KR101317668B1 - Pharmaceutical composition for treating and preventing arthritis comprising stauntonia hexaphylla leaf extract - Google Patents

Abstract

The present invention relates to a composition for treating, improving or preventing arthritis, comprising a honey leaf extract as an active ingredient. The honey leaf extract is not only cytotoxic, but also confirmed the inhibitory activity of the cytokine (cytokine) mRNA associated with inflammation, the amount of NO secretion and COX-2 enzyme causing inflammation, effectively inhibiting inflammation In addition, it was confirmed through animal experiments that can effectively inhibit the differentiation of osteoclasts with respect to arthritis, and the pharmaceutical composition or arthritis prevention or improvement effect of the arthritis treatment or prevention comprising the same as an active ingredient It can be applied to such a food composition.

Description

Pharmaceutical composition for treating or preventing arthritis comprising honey leaf extract as an active ingredient {PHARMACEUTICAL COMPOSITION FOR TREATING AND PREVENTING ARTHRITIS COMPRISING STAUNTONIA HEXAPHYLLA LEAF EXTRACT}

The present invention relates to a composition for treating, improving or preventing arthritis, comprising a plant extract having an effect of treating, preventing or improving arthritis, specifically, a honey leaf extract as an active ingredient.

Inflammatory reactions are caused by various factors, such as exposure to harmful substances or organisms from outside, including external infectious agents such as bacteria, fungi, viruses, or various types of allergens, or exposure to harmful substances or organisms from outside. When this damage is done or destroyed, it means a locally occurring immune response to minimize the damage and restore the damaged area.

In addition, various factors that cause inflammation include physical factors such as trauma, burns, frostbite, radioactivity, chemical factors such as acids, and immunological factors due to antibody reactions. It can also be caused by imbalance.

The inflammatory response is a protective mechanism useful for protecting the living body and removing products generated by tissue damage. The inflammatory response is associated with various inflammatory mediators and immune cells present in local blood vessels and body fluids, such as enzyme activation, inflammatory mediator secretion, fluid infiltration, Symptoms such as cell migration, tissue destruction, erythema, edema, fever or pain may appear, and dysfunction may be caused by the above symptoms.

In the normal case, the inflammation removes an external infectious agent or neutralizes or removes a pathogen, and regenerates upper tissues, thereby restoring normal structure and function. However, if the antigen is not removed continuously or a specific internal substance causes the degree of inflammation to be above a certain level or becomes chronic, it becomes a problem when progressing to a disease state such as irritability or chronic inflammation. In addition to observing the inflammatory response in almost all diseases, it is known that enzymes related to the inflammatory response play an important role in carcinogenesis. It is also an obstacle in the treatment process such as blood transfusion, drug administration or organ transplantation.

In vivo, the inflammatory response involves various biochemical phenomena, and in particular, the reaction is initiated or regulated by various enzymes related to the inflammatory response produced by immune cells.

Recently, it has been known that the progress of the inflammatory reaction in the body is related to COX (cyclooxygenase) enzyme activity. The COX enzyme is a major enzyme involved in the biosynthesis of prostaglandin in vivo (Smith et al., J. Biol. Chem., 271, 33157 (1996)), and two kinds of isozyme enzymes, COX-1 and COX -2 is known to exist. The COX-1 is constantly present in tissues such as the stomach and kidneys, and is involved in maintaining normal homeostasis, while the COX-2 is involved in mitogens and cytokines during inflammation or other immune responses. Is an enzyme that is transiently and rapidly expressed in cells.

Another strong inflammatory mediator, nitric oxide (NO), is produced from L-arginine by NO synthase (NOS), and is produced by a variety of substances, such as UV, external stress, endotoxin or cytokines Lt; / RTI > cells. Such inflammatory stimuli can increase the expression of inducible NOS (iNOS) in the cell, thereby inducing NO production in the cell, thereby inducing an inflammatory response by activating macrophages.

Therefore, in order to effectively alleviate inflammation, researches on a substance capable of inhibiting the production of NO are under way. However, some side effects are problematic for anti-inflammatory substances developed by these studies. For example, nonsteroidal anti-inflammatory drugs used in the treatment of acute or chronic inflammatory diseases are known to exhibit side effects such as gastrointestinal disorders by inhibiting COX-2 enzyme as well as COX-1 enzyme.

Meanwhile, as the population ages, the number of patients with degenerative diseases such as dementia, arthritis, and arteriosclerosis is increasing. It is a problem.

Among these, arthritis is the most prevalent disease among humans. Especially, degenerative arthritis is 70% to 80% of people over 65 years old, and nearly 100% of people over 75 years old have it. It is a representative senile disease.

According to a recent survey, more than 10% of the population in Korea suffers from degenerative arthritis, and the prevalence is increasing very rapidly due to the rapid aging of Korean society. Specifically, in Korea, 80% of the population over the age of 55 suffer from osteoarthritis, and more than 75 years old is known to have a bone joint disease.

In addition, it is estimated that there are more than 500 million patients with degenerative arthritis worldwide. Among the elderly diseases that need to be overcome urgently in terms of the quality of human life as the average life expectancy of humans increases and the length of social activities of humans increases. Occupies an important position. In particular, knee osteoarthritis is a disease with a high incidence and inflammation continues to cause atrophy of the surrounding muscles, resulting in malformations of the joints, and not only treatment costs but also various social losses such as impaired social activities of the elderly population.

The arthritis, specifically, osteoarthritis or degenerative arthritis may be caused by the onset of the lesion based on the degenerative change of the articular cartilage, causing pain and swelling, joint stiffness and gradual movement disorder during exercise.

In the treatment of osteoarthritis, exercise therapy such as weight loss, muscle strengthening exercise, simple analgesic agent, nonsteroidal anti-inflammatory agent, intra-articular steroid injection and surgery, etc. are currently being performed. However, steroidal anti-inflammatory drugs can cause complications such as gastrointestinal disorders, and in addition, intra-articular injection therapy of steroids has limitations due to aggressive cartilage destruction and risk of infection due to systemic side effects and repeated injections of steroids.

On the other hand, the regulation of osteoclasts is essential for balancing bone remodeling, ie, bone resorption by osteoclasts and bone formation by osteoblasts, and is important in the treatment of bone diseases.

Specifically, bone-resorbing osteoclasts are derived from hematopoietic cells of monocyte-macrophage lineage and differentiate into multinuclear cells through a number of processes. The formation and activity of the osteoclasts is regulated by local factors or stromal and osteoblasts in the bone.

In relation to the differentiation of osteoclasts, systemic modifications such as increased expression of osteotropic and osteoclastogenic factors or lack of estrogen can lead to an increase in osteoclast number and activity. Increases in number and activity can result in inflammatory diseases such as rheumatoid arthritis or periodontitis and bone diseases such as osteoporosis.

Recently, in order to effectively alleviate inflammation, studies on substances capable of inhibiting NO production have been conducted. However, some side effects are problematic for anti-inflammatory substances developed by these studies. For example, nonsteroidal anti-inflammatory drugs used in the treatment of acute or chronic inflammatory diseases are known to exhibit side effects such as gastrointestinal disorders by inhibiting COX-2 enzyme as well as COX-1 enzyme.

In addition, there is a lack of therapeutic drugs that can effectively inhibit or block the progression of arthritis such as osteoarthritis without additional side effects, and there are almost no preventive health functional foods.

For example, even in the case of anti-rheumatitis drugs currently used, bulamamine, which has been proven to be safe and effective, shows effective disease suppression effect from 4 weeks after administration, resulting in a late effect. It is not effective, and has the disadvantage that it does not suppress painful fever, swelling, and painful symptoms at once. As a side effect, adverse effects may occur on skin side effects, digestive system, blood, musculoskeletal system, liver, kidney and endocrine system.

In addition, according to the 2008 Food and Drug Administration data, even in the case of glucosamine-containing products that are steadily sold in the elderly, the opinions of the medical community and the related industries differ in relation to the efficacy of the preventive treatment of arthritis.

Therefore, as a substance derived from natural substances, it can effectively inhibit NO production, inhibit iNOS and TNF-α expression, and effectively inhibit COX-2 enzyme activity, without any risk of side effects or cytotoxicity. In addition, it is possible to suppress the differentiation of osteoclasts, and to develop materials excellent in treating, preventing or improving arthritis.

The disclosures of patent documents or articles cited throughout this specification are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

KR 0530843 B KR 0614465 B KR 0941133 B KR 0954984 B KR 1023487 B KR 1150900 B KR 1150528 B KR 1162336 B

In order to meet the above demands, the present invention aims to provide a composition for treating or preventing arthritis with an active ingredient of a plant extract which is less likely to cause problems related to side effects.

In addition, unlike the existing anti-inflammatory substances, side effects such as natural plants, which can be used for food, are not a problem, and safety is excellent, and it is possible to inhibit NO production related to inflammation and effectively inhibit COX-2 enzyme. It can be, and to provide a composition for treating or improving arthritis comprising a plant extract that can inhibit the differentiation of osteoclasts as an active ingredient.

In order to achieve the above object, the present invention provides a composition for treating or preventing arthritis, comprising a honey leaf extract as an active ingredient. The composition for treating or preventing arthritis may be a pharmaceutical composition.

The honey leaf extract can effectively inhibit NO secretion by inhibiting the expression of iNOS related to NO production, and promotes an inflammatory response in relation to the biosynthesis of prostaglandin present in the body (cyclooxygenase-2). In addition to inhibiting the synthesis of), animal experiments with arthritis have been shown to effectively inhibit the differentiation of osteoclasts.

Specifically, the present inventors have an anti-inflammatory effect because the honey leaf extract can effectively inhibit the mRNA transcription of cytokines associated with the inflammation and inflammation-induced NO, and animal experiments related to arthritis ( In In vivo ), it is possible to effectively inhibit the differentiation of osteoclasts and to inhibit the edema of joints in the experimental animal model of collagen rheumatoid arthritis, and it was confirmed that the honey leaf extract is excellent in treating, improving or preventing arthritis. The present invention was completed.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a composition for treating or preventing arthritis, comprising a honey extract, preferably a honey leaf extract as an active ingredient.

The nectar ( Stauntonia hexapillya ) is an evergreen vine plant with the dicotyledon plant Buttercup hyacinth vine, also known as the beech tree. The honey is one male and female, the main stem extends about 5 m, the leaves are shifted, palm-like double leaf of 5 to 7 small leaves. Small leaves are thick, egg-shaped or oval, and have flat edges. Petioles about 6 cm to 8 cm long, petioles about 3 cm long. Flowers blossom in May, yellowish white, hanging on inflorescences. Small flower branch of female flower becomes reddish brown in autumn, and there are many big trees and is rough. Fruits are berry, egg-shaped or oval, 5 cm to 10 cm long, ripened in reddish brown in October, and pulp tastes better than erosion. Seeds are oval-shaped oval black. The honey is mainly distributed in Korea, Japan, Taiwan or China. In Korea, it grows well in the valleys and forests of southern regions such as Jeollanam-do, Gyeongsangnam-do, and Chungcheongnam-do.

The honey extract may be prepared according to a conventional method for producing a plant extract, for example, leaves, branches, stems, roots or bark of honey, or its pulverized, preferably in terms of the inflammatory treatment effect on the leaves of honey The extract may be prepared by adding an extraction solvent or may be prepared by adding a fractional solvent to the crude extract prepared by extracting with the extraction solvent.

The extraction solvent may be at least one selected from the group consisting of water and an organic solvent. The organic solvent may be a polar solvent such as an alcohol having 1 to 5 carbon atoms, the alcohol dilution water or acetone, a nonpolar solvent of ether, chloroform, benzene, hexane, ethyl acetate or dichloromethane, or a mixed solvent thereof. The alcohol having 1 to 5 carbon atoms may be methanol, ethanol, propanol, butanol, or isopropanol, but is not limited thereto. In addition, the alcohol dilution water may be one obtained by diluting the alcohol in water at 50% (v / v) to 99.9% (v / v).

Extraction solvent of the honey leaf extract of the present invention is preferably at least one selected from the group consisting of water, alcohol having 1 to 5 carbon atoms, alcohol dilution water and mixtures thereof, more preferably water, 1 to 4 carbon atoms It may be any one selected from the group consisting of alcohols and mixtures thereof, and even more preferably may be water. For example, the extraction process may be performed at 50 ° C. to 150 ° C., or 75 ° C. to 120 ° C., or 90 ° C. to 115 ° C., but is not limited thereto. In addition, the extraction time is not particularly limited, but may be 10 minutes to 12 hours, or 30 minutes to 8 hours, or 2 hours to 6 hours.

The honey leaf extract of the present invention may be prepared according to a conventional method of producing a plant extract, and specifically, may be a heat extraction method including a hot water extraction method, a cold extraction method, a warm extraction method, an ultrasonic extraction method, etc. Grinding extractors or fractionators may be used.

In addition, the extract extracted with the solvent may be further subjected to the fractionation process with any one solvent selected from the group consisting of hexane, chloroform, methylene chloride, ethyl acetate, ethyl ether, acetone, butanol, water and mixtures thereof. . Two or more solvents may be used in the fractionation, and each solvent extract may be prepared by sequentially using or mixing the solvent according to the polarity of the solvent.

The thus-prepared extract or the fraction obtained by performing the fractionation process may be filtered, concentrated or dried to remove the solvent, and may be subjected to both filtration, concentration and drying. Specifically, the filtration may be using a filter paper or a reduced pressure filter, the concentration may be concentrated under reduced pressure using a reduced pressure concentrator, for example, a rotary evaporator, the drying may be carried out by freeze drying as an example.

The composition for treating, improving or preventing arthritis may be used as a medicament, or may be used for medicinal or pharmaceutical use. In this aspect, the composition for treating or preventing arthritis may be a medicinal composition, and for example, may be a pharmaceutical composition. .

Arthritis is a common disease in the joints and includes, for example, osteoarthritis, rheumatoid arthritis, degenerative arthritis, or polyarthritis.

The osteoarthritis is the most common cause of chronic inability after middle age due to pain and stiffness, and the basic pathological process is degenerative, unlike inflammatory rheumatoid arthritis.

The incidence of rheumatoid arthritis does not differ in race and geographic location, but the incidence of females is higher than that of males, especially between 35 and 45 years of age, and the difference in gender between men and women decreases with age.

When the composition for treating or preventing arthritis is used for medicinal or pharmaceutical use, the composition for treating or preventing arthritis is used to inhibit or treat or prevent inflammation, and in particular to treat, improve, prevent or alleviate arthritis. Can be.

In this aspect, the present invention may be directed to a pharmaceutical composition for treating or preventing visceritis including honey leaf extract as an active ingredient. The pharmaceutical composition comprising the honey leaf extract as an active ingredient may be used to inhibit, treat or prevent arthritis.

In the composition for treating or preventing arthritis comprising the honey leaf extract as an active ingredient or an anti-inflammatory agent comprising the honey leaf extract as an active ingredient, the honey leaf extract may be a honey leaf hydrothermal extract.

Arthritis treatment or prevention composition comprising the honey leaf extract as an active ingredient can be applied directly to animals, including humans. The animal is a group of organisms corresponding to plants. It mainly consumes organic matter as nutrients, and differentiates digestive organs, excretory or respiratory organs. Preferably, it is a mammal, more preferably a human.

The honey leaf extract may be used alone in the composition for treating or preventing arthritis, and may further include other pharmacologically acceptable carriers, excipients, diluents or subcomponents.

More specifically, when the composition comprising the honey leaf extract is used as a medicament, or for medicinal or pharmaceutical use, the honey leaf extract is mixed with a pharmaceutically acceptable carrier or excipient according to a conventional method or It can be used diluted with a diluent.

In this case, the content of the honey leaf extract in the composition may be 0.001% by weight to 99.9% by weight, 0.1% by weight to 99% by weight or 1% by weight to 50% by weight, but is not limited thereto. Depending on the method, the content of the extract can be used by appropriately adjusting to the desired content.

Examples of the pharmaceutically acceptable carrier, excipient or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , Dextrin, calcium carbonate, propylene glycol, liquid paraffin, and one or more selected from the group consisting of physiological saline, but is not limited thereto, and all common carriers, excipients or diluents can be used. Carrier or excipient may be used two or more kinds.

In addition, the pharmaceutical composition is conventional fillers, extenders, binders, disintegrants, anti-coagulants, lubricants, wetting agents, pH adjusting agents, nutrients, vitamins, electrolytes, alginic acid and salts thereof, pectic acid and salts thereof, protective colloids, glycerin , Fragrances, emulsifiers or preservatives may be further included. The components may be added independently or in combination to the honey leaf extract is the active ingredient.

In addition, the composition for treating or preventing arthritis of the present invention is a substance recognized to have an effect of treating, preventing or improving arthritis in addition to the active ingredient, for example, an arthritis agent, an osteoclast differentiation inhibitor, an anti-inflammatory agent, an NO inhibitor, or COX-2. It may further include a substance used as an inhibitor.

In addition, the composition for treating or preventing arthritis of the present invention may further include a compound or plant extract having a known arthritis treatment, prophylaxis or improvement effect, in addition to the active ingredient, each 0.1 part by weight based on 100 parts by weight of the active ingredient. To 99.9 parts by weight or 0.5 to 20 parts by weight.

When the composition is used as a medicine, it can be administered orally or parenterally. In one example, it can be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular.

In addition, the formulations of the compositions may vary depending on the method of use and may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal .

In general, solid dosage forms for oral administration include tablets, pills, soft or hard capsules, pills, powders and granules, which may contain one or more Excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, Sweeteners, fragrances, preservatives, and the like.

Forms for parenteral administration include creams, looses, ONITMENTS, PLASTERS, LIQUIDS AND SOULTIONS, AEROSOLS, FRUIDEXTRACTS, (ELIXIR), INFUSIONS, SACHET, PATCH or INJECTIONS.

Furthermore, the compositions of the present invention may be formulated using any suitable method known in the art or using methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA .

The dosage of the composition may be determined in consideration of the administration method, the age, sex, the severity of the patient, the condition of the patient, the degree of absorption of the active ingredient in the body, the inactivity and the drug to be used in combination. 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, And may be administered once or several times in divided doses. The dose is not intended to limit the scope of the invention in any way.

In addition, the composition for improving or preventing arthritis of the present invention can be applied as a food composition for preventing or improving arthritis. The food composition may be, for example, a health functional food composition for preventing or improving the arthritis.

In this aspect, the present invention may be a health functional food composition for improving or preventing arthritis, including a honey leaf extract as an active ingredient.

The health functional food is a physical, biochemical, biotechnological method, such as using the physical, biochemical, biotechnological techniques, such as food groups or food composition that has added value to give a function and expression of the function of the food for a specific purpose, disease prevention and recovery, etc. It refers to a food that is designed and processed to fully express the function of the gymnastics. The health functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of health functional foods.

Health functional food composition for preventing or improving arthritis of the present invention is the honey leaf extract 0.001% to 99.9% or 0.01% to 50% or 0.1% to 30% or 0.1% by weight of the total food weight To 15% by weight may be included.

Therefore, the arthritis improvement or prevention composition or honey leaf extract containing the honey leaf extract as an active ingredient may be used as an active ingredient of a food composition having an arthritis improvement and relief effect.

The honey leaf extract of the present invention is a plant-derived extract used for edible, and has no problems with side effects or safety, and as a result of MTT analysis, there is no cytotoxicity, and it is easy to produce because the yield is higher than that of the honey fruit. It is possible not only to inhibit related anti-inflammatory effects, specifically NO secretion, to effectively inhibit the expression of iNOS, to significantly inhibit the activity of COX-2 enzyme, but also to inhibit the differentiation of osteoclasts in connection with the treatment, improvement or prevention of arthritis. That is, it was confirmed that osteoclasts can inhibit TRAP activity and inhibit differentiation.

In this aspect, the composition for treating, improving or preventing arthritis, comprising the honey leaf extract as an active ingredient, inhibits inflammation and inhibits the differentiation of osteoclasts, thereby improving or preventing a pharmaceutical composition or arthritis for treating or preventing arthritis. It can be used as a functional food composition for.

Therefore, the present invention can be widely used in the medical industry related to the treatment for arthritis and in the field related to the functional food related to arthritis improvement or prevention.

1 is a schematic diagram showing a process for preparing a honey leaf hydrothermal extract and a solvent fraction of the hydrothermal extract according to an embodiment of the present invention.
Figure 2 is a graph showing the results of measuring the cytotoxicity of the extract of each part of the honey by MTT assay, using the RAW264.7 cell line according to an embodiment of the present invention, the horizontal axis in the graph is the extract for each part of the honey Is the numerical value representing the dose (μg / mL), and the vertical axis is the numerical value (%) indicating the number of cells compared to the control group to which no sample was administered.
3 is a graph showing the results of measuring inflammation-related cytokine iNOS mRNA level in order to confirm the anti-inflammatory effect of the honey leaf hot water extract according to an embodiment of the present invention, + is LPS (1 μg / mL) or Means that the solvent fraction has been treated,-means that the sample has not been treated, and the horizontal axis value indicates the dose of the honey leaf hydrothermal extract (μg / mL).
Figure 4 is a graph showing the results of measuring the expression level of iNOS and COX-2, in order to confirm the anti-inflammatory effect of the honey leaf hot water extract according to an embodiment of the present invention, + is LPS (1 ㎍ / mL) Or means that the solvent fraction has been treated,-means that the sample has not been treated, and the horizontal axis value indicates the dose of the honey leaf hydrothermal extract (μg / mL).
Figure 5 is a graph showing the results of measuring the inhibitory activity of COX-2 through the activity of COX-2 in order to confirm the anti-inflammatory effect of each solvent fraction of the honey leaf hot water extract according to an embodiment of the present invention , The solvent that separates each graph means a fraction solvent, the horizontal axis represents the elapsed time after treatment, and the vertical axis represents the activity of COX-2.
Figure 6 is a graph showing the results of measuring the inhibitory activity of COX-2 through the activity of COX-2, in order to confirm the anti-inflammatory effect of the extract for each honey site according to an embodiment of the present invention, each graph is divided One solvent refers to the fractional solvent, the horizontal axis represents the elapsed time after treatment, and the vertical axis represents the activity of COX-2.
7 is a graph showing the pharmacological efficacy according to the yield per g of the extract according to an embodiment of the present invention, extracts for each honey site, from the inhibitory activity of COX-2 measured through the activity of COX-2 It is a graph showing the calculated results, the horizontal axis shows the extract type of each region, the vertical axis shows the pharmacological effect compared to the honey leaf hydrothermal extract with the best effect.
8 is a graph measuring TRAP activity of osteoclasts in order to confirm the therapeutic effect of osteoarthritis of honey leaf hot water extract according to an embodiment of the present invention, in which the horizontal axis is the dose of honey hot water extract (μg) / mL), and the vertical axis represents% of activity compared to the control group to which no sample was administered.
9 is a photograph of measuring the degree of differentiation of osteoclasts in order to confirm the therapeutic effect of osteoarthritis of honey leaf hot water extract according to an embodiment of the present invention, the numerical value described at the bottom of the photo is the dose of honey hot water extract It is a numerical value showing (microgram / mL).
10 is a photograph showing the therapeutic effect of arthritis in the experimental animal according to the administration of collagen and honey leaf hydrothermal extract, in order to confirm the therapeutic effect of the honey leaf hydrothermal extract according to an embodiment of the present invention, + Or means that the sample was administered,-means that no drug or sample was treated, and the value means the dosage (μg / mL) of the honey leaf hydrothermal extract.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to specific examples and comparative examples in order to help understanding of the present invention. However, the following examples are only intended to more clearly understand the present invention, and are not limited to the following examples of the present invention, the protection scope of the present invention should be interpreted by the claims, and within the equivalent range All technical thoughts should be construed as being included in the scope of the present invention.

< Example  1> Honey  Leaf extract and Fraction  Produce

1-1. Honey  Leaf extract manufacturer

A hot water extract was prepared by performing hot water extraction at 110 ° C. using 10 kg of leaves of Stauntonia hexaphylla .

More specifically, after adding 200 L of distilled water to 10 kg of dried honey leaf dried water washed with distilled water, hot water extraction was performed while heating at 110 ° C. for 4 hours using an electric bath. After performing the extraction, the resultant was filtered through a 400 mesh filter cloth, and the filtrate was concentrated using a vacuum concentrator. After filtration, using the same amount of distilled water again to the remaining residue was performed twice more extraction, filtration and reduced pressure in the same process.

The honey leaf hot water extract prepared through the above process was lyophilized in a freeze dryer. Through the lyophilization, 1 kg of honey leaf hydrothermal extract was obtained, and as a result, the yield by the honey leaf hydrothermal extraction method was found to be 19.56%.

In addition, other parts of the honey, that is, the honey fruit, honey root and honey bees were obtained by the same method, washed and dried, and then the extract was prepared in the same manner as described above. As a result of the manufacturing method, the yield of the honey fruit was 7.05%, 7.91% for the stem, 3.05% for the root was confirmed by the extract.

1-2. Honey  Of leaf extract Fraction  Produce

Preparation of fractions of the honey leaf hot water extract was carried out by the production method shown in FIG.

Specifically, 250 g of the hot water extract is completely dissolved in 5 L of distilled water, and then put into a fraction filter and 5 L of hexane (Hexane) is added and mixed, and then fractionated. The hexane soluble layer, the hexane layer and the hexane insoluble layer, are separated. The hexane fraction was prepared by separating and obtaining only the hexane layer.

5 L of chloroform was added to the remaining solution (aqueous layer), followed by mixing. The chloroform fraction was prepared by separating the chloroform layer, which is a chloroform soluble layer, and the aqueous layer, which is a chloroform insoluble layer, to obtain only a chloroform layer.

5 L of ethyl acetate was added to the remaining solution (aqueous layer), followed by mixing, fractionating, separating an ethyl acetate layer as an ethyl acetate soluble layer and an aqueous layer as an ethyl acetate insoluble layer, and obtaining only an ethyl acetate layer to prepare an ethyl acetate fraction. It was.

Butanol fraction was prepared by adding 5 L of butanol to the remaining solution (aqueous layer), followed by mixing, separating the butanol layer as the butanol soluble layer and the water layer as the butanol insoluble layer, and obtaining only butanol layer.

The butanol soluble layer was fractionated to separate the remaining butanol insoluble layer to remove the remaining organic solvent, thereby preparing a water fraction.

Each of the obtained fractions was concentrated by filtration through a vacuum filter, and then freeze-dried at -20 ° C to completely remove the solvent, and then used in this experiment. Throughout this process 0.02 g of hexane fraction (0.015%), 0.67 g of chloroform fraction (0.27%), 2 g of ethyl acetate fraction (1.05%), 68.75 g of butanol fraction (27.5%) and 150.14 g of water fraction (60.06%) was obtained and used as a sample. The butanol fraction and the water fraction in the manufacturing process is confirmed that the excellent yield, because the economic efficiency is recognized due to the high yield in the efficiency of fraction production is evaluated to be industrially excellent.

The extracts and fractions obtained were cryopreserved until use in experiments.

< Example  2> Cytotoxicity test of extract

In order to measure the cytotoxicity of hot water extract of each site of the honey prepared in Example 1, rat macrophage RAW264.7 cells were purchased from ATCC.

DMEM / F12 (Dulbecco's modified Eagle's medium / Nutrient Mixture Ham's F12), FBS (fetal bovine serum), L-glutamine (L-glutamine) and penicillin-streptomycin used in the cell culture were Gibco / BRL (USA).

The RAW264.7 cells were cultured using a medium containing 10% FBS, 1% penicillin streptomycin and 1% L-glutamine in DMEM / F12 medium, and a 37 ° C. wet CO₂ incubator (5% CO₂ / 95% air). Incubated).

After about 80% of the cells were incubated with the cells, the monolayers of cells were washed with PBS (pH 7.4), washed, and subcultured with 0.25% trypsin and 2.56 mmol / L EDTA. Medium was changed every two days.

The cultured cells were aliquoted into 48 well-plates at a density of 50,000 cells / well and further cultured for 24 hours. After 24 hours, the control group treated only LPS without any treatment and the hot water extract of each part of the honeycomb of LPS and each part of Example 1 at various concentrations using DMSO confirmed to have no effect on cell survival. Divided into experimental groups treated with hot water extract for each part of the prepared honey, further cultured for 24 hours, the culture solution was removed and the number of living cells was measured by MTT assay (MTT assay) method. The MTT analysis was performed in the following manner.

First, the cell culture medium was removed, and then treated with 1 ml per well of DMEM / F12 medium containing MTT at 1 mg / ml, and further incubated in a 37 ° C. wet CO 2 incubator for 4 hours. After removing the medium, the tetrazolium bromide salt was removed, and 200 μl of DMSO was dissolved to dissolve the formazan crystal produced in each well, and the cell viability was measured by measuring absorbance at a wavelength of 540 nm in a microplate reader (BIO-RAD). Confirmed.

The results of treatment with the hydrothermal extract for each part of the honey was performed in the same manner three times, shown in Figure 2 as the average value of the measured value.

As shown in FIG. 2, the honey leaf hydrothermal extract prepared in Example 1-1 was treated at various concentrations, specifically, the honey leaf hydrothermal extract at a concentration of 10 μg / ml to 200 μg / ml and elapsed for 24 hours. In any case, it was confirmed that all samples did not show any effect on the proliferation of the cells as compared with the control without LPS treatment. Therefore, it was confirmed from the results that the honey leaf extract is not cytotoxic up to 200 ㎍ / ㎖.

In addition, the extracts of other parts did not show any toxicity until 50 ㎍ / ml treatment, and 100 ㎍ / ml and 200 ㎍ / ml showed weak toxicity in fruits and branches. It was confirmed that it did not show a great influence on the growth of.

< Example 3 > Inflammatory cytokines mRNA  Determination of anti-inflammatory effect by measuring level

In order to confirm the anti-inflammatory effect of the honey leaf hydrothermal extract was confirmed that the toxicity is not a problem in Example 2, the change in the mRNA content of cytokines (cytokine), specifically iNOS associated with the inflammatory response macrophages (primary cell) It was confirmed using.

In order to obtain the primary cells, 32 rats (ICR mouse, male) and Sprague-Dawley rats weighing 15 g to 20 g each were purchased from Samtaco Inc. (Korea), Each group was divided into 16 groups, and 4 animals were placed in each group for breeding. The breeding of the experimental animals was carried out under a temperature condition of 20 ℃ to 24 ℃, humidity conditions of 60% to 70% and day and night lighting conditions of 12 hours cycle, water and diet were to be ingested freely. The diet used a solid feed (Samyang feed, South Korea). The experimental animals were bred under the above conditions for 7 days to adapt to the laboratory environment, and then used for the experiment.

Macrophages obtained from the experimental animals (Macrophage primary cells, 2 X 10 ⁶ cells / ml) were incubated with serum starvation medium for 24 hours. After the incubation, and treated with LPS (0.5 mg / ml) or LPS (0.5 mg / ml) and honey leaf hydrothermal extract of various concentrations, it was incubated for 24 hours. After 24 hours, RNA was isolated from each of the cultured cells. Isolation of the RNA was carried out in the following manner.

Specifically, the cultured cells were lysed with GIT solution (easy BLUE Total RNA extraction kit, Intron Biotechnology Co., Ltd.), centrifuged at 10,000 rpm at room temperature for 5 minutes, and then the supernatant was removed and pelleted. (pellet) was obtained. 1 ml of 0.1% DEPC solution (Sigma, USA) was added to the pellet, and centrifuged again at 12,000 rpm for 2 minutes to remove the supernatant, and then pellets were obtained. 0.5 ml of guanidinium was added to the obtained pellets, followed by vortexing. Further, 0.5 ml of a phenol / chloroform / iso-amylalchol mixed solution (25: 24: 1) was added, vortexed, and centrifuged at 12,000 rpm for 3 minutes to obtain a supernatant. The supernatant and the same amount of isopropyl alcohol (iso-propylalcol) was added and mixed well, and left to stand at -20 ° C for 30 minutes. Thereafter, the supernatant was removed by centrifugation at 12,000 rpm for 10 minutes, and then the pellet was washed with 70% aqueous ethanol solution and dried under vacuum to separate RNA.

The isolated RNA was dissolved in 1 ml of 0.1% DEPC solution and used to measure mRNA content of inflammation-related cytokines. The mRNA content of iNOS, an inflammation-related cytokine, was measured by the following method.

Superscript II reverse transcriptase (Invitrogen, USA) was added to 3 μg of the isolated RNA, incubated at 42 ° C. for 1 hour and 45 minutes, and then incubated at 70 ° C. for 15 minutes to obtain cDNA. The obtained cDNA was quantified by real-time PCR. Sequence and experimental conditions of the primer for performing the real-time PCR are described in Table 1 below.

Target mRNA primer sequence Annealing Tm (℃) iNOS sense CAGAGGACCCAGAGACAAG 50.8 anti-sense ACCTGATGTTGCCATTGTTG

The real-time PCR result is shown in Figure 3 the result taken with a picture compared with the β-actin content.

As shown in FIG. 3, in the control group not treated with LPS, mRNA of iNOS, a cytokine associated with inflammation, was not identified at all, whereas mRNA of iNOS was significantly higher when only LPS was treated. In addition, despite the LPS treatment, when the honey leaf hot water extract prepared in Example 1 was treated, it was confirmed that the mRNA content of iNOS was reduced depending on the concentration. When the honey leaf hydrothermal extract was treated, the mRNA content of iNOS was reduced in a concentration-dependent manner, and the honey leaf hydrothermal extract was found to have an excellent anti-inflammatory effect.

< Example 4 > Inflammation iNOS  And COX -2 Expression Inhibition Activity Confirmation

In order to confirm the anti-inflammatory effect of the honey leaf hydrothermal extract was confirmed that the anti-inflammatory effect is excellent through the mRNA content reduction effect of iNOS in Example 3, iNOS and COX2 expression inhibitory activity was confirmed.

Specifically, the macrophage (Macrophage primary cell) obtained in Example 3 was adjusted to 1 X 10 ⁵ cells / ml using DMEM medium and then inoculated in a 24 well plate, 18 hours ago in a 5% CO ₂ thermostat Incubated. After the cultivation, the honey leaf hydrothermal extract was treated by concentration (0.1 ㎍ / ㎖, 1 ㎍ / ㎖, 10 ㎍ / ㎖, 100 ㎍ / ㎖ and 200 ㎍ / ㎖), and after 1 hour incubation LPS (1 ㎍ / ㎖) Ml) was treated and cultured under the same conditions as the pre-culture. After 24 hours, the cultured cells were collected and washed three times with phosphate buffered saline (PBS), followed by cell lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA). , 1 mM EGTA, 1 mM NaVO ₃ , 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 μg / ml aprotinin, 25 μg / ml leupeptin) were added and dissolved at 4 ° C. for 30 minutes at 4 ° C. and 15,000 rpm. Cell membrane components and the like were removed by centrifugation for 15 minutes at.

Protein concentration was quantified using the Bio-Rad Protein Assay Kit by standardizing bovine serum albumin (BSA). 20 μg of the isolated protein was loaded onto 10% mini gel SDS-PAGE and denatured, and transferred to nitrocellulose membrane (membrane, BIO-RAD, Richmond, CA, USA) for 1 hour at 350 mA. The blocking of the membrane to which the protein was moved was performed at room temperature for 2 hours in a TTBS (0.1% Tween 20 + TBS) solution containing 5% skim milk.

Anti-mouse iNOS (Calbiochem, La Jolla, USA) was used as an antibody to examine the expression level of iNOS, and anti-mouse COX-2 (BD Biosciences Pharmingen, SanJose was used as an antibody to examine the expression level of COX-2. , USA) was diluted 1: 1000 in a TTBS solution and reacted at room temperature for 2 hours, and then washed three times with TTBS. As a secondary antibody, HRP (horse radish peroxidase) conjugated anti-mouse IgG (Amersham Pharmacia Biotech, LittleChalfont, UK) was diluted 1: 5000, reacted at room temperature for 30 minutes, washed three times with TTBS, and then ECL substrate. After 30 seconds of reaction with (Amersham Biosciences, Piscataway, NJ, USA), the amount of expression was measured using a chemiluminescence imaging system (ATemi AE-9150 EZ-Capture II, Japan). The result of measuring the expression amount is shown in FIG.

As shown in FIG. 4, the control group not treated with LPS, i.e., iNOS and COX-2, was not identified at all, whereas the treatment with LPS alone showed significantly higher iNOS and COX-2. In addition, despite the LPS treatment, it was confirmed that the iNOS and COX-2 contents were reduced depending on the concentration when the honey leaf hydrothermal extract prepared in Example 1 was treated. When the honey leaf hydrothermal extract was treated, it was confirmed that the honey leaf hydrothermal extract had an excellent anti-inflammatory effect from concentration-dependently reduced iNOS and COX-2 contents.

< Example 5 > Fraction COX -2( Cyclooxygenase enzyme -2) Confirmation of inhibitory effect

In order to confirm the anti-inflammatory effect of the fractions of each fraction solvent of the honey leaf hot water extract was confirmed to have excellent anti-inflammatory effect in Example 3 and Example 4, COX-2 enzyme inhibitory activity was confirmed.

First, five-week-old Sprague-Dawley male rats (Samtako, South Korea) were used in the experiment after adapting to the environment for one week. The breeding of the experimental animals was carried out under a temperature condition of 20 ℃ to 24 ℃, humidity conditions of 50% to 55% and day and night lighting conditions of 12 hours cycle, water and diet were to be ingested freely. The diet used a solid feed (Samyang feed, South Korea). The experimental animals were bred under the above conditions for 7 days to adapt to the laboratory environment, and then used for the experiment.

10 ml of 4% thioglycolate was injected into the abdominal cavity of the experimental animals (SD male rats), and the peritoneal macrophages were propagated for 3 days, followed by cervical dislocation. Peritoneal macrophages were collected from SD male rats prepared by cervical dislocation.

Specifically, 10 ml of HBSS was injected into the abdominal cavity, and then the abdominal macrophages were taken using a syringe (syringe), and then transferred to a conical tube. Peritoneal macrophages were centrifuged at 13,000 rpm for 5 minutes, washed twice with DMEM medium, aliquoted in a 60 mm diameter petri dish, and incubated in a CO ₂ cell incubator for 4 hours. After incubation, the suspended cells were removed and the adhered cells were stabilized for 24 hours, after which the proteins were separated and used.

50 mg / ml each of the fractions of the honey leaf hot water extract obtained in Example 1 was treated to the separated protein, stabilized for 30 minutes, and then in a method according to COX Fluorescent Activity Assay Kit (Cayman ChemiaclCpmpany, Item No. 700200). Cyclooxygenase enzyme activity was measured. 5 shows the results of enzyme activity measurement.

As shown in FIG. 5, the water fraction of the honey leaf hydrothermal extract was found to have a low inhibitory effect, and the inhibitory activity was also decreased in the hexane and butanol fractions, whereas the ethyl acetate fraction and the chloroform fraction were It was confirmed that the inhibitory activity was remarkably excellent. In particular, it was confirmed that the difference in the inhibitory activity is remarkable with time. In particular, the ethyl acetate fraction of the honey leaf hydrothermal extract was found to have the best COX-2 inhibitory activity.

< Example 6 > Extraction by Part COX -2( Cyclooxygenase enzyme -2) Confirmation of inhibitory effect

In relation to the honey leaf hot water extracts confirmed to have excellent anti-inflammatory effects in Examples 3 and 4, in order to compare the anti-inflammatory effects with other sites, COX-2 enzyme inhibitory activity was confirmed.

The breeding of five-week-old Sprague-Dawley male rats (Samtako, Korea) and the method of collecting celiac macrophages from the experimental animals were performed in the same manner as in Example 5.

10 mg / ml or 100 mg / ml of the hot water extract for each part of the honey honey obtained in Example 1 was treated to the separated protein and stabilized for 30 minutes, followed by COX Fluorescent Activity Assay Kit (Cayman ChemiaclCpmpany, Item No. 700200). Cyclooxygenase enzyme activity was measured by the method according to the following steps. 6 shows the results of enzyme activity measurement. In addition, the efficacy associated with the anti-inflammatory effect from the above results are shown as IC ₅₀ values, and are shown in Table 2 below.

Extract Target Area IC 50 (ug / ml) Fruit 70.41 Leaf 8.46 Stem 49.74 Root 66.04

As shown in FIG. 6 and Table 2, the honey leaf hydrothermal extract was found to be remarkably excellent anti-inflammatory effect compared to other sites, in particular, the anti-inflammatory effect of the known honey root extract, as well as remarkably excellent compared to other sites. It was confirmed that there is.

Specifically, IC ₅₀ inhibits the activity of COX-2 enzymes. As a result, the honey leaf hydrothermal extract was 8.3 times higher than the honey fruit hydrothermal extract, 5.8 times higher than the honey stem hydrothermal extract and 7.8 times higher than the honey root hydrothermal extract.

Compared in consideration of the production yield of the extract confirmed in Example 1, when the efficacy of the honey leaf to 100%, the amount of raw materials required to show the same anti-inflammatory effect for each site raw material for each site of the honey site g Results shown in terms of sugar physiological efficacy are shown in FIG. 7. As shown in FIG. 7, it was confirmed that the honey leaf leaves 8 times higher in pharmacological efficacy than other parts, and the honey leaf is not only excellent in anti-inflammatory effect compared to other parts, but also in industrial aspects such as raw material requirements and manufacturing cost. The effect was found to be very good.

< Example 7 > Confirmation of osteoarthritis treatment effect using osteoclasts

Confirmation of arthritis treatment effect of the honey leaf hot water extract was confirmed that the anti-inflammatory effect is excellent in the above embodiment.

Specifically, osteoclasts closely related to the cause of arthritis have a tartrate-resistant acid phosphatase (TRAP), an acid phosphatase that is characteristically resistant to tartrate, which is associated with other bone tissue cells. It is known to be used as a cytochemical marker for distinguishing osteoclasts. Therefore, the effect of treating osteoarthritis was confirmed through inhibition of TRAP activity of the osteoclasts.

First, 5 weeks old ICR Mouse (Samtako, South Korea) was used in the experiment after adapting to the environment for one week. The breeding of the experimental animals was carried out under a temperature condition of 20 ℃ to 24 ℃, humidity conditions of 50% to 55% and day and night lighting conditions of 12 hours cycle, water and diet were to be ingested freely. The diet used a solid feed (Samyang feed, South Korea). The experimental animals were bred under the above conditions for 7 days to adapt to the laboratory environment, and then used for the experiment.

Tibia of the ICR mouse was extracted, both ends were cut and bone marrow cells were collected by passing through α-MEM essential medium and treated with 50 ng / mL macrophage-colony stimulation factor (M-CSF). Incubated for 24 hours. Unattached cells were washed with α-MEM and then dispensed into 96 wells at 5 × 10 ⁴ cells / well and incubated in α-MEM medium to which 50 ng / mL of M-CSF was added for 3 days. Thereafter, 50 ng / mL of MCSF and 100 ng / mL of RANKL (Receptor Activator for Nuclear Factor κB Ligand) were treated together, and the samples were treated by concentration and incubated for 4 days.

After removing the medium and washing with PBS, 100 μL of substrate solution (1.36 mg / mL 4-nitrophenyl phosphate disodium salt, 10 mM tartrate, 50 mM citrate buffer pH 4.6) was added to the cell fixed with 10% formaldehyde at 37 ° C. The reaction was carried out for 30 minutes. After the reaction, the enzyme reaction solution was transferred to a new plate and the absorbance was measured at 405 nm by stopping the reaction with 0.1 N NaOH. TRAP activity is shown in FIG. 8 by expressing the absorbance of the sample as a percentage of the absorbance of the control.

As shown in FIG. 8, it was confirmed that the honey leaf extracts of the present invention acted to inhibit the production of osteoclasts in a concentration-dependent manner. Specifically, TRAP activity was hardly confirmed in the control group that did not undergo differentiation due to RANKL treatment, while remarkably high TRAP activity was confirmed when differentiated through RANKL treatment. In addition, even when differentiated through RANKL treatment, when the honey leaf hot water extract prepared in Example 1 was treated, it was confirmed to decrease TRAP activity in a concentration-dependent manner, in particular, even when only 100 ug / ml treatment It was found that the TRAP activity was reduced to about the same level as, and from the above results, the honey leaf hydrothermal extract was found to have an excellent arthritis therapeutic effect.

< Example 8 > Confirmation of arthritis treatment effect by suppressing osteoclast differentiation

After treating the differentiation factor and the sample in Example 7 and incubating the honey leaf hydrothermal extract to the osteoclasts induced differentiation, the degree of differentiation of the cells was confirmed, and the arthritis treatment effect was confirmed by inhibiting the differentiation of the osteoclasts. It was.

First, after treating the differentiation factor and the sample to the cells isolated in Example 7 and incubated for 4 days, the medium was removed, washed with PBS and then the substrate solution (1.36 mg / mL 4-nitrophenyl) in the cell fixed with 10% formaldehyde Phosphate disodium salt, 10 mM tartrate, 50 mM citrate buffer pH 4.6) was dispensed in 100 μL each reaction for 30 minutes at 37 ℃. Thereafter, the medium was removed, washed with PBS, fixed with cells for 10 minutes with 10% formaldehyde (formaldehyde), and washed three times with PBS.

TRAP staining was performed on the fixed cells. TRAP staining was prepared by adding 1 mg / mL naphtol AS-MX phosphate and 100 μL of N, N-dimethylformamide to 10 mL of 50 mM sodium acetate buffer containing 50 mM tartrate acid, and then using 10% formaldehyde. 45 μL of the staining solution was dispensed to the fixed cells and left to stand at 37 ° C. for 30 minutes. After the staining, the stained cells were photographed by observing the microscope under a microscope.

As shown in FIG. 9, TRAP-positive multinucleated osteoclasts were formed in the control cells, but it was found that differentiation of osteoclasts was inhibited in a concentration-dependent manner in the experimental group treated with honey leaf hydrothermal extract. From the above results, it can be confirmed that the honey leaf hydrothermal extract of the present invention acts to inhibit the production of osteoclasts in a concentration-dependent manner.

< Example 9 > Effect of Collagen-Induced Arthritis on Animals

Using the honey leaf hydrothermal extract confirmed that the osteoarthritis-related arthritis treatment effect was excellent in the above example, the arthritis treatment effect of the experimental animals induced by the treatment of collagen II (Collagen II) was confirmed.

First, 8-week-old male DBA1 / J mice (Central Laboratory Animal Lab, Korea) were used in the experiment after being adapted to the environment for 1 week. The breeding of the experimental animals was carried out under a temperature condition of 20 ℃ to 24 ℃, humidity conditions of 60% to 70% and day and night lighting conditions of 12 hours cycle, water and diet were to be ingested freely. The diet used a solid feed (Samyang feed, South Korea). The experimental animals were bred under the above conditions for 7 days to adapt to the laboratory environment, and then used for the experiment. The experimental animals were divided into a total of three groups to perform the experiment.

Collagen-induced arthritis (CIA) is mixed with bovine type II collagen with 100 mM acetic acid and complete Freund's adjuvant (CFA), followed by subcutaneous injection of 100 μl (1.5 mg / ml) for each experimental animal. After 21 days, 100 μl of the same amount was induced by subcutaneous injection (boosting) again. According to the collagen-induced arthritis or not, five experimental groups that did not cause arthritis, five animals that did not cause any arthritis, and treated with arthritis, and then divided into experimental groups administered with 200 mg / kg honey leaf hydrothermal extract Was carried out. The experimental animals that did not receive the sample were administered orally once daily each physiological saline, and the experimental group was orally administered at 10 am daily for 24 days at 200 mg / kg concentration of honey leaf hot water extract every day. For 42 days after the experimental animals, the progress of arthritis was observed, and the results are shown in FIG. 10.

As shown in FIG. 10, when the honey leaf hot water extract was orally administered daily (200 mg / kg), edema was hardly generated as compared to the group in which arthritis was induced by collagen administration and did not cause arthritis. It was confirmed to be similar to the animal, it was confirmed that the honey leaf hot water extract of the present invention has an action to inhibit arthritis.

According to the results of the experiment, the extract of honey leaves that can be used for food, as expected, was found to have no cytotoxicity in the MTT assay, and related to the mRNA transcription and NOS expression levels of iNOS that cause inflammation in relation to the anti-inflammatory effect In consideration of the results, it was confirmed that it can effectively inhibit inflammation, in particular, the anti-inflammatory effect is very excellent in the honey leaf compared to other sites, and in addition to the therapeutic effect of arthritis, TRAP inhibitory activity and osteoclasts of osteoclasts It has been confirmed that the differentiation inhibitory activity of the cells, and effectively inhibit the collagen-induced arthritis through experimental animals, has been found to have a remarkably excellent treatment, prevention or improvement of arthritis, the honey leaf extract is an existing inflammatory treatment To replace anti-inflammatory agents, especially for the treatment, prevention or amelioration of arthritis It is expected to be used.

Claims (4)

Honey ( Stauntonia) Hexaphylla ) A pharmaceutical composition for treating or preventing arthritis, comprising the leaf extract as an active ingredient. The method of claim 1,
The honey leaf extract is a pharmaceutical composition for treating or preventing arthritis, which extracts one or more selected from the group consisting of water, alcohol having 1 to 5 carbon atoms, and mixtures thereof. Honey ( Stauntonia) Hexaphylla ) Food composition for improving or preventing arthritis comprising leaf extract as an active ingredient. The method of claim 3,
The honey leaf extract is a food composition for improving or preventing arthritis that extracts one or more selected from the group consisting of water, alcohol having 1 to 5 carbon atoms, and mixtures thereof.

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