Composition for preventing, ameliorating or treating arthritis, containing hydrangea macrocarpa extract or hydrangeol as active ingredient

Abstract

Disclosed is a composition for preventing, ameliorating or treating arthritis, which contains a hydrangea crassifolia extract or hydrangeol as an active ingredient. The present invention not only inhibits the expression of MMP-1 and MMP-9, increases the synthesis of type II procollagen, and alleviates inflammation, but also has an effect of preventing, ameliorating or treating arthritis by inhibiting inflammatory pain.

Description

TECHNICAL FIELD

[0001] The present application relates to a composition for preventing, ameliorating or treating arthritis. BACKGROUND

[0002] Arthritis is a disease in which inflammation and pain are generated due to damage or degenerative changes of cartilage protecting joints, and is very common among inflammatory diseases of joints. It is classified into primary or idiopathic arthritis occurring with factors such as age, sex, genetic factors, obesity, specific joint sites, etc., and secondary arthritis occurring due to causes such as trauma, diseases and deformities that can cause damage to joint cartilage.

[0003] It is known that most patients are primary arthritis, and it accounts for 10 to 15% of the world population. Also, it is a typical degenerative disease in which 31% of the adult population in Korea has symptoms. The number of patients with arthritis is continuously increasing with the extension of human lifespan and the aging of the population.

[0004] Arthritis is a disease in which, as chondrocytes constituting joints are degenerated by aging, etc., the synthesis of type-II collagen and proteoglycan, etc., which are matrix substances of joints, is inhibited in chondrocytes, at the same time, as interleukin-1β and tumor necrosis factor-α, etc., inflammatory cytokines are generated, the synthesis and activity of matrix metalloproteinase (MMP) that decomposes joint matrix are increased in joint cells, thereby inducing destruction of joint tissues.

[0005] Also, by generating nitric oxide (NO) through inflammatory cytokines, self-amplification of cytokine generation is induced due to the generated nitric oxide, the synthesis of more MMP is induced, decomposition of joint matrix is promoted, thereby further deteriorating arthritis. At the same time, inflammatory cytokines induce inflammatory responses in arthritis by increasing the generation of prostaglandin E2, which is a lipid metabolite.

[0006] Materials of plant origin are excellent in safety, and are used for a long time, and especially in Korea, development of functional materials mainly using plants and herbal ingredients used in folk or Korean medicine is actively conducted.

[0007] However, no one has known the use of the hydrangea serrata extract or hydrangenol for the prevention, improvement or treatment of arthritic diseases, and no study has been conducted on the mechanism thereof. Therefore, the present inventors have conducted a direct efficacy study on the prevention, improvement or treatment of arthritic diseases on the extract.

[0008] Accordingly, as a result of the present inventors' efforts to develop a composition for the prevention, improvement or treatment of arthritic diseases, it was confirmed that the hydrangea serrata extract or hydrangenol can prevent, improve or treat arthritic diseases, thereby completing the present invention. SUMMARY

[0009] TECHNICAL PROBLEM

[0010] An embodiment of the present invention provides a health functional food composition for preventing or improving arthritis, comprising a hydrangea serrata Seringe extract or hydrangenol represented by Chemical Formula 1 or a food-acceptable salt thereof as an effective ingredient.

[0011] Chemical Formula 1:

[0012]

[0013] Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating arthritis, comprising a hydrangea serrata Seringe extract or hydrangenol represented by Chemical Formula 1 or a food-acceptable salt thereof as an effective ingredient.

[0014] Still another embodiment of the present invention provides a quasi-drug composition for preventing or improving arthritis, comprising a hydrangea serrata Seringe extract or hydrangenol represented by Chemical Formula 1 or a food-acceptable salt thereof as an effective ingredient.

[0015] Still another embodiment of the present invention provides a skin external agent composition for preventing or improving arthritis, comprising a hydrangea serrata Seringe extract or hydrangenol represented by Chemical Formula 1 or a food-acceptable salt thereof as an effective ingredient.

[0016] Still another embodiment of the present invention provides a method for preventing, improving or treating arthritis, comprising the step of administering a hydrangea serrata Seringe extract or hydrangenol represented by Chemical Formula 1 or a food-acceptable salt thereof to a subject in need thereof.

[0017] Still another embodiment of the present invention provides the use of a hydrangea serrata Seringe extract or hydrangenol represented by Chemical Formula 1 or a food-acceptable salt thereof for the preparation of a composition for preventing, improving or treating arthritis.

[0018] TECHNICAL SOLUTION

[0019] An embodiment of the present invention provides a health functional food composition for preventing or improving arthritis, comprising an extract of Hydrangea serrata, or a hydrangenol represented by Chemical Formula 1 or a food-acceptable salt thereof as an effective ingredient.

[0020] Chemical Formula 1:

[0021]

[0022] It can be used by being purified from a natural substance of Hydrangea genus of Hydrangeaceae family. Hydrangea serrata can use one or more selected from the group consisting of all, woody roots, stems, branches, leaves, seeds, or fruits, and preferably, leaves can be used.

[0023] The extract of Hydrangea serrata can be extracted by any extraction method such as hot water extraction, solvent extraction, distillation extraction, supercritical extraction, etc. that are conventionally used to extract natural plants, and preferably, extraction using water, an organic solvent, or a mixed solvent thereof is characteristic. The organic solvent can use an alcohol having 1 to 4 carbon atoms, and for example, one or more selected from the group consisting of ethanol, methanol, isopropyl alcohol, and butanol, etc. can be used. Also, the alcohol can include alcohol. The alcohol concentration of the alcohol aqueous solution can be 1 to 99.5 (v / v)%, for example, 10 to 99.5 (v / v)%, 1 to 70 (v / v)%, 1 to 40 (v / v)%, 5 to 50 (v / v)%, 5 to 40 (v / v)%, 10 to 50 (v / v)%, or 10 to 40 (v / v)%.

[0024] In the extraction, 3 to 50 (w / w) times of the extraction solvent can be added with respect to Hydrangea serrata, for example, 3 to 40 (w / w) times, 3 to 30 (w / w) times, 5 to 50 (w / w) times, 5 to 40 (w / w) times, 5 to 30 (w / w) times, or 4 to 40 (w / w) times of the extraction solvent can be added. For example, with respect to 1 kg of material derived from the Hydrangea serrata, 3 to 50 kg of the extraction solvent can be added.

[0025] The extraction can use a method such as hot water extraction, immersion extraction, supercritical extraction, subcritical extraction, reflux cooling extraction, steam distillation, high-pressure enzyme decomposition, ultrasonic extraction, elution, pressing, etc.

[0026] The extraction can be performed at a temperature of 4°C to 90°C, for example, at a temperature of 4°C to 80°C, 4°C to 70°C, 5°C to 80°C, 10°C to 70°C, 15°C to 70°C, or 20°C to 70°C. The extraction time can vary depending on the temperature selected, and can be 1 hour to 2 months, for example, 1 hour to 1 month, 1 hour to 10 days, 1 hour to 5 days, 1 hour to 3 days, 1 hour to 2 days, 1 hour to 1 day, 1 hour to 10 hours, 2 hours to 1 month, 2 hours to 15 days, 2 hours to 10 days, 2 hours to 5 days, 2 hours to 3 days, 2 hours to 2 days, 2 hours to 1 day, or 2 hours to 10 hours. The extraction can include a step of placing after mixing the entire rough-toothed bedstraw, a part thereof, or a material derived therefrom in the solvent for a prescribed time. The placing can include appropriate agitation. The extraction can be repeated one or more times, for example, 1 to 5 times.

[0027] The extraction can be isolated by a known method such as filtration of plant body residues and the extract. Also, the extraction can include a step of removing the solvent from the extract by a known method such as concentration under reduced pressure. Also, the extraction can include a step of preparing the obtained extract as a dry extract by a drying method such as freeze-drying.

[0028] The globulol can be isolated from the rough-toothed bedstraw extract, and specifically, can be a fraction of the rough-toothed bedstraw extract. The fraction refers to a substance containing a specific component isolated from the extract. The globulol can be isolated and purified using column chromatography. The chromatography can use silicagel column chromatography, HP-20 column chromatography, RP-18 column chromatography, LH-20 column chromatography, High-performance liquid chromatography, or a combination thereof.

[0029] In one embodiment, the extract of H. macrophylla or the hydrangol can be contained in an amount of 0.0001 to 90.0% by weight, for example, 0.01 to 60% by weight, 0.01 to 40% by weight, 0.01 to 30% by weight, 0.01 to 20% by weight, 0.01 to 10% by weight, 0.01 to 5% by weight, 0.05 to 60% by weight, 0.05 to 40% by weight, 0.05 to 30% by weight, 0.05 to 20% by weight, 0.05 to 10% by weight, 0.05 to 5% by weight, 0.1 to 60% by weight, 0.1 to 40% by weight, 0.1 to 30% by weight, 0.1 to 20% by weight, 0.1 to 10% by weight, 0.1 to 5% by weight, 5 to 20% by weight, 5 to 18% by weight, 6 to 15% by weight, 8 to 15% by weight, or 7 to 10% by weight, with respect to the total weight of the composition. However, in the case of long-term ingestion for the purpose of health and hygiene or for the purpose of health regulation, the amount can be less than the above range, and there is no problem in terms of safety, and thus, the amount can be used in an amount greater than the above range.

[0030] The extract of H. macrophylla or the hydrangol can be directly added or used together with other food or food ingredients, and can be appropriately used according to the general method.

[0031] In one embodiment, the extract of H. macrophylla or the hydrangol can inhibit the expression or activity of one or more selected from the group consisting of IL-1β and COX-2.

[0032] In one embodiment, the extract of H. macrophylla or the hydrangol can inhibit the generation of nitric oxide (NO).

[0033] The extract of H. macrophylla or the hydrangol inhibits the expression or activity of inflammatory cytokines such as IL-1β and cyclooxygenase-2 (COX-2), and the generation of nitric oxide (NO) as an inflammatory mediator, and thus, can prevent, improve, or treat arthritis.

[0034] In one embodiment, the extract of H. macrophylla or the hydrangol can inhibit the expression or activity of one or more selected from the group consisting of MMP-1 and MMP-9.

[0035] The rough-hairy ball extract or hydrangol inhibits the expression or activity of matrix metalloproteinases (MMPs) which are enzymes that destroy the matrix structure of cartilage, inhibits pain caused by inflammation of joints, and thus can prevent, improve or treat arthritis.

[0036] In a specific example, the arthritis can be one or more selected from the group consisting of osteoarthritis, degenerative arthritis, rheumatoid arthritis, pyogenic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and Still's disease.

[0037] The term "prevention" in the present specification is a general term of all acts of partially or completely delaying or preventing the onset or recurrence of a disease, disorder or its accompanying symptoms, or preventing the acquisition or re-acquisition of a disease or disorder, or reducing the risk of the acquisition of a disease or disorder. The prevention refers to all acts of inhibiting or delaying the onset of arthritis or an arthritis-related disease, disorder or symptom by administering the composition of the present application.

[0038] The term "improvement" in the present specification refers to all acts of at least reducing a parameter related to a state to be treated, for example, at least reducing the degree of a symptom.

[0039] The term "treatment" in the present specification refers to all acts of changing a disease, disorder or its accompanying symptoms to be better or cured.

[0040] The term "health functional food" in the present specification refers to a food prepared or processed with a specific ingredient as a raw material or a raw material containing a specific ingredient by extraction, concentration, purification, mixing, etc. for the purpose of health assistance, and refers to a food designed and processed in a manner that can sufficiently exert biological regulation functions such as biological defense, regulation of biological rhythm, prevention and recovery of diseases, etc. on an organism by the ingredient. The health functional food composition can exert a function related to the prevention and improvement of arthritis, etc.

[0041] The kind of the food is not particularly limited. Examples of the food to which the extract can be added include a dosage form such as a powder, a granule, a tablet, a capsule, a pill, a gel, a jelly, a suspension, an emulsion, a syrup, a tea bag, an infusion tea, chewing gum, a candy, and a health drink, etc. selected from the group consisting of all generally accepted health foods.

[0042] As with a general beverage, the health beverage can include various sweetening agents, flavoring agents, or natural carbohydrates, etc. as additional ingredients. The sweetening agent can be a natural sweetening agent or a synthetic sweetening agent. The natural sweetening agent can be taumatin or a stevia extract, and the synthetic sweetening agent can be saccharin or aspartame.

[0043] The natural carbohydrate can be a monosaccharide, a disaccharide, a polysaccharide, xylitol, sorbitol, or erythritol. The monosaccharide can be glucose or fructose, and the disaccharide can be maltose or sucrose. The polysaccharide can be dextrin or cyclodextrin. Generally, the ratio of the natural carbohydrate can be about 0.01 g to 10 g per 100 ml of the composition of the present application, for example, about 0.01 g to 0.1 g.

[0044] The health functional food can include a food-acceptable food adjuvant, and can include a suitable carrier generally used in the preparation of a health functional food.

[0045] In addition to the above ingredients, the composition of the present application can include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the composition of the present application can include natural fruit juice, and pulp used in the preparation of fruit juice beverages and vegetable beverages. Such ingredients can be used alone or in combination. The ratio of the above additives is not critical, and generally, it can be selected in the range of 0.01 parts by weight to 0.1 parts by weight per 100 parts by weight of the composition of the present application.

[0046] Still another embodiment of the present application provides a pharmaceutical composition for preventing or treating arthritis, which comprises a rough-hairy-hydrangea extract, or a hydrangenol represented by the following Chemical Formula 1 or a food-acceptable salt thereof as an effective ingredient.

[0047] Chemical Formula 1:

[0048]

[0049] The rough-hairy-hydrangea extract or hydrangenol and their effects on arthritis are as described above.

[0050] Still another embodiment of the present application provides a method for preventing, ameliorating or treating arthritis, comprising the step of administering an extract of C. japonica or hydrangol represented by Chemical Formula 1 below or a food-acceptable salt thereof to a subject in need thereof.

[0051] Chemical Formula 1:

[0052]

[0053] Still another embodiment of the present application provides a use of an extract of C. japonica or hydrangol represented by Chemical Formula 1 below or a food-acceptable salt thereof for the preparation of a composition for preventing, ameliorating or treating arthritis.

[0054] Chemical Formula 1:

[0055]

[0056] In an embodiment, the pharmaceutical composition can be formulated as a preparation selected from the group consisting of tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections, and granules.

[0057] In an embodiment, the pharmaceutical composition can be used for oral or parenteral administration.

[0058] The pharmaceutical composition can include a general filler, extender, binder, disintegrant, anticoagulant, lubricant, humectant, pH adjuster, nutrient, vitamin, electrolyte, alginic acid and its salt, pectic acid and its salt, protective colloid, glycerin, flavor, emulsifier, or preservative, etc.

[0059] The pharmaceutical composition can include a pharmaceutically acceptable carrier, for example, such a carrier can be one or more selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil, propyl paraben, talc, magnesium stearate, and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline.

[0060] The dosage form of the pharmaceutical composition can vary depending on the method of use, and in order to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal, methods widely known in the art to which the present application pertains can be used for formulation.

[0061] Formulations for oral administration include tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections, granules, and the like, which can be prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition, lubricants such as magnesium stearate, talc, and the like can be used in addition to the simple excipients. Formulations for parenteral administration can be creams, emulsions, ointments, pastes, plasters, liquids, aerosols, flowable extracts, elixirs, infusions, sachets, patches, or injections, and the like.

[0062] The animal can be any animal, and includes not only humans and primates, but also domestic animals such as cows, pigs, sheep, horses, dogs, and cats.

[0063] The amount of the composition for treatment, prevention, or improvement to be administered can be determined taking into account the method of administration, the age, sex, severity of the patient's disease, state, the absorption rate and inactivation rate of the active ingredient in the body, and the drugs used in combination, and can be administered at 0.1 mg / kg (body weight) to 500 mg / kg (body weight), 0.1 mg / kg (body weight) to 400 mg / kg (body weight), or 1 mg / kg (body weight) to 300 mg / kg (body weight) based on the effective ingredient per day, and can be administered at once or in divided doses, but is not limited thereto.

[0064] Still another embodiment of the present application provides a medical external preparation composition for preventing or improving arthritis, which comprises a rough-leaved hydrangea extract, or hydrangol represented by the following Chemical Formula 1 or a food-acceptable salt thereof as an effective ingredient.

[0065] Chemical Formula 1:

[0066]

[0067] The rough-leaved hydrangea extract or hydrangol and their effects on arthritis are as described above.

[0068] The term "medical external preparation" as one of a fiber, a rubber product, or the like, which is used for the purpose of treating, alleviating, handling, or preventing a disease of a human or an animal, or a preparation which is used for the purpose of sterilizing, insecticidal, or the like, for preventing infection, and which has a weak effect on a human body or does not directly act on a human body, or an appliance or the like, or a device or the like, or an article which is used for the purpose of bringing a pharmacological effect to a structure and a function of a human or an animal, means an article other than an appliance, a device, or a means, which is used for the purpose of diagnosing, treating, alleviating, handling, or preventing a state or a disease of a human or an animal, and an article other than an appliance, a device, or a means, which is used for the purpose of bringing a pharmacological effect to a structure and a function of a human or an animal, and can include a personal hygiene article.

[0069] In the case where the extract is added to a composition for a non-medical product, the extract can be added directly or used together with other ingredients of the non-medical product, and can be appropriately used according to the usual method. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use (prophylactic, health care, or therapeutic treatment).

[0070] The kind or dosage form of the composition for a non-medical product of the present application is not particularly limited, and can be a bandage, gauze, absorbent cotton, a band-aid, a disinfectant cleaner, a body wash, a mouthwash, a wet wipe, laundry soap, a hand sanitizer, a humidifier filler, a mask or a filter filler, or the like.

[0071] Still another embodiment of the present application provides a composition for preventing or ameliorating arthritis, which contains an extract of C. japonica or hydrangol represented by Chemical Formula 1 below or a food-acceptable salt thereof as an active ingredient.

[0072] Chemical Formula 1:

[0073]

[0074] The extract of C. japonica or hydrangol and their effects on arthritis are as described above.

[0075] The skin external agent can be a cream, a gel, an ointment, a skin emulsion, a skin suspension, a transdermal absorption patch, a lotion, or a combination thereof. The skin external agent can be appropriately combined with ingredients used in skin external agents such as general cosmetics or pharmaceuticals, for example, with an aqueous ingredient, an oily ingredient, a powder ingredient, an alcohol, a humectant, a thickening agent, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a perfume, a color agent, various skin nutrients, or a combination thereof, as needed. The skin external agent can also be appropriately combined with a metal chelating agent such as disodium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, caffeine, tannin, verapamil, licorice extract, glycyrrhizin, carapichea ipecacuanha fruit hot water extract, various crude drugs, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts, vitamin C, ascorbic acid phosphate magnesium, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, trehalose, and the like.

[0076] In the case where the extract is added to a composition for an external agent, the extract can be added directly or used together with other ingredients of the external agent, and can be appropriately used according to the usual method. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use (prophylactic, health care, or therapeutic treatment).

[0077] The skin includes all skin sites such as the face, the hands, the arms, the legs, the feet, the chest, the abdomen, the back, the hips, and the scalp.

[0078] Effects of the Invention

[0079] The composition of one embodiment of the present application inhibits the expression of MMP-1 and MMP-9, inhibits swelling and relieves pain caused by inflammation, thereby having an effect of preventing, improving or treating arthritis. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 A graph showing the amount of NO production from LPS treated with globularia cordifolia extract and globularin.

[0081] Figure 2 A graph showing the mRNA expression amount of IL-1β from LPS treated with globularin.

[0082] Figure 3 A graph showing the mRNA secretion amount of COX-2 from LPS treated with globularia cordifolia extract and globularin.

[0083] Figure 4 A graph showing the mRNA expression amount of TNF-α from LPS treated with globularia cordifolia extract and globularin.

[0084] Figure 5 A graph showing the expression amount of MMP-1 from PMA treated with globularia cordifolia extract and globularin.

[0085] Figure 6 A graph showing the secretion amount of MMP-9 from PMA treated with globularia cordifolia extract and globularin.

[0086] Figure 7 A graph showing the foot swelling inhibition effect of globularia cordifolia extract treated with carrageenan-induced foot swelling at different times.

[0087] Figure 8 A graph showing the results of pain response measurement (Randall-Selitto) and mechanical allodynia (Von-Frey) measurement of animals treated with globularia cordifolia extract using carrageenan-induced inflammatory pain.

[0088] Figure 9 A graph showing the foot swelling inhibition effect of globularia cordifolia extract in an adjuvant-induced arthritis (AIA) animal model. DETAILED DESCRIPTION

[0089] Hereinafter, the present application will be described in more detail by examples. However, these examples are only for illustrative purposes, and the scope of the present application is not limited to these examples.

[0090] The terms used in the present specification are used only to explain embodiments, and are not intended to limit the present application, and are included in the singular form as well as the plural form unless it is specifically stated in the context. The "comprises" and / or "comprising", used in the specification, refer to the presence of stated elements, steps, operations, and / or elements, but do not preclude the presence or addition of another element, step, operation, and / or element.

[0091] The "embodiment", "example", "aspect", "exemplary", and the like used in the present specification should not be interpreted as meaning that the described embodiment or aspect is better or has an advantage than other embodiments or aspects.

[0092] Also, the term "or" means an inclusive or "same or" rather than an exclusive or "either / or". That is, the expression "x utilizes a or b" means any one of the natural inclusive permutations, unless otherwise stated or clearly contradicted by context.

[0093] Also, the singular expression ("a" or "an") used in the present specification and the claims should be interpreted as meaning "one or more" unless otherwise stated or clearly contradicted by context regarding the singular form.

[0094] Also, when referring to a part of a film, layer, region, structural element, etc. being "on" or "upper" another part, it not only means the case of being directly on the other part, but also includes the case of intervening other films, layers, regions, structural elements, etc. therebetween.

[0095] Example 1. Preparation of a crude toothed cup extract

[0096] Hydrangea serrata (Thunb.) Ser. dried material was put into 1500 kg of pure water equivalent to 15-20 times of 100 kg of the dried material of Hydrangea serrata (Thunb.) Ser. and refluxed at a temperature of 98°C for 5 hours, and then an extract filtrate was obtained by reduced pressure filtration (cylindrical filter 10-50 μm). The extract filtrate was concentrated under reduced pressure at a temperature of 60°C to obtain a concentrate, and then a Hydrangea serrata extract was obtained by spray drying at an inlet temperature of 180-200°C and an outlet temperature of 80-100°C (yield 23%). From the Hydrangea serrata extract, a hydrangenol was obtained by separation and purification, and the concentration (mg / g) of the hydrangenol was expressed as the weight of the hydrangenol obtained based on the weight of the Hydrangea serrata extract. LOT 1: 8.03 mg / g, LOT 2: 7.50 mg / g, LOT 3: 7.51 mg / g.

[0097] Experimental Example 1. Inhibitory effect of Hydrangea serrata extract and hydrangenol on nitric oxide (NO) production derived from lipopolysaccharide (LPS)

[0098] To confirm the NO reducing effect of the Hydrangea serrata extract and hydrangenol of Example 1, the change in NO was confirmed after causing an inflammatory response using LPS.

[0099] Specifically, RAW264.7 cells, which are macrophages, were obtained from the American Type Culture Collection (ATCC, MD, USA), and the RAW264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic under conditions of 5% CO2 and 37°C. Then, 8.0 x 105 cells / well were seeded in a 6-well culture plate, and the cells were incubated for 4 hours. After the incubation, the cells were treated with 10 ng / ml of LPS, and then the cells were incubated for 24 hours. After the incubation, the culture medium was collected, and the amount of NO was measured using a Griess reagent (Promega, WI, USA). 5After 24 hours of attachment culture of cells at a concentration of 1.5 x 105cells / 1.5 ml / well, the cells were treated with 500 ng / ml of LPS to induce an inflammatory response. Then, the cells were treated with the crude dentate hydrangea extract of Example 1 at concentrations of 0.78 ppm, 1.56 ppm, and 3.12 ppm, and with the hydrangenol at concentrations of 50 μM, 100 μM, and 200 μM, and then cultured for 24 hours. Then, 100 μl of the culture supernatant was dispensed into each well of a 96-well culture plate, 100 μl of Griess (0.1% N-(1-naphthyl)ethylenediamine, 1% sulfanilamide) reagent was added thereto, and allowed to react for 10 minutes, and then the absorbance of the colored azo-derivative molecules was measured at 540 nm using a microplate reader (BioTek, Vermont, USA). Using the measured absorbance, a standard curve was prepared for converting the dilution range of sodium nitrite into the amount of nitrite in each sample, and the amount of NO production was calculated. The results are shown in Table 1. Figure 1

[0100] Figure 1 Part (a) of FIG. 1 is a graph showing the amount of NO production when treated with different concentrations of the crude dentate hydrangea extract, Figure 1 Part (b) of FIG. 1 is a graph showing the amount of NO production when treated with different concentrations of hydrangenol.

[0101] As shown in Table 1 and FIG. 1, the crude dentate hydrangea extract and hydrangenol significantly inhibited the production of NO in a concentration-dependent manner. Figure 1

[0102] The above results indicate that the crude dentate hydrangea extract and hydrangenol of the present application effectively inhibit the production of NO and have an effect of preventing, ameliorating, or treating arthritis.

[0103] Experimental Example 2. Confirmation of the inhibitory effect of hydrangenol on the mRNA expression of IL-1β derived from LPS

[0104] In order to confirm the inhibitory effect of the hydrangenol of Example 1 on the mRNA expression of IL-1β derived from LPS, the amount of mRNA expression of IL-1β was confirmed using real-time polymerase chain reaction (PCR).

[0105] ​​Specifically, RAW264.7 cells, used as macrophages, were obtained from the American Type Culture Collection (ATCC, Maryland, USA) and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% antibiotic-antifungal agent at 5% CO2 and 37°C. Then, the cells were cultured in 6-well plates at 8.0 × 10⁻⁶ cells / well. 5 After RAW264.7 cells were attached at a concentration of 1.5 ml / well for 24 hours, they were treated with 500 ng / ml LPS in DMEM to induce an inflammatory response. Then, the cells were treated with 50 μM, 100 μM, and 200 μM of physalisol from Example 1 and cultured for 24 hours. After 24 hours, IL-1β mRNA was extracted from the cells to synthesize cDNA. Real-time quantitative PCR was performed using the target template (primers) to finally assess the expression level of the IL-1β gene. The results are as follows: Figure 2 As shown.

[0106] Figure 2 A graph showing the mRNA expression levels of LPS-derived IL-1β after treatment with different concentrations of hygrophoretic acid.

[0107] like Figure 2 As shown, it was confirmed that physalisol significantly inhibited the expression of IL-1β mRNA in a manner proportional to its concentration.

[0108] The above results indicate that physalisol in a specific example effectively inhibits the mRNA expression of IL-1β, and has the effect of preventing, improving or treating arthritis.

[0109] Experiment 3. Confirming the inhibitory effect of *Hydrangea macrophylla* extract and hydrangeol on the mRNA expression of COX-2 derived from LPS.

[0110] To confirm the inhibitory effect of the coarse-toothed hydrangea extract and hydrangea phenols from Example 1 on the mRNA expression of COX-2 derived from LPS, the mRNA expression level of COX-2 was confirmed using real-time polymerase chain reaction.

[0111] Specifically, RAW264.7 cells, used as macrophages, were obtained from the American Type Culture Collection (ATCC, Maryland, USA) and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% antibiotic-antifungal agent at 5% CO2 and 37°C. Cells were then cultured in 6-well plates at 8.0 × 10⁻⁶ cells / well. 5The concentration of cells / 2.0 ml / well was attached to RAW264.7 cells for 24 hours, and then treated with 500 ng / ml of LPS to induce an inflammatory response. Then, the crude dentate ball extract of Example 1 was treated at a concentration of 0.78 ppm, 1.56 ppm, and 3.12 ppm, and the hydrangol was treated at a concentration of 50 μM, 100 μM, and 200 μM, and then cultured for 24 hours. After 24 hours, the mRNA of COX-2 was extracted from the cells to synthesize cDNA, and the expression degree of the COX-2 gene was finally evaluated by performing real-time quantitative PCR using a target template (primer), and the results are shown in FIGS. 1 and 2. Figure 3

[0112] Figure 3 A graph showing the mRNA expression of LPS-derived COX-2 treated with the crude dentate ball extract and hydrangol is shown in FIG. 1.

[0113] Figure 3 Part (a) of FIG. 2 is a graph showing the mRNA expression of COX-2 treated with different concentrations of the crude dentate ball extract, Figure 3 Part (b) of FIG. 2 is a graph showing the mRNA expression of COX-2 treated with different concentrations of hydrangol.

[0114] As shown in FIGS. 1 and 2, it was confirmed that the crude dentate ball extract and hydrangol significantly inhibited the expression of mRNA of COX-2. Figure 3

[0115] The above results show that the crude dentate ball extract and hydrangol of the present application effectively inhibit the mRNA expression of COX-2, and have an effect of preventing, improving, or treating arthritis.

[0116] Experimental Example 4: Confirmation of the inhibitory effect of the crude dentate ball extract and hydrangol on the mRNA expression of LPS-derived TNF-α

[0117] In order to confirm the inhibitory effect of the crude dentate ball extract and hydrangol of Example 1 on the mRNA expression of LPS-derived TNF-α, the mRNA expression amount of TNF-α was confirmed using a real-time polymerase chain reaction.

[0118] Specifically, RAW264.7 cells, which are macrophages, were obtained from the American Type Culture Collection (ATCC, Maryland, USA), and cultured in a DMEM medium containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic agent at 5% CO2 and 37°C. RAW264.7 cells were seeded at a concentration of 8.0 x 105 cells / 2.0 ml / well in a 6-well culture plate, and then treated with 500 ng / ml of LPS to induce an inflammatory response. Then, the crude dentate ball extract of Example 1 was treated at a concentration of 0.78 ppm, 1.56 ppm, and 3.12 ppm, and the hydrangol was treated at a concentration of 50 μM, 100 μM, and 200 μM, and then cultured for 24 hours. After 24 hours, the mRNA of TNF-α was extracted from the cells to synthesize cDNA, and the expression degree of the TNF-α gene was finally evaluated by performing real-time quantitative PCR using a target template (primer), and the results are shown in FIGS. 3 and 4. 5 ​​The concentration of cells / 2.0 ml / well was attached to RAW264.7 cells for 24 hours, and then treated with 500 ng / ml of LPS to induce an inflammatory response. Then, the crude dentate ball extract of Example 1 was treated at a concentration of 0.78 ppm, 1.56 ppm, 3.12 ppm, and the hydrangol was treated at a concentration of 50 μM, 100 μM, 200 μM, and then cultured for 24 hours. After 24 hours, mRNA of TNF-α was extracted from the cells to synthesize cDNA, and real-time quantitative PCR was performed using a target template (primer) to finally evaluate the expression degree of the TNF-1β gene, and the results are shown in FIGS. 1 and 2. Figure 4

[0119] Figure 4 FIG. 1 is a graph showing the mRNA expression of TNF-α treated with different concentrations of the crude dentate ball extract of Example 1.

[0120] Figure 4 FIG. 1 is a graph showing the mRNA expression of TNF-α treated with different concentrations of the crude dentate ball extract of Example 1. Figure 4 FIG. 2 is a graph showing the mRNA expression of TNF-α treated with different concentrations of hydrangol.

[0121] As shown in FIGS. 1 and 2, it was confirmed that the crude dentate ball extract and hydrangol significantly inhibited the expression of mRNA of TNF-α. Figure 4

[0122] The above results show that the crude dentate ball extract and hydrangol of the specific example effectively inhibit the mRNA expression of TNF-α, and have an effect of preventing, improving, or treating arthritis.

[0123] Experimental Example 5. Confirmation of the inhibitory effect of the crude dentate ball extract and hydrangol on the expression of MMP-1 derived from phorbol 12-myristate 13-acetate (PMA) in chondrocytes

[0124] In order to confirm the inhibitory effect of the crude dentate ball extract and hydrangol of Example 1 on the production of MMP-1 derived from PMA in chondrocytes, the secretion of MMP-1 was measured using a human MMP-1 enzyme-linked immunosorbent assay kit (Human MMP-1 ELISA kit) (ab100603, abcam, US).

[0125] Specifically, SW-1353 cells, which are chondrocytes, were obtained from the American Type Culture Collection (ATCC, Maryland, US), and were cultured in a DMEM medium containing 10% FBS and 1% antibiotic-antimycotic agent at 5% CO2 and 37℃. Then, 2.0 × 105 cells / well were seeded in a 6-well culture plate, and then treated with 100 ng / ml of PMA to induce an inflammatory response. Then, the crude dentate ball extract of Example 1 was treated at a concentration of 0.78 ppm, 1.56 ppm, 3.12 ppm, and the hydrangol was treated at a concentration of 50 μM, 100 μM, 200 μM, and then cultured for 24 hours. After 24 hours, the culture medium was collected, and the amount of MMP-1 secreted from the cells was measured using a human MMP-1 enzyme-linked immunosorbent assay kit (ab100603, abcam, US).​​5 The SW-1353 cells were attached at a concentration of 2.0 ml / well for 24 hours, and then treated with 50 ng / ml of PMA in DMEM. Then, the crude dentate hydrangea extract of Example 1 was treated at concentrations of 0.78 ppm, 1.56 ppm, and 3.12 ppm, and the hydrangenol was treated at concentrations of 50 μM, 100 μM, and 200 μM, and then incubated for 24 hours. After 24 hours, the MMP-1 of the supernatant was quantified using an enzyme-linked immunoassay kit (elisa kit), and the results are shown in FIG. 2. Figure 5

[0126] Figure 5 A graph showing the PMA-induced MMP-1 expression level treated with the crude dentate hydrangea extract and hydrangenol is shown in FIG. 2.

[0127] Figure 5 Part (a) of FIG. 2 is a graph showing the MMP-1 secretion level treated with the crude dentate hydrangea extract at different concentrations, Figure 5 Part (b) of FIG. 2 is a graph showing the MMP-1 secretion level treated with hydrangenol at different concentrations.

[0128] As shown in FIG. 2, it was confirmed that the crude dentate hydrangea extract and hydrangenol significantly inhibited the secretion of MMP-1 in a concentration-dependent manner. Figure 5

[0129] The above results show that the crude dentate hydrangea extract and hydrangenol of the present application effectively inhibit the secretion of MMP-1 and have an effect of preventing, ameliorating, or treating arthritis.

[0130] Experimental Example 6. Confirmation of the inhibitory effect of the crude dentate hydrangea extract and hydrangenol on phorbol 12-myristate 13-acetate-induced MMP-9 expression in chondrocytes

[0131] To confirm the inhibitory effect of the hydrangenol of Example 2 on PMA-induced MMP-1 production in chondrocytes, the secretion of MMP-9 was measured using a human MMP-9 enzyme-linked immunosorbent assay kit (Human MMP-9 LISA kit) (ab246539, abcam, USA).

[0132] Specifically, SW-1353 cells, which are chondrocytes, were obtained from the American Type Culture Collection (ATCC, Maryland, USA) and cultured in a DMEM medium containing 10% FBS and 1% antibiotic-antimycotic at 5% CO2 and 37°C. Then, 2.0 x 105 cells / well were seeded in a 6-well culture plate, and the cells were allowed to adhere for 24 hours. After 24 hours, the cells were treated with 50 ng / ml of PMA in DMEM, and then treated with 0.78 ppm, 1.56 ppm, and 3.12 ppm of the crude dentate hydrangea extract of Example 1, and 50 μM, 100 μM, and 200 μM of hydrangenol, and then incubated for 24 hours. After 24 hours, the secretion of MMP-9 was measured using a human MMP-9 enzyme-linked immunosorbent assay kit (Human MMP-9 LISA kit) (ab246539, abcam, USA). 5 ​​After SW-1353 cells were attached at a concentration of 2.0 ml / well for 24 hours, they were treated with DMEM containing 50 ng / ml PMA. Then, the *Hydrangea macrophylla* extract from Example 1 was treated with 0.78 ppm, 1.56 ppm, and 3.12 ppm of *Hydrangea macrophylla* extract, and with 50 μM, 100 μM, and 200 μM of *Hydrangea macrophylla* phenol, followed by 24 hours of culture. After 24 hours, the MMP-9 content in the supernatant was quantified using an ELISA kit. The results are as follows: Figure 6 As shown.

[0133] Figure 6 A graph showing the expression levels of MMP-9 derived from PMA in the treatment of Hydrangea dentata extract and hydrangeol.

[0134] Figure 6 Part (a) is a graph showing the MMP-9 secretion levels after treatment with different concentrations of Hydrangea rotundifolia extract. Figure 6 Part (b) is a graph showing the amount of MMP-9 secreted by different concentrations of hygrophoretic acid.

[0135] like Figure 6 As shown, it was confirmed that the extract of Hydrangea dentata and Hydrangea phenol significantly inhibited the secretion of MMP-9 in a manner proportional to concentration.

[0136] The above results indicate that the extract of *Hydrangea dentata* and *Hydrangea dentata* phenol in a specific example effectively inhibits the secretion of MMP-9, and has the effect of preventing, improving or treating arthritis.

[0137] Example 7. Confirmation of the inhibitory effect of *Hydrangea macrophylla* extract on foot swelling in a carrageenan-induced inflammation model in animals.

[0138] In male SD rats (Sprague Dawley Rat, 200g ± 10%), paw volume was measured using a paw edema meter for animals not injected with carrageenan. Animals were orally administered sterile distilled water or *Hydrangea macrophylla* extract (100 mg / kg or 300 mg / kg, N=5 per group). 2% carrageenan was subcutaneously injected into both paws. Paw volume changes were measured using a paw edema meter 1, 3, and 5 hours after injection. Figure 7 (Part A). Foot volume was measured 3 hours after carrageenan injection, followed by measurement of inflammatory pain. Figure 7 (Part B)

[0139] The use of a pain response meter (Randall-Selitto) confirmed that inflammatory pain was suppressed in the left foot. Figure 8 (Part A).

[0140] Using the animal's vony-frey mechanical symptom, suppression of inflammatory pain was confirmed in the right foot. Figure 8 (Part B)

[0141] Experimental Example 8. Confirmation of the inhibitory effect of *Hydrangea macrophylla* extract on foot swelling in an adjuvant-induced arthritis animal model in animals.

[0142] For male SD rats (200g ± 10%), the volume of both paws was measured using a paw edema meter. Animals were orally administered sterile distilled water or ibuprofen (30 mg / kg) or *Hydrangea macrophylla* extract (100 mg / kg, 300 mg / kg, 6 rats per group). Following an injection of Complete Freund's adjuvant (CFA) into the left ankle joint, paw volume changes were measured using a paw edema meter at approximately 3-day intervals for 3 weeks. Figure 9 (Part A) , using calipers to measure the thickness of both feet ( Figure 9 (Part B). Pain response was measured in the uninjected right foot using Randall-Selitto immediately before CFA injection and 20 days after CFA injection.

[0143] The use of a pain response meter (Randall-Selitto) confirmed that inflammatory pain was suppressed in the left foot. Figure 9 (Part C)

[0144] Preparation Example 1. Tablet Preparation

[0145] The coarse-toothed hydrangea extract and hydrangea phenols of Example 1 were mixed according to the component ratios shown in Table 1 below, and tablets were prepared by compression according to conventional tablet preparation methods. The prepared tablets were confirmed to be safe under all dosage form test conditions.

[0146] Table 1

[0147]

[0148]

[0149] Preparation Example 2. Preparation of Capsules

[0150] The coarse-toothed hydrangea extract and hydrangea phenols from Example 1 were mixed according to the component ratios shown in Table 2 below and then filled into gelatin capsules to prepare soft capsules. The prepared capsules were confirmed to be safe under all dosage form test conditions.

[0151] Table 2

[0152] Raw material name Unit weight (mg) Raw material name Unit weight (mg) Example 1 50 Example 2 10 Corn starch 100 Corn starch 100 Lactose 100 Lactose 100 Stearic acid 2 Stearic acid 2

[0153] Preparation Example 3. Preparation of a liquid preparation

[0154] The crude toothed bedstraw extract and globulol of Example 1 were mixed in the ingredient ratio according to Table 3 below, and filled into a glass bottle or a soft bag according to a beverage preparation method suitable for preference to prepare a liquid preparation. It was confirmed that the prepared liquid preparation was safe under all the dosage form example test conditions.

[0155] Table 3

[0156] Raw material name Unit weight (g) Raw material name Unit weight (g) Example 1 2.5050 Example 2 0.5050 Xanthan gum 0.0075 Xanthan gum 0.0075 Fructooligosaccharide solution 0.7500 Fructooligosaccharide solution 0.7500 Coconut flower juice powder 1.0500 Leaf flower juice powder 1.0500 Double flower concentrate 1.5000 Double flower concentrate 1.5000 Red ginseng flavor 0.0450 Red ginseng flavor 0.0450 Pure water 9.1425 Pure water 11.1425

[0157] Preparation Example 4. Preparation of a jelly

[0158] The crude toothed bedstraw extract and globulol of Example 1 were mixed in the ingredient ratio according to Table 4 below, and filled into a three-side cloth according to a jelly preparation method suitable for preference to prepare a jelly. It was confirmed that the prepared jelly was safe under all the dosage form example test conditions.

[0159] Table 4

[0160]

[0161] The ingredient ratio of the preparation examples 1 to 4 was prepared by mixing appropriate ingredients as a dosage form example, but the compounding ratio and raw materials can be arbitrarily changed as needed. The extract of the present application and the ingredient derived therefrom are safe under all the dosage form example test conditions, and thus there is no safety problem of the dosage form.

[0162] The present application has been described above by the defined examples and the accompanying drawings, but the present application is not limited to the described examples, and a person having ordinary skill in the art to which the present application pertains can implement various modifications and alterations from such description. Therefore, the scope of the present application is not limited to the described examples, but should be determined by the scope of the above invention claim and its equivalent content.

Claims

1. A health functional food composition for preventing or improving arthritis, characterized by comprising a mixture of the following components: A health functional food composition comprising a rough-hairy-hydrangea extract, or a hydrangenol represented by the following Chemical Formula 1 or a food-acceptable salt thereof as an effective ingredient: ​ Chemical Formula 1:

2. The health functional food composition for preventing or improving arthritis according to claim 1, wherein the composition is a food composition for preventing or improving arthritis. The rough-hairy-hydrangea extract is extracted by water, C1 to C4 alcohol, or a mixed solvent thereof.

3. The health functional food composition for preventing or improving arthritis according to claim 1, wherein the composition is a food composition for preventing or improving arthritis. The hydrangenol is separated from the rough-hairy-hydrangea extract.

4. The health functional food composition for preventing or improving arthritis according to claim 1, wherein the composition is a food composition for preventing or improving arthritis. The rough-hairy-hydrangea extract or the hydrangenol is contained in an amount of 0.0001 to 90% by weight, relative to the total weight of the health functional food composition.

5. The health functional food composition for preventing or improving arthritis according to claim 1, wherein the composition is a food composition for preventing or improving arthritis. The rough-hairy-hydrangea extract or the hydrangenol inhibits the expression or activity of one or more selected from the group consisting of IL-1β and COX-2.

6. The health functional food composition for preventing or improving arthritis according to claim 1, wherein the composition is a food composition for preventing or improving arthritis. The rough-hairy-hydrangea extract or the hydrangenol inhibits the production of nitric oxide.

7. The health functional food composition for preventing or improving arthritis according to claim 1, wherein the composition is a food composition for preventing or improving arthritis. The rough-hairy-hydrangea extract or the hydrangenol inhibits the expression or activity of one or more selected from the group consisting of MMP-1 and MMP-9.

8. The health functional food composition for preventing or improving arthritis according to claim 1, wherein the composition is a food composition for preventing or improving arthritis. The health functional food has a dosage form selected from the group consisting of a powder, a granule, a tablet, a capsule, a pill, a gel, a jelly, a suspension, an emulsion, a syrup, a tea bag, an infusion tea, chewing gum, a candy, and a health drink.

9. A pharmaceutical composition for preventing or treating arthritis, characterized by, A health functional food composition comprising a rough-hairy-hydrangea extract, or a hydrangenol represented by the following Chemical Formula 1 or a food-acceptable salt thereof as an effective ingredient: Chemical Formula 1:

10. A quasi-drug composition for preventing or ameliorating arthritis, characterized by comprising the compound of claim 1. A health functional food composition comprising a rough-hairy-hydrangea extract, or a hydrangenol represented by the following Chemical Formula 1 or a food-acceptable salt thereof as an effective ingredient: Chemical Formula 1:

11. A skin external preparation composition for preventing or ameliorating arthritis, characterized by comprising the compound according to any one of claims 1 to 10. A health functional food composition comprising a rough-hairy-hydrangea extract, or a hydrangenol represented by the following Chemical Formula 1 or a food-acceptable salt thereof as an effective ingredient: Chemical Formula 1:

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