JP7494232B2 - Anti-inflammatory for joints - Google Patents

Description

The present invention relates to an anti-inflammatory agent for joints.

Inflammation is a biological defense reaction that is primarily induced by external stimuli. When inflammation occurs, symptoms such as redness, swelling, heat, and pain occur. Because inflammation also damages living tissue, excessive or chronic inflammatory reactions can be harmful.

Non-patent literature 1 discloses that cartilage damage was suppressed in mice administered apple-derived procyanidins.

Masuda et al., "Apple procyanidins promote mitochondrial biogenesis and proteoglycan biosynthesis in chondrocytes", Sci. Rep. 2018 May 8;8(1):7229. doi:10.1038/s41598-018-25348-1.

The present invention aims to provide a new anti-inflammatory agent for joints.

The present invention relates to the following exemplary items.
[1] An anti-inflammatory agent for joints containing fruit-derived polyphenols.
[2] An anti-inflammatory agent for synovial membrane of joints containing apple-derived polyphenols.
[3] An agent for suppressing joint pain, comprising apple-derived polyphenols, and suppressing joint pain through anti-inflammatory effects.
[4] The agent according to any one of [1] to [3], wherein the joint is a knee joint.
[5] The agent according to any one of [1] to [4], which suppresses joint inflammation by promoting IL-10 expression, suppressing TNF-α expression, or suppressing MMP-13 expression.
[6] The agent according to [2] or [3], wherein the apple-derived polyphenols include procyanidins.
[7] The agent described in [2] or [3], wherein the apple-derived polyphenol is derived from unripe apples.
[8] The agent according to any one of [1] to [7], which is an oral agent.
[9] The agent according to [8], which is a health food, dietary supplement or supplement.
[10] The agent according to any one of [1] to [9], which is used for people suffering from joint pain.

The present invention provides a new joint anti-inflammatory agent and joint pain suppressant.

1 is a graph showing the gene expression level of IL-10 in Experiment 1. 1 is a graph showing the gene expression level of TNF-α in Experiment 2. 1 is a graph showing the gene expression level of MMP-13 in Experiment 2. 1 is a graph showing the gene expression level of SOD1 in Experiment 2. 1 is a graph showing the antioxidant effect of test substances in Experiment 2.

One embodiment of the agent according to the present invention is an anti-inflammatory agent for joints that contains fruit-derived polyphenols. The anti-inflammatory agent for joints can suppress systemic or localized inflammation of joints. By suppressing inflammation, joint redness, swelling, heat, pain, and the like can be prevented, suppressed, or alleviated. One embodiment of the agent according to the present invention is an anti-inflammatory agent for joint pain that contains fruit-derived polyphenols and suppresses joint pain through its anti-inflammatory effect.

In this specification, the term "joint" includes various types of joints such as knee joints, shoulder joints, neck joints, hip joints, spinal joints, temporomandibular joints, finger joints, elbow joints, wrist joints, and ankle joints. Joint inflammation includes osteoarthritis, rheumatoid arthritis, articular cartilage damage, osteonecrosis of the knee joint, osteonecrosis of the femur, shoulder arthritis, bacterial arthritis, viral arthritis, and neuropathic arthropathy.

One aspect of the agent according to the present invention is an anti-inflammatory agent for the synovial membrane of joints, which contains fruit-derived polyphenols. The synovial membrane adheres to the periphery of articular cartilage, secretes synovial fluid, and contributes to the stability of the joint. Fruit-derived polyphenols can suppress inflammation in the joints, particularly by suppressing inflammation of the synovial membrane.

Anti-inflammatory agents or joint pain suppressants suppress inflammation of joints, preferably synovium, by promoting interleukin 10 (IL-10) expression, inhibiting tumor necrosis factor alpha (TNF-α) expression, or inhibiting matrix metalloproteinase 13 (MMP-13) expression. IL-10 is an anti-inflammatory cytokine, TNF-α is an inflammatory cytokine, and MMP-13 is a collagen degrading enzyme. One aspect of the present invention can be said to be an IL-10 expression promoter, TNF-α expression suppressor, or MMP-13 expression suppressor that contains fruit-derived polyphenols. Fruit-derived polyphenols have antioxidant properties and can suppress the expression of Cu/Zn superoxide dismutase 1 (SOD1).

The joint anti-inflammatory agent or joint pain suppressant can be used to prevent, treat, or suppress the worsening of joint inflammation. The joint anti-inflammatory agent or joint pain suppressant may be used in people who do not have joint pain, or in people who have joint pain.

Fruits that are the source of fruit-derived polyphenols include berries such as blueberries, cranberries, blackberries, and raspberries, strawberries, black currants, cherries, grapes, nectarines, plums, apricots, kiwi, avocado, mango, dates, and bananas, as well as fruits of the Rosaceae family, such as apples, pears, and peaches. The fruit-derived polyphenol is preferably apple-derived polyphenol.

The apple-derived polyphenols may be an extract of apples, and may be an extract of the skin and/or pulp. The apple-derived polyphenols may be derived from unripe apples. The apple-derived polyphenols may be obtained by purification, for example, by the following method. Unripe apples are washed, crushed, and squeezed to obtain fruit juice. Insoluble components such as pectin are removed from the fruit juice by enzymatic hydrolysis and/or filtration. The fruit juice is passed through a column, and the polyphenol components are retained in the column, and sugars, organic acids, etc. are removed by washing with water. The polyphenol components are eluted from the column using an aqueous alcohol solution to obtain a polyphenol fraction. The polyphenol fraction may be concentrated under reduced pressure and then spray-dried.

The apple-derived polyphenols preferably contain procyanidins. For example, the procyanidins account for 10% by mass or more or 15% by mass or more of the apple-derived polyphenols. In addition to procyanidins, the apple-derived polyphenols may contain catechins, phenolic carboxylic acids, phloretin glycosides, and the like.

Apple-derived polyphenols are readily available commercially. Examples of commercially available products include "Apple Polyphenol Powder" from Frontier Foods, Inc., "ApplePhenon (registered trademark) Apple Phenon SH," "ApplePhenon (registered trademark) Apple Procyanidin," and "ApplePhenon (registered trademark) Apple Phenon C-100" from BGG Japan, Inc., "Apple Polyphenol" from Access One, Inc., and "Apple Extract Powder" from Vidya Japan, Inc.

The agent according to the present invention may be an oral agent or a parenteral agent. Parenteral agents can be administered, for example, by external application or injection. There are no particular limitations on the subjects to which the agent is administered, and examples include humans, as well as mammals such as cows, horses, pigs, sheep, dogs, rabbits, mice, and rats, and birds such as chickens. Subjects to which the agent is administered may also be livestock, poultry, pets, laboratory animals, etc.

Oral preparations may be foods, medicines, or quasi-drugs. Foods include beverages and feeds. Foods include foods for special dietary uses, foods with health claims (foods for specified health uses, foods with nutrient functions, foods with functional claims), dietary supplements, and supplements. Medicines include therapeutic or veterinary medicines, preventive medicines, and the like.

The amount of fruit-derived polyphenols in the oral preparation may be 0.01% by mass or more and 50% by mass or less, or 0.1% by mass or more, 1% by mass or more, or 3% by mass or more, and may be 30% by mass or less, 20% by mass or 10% by mass or less. The amount of fruit-derived polyphenols can be analyzed, for example, by high performance liquid chromatography.

When the agent according to the present invention is an oral agent, the dosage can be adjusted widely depending on the sex, age, and weight of the person taking the agent, as well as the severity of symptoms, but generally the daily intake of fruit-derived polyphenols per person may be 1 mg or more and 2000 mg or less, or may be 100 mg or more, 200 mg or more, or 500 mg or more, or may be 1500 mg or less. The oral agent may be taken once a day or in several divided doses.

Examples of foods include dairy products such as cheese, modified milk powder, ice cream, and yogurt; frozen desserts, chocolate, tablets, gummies, cookies, biscuits, candies, Japanese sweets, rice crackers, cakes, pies, puddings, and other confectioneries; flour products such as bread and noodles; rice products such as porridge and cooked rice; and seasonings such as soy sauce, miso, mayonnaise, and dressing. Foods may also be processed marine products, agricultural products, or livestock products.

Beverages include tea, coffee, milk, dairy drinks, fruit juice drinks, juice, lactic acid drinks, soft drinks, nutritional drinks, beauty drinks, etc.

The food may be a food additive in the form of liquid, paste, powder, flake, granule, etc. Food additives also include additives for beverages. Food additives can be manufactured according to general manufacturing methods for food additives. Oral preparations containing fruit-derived polyphenols may be added to other foods.

Foods may contain additives suitable for oral consumption depending on the type of food. Additives include sweeteners. Sweeteners include glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, lactose, maltitol, etc.

Other additives include citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid esters, magnesium stearate, calcium stearate, cellulose derivatives such as hydroxypropyl cellulose, crystalline cellulose, silicon dioxide, polyglycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamins, nicotinamide, calcium pantothenate, amino acids, calcium salts, colorings, flavorings, preservatives, etc.

The dosage form of the oral preparation is not particularly limited, and may be a tablet, capsule, granule, powder, fine granule, chewable tablet, pill, lozenge, sublingual tablet, ointment, cream, emulsion, suspension, jelly, syrup, liquid, etc.

The preparation of oral preparations can be carried out by known methods using additives such as non-toxic excipients, binders, lubricants, disintegrants, preservatives, isotonicity agents, stabilizers, dispersants, antioxidants, colorants, flavorings, buffers, pH adjusters, thickeners, and gloss agents. Examples of non-toxic additives contained in these preparations include starch, gelatin, glucose, dextrin, hyaluronic acid, lactose, fructose, maltose, magnesium carbonate, silicon dioxide, talc, magnesium stearate, calcium stearate, methylcellulose, carboxymethylcellulose, crystalline cellulose, gum arabic, polyethylene glycol, propylene glycol, petrolatum, glycerin, ethanol, syrup, sodium chloride, sodium sulfite, sodium phosphate, citric acid, polyvinylpyrrolidone, water, shellac, and carnauba wax. In order to enhance the usefulness of the agent according to the present invention, other ingredients, such as bone crushed extract, steroidal or non-steroidal anti-inflammatory compounds, may be included in the preparation. One aspect of the present invention is the use of fruit-derived polyphenols in the production of the above-mentioned agent.

Parenteral preparations may be medicines, quasi-drugs, or cosmetics. Topical preparations may be, for example, directly applied or attached to the skin, and may be liquids, creams, emulsions, lotions, ointments, gels, aerosols, packs, microneedle patches, poultices, etc. Injectables may be, for example, administered into joints, veins, subcutaneously, or muscles.

The content of each component in the parenteral preparation may be the same as or different from that in the oral preparation. The amount of fruit polyphenols in the parenteral preparation may be, for example, 0.001% by mass or more and 20% by mass or less, 0.01% by mass or more or 0.05% by mass or more, and 10% by mass or less or 5% by mass or less. The parenteral preparation may be administered once a day or in several divided doses.

Parenteral preparations can be formulated by mixing fruit-derived polyphenols with bases or carriers that are commonly used in pharmaceuticals, quasi-drugs, or cosmetics in the usual manner. Examples of bases or carriers include fats and oils such as coconut oil, olive oil, rice bran oil, and shea butter; hydrocarbons such as petrolatum and liquid paraffin; waxes such as jojoba oil, meu wax, candelilla wax, and lanolin; higher alcohols such as cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, octyldodecanol, isostearyl alcohol, phytosterol, and cholesterol; silicones such as dimethicone, cyclic silicone, and modified silicone; cellulose derivatives such as ethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methylcellulose; polyvinylpyrrolidone; carrageenan; polyvinyl butyrate; polyethylene glycol; and dioxane. ; butylene glycol adipic acid polyester; esters such as diisopropyl adipate, isopropyl myristate, octyldodecyl myristate, isopropyl palmitate, cetyl palmitate, isononyl isononanoate, and pentaerythritol tetra 2-ethylhexanoate; polysaccharides such as dextrin and maltodextrin; vinyl polymers such as carboxyvinyl polymers and alkyl-modified carboxyvinyl polymers; lower alcohols such as ethanol and isopropanol; polyhydric alcohols such as propylene glycol, 1,3-butylene glycol, glycerin, and polyethylene glycol; glycol ethers such as diethylene glycol monoethyl ether; and water. These may be used alone or in combination of two or more, and the amount of each may be appropriately selected from the range known to those skilled in the art. In order to enhance the usefulness of the agent according to the present invention, other ingredients such as bone crushing extract, steroidal or nonsteroidal anti-inflammatory compounds, etc. may be contained in the formulation.

To the parenteral preparation, known additives such as surfactants, stabilizers, antioxidants, colorants, dispersants, chelating agents, pH adjusters, preservatives, thickeners, irritation reducers, fragrances, ultraviolet absorbers, moisturizers, vasodilators, etc. can be added within the scope of the invention, so long as they do not impair the effects of the invention. These additives can be used alone or in combination of two or more.

Examples of surfactants include sorbitan fatty acid esters such as sorbitan monoisostearate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, diglycerol sorbitan penta-2-ethylhexyl acid, and diglycerol sorbitan tetra-2-ethylhexyl acid; propylene glycol fatty acid esters such as propylene glycol monostearate; and hydrogenated castor oils such as polyoxyethylene hydrogenated castor oil 40 (HCO-40), polyoxyethylene hydrogenated castor oil 50 (HCO-50), polyoxyethylene hydrogenated castor oil 60 (HCO-60), and polyoxyethylene hydrogenated castor oil 80. Derivatives include polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene (20) sorbitan monolaurate (polysorbate 20), polyoxyethylene (20) sorbitan monostearate (polysorbate 60), polyoxyethylene (20) sorbitan monooleate (polysorbate 80), and polyoxyethylene (20) sorbitan isostearate; polyoxyethylene monococoate glyceryl; glycerin alkyl ethers; alkyl glucosides; polyoxyalkylene alkyl ethers such as polyoxyethylene cetyl ether; and amines such as stearylamine and oleylamine.

Examples of stabilizers include sodium polyacrylate, dibutylhydroxytoluene, butylhydroxyanisole, sodium phosphate, sodium sulfite, sodium hydrogen sulfite, sodium pyrosulfite, sodium thiosulfate, cysteine hydrochloride, phosphates, sodium ascorbate, sodium chloride, sodium bicarbonate; glycine, various amino acids and their derivatives.

Examples of antioxidants include dibutylhydroxytoluene, sodium pyrosulfite, sodium edetate, butylhydroxyanisole, sorbic acid, sodium sulfite, ascorbic acid, erythorbic acid, and L-cysteine hydrochloride.

Colorants include, for example, inorganic pigments and natural dyes.

Dispersants include, for example, sodium pyrophosphate, sodium hexametaphosphate, polyvinyl alcohol, polyvinylpyrrolidone, methyl vinyl ether/maleic anhydride crosslinked copolymer, and organic acids.

Examples of chelating agents include EDTA disodium salt and citric acid.

Examples of pH adjusters include inorganic acids such as hydrochloric acid and sulfuric acid; organic acids such as lactic acid, sodium lactate, citric acid, sodium citrate, succinic acid, and sodium succinate; inorganic bases such as potassium hydroxide and sodium hydroxide; and organic bases such as triethanolamine, diisopropanolamine, and triisopropanolamine.

Preservatives include, for example, benzoic acid, sodium benzoate, dehydroacetic acid, sodium dehydroacetate, parabens, and phenoxyethanol.

Examples of thickeners include cellulose-based thickeners such as methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, and carboxyethylcellulose, white sugar, guar gum, pectin, pullulan, gelatin, locust bean gum, carrageenan, agar, xanthan gum, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, acrylate/alkyl methacrylate copolymer, polyethylene glycol, bentonite, alginic acid, propylene glycol alginate, macrogol, hyaluronic acid, sodium hyaluronate, (hydroxyethyl acrylate/sodium acryloyldimethyltaurate) copolymer, and (ammonium acryloyldimethyltaurate/vinylpyrrolidone) copolymer.

Irritation reducers include, for example, licorice extract, sodium alginate, gum arabic, polyvinylpyrrolidone, licorice extract, and sodium alginate.

Examples of ultraviolet absorbers include ethyl paraaminobenzoate, ethylhexyl paradimethylaminobenzoate, amyl salicylate and its derivatives, 2-ethylhexyl paramethoxycinnamate, octyl cinnamate, oxybenzone, 2,4-dihydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone-5-sulfonate, 4-tert-butyl-4-methoxybenzoylmethane, 2-(2-hydroxy-5-methylphenyl)benzotriazole, urocanic acid, ethyl urocanate, and aloe extract.

Examples of moisturizing agents include sugars such as glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol, polyethylene glycol, sorbitol, xylitol, sodium pyrrolidone carboxylate, and trehalose, mucopolysaccharides (e.g., sodium hyaluronate, heparin), elastin, collagen, NMF-related substances, lactic acid, urea, higher fatty acid octyldodecyl, seaweed extract, and striated serrata root (white and red) extract.

Examples of vasodilators include carpronium chloride, benzyl nicotinate, Swertia japonica extract, minoxidil, ginseng extract, and capsicum tincture.

[Experiment 1: Animal study (gene expression analysis of synovial membrane in a rat traumatic arthropathy model)]
For the experiment, 10-week-old male Wister rats (Crij:WI) were used. An OA-free model was prepared by sham surgery (sham surgery, only incision of the skin and muscle layer was performed) and an OA-onset model was prepared by medial collateral ligament and medial meniscus resection (MTT surgery). Surgery was performed only on the right knee of all rats, and the left knee was left untreated. Two days after surgery, the rats were introduced into the animal breeding room, and after one week of preliminary breeding, they were assigned so that there was no bias in the average weight of each test group. The rats were kept under a 12-hour light-dark cycle and at a room temperature of 24±2°C. They were kept using solid feed for general breeding (Labo MR Stock, manufactured by Nippon Nosan Kogyo Co., Ltd.) and were fed and watered at all times.

The test conditions are shown in Table 1. As the fruit-derived polyphenol, "Applephenon (registered trademark)" (AP) (manufactured by BGG JAPAN), a polyphenol extract derived from unripe apples, was administered. N-acetylcysteine (NAC) was administered as a positive control. Sterile water was administered to the sham and control groups. The test substance was orally administered once a day during the test period using a stomach tube.

After 8 weeks of treatment, the rats were dissected. Under anesthesia with Sebofren (registered trademark) inhalation anesthetic (Maruishi Pharmaceutical Co., Ltd.), the rats were euthanized by collecting blood from the inferior vena cava, and the synovial tissue below the patella of the right knee and the knee joint of the right hind leg were collected. Tissue sections were prepared from the area surrounding the articular cartilage of the right hind leg knee joint, and it was confirmed that osteoarthritis of the knee had developed.

For gene expression analysis of synovial membrane, synovial tissue was collected from the right knee joint. The isolated synovial tissue was washed with PBS, immersed in RNAlater (Invitrogen) overnight at 4°C, and then treated with TRIzol (registered trademark) reagent (Invitrogen) to purify total RNA. Using this as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Perfect Real Time, Takara Bio Inc.). Using this cDNA as a template, quantitative real-time PCR was performed using TB Green Premix Ex Taq II (Takara Bio Inc.) and Thermal Cycler Dice Real Time System TP800 (Takara Bio Inc.). The base sequences of the primers used are shown in Table 2 and SEQ ID NOs: 1 to 4.

After the reaction was completed, the gene expression level was analyzed using dedicated software (Thermal Cycler Dice Real Time System TP800 Software Ver. 1.02A). The gene expression level of each sample was standardized with amplified β-Actin, and the results were shown as the mean ± standard error (S.E.).

The results of gene expression analysis of IL-10 in the synovium are shown in Figure 1. In the control group, the gene expression level of IL-10 was relatively low compared to the sham group. In the AP administration group, the gene expression level of IL-10 was significantly higher compared to the control group. IL-10 is known as an anti-inflammatory cytokine. It has been suggested that fruit-derived polyphenols strongly promote the gene expression of IL-10, and may reduce joint inflammation, especially inflammation of the synovium.

[Experiment 2: Animal study (gene expression analysis of synovial membrane in a rat traumatic arthropathy model)]
In Experiment 2, the test substance was administered to rats in the same manner as in Experiment 1, except that the dose of AP was different. The test conditions are shown in Table 3. cDNA was obtained from the synovial tissue of the knee joint in the same manner as in Experiment 1, and quantitative real-time PCR was performed. The nucleotide sequences of the primers used are shown in Table 4 and SEQ ID NOs: 3 to 10.

The results of gene expression analysis of TNF-α in synovium are shown in Figure 2. The control group had significantly higher levels of TNF-α gene expression than the sham group. On the other hand, the AP-administered group had significantly lower levels of TNF-α gene expression than the control group, and was equivalent to that of the sham group. TNF-α is one of the most representative inflammatory cytokines. It has been suggested that fruit-derived polyphenols may suppress TNF-α gene expression and reduce joint inflammation, particularly inflammation of the synovium.

The results of gene expression analysis of MMP-13 in the synovium are shown in Figure 3. The control group had significantly higher MMP-13 gene expression levels than the sham group. On the other hand, the AP-administered group had lower MMP-13 gene expression levels than the control group, and were almost the same as the positive control NAC-administered group. MMP-13 has the function of breaking down collagen, one of the main components of cartilage. It was suggested that fruit-derived polyphenol extract may suppress MMP-13 gene expression and act to inhibit cartilage degradation.

The results of gene expression analysis of SOD1 in the synovium are shown in Figure 4. The control group had significantly higher SOD1 gene expression levels than the sham group. On the other hand, the AP-administered group had significantly lower SOD1 gene expression levels than the control and NAC-administered groups, and were equivalent to those of the sham group. It is speculated that the antioxidant effect of AP reduces oxidative stress in the synovium, which is enhanced by inflammation, resulting in a decrease in SOD1 gene expression levels.

The antioxidant activity (ORAC value) of NAC and AP was measured based on the method described in Cao G et al., Free Radical Biology and Medicine, vol. 14, Issue 3, 303-311, March 1993. It was shown that the antioxidant activity of NAC and AP is higher than that of vitamin C (L-ascorbic acid) used as a standard substance, and that there is a correlation between the level of the antioxidant activity of NAC and AP and the level of the inhibitory activity of SOD1 shown in Figure 4 (Figure 5).

Claims (7)

An IL-10 expression promoter which is an oral agent and which is used for suppressing joint inflammation or joint pain and which contains apple- derived polyphenols. The method according to claim 1 , wherein the joint is a knee joint. The method according to claim 1 or 2 , wherein the joint is a synovial membrane. The agent according to any one of claims 1 to 3 , wherein the apple-derived polyphenols include procyanidins. The agent according to any one of claims 1 to 3 , wherein the apple-derived polyphenol is derived from unripe apples. The agent according to any one of claims 1 to 5 , which is a health food, a dietary supplement or a supplement. The agent according to any one of claims 1 to 6 , which is used for people suffering from joint pain.

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