / mol. The structure of naringenin is shown below. [ka]

[0019] In the technology of this disclosure, genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin may optionally take the form of glycosides bound to a sugar. The sugar constituting the glycoside is not particularly limited and may be a monosaccharide, disaccharide, or trisaccharide composed of one or more monosaccharides selected from the group consisting of glucose, galactose, mannose, ribose, deoxyribose, arabinose, xylose, rhamnose, fucose, and fructose. The aforementioned monosaccharide may be the L-form, the D-form, or a mixture of the L-form and D-form, and it is preferable that it be the enantiomer which is more abundant in nature. Therefore, the sugar constituting the glycoside is preferably a monosaccharide, disaccharide, or trisaccharide composed of one or more monosaccharides selected from the group consisting of, for example, D-glucose, D-galactose, D-mannose, D-ribose, D-deoxyribose, L-arabinose, D-xylose, L-rhamnose, L-fucose, and D-fructose. Furthermore, the aforementioned monosaccharide may be in the α-form, the β-form, or a mixture of the α-form and β-form, and it is preferable that it be the anomeric which is more abundant in nature.

[0020] More specifically, disaccharides include maltose, sucrose, lactose, rutinose, cellobiose, trehalose, lactulose, cellobiose, isomaltose, isotrehalose, neotrehalose, turanose, palatinose, and mannobiose. More specifically, trisaccharides include maltotriose, melegitose, raffinose, nigerotriose, and kestose.

[0021] Genistin is an example of a glycoside of genistein. Genistin has a structure in which D-glucose is O-glycosidically bonded to the hydroxyl group at position 7 of genistein. Genistin is the 7-O-glucoside of genistein.

[0022] Examples of quercetin glycosides include rutin, quercitrin, and isoquercitrin. Rutin has a structure in which rutinose, composed of D-glucose and L-rhamnose, is O-glycosidically bonded to the hydroxyl group at position 3 of quercetin. Rutin is the 3-O-rutinoside of quercetin. Quercitrin has a structure in which L-rhamnose is O-glycosidically bonded to the hydroxyl group at position 3 of quercetin. Quercitrin is the 3-O-rhamnoside of quercetin. Isoquercitrin has a structure in which D-glucose is O-glycosidically bonded to the hydroxyl group at position 3 of quercetin. Isoquercitrin is the 3-O-glucoside of quercetin.

[0023] Examples of glycosides of kaempferol include astragalin and kaempferritrin. Astragalin has a structure in which D-glucose is O-glycosidically bonded to the hydroxyl group at position 3 of kaempferol. Astragalin is the 3-O-glucoside of kaempferol. Kaempferritrin has a structure in which L-rhamnose is O-glycosidically bonded to the hydroxyl groups at positions 3 and 7 of kaempferol, respectively. Kaempferritrin is the 3,7-O-diramnoside of kaempferol.

[0024] Examples of apigenin glycosides include vitexin and isovitexin. Vitexin has a structure in which D-glucose is C-glycosidically bonded to the carbon atom at position 8 of apigenin. Vitexin is the 8-C-glucoside of apigenin. Isovitexin has a structure in which D-glucose is C-glycosidically bonded to the carbon atom at position 6 of apigenin. Isovitexin is the 6-C-glucoside of apigenin.

[0025] Naringin is an example of a glycoside of naringenin. Naringin has a structure in which neohesperidinose, composed of D-glucose and L-rhamnose, is O-glycosidically bonded to the hydroxyl group at position 7 of naringenin. Naringin is the 7-O-neohesperidinoside of naringenin.

[0026] The content of the flavonoids in this disclosure is not particularly limited and may be, for example, 0.000001 to 1% by mass of the total composition. The upper or lower limits of the range are 0.000001, 0.000005, 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.002, 0.005, 0.008, 0.01, 0.012, 0.014, 0.016, 0.018, 0.02, 0.022, 0.024, 0.026, 0.028, 0.03, 0.032, 0.034, 0.036, 0.038, 0.04, 0.042, 0.044, 0 The amount may be 0.046, 0.048, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% by mass. The flavonoid content of this disclosure is preferably 0.00001 to 0.5% by mass, more preferably 0.0001 to 0.2% by mass, and even more preferably 0.001 to 0.1% by mass, based on the total composition.

[0027] The compositions of this disclosure contain at least one selected from the group consisting of genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin, and their glycosides, and may further contain other components insofar as the desired effect is obtained. Examples of such other components include pharmacologically or food-hygiene-acceptable bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, antioxidants, preservatives, coatings, colorants, etc. They may also contain flavonoids and flavonoid glycosides other than the flavonoids of this disclosure. These components may be used individually or in combination of two or more.

[0028] The compositions of this disclosure are not particularly limited, but may include, for example, oral compositions. The compositions of this disclosure may also include pharmaceutical compositions or food and beverage compositions. If they are food and beverage compositions, they may include, for example, processed foods, beverages, health foods (nutrient function foods, foods with functional claims, foods for specified health uses, etc.), supplements, and foods for the sick (hospital meals, sick person meals, or nursing care meals, etc.).

[0029] The compositions of this disclosure can be prepared by conventional methods by combining the flavonoids of this disclosure with other components as needed.

[0030] II. Application As described above, the inventors have found that at least one selected from the group consisting of genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin, and their glycosides, can exert anti-inflammatory effects, particularly in senescent cells. More specifically, the inventors have found that the flavonoids of this disclosure can suppress the expression of inflammation-related factors such as CCL2 (CC motif chemokine ligand 2), PAI-1 (Plasminogen activator inhibitor-1), MMP-1 (Matrix metalloproteinase-1), IL-8 (Interleukin-8), E-selectin (Endothelial-selectin), and CXCL1 (CXC motif chemokine ligand 1), particularly in senescent cells.

[0031] Furthermore, the inventors have confirmed that senescent cells are more sensitive to endotoxins than normal cells (i.e., non-senescent cells), and that the expression of inflammation-related factors increases significantly when endotoxins are added to senescent cells. They have also found that the flavonoids of this disclosure can suppress the expression of inflammation-related factors induced by endotoxins in senescent cells. Endotoxins are pathogenic factors also known as lipopolysaccharides (LPS), and are components of the outer membrane of the cell wall of Gram-negative bacteria such as Porphyromonas gingivalis and Escherichia coli.

[0032] Based on the effects of the flavonoids of this disclosure, the compositions of this disclosure can be used for anti-inflammatory applications in aging tissue (preferably aged vascular endothelial tissue), and for suppressing the expression of at least one inflammation-related factor selected from the group consisting of CCL2, PAI-1, MMP-1, IL-8, E-selectin, and CXCL1 in aging tissue (preferably aged vascular endothelial tissue).

[0033] In one preferred embodiment, though not particularly limited, the compositions of the disclosure are used to suppress endotoxin-induced inflammatory responses. In another preferred embodiment, the compositions of the disclosure are used to suppress the expression of endotoxin-induced inflammation-related factors such as CCL2, PAI-1, MMP-1, IL-8, E-selectin, and CXCL1.

[0034] In one preferred embodiment, though not particularly limited, the aging tissue is the tissue of a subject aged 40 years or older. More preferably, the aging tissue is the tissue of a subject aged 45 years or older, even more preferably, the tissue is the tissue of a subject aged 50 years or older or 55 years or older, and particularly preferably, the tissue is the tissue of a subject aged 60 years or older. The lower limit of the range may be 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 years.

[0035] In this disclosure, senescent tissue refers to tissue containing senescent cells. Senescent cells are known to exhibit several characteristics that differ from normal cells. One such characteristic of senescent cells is that they are SA-β-gal (senescence-associated beta galactosidase) positive.

[0036] The anti-inflammatory effect of the compositions disclosed can be confirmed, for example, by adding the compositions to an evaluation system (preferably an evaluation system including LPS) that includes senescent cells (preferably senescence-induced vascular endothelial cells), and observing a decrease in the expression level of at least one inflammation-related factor selected from the group consisting of CCL2, PAI-1, MMP-1, IL-8, E-selectin, and CXCL1, compared to the case where the compositions are not added. In this disclosure, the term "factor" is used to include both genes and proteins.

[0037] The method of administering the compositions of this disclosure is not particularly limited and may be administered, for example, orally or parenterally. Parenteral administration may include, for example, intravenous, arterial, intramuscular, subcutaneous, abdominal, rectal, and topical administration, as well as transdermal administration. The compositions of this disclosure are preferably administered orally.

[0038] The dosage (intake) of the composition disclosed herein is not particularly limited and can be appropriately selected depending on the form and method of administration of the composition disclosed herein. For example, when administered orally to a human (adult), the total intake of the flavonoids disclosed herein may be 0.01 to 10,000 mg per day, preferably 0.1 to 50,000 mg, more preferably 0.3 to 2,000 mg, even more preferably 1 to 1,000 mg, and particularly preferably 1 to 500 mg. The above amount may be administered or ingested to the subject in, for example, 1 to 3 divided doses per day.

[0039] The frequency of administration (ingestion) of the compositions disclosed herein is not particularly limited. For example, they may be administered or ingested once or more times a day, once or more times a week, or once or more times a month. Furthermore, the duration of application of the technology disclosed herein is not particularly limited.

[0040] The subjects to whom the compositions of this disclosure are administered or ingested are not particularly limited, and may be, for example, humans or other mammals. Examples of other mammals include rats, mice, rabbits, cattle, pigs, dogs, cattle, sheep, and monkeys.

[0041] While not particularly limited, the compositions of this disclosure are preferably applied to persons aged 40 years or older, more preferably to persons aged 45 years or older, even more preferably to persons aged 50 years or older or 55 years or older, and particularly preferably to persons aged 60 years or older. The lower limit of the range may be 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 years.

[0042] The compositions disclosed herein are preferably applied to people who want to prevent aging, people who want to suppress aging, people who want to improve aging, people who want to prevent aging of blood vessels (especially vascular endothelium), people who want to suppress aging of blood vessels (especially vascular endothelium), people who want to improve aging of blood vessels (especially vascular endothelium), people who want to maintain and / or improve vascular function, people who want to maintain and / or improve the flexibility and suppleness of blood vessels that decline with age, people who want to prevent inflammation (especially inflammation in the vascular endothelium), people who want to suppress inflammation (especially inflammation in the vascular endothelium), and people who want to improve inflammation (especially inflammation in the vascular endothelium).

[0043] In this specification, the term “comprising” includes not only “containing” but also “essentially consisting of” and “consisting of.” Furthermore, this disclosure encompasses all combinations of the constituent elements described herein.

[0044] Furthermore, the various characteristics (properties, structure, numerical values, functions, etc.) described for each embodiment of this disclosure described above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein. [Examples]

[0045] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below. The density of the culture medium used for cell culture was approximately 1.0 g / mL in all cases.

[0046] Experiment 1. Induction of aging in human vascular endothelial cells and confirmation of aging characteristics. 1-1. Method 1-1-1. Evaluation of SA-β-gal-positive cells (Staining - Microscope) Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage 4, hereafter p.4) or passage 21 (Passage 21, hereafter p.21). Subsequently, p.4 cells measured 2.0 × 10⁶ cells. 4 At a density of cells / 1 mL / well, P.21 is 2.5 × 10⁻⁶ 4 Cells were seeded at a density of 1 mL / well into 12-well plates and cultured for 3 days in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)). After washing once with PBS(-), Bafilomycin A1 (Focus Biomolecules), diluted to 100 nM in evaluation medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), and 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation)), was added and incubated for 1 hour.

[0047] After 1 hour, the cells were washed twice with PBS(-) and fixed for 3 minutes using 4% paraformaldehyde-phosphate buffer (FUJIFILM Wako Pure Chemical). After washing three times with PBS(-), SPiDER-βGal (Dojindo), dissolved in DMSO and diluted with McIlvaine buffer (pH 6.0), was added and incubated for 30 minutes. Then, Hoechst 33342 (Thermo Scientific), diluted with PBS(-) containing 0.3% Triton X-100 (Sigma-Aldrich), was added and incubated for 15 minutes. After washing three times with PBS(-), fluorescence images were acquired using the EVOS M7000 Imaging System (Thermo Scientific). Fluorescence intensity was quantified by processing the fluorescence images using Celleste 5 Image Analysis Software (Thermo Scientific).

[0048] 1-1-2. Evaluation of SA-β-gal-positive cells (Staining-Flow cytometry) Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage 4, hereafter p.4) or passage 21 (Passage 21, hereafter p.21). Subsequently, p.4 cells measured 2.0 × 10⁶ cells. 4 At a density of cells / 1 mL / well, P.21 is 2.5 × 10⁻⁶ 4Cells were seeded at a density of 1 mL / well into 12-well plates and cultured for 3 days in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)). After washing once with HBSS, Bafilomycin A1 (Focus Biomolecules), diluted to 100 nM in evaluation medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), and 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation)), was added and incubated for 1 hour.

[0049] After 1 hour, SPiDER-βGal (Dojindo), dissolved in DMSO and diluted in evaluation medium, was added and incubated for 30 minutes. After washing once with HBSS, the cells were detached from the plate using trypsin and collected. The collected cells were centrifuged at 4°C (1,000 rpm, 5 minutes), and the supernatant was aspirated and removed. The cells were washed again with HBSS, centrifuged at 4°C (1,000 rpm, 5 minutes), and the supernatant was aspirated and removed. Subsequently, the precipitated cells were dispersed in HBSS, and then dispersed individually using a cell strainer (40 μm, Corning). The dispersed cells were analyzed by fluorescence measurement using a BD Accuri C6 Plus Flow Cytometer (BD Biosciences).

[0050] 1-1-3. Cell cycle evaluation (Staining-flow cytometry) Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage 4, hereafter p.4) or passage 21 (Passage 21, hereafter p.21). Subsequently, p.4 cells measured 2.0 × 10⁶ cells. 4 At a density of cells / 1 mL / well, P.21 is 2.5 × 10⁻⁶ 4 Cells were seeded at a density of 1 mL / well into 12-well plates and cultured for 3 days in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)). After reaching confluence, the cells were washed once with PBS(-), detached from the plate using trypsin, and collected. The collected cells were centrifuged at 20°C (300 g, 5 min), the supernatant was removed, and the cells were dispersed in PBS(-). A predetermined number of cells were mixed with Cell Cycle Assay Solution Deep Red (Dojindo) and incubated at 37°C under light-shielded conditions for 15 minutes. After dispersing the cells individually using a cell strainer (40 μm, Corning), fluorescence measurements were performed and analyzed using a BD Accuri C6 Plus Flow Cytometer (BD Biosciences).

[0051] 1-1-4. Evaluation of the expression of inflammation-related genes, cell cycle regulatory genes, and mitochondrial function-related genes (qPCR) Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage 4, hereafter p.4) or passage 21 (Passage 21, hereafter p.21). Subsequently, p.4 cells measured 2.0 × 10⁶ cells. 4 At a density of cells / 1 mL / well, P.21 is 2.5 × 10⁻⁶ 4Cells were seeded at a density of 1 mL / well into 12-well plates and cultured for 3 days in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)). After reaching confluence, the cells were switched to evaluation medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), and 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation)) and cultured for 30 hours.

[0052] Total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted total RNA using the PrimeScript RT reagent Kit (Takara Bio). Using the synthesized cDNA as a template, gene expression was quantified using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) with primers specific to each gene (p16, p21, p53, CDK2, CDK4, SIRT1, NRF-1, TFAM, and RPS18) and an intercalator method using TB Green Premix Ex Taq II FAST qPCR (Takara Bio). The expression levels of each gene were corrected to the expression level of RPS18, and the relative value at p21 was determined when the expression at p4 was set to 1. The primer sets used to quantify each gene expression in this example are described in "4. Appendix".

[0053] 1-1-5. Evaluation of the expression levels of inflammation-related proteins, cell cycle regulatory proteins, and mitochondrial function-related proteins (Western Blotting) Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until passage 4 (Passage 4, hereinafter p.4) or passage 21 (Passage 21, hereinafter p.21) was reached. Thereafter, p.4 was seeded at a density of 2.0 × 10 4 cells / 1 mL / well, and p.21 was seeded at a density of 2.5 × 10 4 cells / 1 mL / well in 12-well plates, respectively, and cultured in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)) for 3 days. After reaching confluence, the cells were washed twice with cold PBS(-) and harvested on ice with M-PER buffer (Thermo Scientific) supplemented with protease inhibitor (Thermo Scientific) and phosphatase inhibitor (Thermo Scientific). The harvested cells were centrifuged at 4°C (15,000 g, 20 minutes), and the supernatant was collected.

[0054] The total protein concentration of the obtained protein extract was measured by BCA assay (Thermo Scientific). After standardizing the protein concentration, Laemmli Sample Buffer (BIO-RAD) was added, and the proteins were denatured by boiling. The denatured proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes with transferred proteins were blocked with PBS containing 5% skim milk and 0.05% Tween-20. After blocking, the membranes were sequentially reacted with primary and secondary antibodies, and then chemiluminescent membranes were induced using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and SuperSignal West Atto Ultimate Sensitivity Substrate (Thermo Scientific) as substrates. The emission intensity was detected using an Amersham Imager 680 RGB (Cytiva) system. Band intensity quantification was performed using Image J.

[0055] 1-2. Results Senescent cells are known to be positive for SA-β-gal (senescence-associated beta galactosidase). The results regarding SA-β-gal positive cells are shown in Figures 1-3. Figure 1 shows fluorescence microscope images (left) and fluorescence intensity (relative value with fluorescence intensity at p.4 set to 1, right) when SA-β-gal positive cells were detected using the β-galactosidase detection fluorescent reagent SPiDER-βGal. Figure 2 shows the results of flow cytometry analysis of SA-β-gal positive cells. Figure 3 shows the results of flow cytometry analysis of cells that highly express SA-β-gal. As shown in Figures 1-3, it was shown that SA-β-gal expression, a characteristic of aging, is upregulated at p.21.

[0056] Figure 4 shows the results of flow cytometry analysis of the cell cycles of cells p.4 and p.21. As shown in Figure 4, p.21 showed a decrease in G0 / G1 and S phase cells and an increase in G2 phase cells.

[0057] The expression levels of each gene are shown in Figures 5 and 6. All of the genes evaluated are those reported to be associated with aging (aging-related genes). The genes shown in Figure 5 all showed significantly increased expression at p.21. This result is consistent with previous findings that the expression of these genes is induced in senescent cells. The genes shown in Figure 6 are all mitochondrial function-related genes, and their expression was significantly decreased. This result is consistent with previous findings that mitochondrial function is impaired in senescent cells.

[0058] Figure 7 shows the results of analyzing the protein expression levels of p16, p21, and SIRT1 by Western blotting. Consistent with the gene expression levels shown in Figures 5 and 6, the protein expression levels of p16 and p21 were significantly increased in p.21, while the protein expression level of SIRT1 was significantly decreased.

[0059] These results confirm that aging is induced in HUVECs that have undergone repeated subculturing.

[0060] Study 2. Evaluation of inflammation-related gene responses to E. coli LPS. 2-1. Method Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage 4, hereafter p.4) or passage 21 (Passage 21, hereafter p.21). Subsequently, p.4 cells measured 2.0 × 10⁶ cells. 4 At a density of cells / 1 mL / well, P.21 is 2.5 × 10⁻⁶ 4Cells were seeded at a density of 1 mL / well into 12-well plates and cultured for 3 days in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)). After reaching confluence, the cells were switched to evaluation medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), and 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation)) and cultured for 24 hours. After 24 hours, 100 ng / mL of Escherichia coli-derived Lipopolysaccharides (hereinafter referred to as E. coli LPS) diluted in evaluation medium was added, or not added, and the cells were incubated for 6 hours.

[0061] Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted total RNA using the PrimeScript RT reagent Kit (Takara Bio). Using the synthesized cDNA as a template, gene expression was quantified using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) with primers specific to each gene (IL-8, CXCL1, PAI-1, MMP-1, E-selectin, and CCL2) and an intercalator method using TB Green Premix Ex Taq II FAST qPCR (Takara Bio). The expression levels of each gene were corrected for the expression level of RPS18, and the relative values ​​were calculated with the gene expression level when p.4 was cultured in evaluation medium without E. coli LPS set to 1.

[0062] 2-2. Results The results are shown in Figures 8 and 9. IL-8, CXCL1, PAI-1, MMP-1, E-selectin, and CCL2 are all inflammation-related genes. As shown in Figures 8 and 9, the expression levels of IL-8, CXCL1, PAI-1, MMP-1, E-selectin, and CCL2 were significantly higher in p.21 than in p.4 when E. coli LPS was added.

[0063] These results suggest that senescent cells exhibit increased sensitivity to endotoxins and a heightened inflammatory response to endotoxins.

[0064] Test 3. Evaluation of the anti-inflammatory effect of flavonoids. 3-1. Method Genistein, quercetin, fisetin, nobiletin, kaempferol, myricetin, apigenin, and naringenin (all from Tokyo Chemical Industry) were dissolved in DMSO (Dimethyl sulfoxide, FUJIFILM Wako Pure Chemical Corporation) and used as flavonoid samples for subsequent cell tests.

[0065] Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage 4, hereafter p.4) or passage 21 (Passage 21, hereafter p.21). Subsequently, p.4 cells measured 2.0 × 10⁶ cells. 4 At a density of cells / 1 mL / well, P.21 is 2.5 × 10⁻⁶ 4Cells were seeded at a density of 1 mL / well into 12-well plates and cultured for 3 days in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)). After reaching confluence, the medium was changed to evaluation medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), and 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation)), and each flavonoid was added at a concentration of 20 μM, followed by incubation for 24 hours. After 24 hours, 100 ng / mL of Escherichia coli-derived lipopolysaccharides (hereinafter referred to as E. coli LPS), diluted in evaluation medium, was added and incubated for 6 hours. Flavonoid samples and E. coli LPS were not added to the controls on p.4 and p.21, respectively.

[0066] Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted total RNA using the PrimeScript RT reagent Kit (Takara Bio). Using the synthesized cDNA as a template, gene expression was quantified using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) with primers specific to each gene and an intercalator method using TB Green Premix Ex Taq II FAST qPCR (Takara Bio). The expression levels of each gene were corrected for the expression level of RPS18, and the relative value was determined with the gene expression level when p.4 was cultured without the addition of flavonoids and E. coli LPS set to 1. This study was performed with n=2.

[0067] Based on the relative values ​​calculated as described above, for both p.4 and p.21, the percentage reduction in gene expression when E. coli LPS was added but no flavonoids were added was set to 100%, and the percentage reduction in gene expression when both flavonoids and E. coli LPS were added was set to 0%. The calculation formula is shown below. As can be seen from the formula below, when gene expression is suppressed by flavonoid addition, the percentage reduction in gene expression takes a positive value. Furthermore, the greater the gene expression suppression effect, the larger the percentage reduction in gene expression. On the other hand, when gene expression is enhanced by flavonoid addition, the percentage reduction in gene expression takes a negative value.

number

[0068] 3-2. Results For pages 4 and 21, the percentage reduction in gene expression upon addition of each flavonoid is shown in the following tables.

[0069] [Table 1]

[0070] As shown in the table above, the percentage reduction in the expression of the inflammation-related genes CXCL1, PAI-1, and MMP-1 was greater in p.21 than in p.4 when genistein was added. In particular, for PAI-1, although the percentage reduction in expression was negative in p.4 (i.e., gene expression was increased by genistein addition), gene expression decreased by as much as 20.2% in p.21. On the other hand, when myricetin, a type of flavonoid, was added, unlike when genistein was added, no significant decrease in the expression of any inflammation-related genes was observed.

[0071] [Table 2]

[0072] As shown in the table above, in p.4, the rate of decrease in MMP-1 expression was -363.6% with the addition of quercetin (i.e., MMP-1 expression was significantly increased), while in p.21, MMP-1 expression was suppressed with the addition of quercetin. On the other hand, when myricetin was added, MMP-1 expression was increased in both p.4 and p.21.

[0073] [Table 3]

[0074] As shown in the table above, p.21 showed a greater decrease in the expression of IL-8 and PAI-1, inflammation-related genes, when fisetin was added, compared to p.4. On the other hand, when myricetin was added, unlike when fisetin was added, no significant decrease in the expression of any inflammation-related genes was observed.

[0075] [Table 4]

[0076] As shown in the table above, the decrease in expression of inflammation-related genes E-selectin, IL-8, CCL2, PAI-1, and MMP-1 upon nobiletin addition was greater in p.21 than in p.4. In particular, for E-selectin, PAI-1, and MMP-1, the decrease in expression was negative in p.4 (i.e., gene expression was increased by nobiletin addition), while gene expression was decreased in p.21. On the other hand, when myricetin was added, no significant decrease in expression was observed for any of the inflammation-related genes, and MMP-1 expression, in particular, was increased in both p.4 and p.21.

[0077] [Table 5]

[0078] As shown in the table above, the decrease in MMP-1 expression upon addition of kaempferol was greater in p.21 than in p.4. On the other hand, when myricetin was added, MMP-1 expression was increased in both p.4 and p.21.

[0079] [Table 6]

[0080] As shown in the table above, the decrease in expression of the inflammation-related genes CCL2, PAI-1, and MMP-1 was greater in p.21 than in p.4 when apigenin was added. On the other hand, when myricetin was added, no significant decrease in expression was observed for any of the inflammation-related genes, and in particular, MMP-1 expression was increased in both p.4 and p.21.

[0081] [Table 7]

[0082] As shown in the table above, the decrease in expression of the inflammation-related genes CCL2, PAI-1, and MMP-1 was greater in p.21 than in p.4 when naringenin was added. On the other hand, when myricetin was added, no significant decrease in expression was observed for any of the inflammation-related genes, and in particular, MMP-1 expression was increased in both p.4 and p.21.

[0083] 3-3. Discussion The results suggest that genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin may exert anti-inflammatory effects, particularly in aging tissues. Furthermore, it was suggested that certain combinations of flavonoids and certain inflammation-related factors may enhance the expression of these factors in non-senescent cells, while conversely suppressing their expression in senescent cells.

[0084] 4. Appendix The primer sets used to quantify each gene expression in the examples of this disclosure are shown in the table below.

[0085] [Table 8]

Claims

1. An anti-inflammatory composition for aging tissue, comprising at least one selected from the group consisting of genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin, and glycosides thereof.

2. The composition according to claim 1, wherein the aging tissue is vascular endothelial tissue.

3. The composition according to claim 1 or 2, for use against an inflammatory response induced by endotoxins.

4. It contains at least one selected from the group consisting of nobiletin, apigenin, and naringenin, and their glycosides. A composition used to suppress the expression of at least one inflammation-related gene selected from the group consisting of CCL2, PAI-1, and MMP-1 in aging tissue.

5. It contains at least one selected from the group consisting of genistein, nobiletin, apigenin, and naringenin, and their glycosides. A composition used to suppress the expression of at least one inflammation-related gene selected from the group consisting of PAI-1 and MMP-1 in aging tissue.

6. The composition according to claim 4 or 5, wherein the aging tissue is vascular endothelial tissue.

7. The composition according to claim 4 or 5, used to suppress the expression of inflammation-related genes induced by endotoxins.

8. The composition according to claim 1, 2, 4, or 5, wherein the aging tissue is the tissue of a subject aged 40 years or older.

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