KR102856074B1 - Composition for preventing, ameliorating or treating cartilage regeneration, arthritis and pain caused by osteoarthritis comprising Adenocaulon himalaicum Edgew extract as effective component - Google Patents

Abstract

The present invention relates to a composition containing an anchovy extract as an active ingredient for cartilage regeneration, arthritis, and the prevention, improvement, or treatment of pain caused by arthritis. The anchovy extract of the present invention reduces the content of prostaglandin E2 (PGE2), an inflammatory mediator, has an excellent effect of inhibiting MMPs, a cartilage matrix decomposing enzyme, reduces GAG content, exhibits an analgesic effect in an animal model induced with pain, and not only alleviates swelling, but also increases the weight-bearing rate and has the effect of reducing the expression level of inflammatory factors (IL-6, TNF-α, and LTB4). In addition, it was confirmed that the anchovy extract increases the expression of cartilage formation marker genes (aggrecan (ACAN), collagen type II (COL2A1), and collagen type X (COL10A1)) in chondrocytes. Therefore, the composition of the present invention can be usefully used for the prevention, improvement, or treatment of cartilage regeneration, arthritis, or pain caused by arthritis.

Description

Composition for preventing, ameliorating or treating cartilage regeneration, arthritis and pain caused by osteoarthritis comprising Adenocaulon himalaicum Edgew extract as effective component

The present invention relates to a composition for cartilage regeneration, arthritis, and prevention, improvement, or treatment of pain caused by arthritis, containing an anchovy extract as an active ingredient.

As we enter an aging society, inflammatory diseases, including arthritis, are becoming increasingly prevalent, regardless of gender. These inflammatory diseases, caused by this inflammatory response, include gastritis, colitis, arthritis, nephritis, hepatitis, arteriosclerosis, cancer, and degenerative diseases. However, no effective medications or treatments have been developed to prevent or treat arthritis. Arthritis refers to inflammatory changes in joints caused by various factors, including aging, mechanical damage, and immune dysfunction.

Among the above-mentioned arthritis, osteoarthritis is a type of arthritis, also called degenerative arthritis, and refers to arthritis caused by degenerative changes in the cartilage and surrounding bone in synovial joints. In other words, osteoarthritis is a disease characterized by the gradual loss of articular cartilage, along with hypertrophy of the bone located below the cartilage, bone formation at the joint margin, and non-specific synovial inflammation. Osteoarthritis is a disease caused by cartilage damage due to aging or excessive physical pressure (e.g., obesity, trauma, etc.). Therefore, osteoarthritis causes severe pain and movement disorders in weight-bearing joints, such as the knee joint and hip joint, and if left untreated for a long time, it can even lead to joint deformation.

Gouty arthritis is an inflammation caused by the deposition of uric acid in the spaces and tissues within the joints. As the concentration of uric acid (a byproduct of the body's metabolism of purines ingested through food) in the blood increases, urate crystals (uric acid exists in the form of urate in the blood, body fluids, and joint fluid) are deposited in the cartilage, tendons, and surrounding tissues of the joints. This phenomenon causes inflammation of the joints, resulting in recurrent attacks accompanied by severe pain.

Meanwhile, Adenocaulon himalaicum Edgew is a perennial herb of the Asteraceae family in the Campanulate order. It grows in shaded, slightly moist areas. The stem is erect, with a single main stem arising from a short rhizome, growing to 50–100 cm in height and branching. The upper portion has a ridged line, and the stem and leaf backs are densely covered with glandular hairs. The leaves are alternate, triangular, and kidney-shaped. The backs of the leaves are densely covered with fluffy white hairs, and the margins are serrated. The white flowers bloom from August to September, and a single capitulum blooms at the end of each branch. The involucre is hemispherical, with 5–7 bracts. The fruit is an achene, club-shaped, radiating, densely glandular, sticky, and lacking a pappus. The young shoots are eaten as a vegetable. It is distributed in Korea, Japan, China, the Himalayas, the Amur region, and the Ussuri region.

As for prior art related to anchovy, Korean Patent Publication No. 2011-0087363 discloses a cosmetic composition containing an anchovy extract as an active ingredient, and Korean Patent Publication No. 2020-0006883 discloses a composition for preventing or improving skin damage caused by ultraviolet rays, containing at least one of anchovy ( Adenocaulon himalaicum Edgew) extract, campanula takesimana Nakai extract, or filipendula glaberrima Nakai extract as an active ingredient. However, a composition for preventing, improving, or treating cartilage regeneration, arthritis, and pain caused by arthritis, which contains an anchovy extract of the present invention as an active ingredient, has not been disclosed.

The present invention was derived in response to the above-mentioned needs, and the present invention provides a composition for preventing, improving or treating cartilage regeneration, arthritis and pain caused by arthritis, containing an anchovy extract as an active ingredient, and the anchovy ( Adenocaulon himalaicum Edgew) extract of the present invention reduces the content of prostaglandin E2 (PGE2), which is an inflammatory mediator, has an excellent effect of inhibiting MMPs, which are cartilage matrix decomposing enzymes, reduces GAG content, exhibits an analgesic effect in an animal model induced by pain, and not only relieves swelling, but also increases the weight-bearing rate and has an effect of reducing the expression of inflammatory factors (IL-6, TNF-α and LTB4), and the anchovy extract has an effect of increasing the expression of cartilage formation marker genes (aggrecan (ACAN), collagen type II (COL2A1) and collagen type X (COL10A1)) in chondrocytes, thereby completing the present invention.

In order to achieve the above purpose, the present invention provides a health functional food composition for cartilage regeneration, arthritis, or prevention or improvement of pain caused by arthritis, containing an extract of Adenocaulon himalaicum Edgew as an effective ingredient.

In addition, the present invention provides a pharmaceutical composition for cartilage regeneration, arthritis, or prevention or treatment of pain caused by arthritis, containing an anchovy extract as an active ingredient.

In addition, the present invention provides a feed additive for cartilage regeneration, arthritis, or prevention or improvement of pain caused by arthritis, containing an anchovy extract as an effective ingredient.

In addition, the present invention provides a veterinary composition for cartilage regeneration, arthritis, or prevention or treatment of pain caused by arthritis, containing an anchovy extract as an active ingredient.

The present invention relates to a composition for preventing, improving or treating cartilage regeneration, arthritis and pain caused by arthritis, containing an anchovy extract as an active ingredient. The anchovy ( Adenocaulon himalaicum Edgew) extract of the present invention reduces the content of prostaglandin E2 (PGE2), an inflammatory mediator, has an excellent effect of inhibiting MMPs, a cartilage matrix decomposing enzyme, reduces GAG content, exhibits an analgesic effect in an animal model induced with pain, and not only relieves swelling, but also increases the weight-bearing rate and has an effect of reducing the expression level of inflammatory factors (IL-6, TNF-α and LTB4). In addition, the anchovy extract has an effect of increasing the expression of cartilage formation marker genes (aggrecan (ACAN), collagen type II (COL2A1) and collagen type X (COL10A1)) in chondrocytes.

Figure 1 shows the results of confirming the change in PGE2 content according to treatment with the anchovy extract of the present invention. # indicates that the PGE2 content of the IL-1β treatment group significantly increased compared to the untreated control group (Con), p<0.05. * indicates that the PGE2 content of the anchovy extract treatment group significantly decreased compared to the IL-1β treatment group, p<0.05.
Figure 2 shows the results of confirming the change in the content of MMP-1 and MMP-13 according to the treatment with the anchovy extract of the present invention. #### indicates that the MMP-1 and MMP-13 content of the IL-1β treatment group significantly increased compared to the untreated control group (Con), and p<0.0001. **, *** indicate that the MMP-1 and MMP-13 content of the anchovy extract treatment group significantly decreased compared to the IL-1β treatment group, and ** indicates p<0.01, and *** indicates p<0.001.
Figure 3 shows the results of confirming the change in GAG content according to treatment with the anchovy extract of the present invention. ## indicates that the GAG content of the IL-1β treated group significantly increased compared to the untreated control group (Con), and p<0.01. * indicates that the GAG content of the anchovy extract treated group of the present invention significantly decreased compared to the IL-1β treated group, and p<0.05.
Figure 4 shows the results of confirming the analgesic effect on pain according to administration of the anchovy extract of the present invention in an animal model. Indo is indomethacin, a positive control. *, *** indicate that after pain induction, the number of times the body was twisted for 10 minutes was significantly reduced in the positive control group (Indo) or the experimental group administered the anchovy extract of the present invention compared to the control group (con) that was not treated with the anchovy extract of the present invention. * indicates p<0.05, and *** indicates p<0.001.
Figure 5 shows the results of confirming the effect of alleviating ear edema in an animal model induced by arachidonic acid (AA) following administration of the anchovy extract of the present invention. ### indicates that ear edema significantly increased in the arachidonic acid-treated group (AA) compared to the untreated normal group (Nor), p<0.001. * indicates that ear edema significantly decreased in the anchovy extract-treated group of the present invention compared to the arachidonic acid-treated group (AA), p<0.05.
Figure 6 shows the results of the hind limb weight bearing rate (%) of SD rats. Con is the negative control group, MIA (monosodium iodoacetate) is the osteoarthritis-induced group, and JOINS is the positive control group, which is the MIA+JOINS administration group, and Anchovy-100 and Anchovy-200 are groups that were administered MIA and 100 mg/kg or 200 mg/kg of anchovy extract in parallel. #### indicates that the weight bearing rate of the group in which osteoarthritis was induced by MIA was significantly reduced compared to the negative control group, and p<0.0001, and *, ** indicate that the weight bearing rate of the MIA+Joins or MIA+anchovy extract parallel administration group was statistically significantly increased compared to the osteoarthritis-induced group by MIA treatment, and * indicates p<0.05, and ** indicates p<0.01.
Figure 7 shows the results of confirming the expression of inflammatory factors (LTB4, TNF-α, and IL-6) and cartilage degeneration factors (MMP-2 and MMP-9) by anchovy extract in an animal model of osteoarthritis. Con is a negative control group, MIA (monosodium iodoacetate) is an osteoarthritis-induced group, JOINS is a positive control group, which is the MIA+JOINS administration group, and anchovy is a group that was administered MIA and 100 mg/kg or 200 mg/kg of anchovy extract in parallel. #, ##, ### indicate that the expression of inflammatory factors and cartilage degeneration factors significantly increased when MIA was treated compared to Con, the negative control group. # is p<0.05, ## is p<0.01, and ### is p<0.001. *, ** indicate that the expression levels of inflammatory factors (LTB4, TNF-α, and IL-6) and cartilage degeneration factors (MMP-2 and MMP-9) in the MIA+Joins or MIA+Anchovy Extract parallel administration groups were statistically significantly reduced compared to the MIA group. * indicates p<0.05, ** indicates p<0.01.
Figure 8 shows the results of confirming the activity of cartilage formation indicators (aggrecan (ACAN), collagen type II (COL2A1), and collagen type Ⅹ (COL10A1)) by anchovy extract in chondrogenic precursor cells. Con is the negative control group, ITS is the cartilage differentiation promotion induction group, and anchovy is the group treated with ITS and 50 ㎍/㎖, 100 ㎍/㎖ anchovy extract in parallel. #, ##, #### indicate that the expression of cartilage formation indicators in the ITS group significantly increased compared to Con. # is p<0.05, ## is p<0.01, and #### is p<0.0001. *, ***, **** indicate that the expression level of cartilage formation indicators in the ITS + anchovy extract parallel treatment group was statistically significantly increased compared to the ITS group. * indicates p<0.05, *** indicates p<0.001, and **** indicates p<0.0001.

The present invention relates to a health functional food composition for cartilage regeneration, arthritis, or prevention or improvement of pain caused by arthritis, containing an extract of Adenocaulon himalaicum Edgew as an active ingredient.

The above anchovy extract may be used in its entirety, preferably one or more parts selected from the flowers, leaves, stems, and roots, but is not limited thereto. The arthritis is preferably osteoarthritis, but is not limited thereto. The above active ingredient has the characteristic of suppressing inflammatory cytokines.

The above anchovy extract may be manufactured by a method including, but not limited to, the following steps:

1) A step of extracting by adding an extraction solvent to the anchovy;

2) a step of filtering the extract of step 1); and

3) A step of producing an extract by concentrating and drying the filtered extract of step 2) under reduced pressure.

In the above step 1), the extraction solvent is preferably water, a lower alcohol of C ₁ to C ₄ , or a mixture thereof, more preferably a 70% (v/v) ethanol extract, but is not limited thereto.

In the above manufacturing method, the extraction of anchovies can utilize any conventional method known in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. The reduced pressure concentration in step 3) is preferably performed using a vacuum reduced pressure concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably performed using reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.

The above composition is preferably prepared in any one of the following dosage forms: powder, granules, pills, tablets, capsules, candies, syrups, and beverages, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the anchovy extract may be added as is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method. The amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range. There are no particular restrictions on the type of the food. Examples of foods to which the above extract or its fraction can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

When the composition of the present invention is used as a health beverage, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclotensin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 g of the composition of the present invention. 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 adjusters, 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 mixtures. The proportion of these additives is not particularly important, but the composition of the present invention is typically selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

In addition, the present invention relates to a pharmaceutical composition for cartilage regeneration, arthritis, or prevention or treatment of pain caused by arthritis, containing an anchovy extract as an active ingredient.

The above arthritis is preferably osteoarthritis, but is not limited thereto. The composition of the present invention may be prepared in any one formulation selected from, but not limited to, capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols.

The composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above-mentioned effective ingredient, and may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include capsules, powders, granules, tablets, pills, etc., and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, emulsions, syrups, and aerosols. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. For parenteral administration, it is preferable to select a method such as topical application to the skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine, epidural, or intracerebrovascular injection.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment. The level of the effective amount may be determined based on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

The dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity, and the daily dosage is 0.01 to 2,000 mg/kg based on the amount of anchovy extract, preferably 30 to 500 mg/kg, and more preferably 50 to 300 mg/kg, and can be administered 1 to 6 times a day. The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In addition, the present invention relates to a feed additive for cartilage regeneration, arthritis, or prevention or improvement of pain caused by arthritis, containing an anchovy extract as an effective ingredient.

The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. The term "feed" in the present invention may mean any natural or artificial diet, meal, etc., or ingredients of the meal, which are intended for or suitable for animals to eat, ingest, and digest. The type of the feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feeds such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal feeds such as proteins, inorganic substances, oils and fats, mineral substances, oils and fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

In addition, the present invention relates to a veterinary composition for cartilage regeneration, arthritis, or prevention or treatment of pain caused by arthritis, containing an anchovy extract as an active ingredient.

The veterinary composition of the present invention may further comprise suitable excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using methods well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc.

The effective amount of the veterinary composition according to the present invention can be appropriately selected depending on the individual animal. This may be determined based on factors including the severity of the disease or condition, the individual's age, weight, health status, or sex, sensitivity to the active ingredient of the present invention, the route of administration, the duration of administration, other compositions combined with or used concurrently with the composition, and other factors well known in the fields of physiology and veterinary medicine.

Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1. Preparation of anchovy extract

For 1 kg of anchovy, 15 ℓ of 70% (v/v) ethanol was added, extraction was performed at 85°C for 3 hours, and the filtrate was concentrated under reduced pressure and dried at 45°C to obtain an anchovy extract.

Example 2. Evaluation of anti-inflammatory efficacy in chondrocytes

In human chondrocytes (SW1353), changes in the content of prostaglandin E2 (PGE2), an inflammatory mediator, and inhibition of MMPs (MMP-1, MMP-13), which are cartilage matrix degrading enzymes activated by increased inflammatory cytokines, according to extract treatment, and changes in the content of GAG (Glycosaminoglycan), a cartilage proteoglycan degradation product, were confirmed.

[Cell culture]

Human chondrocytes (SW1353) were cultured using a culture medium containing 10% FBS in DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), treated with 25-100 ㎍/㎖ of anchovy extract, cultured for 2 hours, and then treated with IL-1β (10 ㎍/㎖) for 24 hours.

(1) Confirmation of the inhibitory effect of PGE2 production induced by IL-1β

To evaluate the anti-inflammatory effect of anchovy extract, the inhibitory effect on the production of PGE2, an inflammatory mediator, was measured using an ELISA kit (R&D Systems).

As a result, PGE2 production was significantly increased by IL-1β treatment in chondrocytes, and PGE2 production was significantly reduced in cells pretreated with 100 μg/ml of anchovy extract (Fig. 1).

(2) Confirmation of the effect of reducing the expression levels of MMP-1 and MMP-13 mediated by IL-1β

The extracellular matrix (ECM) that constitutes cartilage tissue is decomposed by the activation and synthesis of MMPs following the secretion of inflammatory cytokines. We confirmed using an ELISA kit (R&D Systems) whether the anchovy extract affects the expression of MMPs.

As a result, the expression levels of MMP-1 and MMP-13 increased in the culture supernatant treated with IL-1β for 24 hours, but in cells pretreated with the anchovy extract of the present invention, the expression levels of MMP-1 and MMP-13 by IL-1β significantly decreased in a concentration-dependent manner (Fig. 2).

(3) Confirmation of changes in GAG (Glycosaminoglycan) content

GAG is a component of cartilage tissue and is a substance produced by the breakdown of proteoglycan. To determine whether anchovy extract affects the breakdown of proteoglycan, changes in GAG content were observed. Specifically, after IL-1β treatment for 24 hours, the culture supernatant was reacted with blyscan dye and the absorbance was measured at 656 nm to determine the amount of GAG.

As a result, it was confirmed that the increased GAG content, which was promoted by cartilage degradation due to IL-1β, was reduced by treatment with anchovy extract (Fig. 3).

Example 3. Evaluation of analgesic efficacy in an animal model of pain induced by acetic acid.

Seven-week-old ICR mice were acclimatized for a week, and then orally administered anchovy extract (150 mg/kg, 300 mg/kg) and indomethacin (5 mg/kg), a positive control, for four days each. On the fourth day, one hour after administration, 0.75% (v/v) acetic acid (acetic acid) was administered intraperitoneally at a dose of 10 ml/kg. The mice were fasted for more than 18 hours before intraperitoneal acetic acid administration. Five minutes after acetic acid administration, the number of times the mice twisted their bodies for 10 minutes was observed.

As a result, intraperitoneal administration of acetic acid caused damage to capillaries and pain, which increased the number of times the mouse body was twisted, and when anchovy extract was administered, the number of times the mouse body was twisted significantly decreased (Fig. 4).

Example 4. Evaluation of the efficacy of alleviating ear edema in an animal model of inflammation induced by arachidonic acid.

Seven-week-old ICR mice were acclimatized for one week, and then orally administered anchovy extract (150 mg/kg, 300 mg/kg) and indomethacin (5 mg/kg), a positive control, for four days. On the fourth day, 1 hour after administration, 10 μl of 2% arachidonic acid dissolved in acetone was applied to the inner and outer surfaces of each ear, respectively. The mice were euthanized two hours later, and the ear tissue weight was measured using a 5 mm dermal biopsy punch and fixed in formalin for more than 24 hours. After dehydration, the ear tissue was embedded in paraffin, sectioned into 10 μm sections, and stained with H&E.

As a result, it was confirmed that ear edema was significantly reduced by administration of anchovy extract in an animal model of ear edema induced by arachidonic acid (Fig. 5).

Example 5. Confirmation of the effect of anchovy extract on weight bearing in an animal model of osteoarthritis induced by monosodium iodoacetate (MIA).

In order to confirm the effect of the anchovy extract prepared in Example 1 on weight bearing in an animal model of osteoarthritis induced by MIA, 50 μl of MIA (diluted to a concentration of 60 mg/ml with 0.9% saline), an osteoarthritis-inducing substance, was administered into the right hind limb joint space of 7-week-old SD rats to induce osteoarthritis. Then, 100 mg/kg or 200 mg/kg of the anchovy extract was orally administered once daily for a total of 21 days, and the weight bearing rate was measured at 7-day intervals. JOINS tablets (20 mg/kg) were used as a positive control group.

The hindlimb weight bearing was measured using a paw incapacitance tester (Linton instrument Co., UK, Ser No. 01/45/25). Since the osteoarthritis-induced rats in the holder of the tester stood relying on the normal paw that was not administered MIA due to pain, the weight of both paws was unbalanced, and the weight of the paw administered MIA was measured to be relatively lighter than the weight of the normal paw. When measuring the paw weight, the weight (g) of each paw was measured while the belly of the SD rat did not touch the sensor of the device, and the weight bearing rate (%) was calculated using the method of Equation 1 below using the measured paw weight. The weight bearing is the force of pressing with the paw. In a normal case, the weight of both paws is balanced, and the weight bearing rate of one paw is 50%. However, as the pain caused by the induction of osteoarthritis worsens, the weight bearing rate (%) of the osteoarthritis-induced hindlimb decreases.

[Formula 1]

Weight-bearing ratio (%) = [Weight of hind limb causing osteoarthritis / (Weight of both hind limbs)] × 100

As a result, as shown in Fig. 6, it was confirmed that the weight bearing rate (%) of the group in which osteoarthritis was induced by administering MIA was reduced by about half compared to the control group over time compared to the normal SD rat group. In contrast, the MIA + Joins or MIA + Anchovy Extract administration groups showed an increase in the previously decreased weight bearing rate (%).

Example 6. Confirmation of the effect of anchovy extract on inflammatory and cartilage degeneration factors in an animal model of osteoarthritis induced by monosodium iodoacetate (MIA).

The expression levels of inflammatory factors (IL-6, TNF-α, and LTB4) and cartilage degeneration factors (MMP-2 and MMP-9) in the blood and joints of animals with induced osteoarthritis were confirmed using ELISA.

As a result, as shown in Figure 7, when osteoarthritis was induced by MIA, the expression levels of inflammatory and cartilage degeneration-inducing factors increased, whereas when treated with anchovy extract, the expression levels of these increased factors decreased. This suggests that anchovy extract suppresses inflammation and articular cartilage damage induced by osteoarthritis.

Example 7. Evaluation of the efficacy of promoting chondrocyte differentiation

The efficacy of drugs in promoting cartilage differentiation was evaluated during the differentiation process of chondrogenic progenitor cells (ATDC-5). The efficacy of drugs in promoting the production of aggrecan (ACAN), collagen type II (COL2A1), and collagen type X (COL10A1), which are major components of the extracellular matrix (ECM) of cartilage, was confirmed.

ATDC5 (RCB0565, RIKEN cell bank, Japan), chondrocyte precursor cells, were cultured in DMEM/F12 medium supplemented with 5% FBS, 1% penicillin (100 U/mL), and streptomycin (100 g/mL) at 37°C and 5% _CO2 . After 24 hours of seeding, cell differentiation was induced for 21 days in a medium containing ITS (10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL sodium selenite, I3146, Sigma), during which time each sample was treated together at different concentrations. After 21 days of differentiation induction, the mRNA expression levels of aggrecan, collagen type II, and collagen type X, which are specific markers of chondrocytes, were confirmed.

Primer sequences of chondrogenesis marker genes Gene name direction Sequence (5'-> 3') Sequence number Aggrecan Forward 5′-GCTGCAGTGATCTCAGAAGAAG-3′ 1 Reverse 5′-GATGGTGAGGGAAGACCCTA-3′ 2 collagen type II Forward 5′-AGAGGCGGAGACTACTGGATTG-3′ 3 Reverse 5′-CGTTAGCGGTGTTGGGAG-3′ 4 collagen type Ⅹ Forward 5′-TGGGATGCCTCTTGTCAGTG-3′ 5 Reverse 5′-GTGGGCGTGCCATTCTTAT-3′ 6 GAPDH Forward 5′-AACAGTCCGCCTAGAAGCAC-3′ 7 Reverse 5′-CGTTGACATCCGTAAAGACC-3′ 8

As a result, as shown in Fig. 8, it was confirmed that the expression of cartilage formation markers (aggrecan (ACAN), collagen type II (COL2A1), collagen type Ⅹ (COL10A1)) increased in cells that were induced to differentiate into chondrocytes by treating chondrocyte precursor cells with ITS, and when treated with anchovy extract in parallel, the expression of cartilage formation markers increased. Through this, the cartilage formation promoting effect of anchovy extract was confirmed.

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Claims (11)

A health functional food composition containing an extract of Adenocaulon himalaicum Edgew as an active ingredient for cartilage regeneration, arthritis, or prevention or improvement of pain caused by arthritis. A health functional food composition for cartilage regeneration, arthritis, or prevention or improvement of pain caused by arthritis, characterized in that the anchovy extract in the first paragraph is extracted from one or more parts selected from among flowers, leaves, stems, and roots. A health functional food composition for preventing or improving cartilage regeneration, arthritis or pain caused by arthritis, characterized in that the arthritis in claim 1 is osteoarthritis. In claim 1, the effective ingredient is a health functional food composition for preventing or improving cartilage regeneration, arthritis, or pain caused by arthritis, characterized in that it suppresses inflammatory cytokines. A health functional food composition for preventing or improving cartilage regeneration, arthritis or pain caused by arthritis, characterized in that in claim 1, the extraction solvent of the anchovy extract is water, a lower alcohol of C ₁ to C ₄ or a mixture thereof. A health functional food composition for preventing or improving cartilage regeneration, arthritis or pain caused by arthritis, characterized in that the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup and beverage in the first paragraph. A pharmaceutical composition for cartilage regeneration, arthritis or prevention or treatment of pain caused by arthritis, containing an anchovy extract as an active ingredient. A pharmaceutical composition for preventing or treating cartilage regeneration, arthritis or pain caused by arthritis, characterized in that in claim 7, the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient. A pharmaceutical composition for preventing or treating cartilage regeneration, arthritis or pain caused by arthritis, characterized in that the composition is prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups and aerosols in claim 7. A feed additive containing anchovy extract as an active ingredient for cartilage regeneration, arthritis, or prevention or improvement of pain caused by arthritis. A veterinary composition containing an anchovy extract as an active ingredient for cartilage regeneration, arthritis or prevention or treatment of pain caused by arthritis.