KR20200022422A - Pharmaceutical composition for anti-inflammatory, and improvement of bone tissue generation or cartilage tissue generation comprising purified fraction materials from Stauntonia hexaphylla leaves or flavonoid compounds and caffeic acid compounds separated therefrom as active ingredients - Google Patents

Abstract

The present invention relates to: a manufacturing method of a Stauntonia hexaphylla leaf-derived extract or a fractional purified product; the Stauntonia hexaphylla leaf-derived extract or the fractional purified product manufactured by the manufacturing method; and a pharmaceutical composition for anti-inflammation, or promotion of bone tissue formation or cartilage tissue formation, which contains at least one selected from a group consisting of the Stauntonia hexaphylla leaf-derived extract, the fractional purified product, or a caffeic acid compound and a novel flavonoid compound separated from the extract or the fractional purified product as active components.

Description

Anti-inflammatory, or osteogenic or cartilage tissue-promoting pharmaceutical composition comprising an extract or fraction purified from honey leaf or a novel flavonoid compound and caffeic acid compound isolated therefrom as an active ingredient {Pharmaceutical composition for anti-inflammatory, and improvement of bone tissue generation or cartilage tissue generation comprising purified fraction materials from Stauntonia hexaphylla leaves or flavonoid compounds and caffeic acid compounds separated therefrom as active ingredients}

The present invention provides a method for preparing an extract or fractional purified extract of the honey (Stauntonia hexaphylla) leaf, an extract or fractional purified product derived from the honey leaf prepared by the method, and the extract or fractional purified product derived from the honey leaf, or the extract or fractional purification The present invention relates to a pharmaceutical composition for promoting anti-inflammatory or bone tissue production or cartilage tissue production, comprising as an active ingredient one or more selected from the group consisting of caffeic acid compounds and novel flavonoid compounds separated from water.

Bone tissue is generally made of dense bone with a strong surface of the bone, the center or both ends of the long bone (長 骨) is composed of the spongy bone associated with the bone like mesh. Most bones initially develop as cartilage in connective tissue, which later turns into bone tissue, some of which are made directly from connective tissue. In the epiphysis, the joint surface is in contact with the neighboring bone, and the surface is covered with the articular cartilage, which is a supernatural bone. Sea sponge shochu in the sponge is characterized by a certain arrangement. The wide course of the bony stem is continuous with the small lumen of sponge shochu and all are filled with bone marrow. Hematopoietic bone marrow has a lot of blood vessels distributed in red color, called red bone marrow. In the structure of the bone, dense and spongy bones are overlapped with corrugated plates having a thickness of 5 to 12 μm. In dense material, several layered bone layers (harbor layered plates) are arranged in various directions and at the center of each layered plate. There is a harbor tube and blood vessels go through. Osteoblasts are arranged between the lamellar plates and are connected to other bone cells adjacent to each other by a protoplasma that leads to an irregular star shape. On the surface of the bone is a tough connective tissue periosteum, and nerves and blood vessels are distributed to protect and nourish bone. Defects of the periosteum make it difficult to survive, regenerate, and regenerate bones. The bone component is 20% water, 35% organic matter including cells, 45% minerals, because the constant elasticity of bone is organic. As age increases, minerals (mainly calcium phosphate) increase and bone hardness also increases.

The exact cause and mechanism of chronic inflammation of rheumatoid arthritis is not well known yet, but one of the leading causes of disease identified to date is osteoblasts that produce bone and osteoclasts that destroy bone. It is known that it is caused by bone homeostasis due to numerical imbalance of). While osteoclasts in normal state differentiate from monocytes and macrophages to destroy old bones or to remove damaged bones, the osteoclasts found in rheumatoid patients are characterized by an increase in the number of osteoclasts and the sustained growth of osteoclasts. Survival and excessive activity are identified, resulting in a pathological form in which the bone is significantly damaged. Osteoclasts produce a variety of enzymes to break down bone.Tartrate-resistant acid phosphatase (TRAP), which oxidizes and breaks down bone, and matrix metallopeptidase- (MMP-9), which breaks down the cell support tissue of bone and cartilage. 9) Cathepsin K, which breaks down collagen, is known as the main enzyme, which is used as a special marker for mature osteoclasts. When RANKL was treated to macrophages, precursors of osteoclasts, NF-κB and MAPK kinase were activated to produce inflammatory mediators.

Recently, the progress of the inflammatory response in the body is known to be associated with the activity of the cyclooxygenase (COX) enzyme. The COX enzyme is a major enzyme involved in the biosynthesis of prostaglandin present in vivo (Smith et al., J. Biol. Chem., 271, 33157 (1996)), and two types of isozymes, 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 mitogen or cytokines during inflammation or other immune responses. Is an enzyme that is transiently and rapidly expressed in cells. Another powerful inflammatory mediator, nitric oxide (NO), is produced from L-arginine by NO synthetase (NOS), and can be produced by many types of substances, such as external stresses such as UV, or substances such as endotoxins or cytokines. Produced in 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, 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 enzymes as well as COX-1 enzymes. In addition, the mechanism of treatment related to gastritis is mainly based on H2 inhibitors (H2-Blockers) that block the second histamine receptor (H2 receptor) to reduce the secretion of gastric acid in gastric wall cells. Reduced gastric acid prevents further damage to already damaged gastric parietal cells (such as stomach ulcers). These H2 inhibitors interfere with the metabolism of other drugs in the liver (potent inhibitors of P-450), so they must be used with other drugs and have anti-Androgen effects, resulting in gynecomastia in men. ), Side effects such as impotence and decreased libido may occur. In addition, because it passes through the placenta and cerebrovascular barrier, side effects may be more dangerous for pregnant women and the elderly, and may cause headaches, confusion, confusion, and dizziness.

Therefore, it is possible to effectively inhibit the production of NO, inhibit the expression of iNOS and TNF-α, and effectively inhibit the activity of COX-2 enzyme as a natural-derived substance, which has anti-inflammatory effect, osteoblast ALP activity and In addition to promoting osteoblast differentiation and producing bone or cartilage tissue, there is a need for the development of materials that have little or no risk for such side effects or cytotoxicity as natural-derived materials, and thus have little or no restriction on their content.

Stauntonia hexaphylla is an evergreen vine plant that grows up to 15 m tall in the southern coastal area. It grows up to 15 m in height and blooms white flowers in mid-April to mid-May. Is a hanging plant (Ikuta, 1989; Lee, 1996). The fruit is known as a delicious fruit as its name is derived from 'sweet as honey', but the fruit is not commercialized due to the large number of coarse seeds and the small flesh. Leaves can be wintered in the southern region, thick flesh, high ornamental value, and is increasingly used for gardening, pergola, etc. As a result, seedling production is increasing.

Since the content of caffeic acid and flavonoid compounds contained in the plant of Stauntonia hexaphylla leaves was very small, there was a very inadequate aspect for industrial use.

Accordingly, the present inventors have studied a method for preparing an extract containing a high content of flavonoid compounds and a caffeic acid compound contained in a plant of the Stauntonia hexaphylla leaf, and more active ingredients, in particular a high content of flavonoid compounds and caffeic acid The present invention was completed by confirming that the extract derived from the honey (Stauntonia hexaphylla) containing a large amount of the compound and its purified fraction have excellent anti-inflammatory, or activity for promoting bone tissue production or cartilage tissue production.

[Patent Document 1] Korean Patent Publication No. 1992-0005995 [Patent Document 2] Republic of Korea Patent Publication No. 199-0006596 [Patent Document 3] Korean Patent Publication No. 0465778 [Patent Document 4] Korean Patent Publication No. 2004-0108265 [Patent Document 5] Korean Patent Publication No. 0514916 [Patent Document 6] Korean Patent Publication No. 2005-0074753 [Patent Document 7] Korean Patent Publication No. 10-2010-0043882 [Patent Document 8] Korean Patent Publication No. 10-0930927 [Patent Document 9] Korean Patent Publication No. 10-2016-0047665 [Patent Document 11] Korean Patent Publication No. 10-2011-0021252 [Patent Document 12] Korean Patent Publication No. 10-2010-0079786 [Patent Document 13] Korean Patent Publication No. 10-2016-0182923

Accordingly, one object of the present invention

Relative to the total extract weight, (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3-(((2,3,4,5) -3,4, 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5-hydroxy-4H-chromen-4-one) 0.1 To about 10% by weight, chlorogenic acid (0.5 to 30% by weight), and Cryptochlorogenic acid (Cryptochlorogenic acid), characterized in that it contains a honey leaf extract and fractions thereof To provide.

Still another object of the present invention relates to a method for preparing a honey leaf extract, characterized in that the step of extracting by adding a solvent to the dried honey honey (Stauntonia hexaphylla) leaves by repeating 1 to 20 times.

Another object of the present invention relates to a method for producing a fraction purified product of a honey leaf extract, characterized in that the honey leaf extract is subjected to chromatography.

Another object of the present invention comprises as an active ingredient containing at least one selected from the group consisting of honey leaf-derived extract or fraction purified, or the caffeic acid compound and novel flavonoid compounds isolated from the extract or fraction purified To, anti-inflammatory, or to provide a pharmaceutical composition for promoting bone tissue production or cartilage tissue production.

Another object of the present invention comprises as an active ingredient comprising one or more selected from the group consisting of honey leaf-derived extract or fraction purified, caffeic acid compound and novel flavonoid compounds isolated from the extract or fraction purified To provide a food composition for promoting anti-inflammatory, or bone tissue production or cartilage tissue production.

Another object of the present invention comprises as an active ingredient containing one or more selected from the group consisting of honey leaf-derived extract or fraction purified, or the caffeic acid compound and novel flavonoid compounds isolated from the extract or fraction purified To, anti-inflammatory, or to provide a health functional food for promoting bone tissue production or cartilage tissue production.

Another object of the present invention is a dietary supplement containing at least one selected from the group consisting of honey leaf-derived extract or fraction purified, or a caffeic acid compound and a novel flavonoid compound isolated from the extract or fraction purified To provide.

Still another object of the present invention is to provide a food additive containing at least one selected from the group consisting of honey leaf-derived extract or fraction purified product, or a caffeic acid compound and flavonoid compound isolated from the extract or fraction purified product. It is.

Another object of the present invention as an active ingredient comprising at least one selected from the group consisting of honey leaf-derived extract, fraction purified product thereof, or caffeic acid compound and novel flavonoid compound isolated from the extract or fraction purified product. It provides a pharmaceutical composition for the prevention or treatment of rheumatoid arthritis or degenerative arthritis, comprising.

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

In one embodiment, the present invention is an anti-inflammatory salt comprising a novel honey leaf-derived extract or fraction purified, and the honey leaf-derived extract or fraction purified or a novel flavonoid compound or caffeic acid compound isolated therefrom as an active ingredient, or It relates to a pharmaceutical composition for promoting bone tissue production or cartilage tissue production.

As used herein, the honey honey (Stauntonia hexaphylla) leaf may be purchased commercially, or may be collected or grown in nature, but is not limited thereto, and may be properly dried before being extracted and purified, finely pulverized and effectively extracted and It can be pretreated to make the purification process possible.

The novel extract or fraction purified as defined herein may be an extract extracted from the leaves of Stauntonia hexaphylla with a solvent selected from water, methanol, ethanol, butanol, etc. 1 to 4 lower alcohols or a mixed solvent thereof In addition, the extraction method may be prepared as a fraction purified using a purification method known to those skilled in the art to which the present invention belongs.

In addition, the extract or fraction purified from the leaves of the honey (Stauntonia hexaphylla) of the present invention may be further subjected to a concentration and drying process, such as drying under reduced pressure, freeze drying or hot air drying, and also purified.

The extract or fractional purified product of the present invention is a novel flavonoid compound (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3-(((2,3,4) , 5) -3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5-hydroxy-4H -chromen-4-one), And chlorogenic acid (Clorogenic acid) and Cryptochlorogenic acid (Cryptochlorogenic acid) which is a caffeic acid compound is characterized in that it comprises within a specific range.

Specifically, the extract or fraction purified is 100% by weight of the total extract or fraction purified (w / w), (7-(((2,3,4,5,6) -4,5-dihydroxy-6 -methyl-3-(((2,3,4,5) -3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- 0.1 to 10 wt% of (3,4-dihydroxyphenyl) -5-hydroxy-4H-chromen-4-one), 0.5 to 30 wt% of chlorogenic acid, and 0.5 to Cryptochlorogenic acid. To 30% by weight, and

Preferably, 100% by weight of the total extract or fraction purified (w / w), the novel compound (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3- (((2,3,4,5) -3,4,5- trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3,4- dihydroxyphenyl) -5-hydroxy-4H-chromen-4-one) 0.1 to 5 wt%, 0.5 to 20 wt% chlorogenic acid, 0.5 to 20 wt% Cryptochlorogenic acid It may be characterized by.

In a specific embodiment of the present invention, the crude honey leaf crude extract according to the present invention is a novel flavonoid compound (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl as an indicator component). -3-((((2,3,4,5) -3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3 , 4-dihydroxyphenyl) -5-hydroxy-4H-chromen-4-one) 0.56% by weight, 1.61% by weight chlorogenic acid and 1.07% by weight Cryptochlorogenic acid, fractional purification of this crude extract is a novel flavonoid Compound (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3-(((2,3,4,5) -3,4,5- trihydroxytetrahydro- 2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5-hydroxy-4H-chromen-4-one) 1.14 wt%, chloro It was confirmed that the formulation includes 5.34 wt% of genic acid and 3.85 wt% of kryptochlorogenic acid.

As another aspect, the present invention relates to a method for producing a honey leaf extract, characterized in that the step of extracting by adding a solvent to the honey (Stauntonia hexaphylla) leaf is carried out by repeating 1 to 20 times.

In the above production method, the honey (Stauntonia hexaphylla) leaf may be a pretreated, such as through a washing, drying, cutting or pulverizing process according to a known method, it is preferable to use a dried honey leaf. The honey leaf is extracted using an appropriate extraction solvent, and as the extraction solvent, a solvent selected from lower alcohols having 1 to 4 carbon atoms such as water, methanol, ethanol, butanol, or a mixed solvent thereof may be used, but the present invention is limited thereto. No, water can be used preferably. The extraction temperature may be specifically 10 ° C to 100 ° C, preferably 20 ° C to 100 ° C, and the extraction time may be treated for 30 minutes to 72 hours, preferably 6 hours to 48 hours, but is not limited thereto. It doesn't happen. The extraction method may be an extraction method selected from cold needle extraction method, ultrasonic extraction method, reflux cooling extraction method, hot water extraction method, room temperature extraction method, reflux extraction method, or a combination of one or more of these extraction methods, but known natural extraction methods may be appropriately selected without limitation. . More preferably, after cold extraction for 30 minutes to 72 hours, preferably 6 hours to 48 hours at a temperature of 10 ℃ to 60 ℃, preferably 20 ℃ to 50 ℃, 40 ℃ to 120 ℃, specifically 60 The reflux cooling extraction method may be repeated for about 1 to 20 times, preferably 2 to 10 times, at a temperature of from 100 ° C. to 100 ° C. for 30 minutes to 72 hours, preferably 6 hours to 48 hours.

Preferably, the honey leaf extract may further include concentrated under reduced pressure after the extraction process.

As another aspect, the present invention relates to a method for producing a fraction purified of a honey leaf extract, characterized in that the honey leaf extract is subjected to chromatography.

In a specific embodiment, the fraction purified product of the honey leaf extract may be prepared by first preparing an extract using the honey leaf and then subjecting it to chromatography to fractionate purification, wherein the method of preparing the honey leaf extract is the honey leaf The same applies to the preparation of the extract.

The chromatography used in the purification process includes (a) reverse phase partition chromatography, (b) normal phase adsorption chromatography, and (c) ion exchange chromatography. chromatography, (d) size exclusion chromatography or one or more combinations thereof, preferably reverse phase partition chromatography or polar substances are eluted first, and the non-polar compound is retained in the stationary phase Chromatography using all resins, more preferably Sephadex, Sephadex LH20, Sephadex G-25, Sephadex G-10, Sepharose, Superdex, Methyl Acrylate resins, carboxymethyl cellulose, sulfopropyl cellulose, carboxymethyl sefadex, sulfopropyl sefadex, sulfopropyl sepharose and carboxy Sephadex-based resins including methyl sepharose; Reverse-phase polymer resins using a family of styrene-divinylbenzen co-polymers such as Polymer X, HP20, and PRP-h1 Polymer, a family of methacrylate supports; Pure silica gels such as Merck's adsorption Bonded Phase Chromatography (BPC) family, Silica family of YMC, Silica family of DAISO, Silica family of ASAHI, Silica family of COSMOSYL, etc .; Stationary phase resins were selected from the reverse phase silica gel family of YMC's ODS family, DAISO's ODS family, ASAHI's ODS family, CHEMCO's ODS family, Merck's ODS family, COSMOSYL's ODS family, and FUJI's ODS family. It includes a method of performing a chromatographic method using.

Specifically, in (a) reverse phase partition chromatography, the stationary phase is a reverse phase column material, and all stationary phases are characterized by eluting polar substances first, and non-polar compounds retained in the stationary phase, where hydrophobic substances can be attached. Fixed phase, preferably a silica gel based stationary phase or a polymer based stationary phase such as polystyrene, more preferably C2, C4, C6, C8, C10, C12, C14, C16, or C18 or silica gels thereof Derivatives or polymer-based stationary phases such as PS-2, Oasis HLB, etc., more preferably, reversed phase silica gel (C18 (IV) -D), (YMC's ODS-A / ODS-AQ family, CHMECO's SP-C-ODS) Family, DAISO's SP-ODS-RPS family, COSMOSYL's 5C18 family, and FUJI's Chromatorex family).

The mobile phase is an alcohol solvent such as distilled water, acetonitrile, methanol, ethanol, butanol, tetrahydrofuran or a mixed solvent thereof, preferably a mixed solvent of distilled water and an alcohol solvent such as methanol, ethanol, butanol, and more preferably, water And starting with 90:10 (v / v) methanol mixed solvent to adjust the ratio of 60:40 to elute the polar material.

Specifically, in (b) the normal phase chromatography, the stationary phase is a normal phase column material, in which all non-polar materials are eluted first, and the polar compounds are retained in the stationary phase. Preferably, silica gel, fluorosyl, alumina based stationary phase, CN, diol, NH ₂ stationary stationary phase, more preferably reverse phase silica gel or a silica gel derivative thereof, even more preferably reverse phase silica gel (C18 ( IV) -D), (YMC's ODS-A / ODS-AQ family, CHMECO's SP-C-ODS family, DAISO's SP-ODS-RPS family, COSMOSYL's 5C18 family, FUJI's Chromatorex family) It is done.

The mobile phase is characterized by using hexane, heptane, ethyl acetate, ethanol, diethyl ether, 2-propanol or a mixed solvent thereof.

Specifically, in (c) Ion Exchange Resin Column Chromatography, "Ion Exchange Chromatography Material" uses all floating high molecular weight solid phases having charged groups covalently bonded as chromatographic functional groups. Possible; Preferably, chromatographic methods using cation exchange resins, anion exchange resins or synthetic adsorbents, more preferably AG 50W-x8, Amberlite IR-120, Doex 50W-x8 and SK1B Strongly acidic cation exchange resins such as these; Weakly acidic cation exchange resins such as Amberlite IRC-50, Bio-Rex 70, Duoolite-436 and WK40; Weakly basic cation exchange resins such as Amberlite IRA-67 and Dowex 3-x4A; DIAION SK1B, DIAION PK216, DIAION CR11, DIAION CR20, DIAION UBK555 (Mitsubishi Chemical co.), TRILITE SPC 160H, TRILITE SPC 180H, TRILITE SPC 400LH (Samyang co.), AMBERLITE 200C Na, AMBERLITE CG50, AMBERLITE AMJBER 10 200H, AMBERLYST 131 WET, AMBERLYST 232 WET (ROHM and HAAS co.), Lewatit VP OC 1800, Lewatit VP OC 1812, Lewatit MDS1368 Na, Lewatit K1221 (Bayer co.), PUROLITE PCR833CA, PUROLITE C145 (Purolite co.), Strongly basic cation exchange resins such as MFG 210 and MFG 250 (Finex co.); Preferably, SK1B type exchange resin which is a strong acid cation exchange resin can be used; Anion exchange resins are strongly basic anion ion exchange resins such as SA11A, SA20A and SA21A, CaptoQ (available from GE Healthcare), or resins with similar characteristics, such as ToyoPearl QEA (available from Tosoh), Q Sepharose FF (Available from GE Healthcare) or Fractogel EMD, Fractogel TMAE or Fractogel HICAP (available from Merck KGaA, Darmstadt Germany), preferably SA21A; The synthetic adsorbent may be SP207, HP20SS, HP20, or the like, preferably HP20.

Specifically in (d) size exclusion chromatography, the gel is typically a dextran-based gel such as Sephadex (eg Sephadex G-25) or a polyacrylamide gel such as For example, Sephacryl (eg Sephacryl-S400), agarose-based gels such as Superose or Sepharose (eg Sepharose CL-4B), and composite gels prepared from two types of gels, For example, it is selected from the group of polymeric gels including but not limited to Superdex 200 Combination Dextran (Sephadex ™) and Crosslinked Agarose (Superose ™) gels; In a preferred embodiment, the buffer is sodium phosphate, ammonium acetate, MES (2- (N-morpholino) ethanesulfonic acid), Bis-Tris (2-bis (2-hydroxyethyl) amino-2- (hydroxy Methyl) -1,3-propanediol), ADA (N- (2-acetamido) iminodiacetic acid), PIPES (piperazin-N, N'-bis (2-ethanesulfonic acid), ACES (N- (2-acetamido) -2-aminoethanesulfonic acid), BES (N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid), MOPS (3- (N-morpholino) Propanesulfonic acid), TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid), HEPES (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid), preferably Is selected from the group consisting of sodium phosphate or ammonium acetate, more preferably ammonium acetate.

In addition, the present invention additionally comprises (a) reverse phase partition chromatography, (b) normal phase partition chromatography, (c) ion exchange chromatography, (d) Gel permeation or gel filtration chromatography using Sephadex LH-20 or Toyopearl HW-40, which can use organic solvents other than size exclusion chromatography or one or more combinations thereof. Gel filtration chromatography is additionally available.

The synthetic adsorbent may be HP20 of 0.5 to 20 times the volume (v / v) of the suspended solvent volume, but is not limited thereto.

As another aspect, the present invention is an active ingredient comprising at least one selected from the group consisting of the honey leaf-derived extract or fraction purified, or the caffeic acid compound and novel flavonoid compounds isolated from the extract or fraction purified It includes, as an anti-inflammatory, or relates to a pharmaceutical composition for promoting bone tissue production or cartilage tissue production.

Matter regarding the extract or fraction purified from the honey leaf in the pharmaceutical composition according to the present invention can be applied to the content of the extract or fraction purified.

The caffeic acid compound is preferably at least one selected from the group consisting of chlorogenic acid and kryptochlorogenic acid.

The flavonoid compound is (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3-(((2,3,4,5) -3,4,5) -trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5-hydroxy-4H-chromen-4-one) is preferred. Do.

In one embodiment of the present invention, it was confirmed that the extract or fraction purified from the honey leaf prepared by the production method inhibits the differentiation inhibitory activity of the osteoclasts (Example 3).

Through the above results, the honey leaf-derived extract or fraction purified from the honeycomb prepared by the production method comprising at least one selected from the group consisting of caffeic acid compounds and novel flavonoid compounds isolated from the extract or fraction purified product is rheumatoid arthritis patients It can be seen that the anti-inflammatory effect is reduced by reducing the amount of secretion and expression of inflammation-related substances of synovial cells.

In addition, through the above results, the honey leaf derived extract or fraction purified product prepared by the method or containing at least one selected from the group consisting of caffeic acid compounds and novel flavonoid compounds isolated from the extract or fraction purified arthritis There is an activity to delay the damage of the joints and bones and the loss of the joints by means, through which the honey leaf-derived extract or fraction purified by the production method has the effect of inhibiting osteoclast activity, and promoting bone tissue production or cartilage tissue production It can be seen that there is an effect.

The present invention also relates to a pharmaceutical composition for preventing or treating diseases that destroy or damage bone tissue or cartilage tissue. The disease that destroys or damages the bone tissue or cartilage tissue, but is not limited thereto, arthritis, destruction of the joint due to autoimmune diseases, multiple myositis, ankylosing spondylitis, systemic lupus erythematosus, multiple myositis, rheumatoid bundle Myalgia (polymyalgia rheumatica), osteoporosis, bone metastasis cancer or Paget's disease. In addition, the arthritis may include various types of arthritis, such as osteoarthritis, rheumatoid arthritis, degenerative arthritis, psoriatic arthritis, reactive arthritis, and the like, but is not limited thereto. Preferably rheumatoid arthritis.

Pharmaceutical compositions comprising extracts or fractional tablets or compounds according to the present invention may be prepared according to conventional methods, respectively, oral formulation external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, suppositories and It can be formulated and used in the form of sterile injectable solutions. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, weights, binders, humectants, sealants, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.

In the above formulations, non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

Preferred dosages of pharmaceutical compositions comprising extracts or fractions of the present invention or compounds vary depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. have. However, for the desired effect, the pharmaceutical composition comprising the extract or fractional tablet of the present invention is preferably administered at 0.0001 to 1000 mg / kg, preferably 0.001 to 500 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

Extracts or fractional purified products or compounds of the invention can be administered to mammals, such as mice, mice, livestock, humans, and the like by a variety of routes. All modes of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or Intracerebroventricular injection.

As another aspect, the present invention comprises as an active ingredient comprising one or more selected from the group consisting of honey leaf-derived extract or fraction purified, caffeic acid compound and novel flavonoid compounds isolated from the extract or fraction purified It relates to an anti-inflammatory, or food composition for promoting bone tissue production or cartilage tissue production.

For the anti-inflammatory, or the pharmaceutical composition for promoting bone tissue production or cartilage tissue production, the following is anti-inflammatory, food composition for promoting bone tissue production or cartilage tissue production, anti-inflammatory, or health functional food for promoting bone tissue production or cartilage tissue production, anti-inflammatory The same may be applied to the health supplement food for promoting bone tissue production or cartilage tissue production, anti-inflammatory, or food additive for promoting bone tissue production or cartilage tissue production.

As another aspect, the present invention as an active ingredient comprising one or more selected from the group consisting of honey leaf-derived extract or fraction purified, or the caffeic acid compound and novel flavonoid compounds isolated from the extract or fraction purified It includes, anti-inflammatory, or health functional food for promoting bone tissue production or cartilage tissue production.

As another aspect, the present invention, the anti-salt containing at least one selected from the group consisting of honey leaf-derived extract or fraction purified, or the caffeic acid compound and novel flavonoid compounds isolated from the extract or fraction purified Or it relates to a health supplement for promoting bone tissue production or cartilage tissue production.

As another aspect, the present invention, the anti-salt containing at least one selected from the group consisting of honey leaf-derived extract or fraction purified, or the caffeic acid compound and novel flavonoid compounds isolated from the extract or fraction purified Or to a food additive for promoting bone tissue production or cartilage tissue production.

As defined herein, "health functional food" means a food prepared and processed using raw materials or ingredients having functional properties useful for the human body according to the Health Functional Food Act No. 6767, and "functional" means It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on structure and function.

The dietary supplement of the present invention comprises 0.01 to 95%, preferably 1 to 80% by weight of the extract or fraction purified based on the total weight of the composition.

In addition, for the purpose of inhibiting anti-inflammatory or osteoclast activity, it is a health functional food in the form of a pharmaceutical dosage form such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, or tea bags, leaches, health drinks, etc. Manufacturing and processing are possible.

In addition, the health functional food may further include food additives, and the suitability as a "food additive" is applied to the item in accordance with the General Regulations and General Test Act of the Food Additives Authority approved by the Ministry of Food and Drug Safety unless otherwise specified. Determined by the relevant standards and standards.

Items listed in the "Food Additives Code" include, for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, cinnamon acid, natural additives such as navy, licorice extract, crystalline cellulose, guar gum, sodium L-glutamate Mixed formulations, such as a preparation, an alkali addition additive, a preservative preparation, and a tar pigment preparation, are mentioned.

Functional foods containing extracts or fractions of the present invention or compounds include bread, rice cakes, dried fruits, candy, chocolates, chewing gum, confectionary products such as ice cream, ice cream products such as ice cream powders, milk powders, low fats Dairy products such as milk, lactose, milk, processed milk, goat milk, fermented milk, buttermilk, concentrated milk, milk cream, buttermilk, natural cheese, processed cheese, milk powder, whey products, processed meat products, eggs, hamburgers and Meat products such as fish paste, processed meats such as fish paste, ham, sausage, bacon Ramen noodles, dried noodles, raw noodles, noodle soups, dehydrated noodles, refined noodles, frozen noodles, pasta noodles Lactic acid bacteria beverages such as soda, soy milk, yogurt, beverages such as mixed drinks, soy sauce, miso, red pepper paste, chunjang, cheonggukjang, mixed soy sauce, vinegar, sauce, tomato ketchup, curry Seasonings, such as dressings, margarine, shortening and pizza, but are not limited thereto.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract or fractional purified product or compound as essential ingredients at the indicated ratios, and additional ingredients such as various flavors or natural carbohydrates, such as ordinary drinks. It may contain as. Examples of the aforementioned natural carbohydrates include monosaccharides (eg, glucose, fructose, and the like); Disaccharides (eg maltose, sucrose and the like); And conventional sugars such as polysaccharides (eg dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The proportion of the natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, extracts or fractions of the present invention, or novel flavonoids and caffeic acid compounds isolated therefrom may be added to food or beverages for the purpose of preventing or ameliorating the desired disease. At this time, the amount of the extract or fractional purified product or compound in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 5 g, preferably 0.3 to based on 100 ml It can be added at a rate of 1 g.

Extracts or fractional purified products or compounds according to the present invention, which are added to foods, including beverages, in the process of preparing the health functional food, may be appropriately added or subtracted as necessary.

In another embodiment, the present invention relates to 100% by weight of the total extract or fraction purified (w / w), (7-(((2,3,4,5,6) -4,5-dihydroxy-6 -methyl-3-(((2,3,4,5) -3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- 0.1 to 10 wt% of (3,4-dihydroxyphenyl) -5-hydroxy-4H-chromen-4-one), 0.5 to 30 wt% of chlorogenic acid, and 0.5 to Cryptochlorogenic acid. It relates to a pharmaceutical composition for the prevention or treatment of rheumatoid arthritis or degenerative arthritis, characterized in that it contains from 30 to 30% by weight.

The present invention provides a method for producing an extract or fractional purified extract of the honey (Stauntonia hexaphylla) leaf, an extract or fractional purified product derived from the honey leaf prepared by the method, and the extract or fractional purified product derived from the honey leaf, or the extract or fractional purification It relates to a pharmaceutical composition for promoting anti-inflammatory or bone tissue production or cartilage tissue production, comprising as an active ingredient comprising at least one selected from the group consisting of caffeic acid compounds and novel flavonoid compounds isolated from water, according to the present invention. New extracts or fractional purified products isolated from honey leaf extracts or new flavonoid compounds and caffeic acid compounds isolated therefrom are not cytotoxic but also cytokines associated with inflammation, such as IL-1β, IL- 6, reduces the secretion of TNF-α, MCP-1, CXCL10, IL-8, VEGF, MMP, activity of NF-κB It is possible to effectively inhibit inflammation by reducing NO and secretion of COX-2 enzyme which causes NO secretion and inflammation, and prevent, improve or treat various diseases related to bone tissue or cartilage destruction such as rheumatoid arthritis. Or as a functional food.

1 is an HPLC analysis of crude honey leaf extract (ATC1) according to an embodiment of the present invention.
2 is a result of HPLC analysis of purified honey leaf fraction (ATC2) according to an embodiment of the present invention.
Figure 3 is a result of the measurement of TRAP activity of osteoclasts of the honey leaf extract (ATC1), fraction purified product thereof (ATC2) according to an embodiment of the present invention.
Figure 4 is a result of inhibiting osteoclast differentiation by honey leaf extract (ATC1), fraction purified product thereof (ATC2) according to an embodiment of the present invention.
5 is an experimental result of confirming the effect of apoptosis in rheumatoid arthritis synovial cells of the fraction purified (ATC2) according to an embodiment of the present invention.
Figure 6 is an experimental result confirming the change in the mobility of rheumatoid arthritis synovial cells when the fraction purified (ATC2) treatment according to an embodiment of the present invention.
7 is an experimental result confirming the change in the amount of secretion of inflammatory secretion factors (cytokines, chemokines, MMPs) during the treatment of fractional purified matter (ATC2) according to an embodiment of the present invention.
8 is an experimental result confirming the expression change of NF-kB in cells when the fraction purified according to an embodiment of the present invention (ATC2).
9 is an experimental result confirming the identification and activation of the activation site of NF-kB inhibited when the fraction purified product (ATC2) according to an embodiment of the present invention.
10 is an experimental result confirming the NO production suppression when the fraction purified product (ATC2) according to an embodiment of the present invention.
11 is an experimental result confirming the change in the activity and expression of the enzyme activity of gelatinase when fractions purified according to an embodiment of the present invention (ATC2).
Figure 12 is an experimental result confirming the location and degree of expression of inflammation and cytoskeleton related proteins during fractional purified product (ATC2) treatment according to an embodiment of the present invention.
FIG. 13 is a screening result of inflammation-related factors in which a difference in secretion amount is observed when a fraction purified product (ATC2) is treated according to an embodiment of the present invention. FIG.
Figure 14 is a result confirming the signal transduction pathways inhibited when the fraction purified product (ATC2) according to an embodiment of the present invention.
Figure 15 is a result confirming the effect of fraction purified product (ATC2) on osteoclast differentiation according to an embodiment of the present invention.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Reference Example 1: Preparation of crude leaf extract of honey and identification of indicator components

1.0 kg of honey (Stauntonia hexaphylla) leaves (GAP cultivation, 565-2, Bujun-ri, Busan-myeon, Jangheung-gun, Jeollanam-do) were dried and cut. Thereafter, reflux extraction was performed with stirring at 80 to 100 ° C. for 12 hours, followed by vacuum filtration to recover the supernatant. The above procedure was repeated twice to recover the supernatant, and the extract was concentrated under reduced pressure with a vacuum condenser (EYELA, N-2100, JAPAN) to obtain 300 g of honey leaf water crude extract (hereinafter referred to as ATC1).

High performance liquid chromatography (HPLC, Agilent 1260, USA) was carried out under the conditions as shown in Table 1 and Table 2 (mobile phase conditions) in order to identify the indicator components contained in the obtained honey leaf crude extract, and the results are shown in FIG. 1 is shown.

Mobile table A in table 2 is 0.025 M phosphate buffer solution (pH = 3.0) and mobile phase B is 90% (w / v) ACN (Acetonitrile).

High performance liquid chromatography analysis conditions Pump Agilent 1260 Series, 1260 quart pump Detector Agilent 1260 Series, 1260 DAD Column INNO column C18 (2.3 × 250 mm, 5 μm) Flow rate 0.5 mL / min UV absorbance 266 nm Injection volume 10 μL

time Mobile phase A (%) Mobile phase B (%) 0 to 20 89 11 20 to 30 79 21 30 to 40 75 25 40 to 45 60 40 45-50 30 70 50 to 56 30 70 56-70 89 11

As a result, as shown in Fig. 1, chlorogenic acid was detected at 8.67 minutes, cryptochlorogenic acid at 9.15 minutes, and a novel compound was detected at 26.89 minutes. Based on the HPLC pattern (remaining time) shown in FIG. The content (%) of the indicator components was analyzed according to Equation 1.

[Equation 1]

A _T : surface area of indicator component in sample solution

A _S : surface area of surface component in standard solution

However, it is used when the amounts taken for the indicator components of the sample solution and the standard solution are the same.

As a result, the crude honey leaf crude extract prepared by the preparation method of Reference Example 1 was 1.61% (w / w) of chlorogenic acid, 1.071% (w / w) of kryptochlorogenic acid, and 0.56% of a novel compound (see Table 3 below). w / w).

Indicator component ATC1 Retention time (minutes) Content (w / w%) Chlorogenic acid 8.67 1.61 Cryptochlorogenic Acid 9.15 1.07 New Flavonoid Compounds 26.89 0.56 Sum 3.24

Example 1 Preparation of Soluble Fractions of Organic Solvents from Honey Leaf Extracts and Identification of Indicator Components

The honey leaf water extract prepared in Reference Example 1 was dissolved in distilled water, adjusted to 23 brix, 700 mL of reversed phase silica gel (HP20, Mitsubishin Co., Ltd. JAPAN) was packed into a column tube, and the packed column was filled in the 23 column. 700 mL of brix honey leaf crude extract was passed through to separate the honey leaf fraction purified product (ATC2). Mobile phase composition for separation was carried out by adjusting the ratio of distilled water: methanol (100: 0 to 30:70). First, 2.1 L of distilled water: methanol (100: 0) eluate was separated, and then 5.6 L of distilled water: methanol (30:70) eluent was separated under the same conditions, followed by distilled water eluent 2-3 times (1.4 L) and 70% ( v / v) Methanol eluate 1-4 times 2.8 L was combined and after removing the solvent through a concentrated under reduced pressure, 55 g of the finally purified honey fraction extract or fraction purified product was isolated. (Hereinafter referred to as ATC2).

In order to identify the indicator components contained in the honey HP20 column extract or fraction purified product, high performance liquid chromatography (HPLC) was performed in the same manner as in Reference Example 1, and the results are shown in FIG. 2.

As a result, as shown in FIG. 2, it was confirmed that chlorogenic acid was detected at 8.67 minutes, cryptochlorogenic acid at 9.15 minutes, and a novel compound was detected at 26.89 minutes.

Based on the HPLC pattern (remaining time) shown in FIG. 2, the content (%) of the indicator components was analyzed according to the equation of Reference Example 1. As a result, the honey bee fraction extract or fraction purified product prepared by the preparation method of Example 1 was 5.34% (w / w) chlorogenic acid, 3.85% (w / w), novel flavonoids as shown in Table 3 It was confirmed that the compound contained 1.14% (w / w).

Indicator component ATC2 Retention time (minutes) content(%) Chlorogenic acid 8.67 5.34 Cryptochlorogenic Acid 9.15 3.85 New Flavonoid Compounds 26.89 1.14 Sum 10.33

Example 2 Separation and Structural Analysis of Caffeic Acid Compound and Flavonoid Compounds The structure of the compound identified in Example 1 was identified by comparing the NMR data of the compound with the physical properties described in the existing literature.

1. Chlorogenic acid; Chlorogenic acid

¹ H NMR (400 MHz, DMSO- d ₆ ) δ: 7.42 (1H, d, J = 16 Hz, H-7 '), 7.03 (1H, d, J = 2 Hz, H-2'), 6.98 ( 1H, dd, J = 2 and 8 Hz, H-6 '), 6.77 (1H, d, J = 8 Hz, H-5'), 6.15 (1H, d, J = 16 Hz, H-8 ') , 5.08 (1H, broad, J = 5 Hz, H-3), 3.94 (1H, broad, H-5), 3.55 (1H, broad, J = 4 Hz, H-4), 1.7-2.1 (2H, m, H-6), 1.98 (2H, broad doublet, J = 5 Hz, H-2).

¹³ C NMR (125 MHz, DMSO- d ₆ ) δ: d 174.9 (C-7 '), 165.7 (C-9'), 148.3 (C-4 '), 145.5 (C-3'), 144.8 (C -7 '), 125.6 (C-1'), 121.2 (C-6 '), 115.7 (C-5'l), 114.7 (C-2'), 114.3 (C-8 '), 73.6 (C- 1), 70.9 (C-3), 70.6 (C-4), 68.3 (C-5), 37.2 (C-6), 36.5 (C-2).

2. Cryptochlorogenic acid; Cryptochlorogenic acid

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 2.00 (1H, dd, J = 11, 13 Hz, H-6ax), 2.06 (1H, ddd, J = 3, 4, 14 Hz, H- 2eq), 2.17 (1H, dd, J = 4, 14 Hz, H-2ax), 2.20 (1H, ddd, J = 3, 5, 13 Hz, H-6eq), 4.27 (1H, ddd, J = 4 , 9, 11 Hz, H-5), 4.28 (1H, ddd, J = 3, 3, 4 Hz, H-3), 4.79 (1H, dd, J = 3, 9 Hz, H-4), 6.37 (1H, d, J = 16 Hz, H-8 '), 6.77 (1H, d, J = 8 Hz, H-5'), 6.96 (1H, dd, J = 2, 8 Hz, H-6 ' ), 7.06 (1H, d, J = 2 Hz, H-2 '), 7.65 (1H, d, J = 16 Hz, H-7').

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 38.4 (C-2), 42.7 (C-6), 65.5 (C-5), 69.6 (C-3), 76.6 (C-1), 79.3 (C-4), 115.1 (C-2 '), 115.4 (C-8 '), 116.5 (C-5'), 123.0 (C-6 '), 127.8 (C-1'), 146.8 (C-3 '), 147.1 (C-7'), 149.6 ( C-4 '), 169.0 (C-9'), 177.3 (C-7 ').

3. Novel Flavonoid Compounds

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 13.65 (1H, s, 5-OH), 7.46 (1H, d, J = 2.0 Hz, H-6 ′), 7.42 (1H, dd, J = 8.5, 2.0 Hz, 2 '), 6.89 (1H, d, J = 8.5 Hz, H-3'), 6.76 (1H, s, H-3), 6.72 (1H, d, J = 2.0 Hz, H -8), 6.35 (1H, d, J = 2.0 Hz, H-6), 5.20 (1H, d, J = 7.2 Hz, H-1 '''), 5.0 (1H, d J = 4.5 Hz, H -1 ''), 4.29 (1H, H-5 ''').

¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 175.8 (C-4), 164.0 (C-7), 161.0 (C-5), 156.1 (C-9), 148.0 (C-5 ') , 147.0 (C-4 '), 145.0 (C-2), 135.7 (C-3), 121.9 (C-1'), 120.0 (C-6 '), 115.6 (C-3'), 115.0 (C -2 '), 105.3 (C-1'''), 103.0 (C-10), 100.5 (C-1 ''), 98.2 (C-6), 93.3 (C-8), 77.6 (C-3 '''), 75.4 (C-2'''), 71.8 (C-4 ''), 70.4 (C-3 '', C-4 '''), 68.3 (C-2''), 66.0 (C-5``), 18.0 (C-6 '').

Example 3. Confirmation of Biological Activity of Inhibiting Osteoclast Differentiation of Honey Extracts and Fractions Purified

3-1. Isolation of Bone Marrow Cells and Osteoclast Differentiation

Tibia were extracted from 4-5 week old ICR mice, and 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). And incubated for 24 hours. Unattached cells were washed with α-MEM, and then divided into 48-wells at 5 × 10 ⁵ 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 MCSF and 100 ng / mL RANKL (Receptor Activator for Nuclear Factor κB Ligand) were treated together.

3-2. Measurement of Changes in TRAP Activity of Osteoclasts by Honey Leaf Extract (ATC1) and Fractionated Purified Products (ATC2)

Osteoclasts express tartrate-resistant acid phosphatase (TRAP), an acid phosphatase that is characteristically resistant to tartrate, which is characterized by the cytochemistry of osteoclasts that can be distinguished from other bone tissue cells. Used as a labeling enzyme (Minkin, C., Calcif. Tissue Int., 34: 285-290, 1982).

In order to confirm the change of TRAP activity of osteoclasts by the honey leaf extract (ATC1) and fraction purified product (ATC2) of the present invention, the following experiment was performed.

Specifically, in the same manner as in Example 3-1, the cells were seeded at 5 × 10 5 cells / well in 48-well, treated with differentiation factors (RANKL, MCSF) and sampled, and then cultured for 4 days. The change was measured.

For measuring TRAP activity, after incubation using the TRAP acivity assay kit (AK04F, COSMO BIO CO., LTD) for 4 days, 30 ul / well of the culture supernatant was dispensed on a new 96-plate, and the chromogenic substrate of the prepared kit was used. 170 μl / well of / Tartrate-containing buffer was added and the absorbance was measured at 540 nm after reaction at 37 ° C. for 30 minutes to 3 hours.

The negative control group was not treated with RNAKL and ATC1 or ATC2, and the cells were in a bone marrow cell state that did not differentiate into osteoclasts.The positive control group was treated with RANKL to differentiate into osteoclasts. (5 uM, or 20 uM) were treated and incubated for 4 days.

TRAP activity expressed the absorbance of the sample as a percentage of the absorbance of the negative control.

Honey leaf extract (ATC1), secondary fraction extract or fraction purified product (ATC2) obtained in Example 3-1 by each concentration, that is, honey leaf extract ATC1 (1, 10, 50, 100 ug / ml), honey leaf Secondary fraction extract or fraction purified ATC2 (0.1, 1, 10, and 50 ug / ml) was treated with osteoclasts, cultured for 4 days, and then TRAP activity (TRAP activity of tartrate showed resistance to tartrate). Mean acid phosphatase activity) and the results are shown graphically in FIG. 3.

Through the results shown in Figure 3, it was confirmed that the honey leaf extract of the present invention (ATC1), secondary fraction extract or fraction purified product (ATC2) inhibits the activity of the osteoclasts.

More specifically, in the case of ATC2 extract extracted through the fractional purification process, 71.7% (0.1 ug) in ATC2 (0.1, 1, 10, and 50 ug / ml), respectively, which is a fraction of purified honey leaf extract compared to the positive control (100%). / ml), 36.4% (1 ug / ml), 21.9% (10 ug / ml) and 221.1% (50 ug / ml) TRAP activity, 1/10 of untreated crude extract (ATC1) Treatment with lower concentrations showed a significant inhibitory effect of TRAP activity of 2 to 3 times or more.

In particular, TRAP activity inhibition rate was 63.6% (inhibition rate: 100% -36.4% = 63.6%) compared to the control group when 1 ug / ml of fraction purified water (ATC2) of the honey leaf extract was treated. When the concentration of the extract (ATC1) 50 ug / ml was treated with TRAP activity inhibition rate of 66.4% (inhibition rate: 100% -33.4% = 66.4%) was equivalent. That is, the honey leaf extract fraction purified (ATC2) showed 50 times more activity than the honey leaf extract (ATC1).

3-3. Inhibitory effect of osteoclast differentiation by honey leaf extract (ATC1) or fractional purified product (ATC2)

In the same manner as in Example 3-1, the cells were seeded at 5 × 10 ⁵ cells / well in a 48-well, and differentiation factors (RANKL, MCSF), honey leaf extract (ATC1), and fractional purified product thereof (ATC2) were prepared. Treated and incubated for 4 days. Remove the medium, wash with PBS and fix 100% of substrate solution (1.36 mg / mL 4-nitrophenyl phosphate disodium salt, 10 mM tartrate, 50 mM citrate buffer pH 4.6) to cells fixed with 10% (w / v) formaldehyde. The reaction was carried out for 30 minutes at 37 ° C. After removing the medium and washing with PBS, the cells were fixed for 15 minutes with 10% (w / v) formaldehyde, and the cells were washed three times with PBS. TRAP staining was performed 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. ) 45 μL of the staining solution was dispensed into cells fixed with formaldehyde, stained at 37 ° C. for 30 minutes, and observed under a microscope. The results are shown in FIG. 4.

As shown in FIG. 4, TRAP-positive multinucleated osteoclasts were formed in the negative control cells, but when the honey leaf extract (ATC1) or fractional purified product (ATC2) was treated, concentration-dependent osteoclast differentiation was confirmed. Could.

More specifically, in the case of ATC2 extract extracted through the first extraction and the second fractionation process, significant osteoclast differentiation (differentiation) of 2 to 3 times or more was achieved even though the concentration was 1/10 lower than that of the non-process extract (ATC1). ) Showed an inhibitory effect.

Example 4 In Vitro Rheumatoid Arthritis Prevention and Treatment Activity

4-1. Isolation and Primary Culture of Synovial Fibroblasts

The synovial tissues obtained from patients with rheumatoid arthritis who meet the diagnostic criteria of the American Rheumatology Society were washed with PBS, chopped to 2-3 mm and dissolved in serum-free DMEM. After treatment with collagenase (type I) of mg / mL for 2 hours in a 37 ℃ incubator and neutralized in DMEM containing FBS, filtered using a strainer and isolated cells were DMEM (10% (v / v) FBS, Rheumatoid arthritis synovial fibroblasts were cultured at 1% (w / v) P / S).

4-2. Measurement of Cell Viability of Rheumatoid Arthritis Synovial Cells

After treating the rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 with ATC2 used in Example 3 at a concentration of 0 to 1000 μg / mL for 1 hour, the LPS was not treated or 1 μg / mL of LPS was applied. After treatment, the cells were incubated at 37 ° C. for 24 hours, and cell viability was measured by CCK-8 assay.

As a result, as shown in FIG. 5, it was confirmed that as the ATC2 was treated at a high concentration, cell viability decreased and cytotoxicity tended to increase. In addition, the LPS treatment was found to increase the number of cells by about 20% compared to the case without the LPS treatment, the higher the concentration of ATC2 treatment, the cell survival rate gradually decreased. In the case of LPS treatment, it was confirmed that the cell survival rate was over 80% up to the 100 μg / mL treatment concentration of ATC2.

4-3. Determination of migration inhibition using rheumatoid arthritis synovial cells

After treating the rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 with ATC2 used in Example 3 at a concentration of 100 μg / mL for 1 hour, 1 μg / mL of LPS was treated for 24 hours at 37 ° C. After culturing, the culture medium was removed and stained with crystal violet to measure synovial cell migration inhibitory activity. The stained photographs and the graphs of the number of transferred cells were shown in FIG. 6.

As a result, as shown in Figure 6, LPS alone treatment of the synovial cells during the development of rheumatoid arthritis increased, but no cell movement was observed when ATC2 treatment, similar to the control (not treated with both LPS and ATC2) Mobility was shown, and the number of migrated cells was quantified. The trend of mobility was similar in the graph to the right of FIG. 6 (*; p <0.05).

4-4. Rheumatoid Arthritis Synovial Cells Determination of Inflammatory Substances

After treating the rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 with ATC2 used in Example 3 at a concentration of 10 μg / mL and 100 μg / mL for 1 hour, 1 μg / mL of LPS was treated. Incubated at 37 ° C. for 24 hours, and cytokines and chemokines (TNF-alpha, IL-1beta, IL-6, IL-17A, IL-8, VEGF, MCP-1, CXCL10, MMP-1,2,3, 9,13) The concentration was measured by ELISA method and the result is shown in FIG.

As a result, as shown in Figure 7, inflammatory related substances (TNF-alpha, IL-6, IL-17A, MCP-1, CXCL10, IL-8, VEGF, MMP-1) during LPS treatment on rheumatoid arthritis synovial cells , MMP-2, MMP-3, MMP-9, and MMP-13) was found to increase significantly. In addition, in the cells treated with ATC2, even when LPS was treated at the same time, it was confirmed that the secretion amount of inflammatory substances related to rheumatoid was reduced ATC2 concentration-dependently.

4-5. Investigation of expression and positional changes of p65 NF-kB using rheumatoid arthritis synovial cells

The rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 were treated with ATC2 used in Example 3 at a concentration of 100 μg / mL for 1 hour, and treated with 1 μg / mL of LPS for 1 hour at 37 ° C. After removal of the culture solution, the result of observing the presence and the expression position of p65 NF-kB using immunofluorescence staining method is shown in FIG.

As a result, as shown in FIG. 8, when LPS was treated to rheumatoid arthritis synovial cells, expression of p65 NF-kB that existed only in the cytoplasm was observed in the cell nucleus, indicating that p65 NF-kB in the cytoplasm was transferred to the cell nucleus. it means. No migration of p65 NF-kB into the nucleus was observed with ATC2 treatment.

Since NF-kB is a major factor in the expression of rheumatoid arthritis, based on the above results, it can be seen that ATC2 has a prophylactic and therapeutic effect of rheumatoid arthritis since ATC2 inhibits nuclear migration of NF-kB.

4-6. Activation site identification of NF-kB using rheumatoid arthritis synovial cells

The rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 were treated with ATC2 used in Example 3 at a concentration of 10 μg / mL and 100 μg / mL for 1 hour, and treated with 1 μg / mL of LPS at 37 ° C. After the reaction for 15 minutes at 4% (w / v) formaldehyde was fixed to the cellulite and Cell-based ELISA was performed, the results are shown in FIG.

As a result, as shown in FIG. 9, when LPS was treated to rheumatoid arthritis synovial cells, residues of Ser468 and Ser536 of NF-k were phosphorylated. However, when ATC2 was treated, it was confirmed that two residues phosphorylation of NF-kB was reduced (activation of NF-kB was reduced).

Through the above results, it can be seen that ATC2 is effective in treating rheumatoid arthritis.

4-7. NO production confirmation test using rheumatoid arthritis synovial cells

RTC arthritis synovial cells obtained in the same manner as in Example 4-1 were treated with ATC2 used in Example 3 at a concentration of 10 μg / mL and 100 μg / mL for 1 hour, and then treated with 1 μg / mL of LPS. Incubated for 24 hours at ℃. NO (Nitric oxide, Nitrate) concentration in the culture was measured by the Griess assay method, the results are shown in Figure 10.

As a result, as shown in FIG. 10, when LPS was treated in rheumatoid arthritis synovial cells, NO production was increased by about 2 times or more compared to the normal group, whereas after LPS treatment, concentrations of 10 μg / mL and 100 μg / mL of ATC2 were observed. In case of treatment, NO production was remarkably suppressed under both conditions.

The results showed that ATC2 has an excellent anti-inflammatory effect.

4-8. Enzymatic Activity and Expression of Gelatinase Using Rheumatoid Arthritis Synovial Cells

The RTC arthritis synovial cells obtained in the same manner as in Example 4-1 were treated with ATC2 used in Example 3 at a concentration of 100 μg / mL for 1 hour, and treated with 1 μg / mL LPS for 24 hours at 37 ° C. The culture solution was recovered and centrifuged to conduct gelatin zymography assay for confirming activation of MMP-2 in the supernatant. The results are shown in FIG. 11.

Gelatinase enzymes are known to have increased activity in patients with rheumatoid arthritis, and gelatinase enzymes are known as MMP-2 and MMP-9.

As a result, as shown in FIG. 11, activation of matrix metalloproteinase-2 (MMP-2) in the form of activation was increased when synovial cells of rheumatoid arthritis patients were treated with LPS, whereas activation of MMP-2 disappeared when ATC2 was treated. As a result of measuring the protein expression level, the expression level of MMP-2 was increased by treatment with LPS, but the expression of the increased MMP-2 was decreased by treatment with LPS and ATC2. In the case of 9 it was confirmed that the expression completely disappeared by the treatment of LPS and ATC2.

4-9. Inflammation and cytoskeleton-related protein expression and expression location identification experiment using rheumatoid arthritis synovial cells

After treating ATC2 used in Example 3 at a concentration of 100 μg / mL for 1 hour to rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1, 1 μg / mL of LPS was incubated at 37 ° C. for 24 hours. After removing the culture medium and fixing the cells with acetone, immunofluorescence staining was used to confirm the presence and expression of inflammation-related proteins (COX-2, MMP-2, IL-1beta, Vimentin and fibronectin), and are shown in FIG. 12.

As a result, as shown in Figure 12, it was confirmed that the expression of COX-2, MMP-2, IL-1beta, vimentin, and fibronetin increased during LPS treatment was significantly reduced by ATC2 treatment, but in the treatment of LPS and ATC2 There was no change in the position of inflammation related protein expression.

4-10. Inflammatory Factor Identification Using Rheumatoid Arthritis Synovial Cells

The rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 were treated with ATC2 used in Example 3 at a concentration of 100 ug / mL for 1 hour. 1 ug / mL LPS was incubated at 37 ° C. for 24 hours and observed using an antibody array kit (ab134003; Abcam) for screening of inflammation-related factors in the culture, and the results are shown in FIG. 13.

As a result, as shown in FIG. 13, it was confirmed that the secretion amount of IL-6, CXCL8, CCL2 and CCL5 was increased when LPS was treated to synovial cells, and when LPS and ATC2 were simultaneously treated to cells, the antibody array resulted in an increase in LPS. It was confirmed that CCL5, a rheumatoid related inflammatory factor, was reduced again to a statistically significant level. (*; p <0.05)

4-11. Inflammation-related protein expression experiment using rheumatoid arthritis synovial cells

The rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 were treated with ATC2 used in Example 3 at a concentration of 100 μg / mL for 1 hour. After treatment with 1 μg / mL LPS for 5 minutes, 15 minutes, 30 minutes and 60 minutes respectively, the protein of the cells was obtained and the p-Erk, p-p38, p-JNK and p-Akt proteins were expressed by Western blot analysis. The amount was measured and shown in FIG.

As a result, as shown in FIG. 14, the expression of p38, JNK and Akt related to rheumatoid arthritis increased at 60 minutes after LPS stimulation, but the signaling of p38, JNK and Akt related to rheumatoid arthritis was increased during ATC2 treatment. It was confirmed that the decrease.

4-12. Inhibition of osteoclast differentiation using rheumatoid arthritis synovial cells

CD14 ⁺ cells obtained by MACS method by Ficoll separation from buffy coat blood of normal people were dispensed in 96 well plates at a concentration of 1 X 10 ^ 5 cells / well and incubated for 20 days with 20 ng / mL M-CSF treatment. It was. At 4 days, RANKL at 40 ng / mL and ATC2 used in Example 3 were treated by concentration to induce osteoclast differentiation for 5 days, and the osteoclast number after TRAP staining was measured and shown in FIG. 15.

As a result, as shown in Figure 15, when ATC2 was treated for each concentration, it was confirmed that the osteoclast differentiation is markedly reduced from a concentration of 50 μg / ml or more.

From the above results, it can be seen that ATC2 used in the present invention has an effect of inhibiting the differentiation of osteoclasts in a concentration-dependent manner.

4-13. In vitro Prevention and Treatment of ATC2 Rheumatoid Arthritis

Based on the above findings, it can be seen that ATC2 used in the present invention exhibits anti-inflammatory therapeutic effect by reducing the amount and expression of inflammation-related substances in synovial cells of rheumatoid arthritis patients.

In addition, the ATC2 reduced the secretion, expression and activity of MMPs that can cause cartilage and bone destruction, and exhibits the ability to inhibit osteoclast differentiation, which delays joint and bone damage and loss of function due to arthritis. It is similar to the properties of DMARD (disease-modifying anti-rheumatic drug), which is known to be active, and it can be seen that the ATC2 has an activity that delays joint and bone damage and loss of function due to arthritis. .

Claims (15)

100% by weight of the total extract or fraction purified (w / w), (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3-(((2, 3,4,5) -3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5- hydroxy-4H-chromen-4-one) 0.1 to 10% by weight, chlorogenic acid (0.5 to 30% by weight) and Cryptochlorogenic acid (Cryptochlorogenic acid) characterized in that it contains 0.5 to 30% by weight Honey leaf extract or fractional purified. Method for producing a honey leaf extract, characterized in that the step of extracting by adding a solvent to the honey (Stauntonia hexaphylla) leaf is carried out by repeating 1 to 20 times. The method of claim 2, wherein the solvent comprises at least one selected from the group consisting of water, methanol, ethanol, butanol, and mixtures thereof. The method of claim 2, wherein the extraction is performed at a temperature of 10 ° C. to 100 ° C. for 30 minutes to 72 hours, and at least one selected from the group consisting of cold needle extraction method, ultrasonic extraction method, reflux extraction method, hot water extraction method, room temperature extraction method, and reflux extraction method. Using a method, a method for producing honey leaf extract. (a) adding a solvent to the leaves of Stauntonia hexaphylla and repeating 1 to 20 times to obtain a honey leaf extract; And
(b) purifying the honey extract obtained in step (a)
Method for producing a fraction purified of honey leaf extract. The method of claim 5, wherein the purifying the honey extract comprises (a) reverse phase partition chromatography, (b) normal phase adsorption chromatography, and (c) ion exchange chromatography. A method for producing a fractional purified product of a honey leaf extract, which is carried out by ion exchange chromatography, (d) size exclusion chromatography, or one or more combinations thereof. Anti-inflammatory, or bone tissue production or cartilage, comprising as an active ingredient extracts from honey leaves, fractions thereof, or one or more selected from the group consisting of caffeic acid compounds and flavonoid compounds isolated from the extracts or fractions Pharmaceutical composition for promoting tissue production. According to claim 7, wherein the honey leaf-derived extract or fractional purified product thereof
100% by weight of the total extract or fraction purified (w / w), (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3-(((2, 3,4,5) -3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5- hydroxy-4H-chromen-4-one) 0.1 to 10% by weight, chlorogenic acid (0.5 to 30% by weight) and Cryptochlorogenic acid (Cryptochlorogenic acid) characterized in that it contains 0.5 to 30% by weight That is, anti-inflammatory, or a pharmaceutical composition for promoting bone tissue production or cartilage tissue production. Honey leaf-derived extract, fractional purified product thereof, caffeic acid compound and flavonoid compound isolated from the extract or fractional purified product comprising at least one selected from the group consisting of anti-inflammatory, or bone tissue production or cartilage tissue Food composition for production promotion. The method of claim 9, wherein the honey leaf-derived extract or fraction purified
100% by weight of the total extract or fraction purified (w / w), (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3-(((2, 3,4,5) -3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5- hydroxy-4H-chromen-4-one) 0.1 to 10% by weight, chlorogenic acid (0.5 to 30% by weight) and Cryptochlorogenic acid (Cryptochlorogenic acid) characterized in that it contains 0.5 to 30% by weight That is, anti-inflammatory, or food composition for promoting bone tissue production or cartilage tissue production. Anti-inflammatory, or bone tissue production or cartilage, comprising as an active ingredient extracts from honey leaves, fractions thereof, or one or more selected from the group consisting of caffeic acid compounds and flavonoid compounds isolated from the extracts or fractions Health functional food for tissue production promotion. The method of claim 11, wherein the extract derived from honey or fractions thereof is
100% by weight of the total extract or fraction purified (w / w), (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3-(((2, 3,4,5) -3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5- hydroxy-4H-chromen-4-one) 0.1 to 10% by weight, chlorogenic acid (0.5 to 30% by weight) and Cryptochlorogenic acid (Cryptochlorogenic acid) characterized in that it contains 0.5 to 30% by weight That is, anti-inflammatory, or health functional food for promoting bone tissue production or cartilage tissue production. Promotes anti-inflammatory, or bone tissue production or cartilage tissue production, containing as a main component one or more selected from the group consisting of honey leaf-derived extracts, fraction purified products thereof, or caffeic acid compounds and flavonoid compounds isolated from the extracts or fraction purified products thereof Food additives. The method of claim 13, wherein the honey leaf-derived extract or fractional purified product thereof
100% by weight of the total extract or fraction purified (w / w), (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3-(((2, 3,4,5) -3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5- hydroxy-4H-chromen-4-one) 0.1 to 10% by weight, chlorogenic acid (0.5 to 30% by weight) and Cryptochlorogenic acid (Cryptochlorogenic acid) characterized in that it contains 0.5 to 30% by weight That is, anti-inflammatory or food additives for promoting bone tissue production or cartilage tissue production. 100% by weight of the total extract or fraction purified (w / w), (7-(((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3-(((2, 3,4,5) -3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5- hydroxy-4H-chromen-4-one) 0.1 to 10% by weight, chlorogenic acid (0.5 to 30% by weight) and Cryptochlorogenic acid (Cryptochlorogenic acid) characterized in that it contains 0.5 to 30% by weight Pharmaceutical composition for the prevention or treatment of rheumatoid arthritis or degenerative arthritis.

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