WO2024219699A1 - Composition for preventing, alleviating or treating arthritis, containing hydrangea macrophylla extract or hydrangenol as active ingredient - Google Patents

Abstract

The present invention provides a composition for preventing, alleviating or treating arthritis, containing a Hydrangea macrophylla extract or hydrangenol as an active ingredient. The present invention inhibits the expression of MMP-1 and MMP-9 and increases the synthesis of procollagen type 2, and also alleviates inflammation and inhibits inflammatory pain, and thus has the effect of preventing, alleviating or treating arthritis.

Description

Composition for preventing, improving or treating arthritis containing hydrangea extract or hydranzenol as an active ingredient

The present invention relates to a composition for preventing, improving or treating arthritis.

Degenerative arthritis is a disease in which inflammation and pain occur due to damage to the bones and ligaments that form the joints, caused by gradual damage or degenerative changes in the cartilage that protects the joints. It is the most common inflammatory disease of the joints. It is classified into primary or idiopathic arthritis, which occurs without a specific organic cause, depending on factors such as age, gender, genetic factors, obesity, and specific joint areas, and secondary or secondary arthritis, which occurs due to trauma, diseases, and deformities that can damage the articular cartilage.

Most patients have primary arthritis, which is known to affect approximately 10-15% of the world's population. It is also a representative degenerative disease with 31% of the domestic adult population having symptoms. The number of patients with arthritis continues to increase due to the extension of human life expectancy and the aging population.

Degenerative arthritis is a disease caused by the destruction of joint tissue as the chondrocytes that make up the joints degenerate due to aging and other factors, which inhibits the synthesis of joint matrix substances such as type II collagen and proteoglycan in the cartilage cells and simultaneously produces inflammatory cytokines such as interleukin-1β and tumor necrosis factor-α, which increases the synthesis and activity of matrix metalloproteinase (MMP), which decomposes the joint matrix, in the joint cells.

In addition, arthritis is further aggravated by the production of nitric oxide (NO) by inflammatory cytokines and the production of auto-amplifying cytokines by the produced NO, which induces the synthesis of more MMPs and promotes the decomposition of the joint matrix. At the same time, inflammatory cytokines increase the production of prostaglandin E2, a lipid metabolite, which induces an inflammatory response in arthritis.

Plant-derived materials have been used for a long time due to their superior safety aspects, and in particular, in Korea, functional materials mainly using plants and herbal ingredients used in the private sector or oriental medicine are being actively developed.

However, the use of hydrangea extract or hydrangeol for preventing, improving, or treating arthritis disease is unknown, and no research on its mechanism has been conducted. Therefore, the inventors of the present invention conducted a direct study on the efficacy of the extract for preventing, improving, or treating arthritis disease.

Accordingly, the inventors of the present invention have endeavored to develop a composition for preventing, improving or treating arthritis disease, and as a result, have confirmed that hydrangea extract or hydrrangenol can prevent, improve or treat arthritis disease, thereby completing the present invention.

One aspect is to provide a health functional food composition for preventing or improving arthritis, which comprises an extract of Hydrangea serrata Seringe; or hydrangeol represented by the following chemical formula 1; or a food-wise acceptable salt thereof, as an active ingredient.

[Chemical formula 1]

Another aspect is to provide a pharmaceutical composition for preventing or treating arthritis, comprising hydrangea extract or hydrangea (Hydrangenol) represented by the chemical formula 1 or a food-chemically acceptable salt thereof as an active ingredient.

Another aspect is to provide a pharmaceutical composition for preventing or improving arthritis, comprising as an active ingredient a hydrangea extract or hydrangea (Hydrangenol) represented by the chemical formula 1 or a food-wise acceptable salt thereof.

Another aspect is to provide a composition for preventing or improving arthritis, comprising hydrangea extract or hydrangea (Hydrangenol) represented by the chemical formula 1 or a food-related acceptable salt thereof as an active ingredient.

Another aspect provides a method for preventing, improving or treating arthritis, comprising a step of administering to a subject in need thereof an extract of hydrangea or hydrangea represented by the chemical formula 1 or a food-wise acceptable salt thereof.

Another aspect provides a use of hydrangea extract or hydrangea (Hydrangenol) represented by the following chemical formula 1 or a food-chemically acceptable salt thereof for the manufacture of a composition for preventing, improving or treating arthritis.

One aspect provides a health functional food composition for preventing or improving arthritis, comprising, as an active ingredient, a hydrangea extract or hydrangea represented by the following chemical formula 1 or a food-wise acceptable salt thereof.

[Chemical formula 1]

It can be used by separating and purifying natural products of the genus Hydrangea of the family Hydrangea . The genus Hydrangea can be at least one selected from the group consisting of the whole, woody roots, stems, branches, leaves, seeds or fruits, and preferably, the leaves can be used.

The above hydrangea extract can be extracted by any extraction method, such as hot water extraction, solvent extraction, distillation extraction, or supercritical extraction, which have been conventionally used to extract natural plants, and is preferably characterized by being extracted with water, an organic solvent, or a mixed solvent thereof. The organic solvent may be at least one selected from the group consisting of alcohols having 1 to 4 carbon atoms, such as ethanol, methanol, isopropanol, and butanol. In addition, the alcohol may include ethanol. The alcohol concentration of the above alcohol aqueous solution may 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)%.

The above extraction may comprise adding the extraction solvent 3 to 50 (w/w) times, 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, to the hydrangea. For example, it may comprise adding 3 to 50 kg of the extraction solvent per 1 kg of the material derived from the hydrangea.

The above extraction can be performed using methods such as hot water extraction, immersion extraction, supercritical extraction, subcritical extraction, reflux cooling extraction, steam distillation, high-pressure enzymatic decomposition, ultrasonic extraction, dissolution, and pressing.

The above extraction may be performed at 4° C. to 90° C., for example, 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 may vary depending on the selected temperature and may 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 may include mixing the whole hydrangea, a part thereof, or materials derived therefrom in the solvent and leaving it for a period of time. The leaving may include appropriate stirring. The extraction may be repeated one or more times, for example, 1 to 5 times.

The above extraction can be performed by separating the plant residue and the extract by a known method such as filtration. The extraction can also include removing the solvent from the obtained extract by a known method such as concentration under reduced pressure. The extraction can also include preparing a dry extract by drying the obtained extract, such as freeze-drying.

The above hydranzenol may be separated from a hydrangea extract, and specifically, may be a fraction of the hydrangea extract. The fraction refers to a substance containing a specific component separated from the extract. The hydranzenol may be separated and purified using column chromatography. The chromatography may be selected from silica gel column chromatography, HP-20 column chromatography, RP-18 column chromatography, LH-20 column chromatography, high-performance liquid chromatography, or a combination thereof.

In one specific example, the hydrangea extract or the hydranzenol is present in an amount of 0.0001 wt% to 90.0 wt%, for example, 0.01 wt% to 60 wt%, 0.01 wt% to 40 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 It may be contained in an amount of from 0.1 wt% to 30 wt%, from 0.1 wt% to 20 wt%, from 0.1 wt% to 10 wt%, from 0.1 wt% to 5 wt%, from 5 to 20 wt%, from 5 to 18 wt%, from 6 to 15 wt%, from 8 to 15 wt%, or from 7 to 10 wt%. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, it may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The above hydrangea extract or hydranzenol can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.

In one specific example, the hydrangea extract or hydranzenol may inhibit the expression or activity of at least one selected from the group consisting of IL-1β and COX-2.

In one specific example, the hydrangea extract or hydranzenol may inhibit the production of nitric oxide (NO).

The above hydrangea extract or hydranzenol may prevent, improve or treat arthritis by inhibiting the expression or activity of inflammatory cytokines such as interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) and inhibiting the production of nitric oxide (NO), an inflammatory mediator.

In one specific example, the hydrangea extract or hydranzenol may inhibit the expression or activity of at least one selected from the group consisting of MMP-1 and MMP-9.

The above hydrangea extract or hydranzenol may prevent, improve or treat arthritis by inhibiting the expression or activity of matrix metalloproteinases (MMPs), which are enzymes that destroy the matrix components of cartilage, and suppressing pain caused by arthritis.

In one specific example, the arthritis may be at least one selected from the group consisting of osteoarthritis, degenerative arthritis, rheumatoid arthritis, septic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and Still's disease.

The term "prevention" as used herein collectively refers to partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its secondary symptoms, preventing the acquisition or reacquisition of a disease or disorder, or reducing the risk of acquiring a disease or disorder. The prevention refers to any act of inhibiting or delaying the onset of arthritis or an arthritis-related disease, disorder, or symptom by administering a composition according to the present invention.

The term "improvement" as used herein refers to any action that at least reduces a parameter associated with the condition being treated, for example, the severity of symptoms.

The term "treatment" as used herein refers to any action by which a disease, disorder, or its accompanying symptoms are improved or beneficially altered.

The term "health functional food" in this specification refers to a food manufactured or processed by using a specific ingredient as a raw material or extracting, concentrating, refining, mixing, etc. a specific ingredient contained in a food raw material for the purpose of health supplementation, and refers to a food designed and processed so that the ingredient can sufficiently exert bioregulatory functions such as biodefense, regulation of biological rhythm, prevention and recovery of disease, etc. on the body. The health functional food composition can perform functions related to prevention and improvement of arthritis, etc.

There is no special limitation on the type of the above food. Examples of foods to which the above extract can be added include formulations selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, gums, candies, and health drinks, and include all health foods in the conventional sense.

The above health beverage composition may contain various sweeteners, flavoring agents, or natural carbohydrates as additional ingredients, like conventional beverages. The sweetener may be a natural sweetener or a synthetic sweetener. The natural sweetener may be taumatin or a stevia extract, and the synthetic sweetener may be saccharin or aspartame.

The natural carbohydrate may be a monosaccharide, a disaccharide, a polysaccharide, xylitol, sorbitol or erythritol. The monosaccharide may be glucose or fructose, and the disaccharide may be maltose or sucrose. The polysaccharide may be dextrin or cyclodextrin. The proportion of the natural carbohydrate may be generally about 0.01 to 10 g, for example, about 0.01 to 0.1 g, per 100 ml of the composition of the present invention.

The above health functional food may contain food additives acceptable from a food science perspective and may contain an appropriate carrier commonly used in the manufacture of health functional foods.

In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Another aspect provides a pharmaceutical composition for preventing or treating arthritis, comprising as an active ingredient a hydrangea extract or hydrangea (Hydrangenol) represented by the following chemical formula 1 or a food-chemically acceptable salt thereof.

[Chemical formula 1]

.

The above-mentioned hydrangea extract or hydranzenol and its effect on arthritis are as described above.

Another aspect provides a method for preventing, improving or treating arthritis, comprising administering to a subject in need thereof an extract of hydrangea or hydrangea represented by the following chemical formula 1 or a food-wise acceptable salt thereof.

[Chemical formula 1]

.

Another aspect provides a use of a hydrangea extract or hydrangea (Hydrangenol) represented by the following chemical formula 1 or a food-wise acceptable salt thereof for the manufacture of a composition for preventing, improving or treating arthritis.

[Chemical formula 1]

.

In one specific embodiment, the pharmaceutical composition may be formulated as a preparation selected from the group consisting of tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections, and granules.

In one embodiment, the pharmaceutical composition may be for oral or parenteral administration.

The above pharmaceutical composition may contain conventional fillers, bulking agents, binders, disintegrants, anticoagulants, lubricants, wetting agents, pH regulators, nutrients, vitamins, electrolytes, alginic acid and salts thereof, pectic acid and salts thereof, protective colloids, glycerin, flavorings, emulsifiers or preservatives.

The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, examples of which include at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and saline.

The formulation of the pharmaceutical composition may vary depending on the method of use and may be formulated using methods well known in the art to which the present invention pertains so as to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

Formulations for oral administration include tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections, and granules, and these formulations can be prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Formulations for parenteral administration can be creams, lotions, ointments, pastes, solutions, aerosols, fluid extracts, elixirs, instillations, sachets, patches, or injections.

The above method is applicable to any animal, and the animals may include not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs and cats.

The dosage of the composition for the above treatment, prevention, or improvement can be determined by considering the administration method, the age and sex of the recipient, the severity and condition of the patient, the absorbability of the active ingredient in the body, the inactivation rate, and the concomitantly administered drug, 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 daily effective ingredient, and can be administered once or in several divided doses, but is not limited thereto.

Another aspect provides a pharmaceutical composition for preventing or improving arthritis, comprising as an active ingredient an extract of hydrangea or hydrangea represented by the following chemical formula 1 or a food-wise acceptable salt thereof.

[Chemical formula 1]

.

The above-mentioned hydrangea extract or hydranzenol and its effect on arthritis are as described above.

The term "quasi-drug" means a fiber, rubber product or similar article used for the purpose of treating, alleviating, managing or preventing diseases of humans or animals; an article that has a weak effect on the human body or does not directly affect the human body and is not an instrument or machine and similar thereto; an article that is one of the preparations used for sterilization, insecticide and similar purposes to prevent infection, excluding articles used for the purpose of diagnosing, treating, alleviating, managing or preventing the condition or disease of humans or animals that are not instruments, machines or devices; and articles used for the purpose of exerting a pharmacological effect on the structure and function of humans or animals that are not instruments, machines or devices; and may also include personal hygiene products.

When adding the above extract to a pharmaceutical composition, the extract may be added as is or used together with other pharmaceutical ingredients, and may be used appropriately according to a conventional method. The mixing amount of the effective ingredients may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment).

The type or formulation of the pharmaceutical composition of the present invention is not particularly limited, but may be a bandage, gauze, cotton wool, adhesive plaster, disinfectant, shower foam, garglin, wet tissue, detergent soap, hand wash, humidifier filler, mask, or filter filler.

Another aspect provides a composition for preventing or improving arthritis, comprising as an active ingredient a hydrangea extract or hydrangea (Hydrangenol) represented by the following chemical formula 1 or a food-chemically acceptable salt thereof.

[Chemical formula 1]

.

The above-mentioned hydrangea extract or hydranzenol and its effect on arthritis are as described above.

The above skin external preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a lotion, or a combination thereof. The above skin external preparation may be appropriately mixed with ingredients commonly used in skin external preparations such as cosmetics or medicines, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof, as needed. The above skin external preparation may also appropriately contain metal sequestrants such as sodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, bellapamil, licorice extract, glablidine, hot water extract of the fruit of Kalin, various crude drugs, tocopheryl acetate, glycyrrhizic acid, tranexamic acid and derivatives or salts thereof; and drugs such as vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

When adding the above extract to an external preparation composition, the extract may be added as is or used together with other external preparation ingredients, and may be used appropriately according to a conventional method. The amount of the active ingredient mixture may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment).

The above skin includes all skin areas of the body, including the face, hands, arms, legs, feet, chest, stomach, back, buttocks, and scalp.

The composition according to the aspect has the effect of preventing, improving or treating arthritis by inhibiting the expression of MMP-1 and MMP-9, suppressing edema and relieving pain caused by inflammation.

Figure 1 is a graph showing the amount of NO produced from LPS according to treatment with hydrangea extract and hydranzenol.

Figure 2 is a graph showing the level of IL-1β mRNA expression derived from LPS according to hydranzenol treatment.

Figure 3 is a graph showing the expression level of COX-2 mRNA derived from LPS according to treatment with hydrangea extract and hydranzenol.

Figure 4 is a graph showing the expression level of TNF-α mRNA derived from LPS according to treatment with hydrangea extract and hydranzenol.

Figure 5 is a graph showing the expression level of MMP-1 derived from PMA according to treatment with hydrangea extract and hydranzenol.

Figure 6 is a graph showing the expression level of MMP-9 derived from PMA according to treatment with hydrangea extract and hydranzenol.

Figure 7 is a graph showing the effect of inducing foot edema over time by carrageenan and suppressing foot edema by treatment with hydrangea extract.

Figure 8 is a graph showing the results of pain response measurement (Randall-Selitto) and mechanical allodynia (Von-Frey) measurement in animals treated with hydrangea extract induced with inflammatory pain by carrageenan.

Figure 9 is a graph showing the effect of hydrangea extract on suppressing foot edema in an AIA (Adjuvant-induced arthritis) animal model of arthritis in animals.

The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

The terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting of the invention. As used herein, the singular includes the plural unless the context clearly dictates otherwise. The words "comprises" and/or "comprising", as used herein, do not exclude the presence or addition of one or more other components, steps, operations and/or elements.

The words “embodiment,” “example,” “aspect,” “example,” and the like, as used herein, are not to be construed as implying that any aspect or design described is better or advantageous over other aspects or designs.

Also, the term "or" implies an inclusive or rather than an exclusive or. That is, unless stated otherwise or clear from the context, the expression "x employs a or b" means any one of the natural inclusive permutations.

Also, as used in this specification and claims, the singular forms “a” or “an” should generally be construed to mean “one or more,” unless otherwise indicated or clear from the context to be in the singular form.

Additionally, when we say that a part, such as a film, layer, region, or component request, is "on top" or "over" another part, this includes not only the case where it is directly on top of the other part, but also the case where there are other films, layers, regions, components, etc. intervening therebetween.

Example 1. Hydrangea ( Hydrangea serrata Preparation of Seringe extract

Dried hydrangea leaves were purchased from Boseong Special Agricultural Products (Hydrangea serrata (Thunb.) Ser.). 1,500 kg of purified water, which is 15 to 20 times the weight of 100 kg of dried hydrangea leaves, was added, refluxed at 98℃ for 5 hours, and then the extract was filtered under reduced pressure (cartridge cylindrical filter 10 to 50 ㎛) to obtain the filtrate. The extract was concentrated under reduced pressure at 60℃ to secure the concentrate, and thereafter, the hydrangea extract was obtained through spray drying at the conditions of an inlet temperature of 180 to 200℃ and an outlet temperature of 80 to 100℃ (yield: 23%). Hydrangenol was obtained through separation and purification from the hydrangea extract, and the concentration of hydrangenol (mg/g) refers to the weight of hydrangenol obtained based on the weight of the hydrangea extract. LOT1: 8.03 mg/g, LOT2: 7.50 mg/g, LOT3: 7.51 mg/g

Experimental example 1. Hydrangea ( Hydrangea serrata ) Extract and hydranzenol LPS (Lipopolysaccharide)-derived nitric oxide (NO) production inhibition effect confirmed

In order to confirm the NO reduction effect of the hydrangea extract and hydranzenol of Example 1 above, the change in NO was confirmed after inducing an inflammatory response with LPS.

Specifically, macrophage RAW264.7 cells were obtained from the American Type Culture Collection (ATCC, MD, USA), and the RAW264.7 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% FBS and 1% antibiotic-antimycotic at 5% _CO2 and 37°C. Then, the cultured cells were attached to a 6-well plate at a concentration of 8.0 × 105 cells/1.5 ml/well for 24 hours, and then 500 ng/ml of LPS in DMEM was treated to induce an inflammatory response. Then, the hydrangea extract of Example 1 was treated at concentrations of 0.78, 1.56, and 3.12 ppm, and hydranzenol was treated at 50, 100, and 200 μM, and cultured for 24 hours. Next, 100 ㎕ of the culture supernatant was dispensed into a 96-well plate, 100 ㎕ of Griess (0.1% N-(1-naphtyl) ethylenediamine, 1% sulfanilamide) reagent was mixed, and the mixture was reacted for 10 minutes. The absorbance of the chromogenic azo-derivative molecule was measured at 540 nm using a microplate reader (BioTek, Vermont, USA). Using the measured absorbance, the dilution range of sodium nitrite was converted into the amount of nitrite in each sample based on a standard curve, and the amount of NO production was calculated. The results are shown in Fig. 1.

Figure 1(a) is a graph showing the amount of NO produced according to the treatment with different concentrations of hydrangea extract, and Figure 1(b) is a graph showing the amount of NO produced according to the treatment with different concentrations of hydranzenol.

As shown in Figure 1, it was confirmed that NO production was significantly inhibited in proportion to the concentration of hydrangea extract and hydranzenol.

The above results imply that the hydrangea extract and hydranzenol according to one specific example effectively inhibit NO production, thereby having a preventive, improving, or therapeutic effect on arthritis.

Experimental Example 2. Confirmation of the inhibitory effect of hydranzenol LPS (Lipopolysaccharide) on IL-1β mRNA expression

In order to confirm the inhibitory effect of hydranzenol of Example 1 on LPS-derived IL-1β mRNA expression, the mRNA expression level of IL-1β was confirmed using real-time PCR (real-time polymerase chain reaction).

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 DMEM medium containing 10% FBS and 1% antibiotic-antimycotic at 5% _CO2 and 37°C. Then, RAW264.7 cells were attached to a 6-well plate at a concentration of 8.0 × 105 cells/1.5 ml/well for 24 hours, and then treated with 500 ng/ml of LPS in DMEM to induce an inflammatory response. Then, hydranzenol of Example 1 was treated at 50, 100, and 200 μM and cultured for 24 hours. After 24 hours, IL-1β mRNA was extracted from the cells, synthesized into cDNA, and quantitative real-time PCR was performed using a target template (primer) to finally evaluate the level of IL-1β gene expression, and the results are shown in Fig. 2.

Figure 2 is a graph showing the expression level of IL-1β mRNA derived from LPS according to treatment with different concentrations of hydranzenol.

As shown in Figure 2, it was confirmed that all hydranzenols significantly inhibited the expression of IL-1β mRNA in proportion to their concentration.

The above results imply that hydranzenol according to one specific example effectively inhibits the expression of IL-1β mRNA, thereby having an effect in preventing, improving, or treating arthritis.

Experimental example 3. Hydrangea ( Hydrangea serrata ) Extract and Hydranzenol LPS (Lipopolysaccharide)-derived COX-2 mRNA expression inhibition effect confirmed

In order to confirm the inhibitory efficacy of the hydrangea extract and hydranzenol of Example 1 above on COX-2 mRNA expression derived from LPS, the mRNA expression level of COX-2 was confirmed using real-time PCR (real-time polymerase chain reaction).

Specifically, RAW264.7 cells, which are macrophages, were obtained from the American Type Culture Collection (ATCC, MD, USA) and cultured in DMEM medium containing 10% FBS and 1% antibiotic-antimycotic at 5% _CO2 and 37℃. RAW264.7 cells were attached to a 6-well plate at a concentration of 8.0 × 105 cells/2.0 ㎖/well for 24 hours, and then treated with 500 ng/ml of LPS in DMEM to induce an inflammatory response. Then, the hydrangea extract of Example 1 was treated at concentrations of 0.78, 1.56, and 3.12 ppm, and hydranzenol was treated at 50, 100, and 200 μM, and cultured for 24 hours. After 24 hours, COX-2 mRNA was extracted from the cells, synthesized into cDNA, and quantitative real-time PCR was performed using target templates (primers) to finally evaluate the level of COX-2 gene expression, and the results are shown in Figure 3.

Figure 3 is a graph showing the expression level of COX-2 mRNA derived from LPS according to treatment with hydrangea extract and hydranzenol.

Figure 3(a) is a graph showing the COX-2 mRNA expression level according to treatment with different concentrations of hydrangea extract, and Figure 3(b) is a graph showing the COX-2 mRNA expression level according to treatment with different concentrations of hydranzenol.

As shown in Figure 3, it was confirmed that hydrangea extract and hydranzenol significantly inhibited the expression of COX-2 mRNA.

The above results imply that the hydrangea extract and hydranzenol according to one specific example effectively inhibit COX-2 mRNA expression, thereby having a preventive, ameliorating, or therapeutic effect on arthritis.

Experimental example 4. Hydrangea ( Hydrangea serrata ) Extract and Hydranzenol LPS (Lipopolysaccharide)-derived TNF-α mRNA expression inhibition effect confirmed

In order to confirm the inhibitory effect of the hydrangea extract and hydrangea serrata of Example 1 on LPS-derived TNF-α mRNA expression, the mRNA expression level of TNF-α was confirmed using real-time PCR (real-time polymerase chain reaction).

Specifically, RAW264.7 cells, which are macrophages, were obtained from the American Type Culture Collection (ATCC, MD, USA) and cultured in DMEM medium containing 10% FBS and 1% antibiotic-antimycotic at 5% _CO2 and 37℃. RAW264.7 cells were attached to a 6-well plate at a concentration of 8.0 × 105 cells/2.0 ㎖/well for 24 hours, and then treated with 500 ng/ml of LPS in DMEM to induce an inflammatory response. Then, the hydrangea extract of Example 1 was treated at concentrations of 0.78, 1.56, and 3.12 ppm, and hydranzenol was treated at 50, 100, and 200 μM, and cultured for 24 hours. After 24 hours, TNF-α mRNA was extracted from the cells, synthesized into cDNA, and quantitative real-time PCR was performed using target templates (primers) to finally evaluate the level of TNF-α gene expression, and the results are shown in Figure 4.

Figure 4 is a graph showing the expression level of TNF-α mRNA derived from LPS according to treatment with hydrangea extract and hydranzenol.

Figure 4(a) is a graph showing the expression level of TNF-α mRNA according to treatment with different concentrations of hydrangea extract, and Figure 4(b) is a graph showing the expression level of TNF-α mRNA according to treatment with different concentrations of hydranzenol.

As shown in Figure 4, it was confirmed that hydrangea extract and hydranzenol significantly inhibited the expression of TNF-α mRNA.

The above results imply that the hydrangea extract and hydranzenol according to one specific example effectively inhibit TNF-α mRNA expression, thereby having a preventive, ameliorating, or therapeutic effect on arthritis.

Experimental example 5. Hydrangea in cartilage cells Hydrangea serrata ) Extract and Hydranzenol PMA (Phorbol 12-myristate 13-acetate) Inhibitory Effect on MMP-1 Expression

To confirm the inhibitory effect of the hydrangea extract and hydranzenol of Example 1 on the production of MMP-1 derived from PMA in chondrocytes, the amount of MMP-1 secretion was measured using a Human MMP-1 ELISA kit (ab100603, abcam, US).

Specifically, SW-1353 cells, which are chondrocytes, were obtained from the American Type Culture Collection (ATCC, MD, USA) and cultured in DMEM medium containing 10% FBS and 1% antibiotic-antimycotic at 5% _CO2 and 37℃. Then, SW-1353 cells were attached to a 6-well plate at a concentration of 2.0 × 105 cells/2.0 ml/well for 24 hours, and then treated with 50 ng/ml of PMA in DMEM. Then, the hydrangea extract of Example 1 was treated at concentrations of 0.78, 1.56, and 3.12 ppm and hydranzenol was treated at 50, 100, and 200 μM, and cultured for 24 hours. MMP-1 in the supernatant obtained after 24 hours was quantified using an ELISA kit, and the results are shown in Fig. 5.

Figure 5 is a graph showing the secretion amount of MMP-1 derived from PMA according to treatment with hydrangea extract and hydranzenol.

Figure 5(a) is a graph showing the amount of MMP-1 secretion according to treatment with different concentrations of hydrangea extract, and Figure 5(b) is a graph showing the amount of MMP-1 secretion according to treatment with different concentrations of hydranzenol.

As shown in Figure 5, it was confirmed that hydrangea extract and hydranzenol significantly inhibited MMP-1 secretion in proportion to all concentrations.

The above results imply that the hydrangea extract and hydranzenol according to one specific example effectively inhibit the secretion of MMP-1, thereby having a preventive, improving, or therapeutic effect on arthritis.

Experimental example 6. Hydrangea in cartilage cells Hydrangea serrata ) Extract and Hydranzenol PMA (Phorbol 12-myristate 13-acetate) Inhibitory Effect on MMP-9 Expression

To confirm the inhibitory effect of hydranzenol of Example 2 on the production of PMA-derived MMP-1 in chondrocytes, the amount of MMP-9 secretion was measured using a Human MMP-9 LISA kit (ab246539, abcam, US).

Specifically, SW-1353 cells, which are chondrocytes, were obtained from the American Type Culture Collection (ATCC, MD, USA) and cultured in DMEM medium containing 10% FBS and 1% antibiotic-antimycotic at 5% _CO2 and 37℃. Then, SW-1353 cells were attached to a 6-well plate at a concentration of 2.0 × 105 cells/2.0 ㎖/well for 24 hours, and then treated with 50 ng/ml of PMA in DMEM. Then, they were treated at the concentrations of 0.78, 1.56, and 3.12 ppm of Example 1 and treated with 50, 100, and 200 μM hydranzenol and cultured for 24 hours. MMP-9 in the supernatant obtained after 24 hours was quantified using an ELISA kit, and the results are shown in Fig. 6.

Figure 6 is a graph showing the secretion amount of MMP-9 derived from PMA according to treatment with hydrangea extract and hydranzenol.

Figure 6(a) is a graph showing the amount of MMP-9 secreted according to the concentration of hydrangea extract, and Figure 6(b) is a graph showing the amount of MMP-9 secreted according to the concentration of hydrangea extract.

As shown in Figure 6, it was confirmed that hydrangea extract and hydranzenol significantly inhibited MMP-9 secretion in proportion to all concentrations.

The above results imply that the hydrangea extract and hydranzenol according to one specific example effectively inhibit the secretion of MMP-9, thereby having a preventive, improving, or therapeutic effect on arthritis.

Experimental example 7 Hydrangea in animals ( Hydrangea serrata ) Inhibition of foot edema in a carrageenan-induced inflammation model for extracts

Animals Sprague Dawley rats (male, 200 g ± 10%) were measured for paw volume using a paw edema meter in animals that were not injected with carrageenan. Sterile distilled water or hydrangea extract (100 mg/kg, hydrangea 300 mg/kg, 5 animals per group) were orally administered to the animals. 2% carrageenan was injected subcutaneously into both paws, and the change in paw volume was measured using a paw edema meter 1, 3, and 5 hours after the carrageenan injection (Fig. 7A). 3 hours after the carrageenan injection, inflammatory pain was measured after measuring the paw volume (Fig. 7B).

Using pain response measurement (Randall-Selitto), it was confirmed that inflammatory pain was suppressed in the left foot (Fig. 8A).

Using mechanical allodynia (Von-Frey) in animals, it was confirmed that inflammatory pain was suppressed in the right paw (Fig. 8B).

Experimental example 8. Hydrangea in animals ( Hydrangea serrata ) Extracts confirmed the effect of inhibiting foot edema in AIA (Adjuvant-induced arthritis) animal models of arthritis

Animals Sprague Dawley rats (male, 200 g ± 10%) were measured for the volume of both paws using a paw edema meter. Sterile distilled water or ibuprofen (30 mg/kg) or hydrangea extract (100 mg/kg, 300 mg/kg, 6 animals per group) were orally administered to the animals. CFA (Complete Freund's adjuvant) was injected into the left ankle joint, and the volume change of both paws was measured using a paw edema meter at approximately 3-day intervals for 3 weeks (Fig. 9A), and the thickness of both paws was measured using a caliper (Fig. 9B). Pain response was measured in the uninjected right paw using Randall-Selitto immediately before and 20 days after CFA injection.

Using pain response measurement (Randall-Selitto), it was confirmed that inflammatory pain was suppressed in the right foot (Fig. 9C).

Manufacturing Example 1. Manufacturing of tablets

The hydrangea extract hydranzenol of Example 1 above was mixed in the ingredient ratio of Table 1 below and tableted according to a conventional tablet manufacturing method. The manufactured tablet was confirmed to be stable under all formulation test conditions.

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

Manufacturing Example 2. Manufacturing of capsules The hydrangea extract and hydranzenol of Example 1 were mixed in the ingredient ratio shown in Table 2 below and filled into gelatin capsules to manufacture soft capsules. The manufactured capsules were confirmed to be stable under all formulation test conditions.

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

Manufacturing Example 3. Manufacturing of a liquid formulation The hydrangea extract and hydranzenol of Example 1 were mixed in the ingredient ratio shown in Table 3 below, and filled into a bottle or pouch according to a beverage manufacturing method suitable for one's taste to manufacture a liquid formulation. The manufactured liquid formulation was confirmed to be stable under all formulation test conditions.

Raw material name	Unit weight (g)	Raw material name	Unit weight (g)
Example 1	2.5050	Example 2	0.5050
Santa gum	0.0075	Santa gum	0.0075
Fructooligosaccharide solution	0.7500	Fructooligosaccharide solution	0.7500
Coconut Flower Extract Powder	1.0500	Coconut Flower Extract Powder	1.0500
Ssanghwa concentrate	1.5000	Ssanghwa concentrate	1.5000
Red ginseng scent	0.0450	Red ginseng scent	0.0450
Purified water	9.1425	Purified water	11.1425

Manufacturing Example 4. Manufacturing of jelly The hydrangea extract and hydranzenol of Example 1 were mixed in the ingredient ratios shown in Table 4 below, and filled into a three-sided bag according to a jelly manufacturing method suitable for the taste to manufacture a jelly. It was confirmed that the manufactured jelly was stable under all formulation test conditions.

Raw material name	Unit weight (g)	Raw material name	Unit weight (g)
Example 1	2.0000	Example 2	0.5000
Food gel	0.3600	Food gel	0.3600
Carrageenan	0.0600	Carrageenan	0.0600
Calcium lactate	0.1000	Calcium lactate	0.1000
Sodium citrate	0.0600	Sodium citrate	0.0600
Compound gold extract	0.0200	Compound gold extract	0.0200
Enzyme-processed stevia	0.0440	Enzyme-processed stevia	0.0440
Fructooligosaccharide solution	5.0000	Fructooligosaccharide solution	5.0000
Red Grape Concentrate	2.4000	Red Grape Concentrate	2.4000
Purified water	13.9560	Purified water	15.4560

The composition ratios of the above Manufacturing Examples 1 to 4 were generally prepared by mixing suitable ingredients into formulation examples, but the mixing ratio and raw materials may be arbitrarily changed as needed. The extract of the present invention and the ingredients derived therefrom were stable under all formulation example test conditions, so there was no problem with the stability of the formulation.

As described above, although the present invention has been described by means of limited embodiments and drawings, the present invention is not limited to the above embodiments, and those skilled in the art to which the present invention pertains can make various modifications and variations based on this description. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be defined not only by the claims described below but also by equivalents of the claims.

Claims (11)

1. A health functional food composition for preventing or improving arthritis, comprising an extract of Hydrangea serrata Seringe; or hydranzenol represented by the following chemical formula 1 or a food-wise acceptable salt thereof as an active ingredient.

   [Chemical formula 1]

   
2. In the first paragraph,

   A health functional food composition for preventing or improving arthritis, wherein the above-mentioned hydrangea extract is extracted with water, C1 to C4 alcohol, or a mixed solvent thereof.
3. In the first paragraph,

   A health functional food composition for preventing or improving arthritis, wherein the above hydranzenol is separated from the above hydrangea extract.
4. In the first paragraph,

   A health functional food composition for preventing or improving arthritis, wherein the hydrangea extract or the hydranzenol is contained in an amount of 0.0001 to 90 wt% based on the total weight of the health functional food composition.
5. In the first paragraph,

   A health functional food composition for preventing or improving arthritis, wherein the above-mentioned hydrangea extract or the above-mentioned hydranzenol inhibits the expression or activity of at least one selected from the group consisting of IL-1β and COX-2.
6. In the first paragraph,

   A health functional food composition for preventing or improving arthritis, wherein the above-mentioned hydrangea extract or the above-mentioned hydranzenol inhibits the production of nitric oxide (NO).
7. In the first paragraph,

   A health functional food composition for preventing or improving arthritis, wherein the above-mentioned hydrangea extract or the above-mentioned hydranzenol inhibits the expression or activity of at least one selected from the group consisting of MMP-1 and MMP-9.
8. In the first paragraph,

   The above health functional food composition for preventing or improving arthritis has a formulation selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, gums, candies, and health drinks.
9. A pharmaceutical composition for preventing or treating arthritis, comprising an extract of Hydrangea serrata Seringe; or Hydrangenol represented by the following chemical formula 1 or a food- or pharmaceutically acceptable salt thereof as an active ingredient.

   [Chemical formula 1]

   
10. A pharmaceutical composition for preventing or improving arthritis, comprising an extract of Hydrangea serrata Seringe; or Hydrangenol represented by the following chemical formula 1 or a food- or pharmaceutically acceptable salt thereof as an active ingredient.

    [Chemical formula 1]

    
11. A composition for external application to the skin for preventing or improving arthritis, comprising an extract of Hydrangea serrata Seringe; or Hydrangenol represented by the following chemical formula 1 or a food- or pharmaceutically acceptable salt thereof as an active ingredient.

    [Chemical formula 1]

    

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