KR102126575B1 - 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 is a method for producing extracts or fractions of extracts derived from leaves of Stauntonia hexaphylla, extracts of fractions of extracts derived from leaves of leaves prepared by the above manufacturing method, and extracts or fractions purified from extracts of fractions of leaves, or extracts or fractions It relates to a pharmaceutical composition for promoting anti-inflammatory, or bone tissue production or cartilage tissue production, including as an active ingredient containing at least one selected from the group consisting of caffeic acid compounds and novel flavonoid compounds separated from water.

Description

Anti-inflammatory, or pharmaceutical composition for promoting bone tissue production or cartilage tissue production, including extracts from mulberry leaves or fractional tablets or novel flavonoid compounds and caffeic acid compounds isolated therefrom 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 is a method for producing extracts or fractions of extracts derived from leaves of Stauntonia hexaphylla, extracts of fractions of extracts derived from leaves of leaves prepared by the above manufacturing method, and extracts or fractions purified from extracts of fractions of leaves, or extracts or fractions It relates to a pharmaceutical composition for promoting anti-inflammatory, or bone tissue production or cartilage tissue production, including as an active ingredient containing at least one selected from the group consisting of caffeic acid compounds and novel flavonoid compounds separated from water.

Bone tissue is usually made of dense bone with a strong bone surface, and both ends of the central or iliac bone are spongy bone associated with a mesh like a 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. At the bony end (骨端), there is an articular surface at the part that comes into contact with the neighboring bone, and the surface is covered with articular cartilage, a supernatural bone. Among the spongy quality, spongy shochu (海綿小柱) is characterized by a constant arrangement. The broad ganglia of the bone extremity is continuous with the small lumen made of cavernous soju and all are filled with bone marrow. Hematopoietic bone marrow that acts as a hematopoietic is red with a large distribution of blood vessels. The structure of the bone is superimposed or spongy, and the corrugated board with a thickness of 5 to 12 µm is overlapped. In the compacted form, the corrugated board (Harbus layer) in which several layers overlap in concentric circles is arranged in various directions. There is a harbor tube, and blood vessels pass through it. Bone cells are arranged between the bone layer plates and are connected to other adjacent bone cells with protoplasmic projections that go into an irregular star shape. On the surface of the bone, there is a tough connective tissue periosteum, and nerves and blood vessels are distributed to protect and nourish the bones. When the periosteum is defective, bone survival, regeneration, and regeneration become difficult. The composition of bone material is 20% of moisture, 35% of organic matter including cells, and 45% of inorganic matter, because the bone's constant elasticity is organic. As age increases, minerals (mainly calcium phosphate) increase, and bone hardness increases.

The exact pathogenesis and mechanism of chronic inflammation, rheumatoid arthritis, is not yet well known, but one of the most prominent pathogens identified to date is osteoblasts that produce bone and osteoclasts that destroy bone. ) Is known to occur due to the collapse of bone homeostasis due to numerical imbalance. Although normal osteoclasts differentiate from monocytes and macrophages to destroy old bones or remove damaged bones, the osteoclasts found in rheumatoid patients are constantly increasing in number due to osteoclastogenesis Survival and excessive activity are confirmed, showing a pathological form in which bone is significantly damaged. Osteoclasts produce a variety of enzymes to break down bones, oxidizing and decomposing by oxidizing bones, TRAP (tartrate-resistant acid phosphatase) and matrix metallopeptidase-MMP-9 (which breaks down cell support tissues of bone and cartilage) 9) Cathepsin K, which breaks down collagen, is known as the main enzyme, and these enzymes are used as special markers for mature osteoclasts. When RANKL is treated on macrophages, which are precursors of osteoclasts, NF-κB and MAPK kinase are activated to produce inflammatory mediators.

According to the recent findings, it is known that the progress of the inflammatory reaction in the body is related to the activity of COX (cyclooxygenase) enzyme. The COX enzyme is a main enzyme involved in the biosynthesis of prostaglandin present in vivo (Smith et al., J. Biol. Chem., 271, 33157 (1996)), two types of isomers, COX-1 and COX It is known that -2 exists. The COX-1 is constantly present in tissues such as the stomach and kidneys, and is involved in maintaining normal homeostasis, whereas the COX-2 is involved in mitogens or cytokines during inflammation or other immune responses. It is an enzyme that is expressed temporarily and rapidly in cells. Another powerful inflammatory mediator, nitric oxide (NO), is produced from L-arginine by NO synthetase (NOS), and has many types due to external stress such as UV or substances such as endotoxin or cytokine. It is produced in cells. The inflammatory stimuli as described above may increase the expression of inducible NOS (iNOS) in the cell, thereby inducing NO production in the cell, thereby activating macrophages to cause an inflammatory response. Therefore, in recent years, studies on substances capable of inhibiting the production of NO have been conducted for effective inflammation relief.

However, in the case of the anti-inflammatory substances developed by these studies, some side effects are problematic. For example, nonsteroidal anti-inflammatory drugs used in the treatment of acute inflammatory diseases or chronic inflammatory diseases are known to exhibit side effects such as gastrointestinal disorders by inhibiting the COX-2 enzyme as well as the COX-1 enzyme. In addition, recently, the mechanism of treatment related to gastritis is mainly composed of H2 inhibitors (H2-Blockers) that block the second histamine receptor (H2 receptor) and reduce gastric acid secretion from gastric wall cells. The reduction of gastric acid prevents further damage of already damaged gastric wall cells (such as stomach ulcers). Because these H2 inhibitors interfere with the metabolism of other drugs in the liver (potent inhibitors of P-450), caution should be taken when taken with other drugs, and have an anti-androgen effect, causing gynecomastia in men. ), side effects such as impotence and decreased libido may occur. In addition, since it passes through the placenta and cerebrovascular barrier, side effects may be more dangerous for pregnant women or the elderly, and may cause headache, confusion, confusion, and dizziness.

Therefore, it is possible to effectively suppress the production of NO as a substance derived from natural substances, and also to suppress iNOS and TNF-α expression, and effectively inhibit the activity of the COX-2 enzyme, thereby anti-inflammatory effect, osteoblast ALP activity, and There is a need to develop a material that promotes osteoblast differentiation and generates bone or cartilage tissue, and is a natural substance-derived material that has little or no risk of side effects or cytotoxicity and thus has little restriction on its use.

Stapling honey (Stauntonia hexaphylla) is an evergreen vine plant that grows up to 15 m tall and grows 15 m tall in mid-April to mid-May, and is a child's fist-sized black-red fruit in autumn. Is a hanging plant (Ikuta, 1989; Lee, 1996). The fruit is known as a fruit that is so delicious that the name was derived from the meaning of'sweet like honey', but the fruit has not been commercialized due to the large number of thick seeds and less flesh. The leaves can be overwintered in the southern regions, the flesh is thick, and the ornamental value is high, so its use is increasing for gardening and pergola, and accordingly, seedling production is increasing.

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

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

[Patent Document 1] Republic of Korea Patent Publication No. 1992-0005995 [Patent Document 2] Republic of Korea Patent Publication No. 1994-0006596 [Patent Document 3] Republic of Korea 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

By weight of total extract, (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 Mulberry leaf extract and fractional tablets thereof, characterized in that it contains from 10 to 10% by weight, chlorogenic acid from 0.5 to 30% by weight, and from Cryptonchlorogenic acid to 0.5 to 30% by weight Is to provide.

Another object of the present invention relates to a method for producing a honeycomb leaf extract, characterized in that the step of extracting by adding a solvent to the dried honeycomb (Stauntonia hexaphylla) leaf is repeated 1 to 20 times.

Still another object of the present invention relates to a method for producing a fractional purified product of honeysuckle leaf extract, wherein the honeysuckle leaf extract is subjected to chromatography.

Another object of the present invention includes as an active ingredient containing at least one selected from the group consisting of extracts or fractions purified from mulberry leaves, or caffeic acid compounds and novel flavonoid compounds separated from the extracts or fractions purified To provide an anti-inflammatory, or pharmaceutical composition for promoting bone tissue production or cartilage tissue production.

Another object of the present invention comprises as an active ingredient containing at least one selected from the group consisting of extracts or fractions purified from mulberry leaves, caffeic acid compounds separated from the extracts or fractions purified and novel flavonoid compounds , Anti-inflammatory, or to provide a food composition for promoting the formation of bone tissue or cartilage tissue.

Another object of the present invention includes as an active ingredient containing at least one selected from the group consisting of mulberry leaf-derived extracts or fractional tablets, or caffeic acid compounds and novel flavonoid compounds isolated from the extracts or fractional tablets. To provide an anti-inflammatory, or health functional food for promoting bone tissue production or cartilage tissue production.

Another object of the present invention is a health supplement food containing at least one selected from the group consisting of mulberry leaf-derived extracts or fractional tablets, or caffeic acid compounds and novel flavonoid compounds separated from the extracts or fractional tablets. Is to provide

Another object of the present invention is to provide a food additive containing at least one selected from the group consisting of extracts or fractions purified from mulberry leaves, or caffeic acid compounds and flavonoid compounds separated from the extracts or fractions purified Is to do.

Another object of the present invention is an active ingredient comprising at least one selected from the group consisting of extracts from mulberry leaves, fractional refinements thereof, or caffeic acid compounds and novel flavonoid compounds isolated from the extracts or fractional refinements. To provide a pharmaceutical composition for the prevention or treatment of rheumatoid arthritis or degenerative arthritis, including.

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

In one embodiment, the present invention is an anti-inflammatory comprising an extract or fraction purified from novel honey leaves and a novel flavonoid compound or caffeic acid compound isolated from the extract or fraction purified from honey leaves, or It relates to a pharmaceutical composition for promoting the formation of bone tissue or cartilage tissue.

The honeysuckle (Stauntonia hexaphylla) leaves used herein may be purchased commercially, or may be harvested or cultivated in nature, but are not limited thereto, and dried appropriately prior to extraction and purification, slicing, and grinding to effectively extract and It can be pretreated to go through the purification process.

The novel extract or fraction purified product as defined herein may be an extract obtained by extracting leaves of Stauntonia hexaphylla with a solvent selected from lower alcohols having 1 to 4 carbon atoms such as water, methanol, ethanol, butanol, or a mixed solvent thereof. 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 pertains.

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

The extract or fraction 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 caffeic acid compounds, chlorogenic acid and cryptochlorogenic acid, within a specific range.

Specifically, the extract or fraction purified is 100% by weight of the whole 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 0.5 It may be characterized as containing from 30 to 30% by weight,

Preferably 100% by weight of the whole extract or fraction purified (w/w), new 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) contains 0.1 to 5% by weight, chlorogenic acid 0.5 to 20% by weight, and kryptochlorogenic acid containing 0.5 to 20% by weight It can be characterized as.

In one specific embodiment of the present invention, the crude honey leaf extract according to 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) 0.56% by weight, chlorogenic acid 1.61% by weight, and cryptochlorogenic acid 1.07% by weight, fractional purification of these crude extracts 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% by weight, chloro It was confirmed that it contained 5.34% by weight of genic acid and 3.85% by weight of cryptochlorogenic acid.

In yet another aspect, the present invention relates to a method for producing a mulberry leaf extract, characterized in that the step of extracting by adding a solvent to the mulberry (Stauntonia hexaphylla) leaf is performed 1 to 20 times.

In the above manufacturing method, the honeysuckle (Stauntonia hexaphylla) leaf may be pre-treated, such as through a washing, drying, cutting or crushing process according to a known method, and preferably a dried honeysuckle leaf may be used. The honeysuckle leaves are extracted using a suitable extraction solvent. 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 is not limited thereto. No, preferably water can be used. Specifically, the extraction temperature may be a temperature of 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 does not work. As the extraction method, an extraction method selected from a cold needle extraction method, an ultrasonic extraction method, a reflux cooling extraction method, a hot water extraction method, a normal temperature extraction method, a reflux extraction method, or a combination of one or more extraction methods can be used, but a known natural product extraction method can be appropriately selected and used without limitation. . More preferably 10 ℃ to 60 ℃, preferably at a temperature of 20 ℃ to 50 ℃ 30 minutes to 72 hours, preferably after 6 hours to 48 hours after cold immersion extraction, 40 ℃ to 120 ℃, specifically 60 The reflux cooling extraction method may be performed repeatedly at about 1 to 20 times, preferably 2 to 10 times, for 30 minutes to 72 hours, preferably 6 hours to 48 hours at a temperature of ℃ to 100 °C.

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

In yet another aspect, the present invention relates to a method for producing a fractional purified product of honeycomb leaf extract, wherein the honeycomb leaf extract is subjected to chromatography.

In a specific embodiment, the fraction purified product of the extract of mulberry leaves can be prepared by first preparing the extract using mulberry leaves and then fractionating it by chromatographic treatment, wherein the method of producing mulberry leaf extract is The same can be applied to the extract manufacturing method.

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 a combination of one or more of these, 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 Sephadex-based resins including acrylate resins, carboxymethyl cellulose, sulfopropyl cellulose, carboxymethyl sephadex, sulfopropyl sephadex, sulfopropyl sepharose and carboxymethyl sepharose; Reverse phase polymer resins using a styrene-divinylbenzen co-polymer family, such as polymer X, HP20, and PRP-h1 polymer, and a methacrylate support family; Normal phase silica gels such as Merck's adsorption Bonded Phase Chromatography (BPC) product line, YMC's Silica product line, DAISO's Silica product line, ASAHI's Silica product line, and COSMOSYL's Silica product line; Stationary phases of resins selected from reverse phase silica gel groups, such as YMC's ODS family, DAISO's ODS family, ASAHI's ODS family, CHEMCO's ODS family, COSMOSYL's ODS family, and FUJI's ODS family, mainly used as HPLC fillers. It includes a method for performing a chromatography method using.

Specifically, in the above (a) reverse phase distribution chromatography, the stationary phase is a reverse phase column material, and all the fixed phases characterized by eluting the polar materials first and retaining the non-polar compound as the stationary phase, to which a hydrophobic material can be attached. Stationary phase, preferably silica gel-based stationary phase or polymer-based stationary phase such as polystyrene, more preferably C2, C4, C6, C8, C10, C12, C14, C16, or C18 or silica gel thereof Derivatives or polymer-based stationary phases such as PS-2, Oasis HLB, and more preferably, reverse phase silica gel (C18(IV)-D), (YMC's ODS-A/ODS-AQ product family, CHMECO's SP-C-ODS Product family, SP-ODS-RPS product family of DAISO, 5C18 product family of COSMOSYL, Chromatorex product family of FUJI).

The mobile phase is distilled water, an alcohol solvent such as 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, more preferably water And starting the mixed solvent of methanol at 90:10 (v/v) to adjust the ratio of 60:40 to elute the polar material.

Specifically, in the (b) normal phase chromatography method, the stationary phase is a normal phase column material, which is eluted with non-polar materials first, and any fixed phase characterized in that the polar compound is retained in the stationary phase. Preferably, silica gel, fluorosyl, alumina-based stationary phase, CN, diol, NH ₂ star 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) Should be

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

Specifically, in the (c) Ion exchange resin column chromatography (Ion Exchange resin columm Chromatography), "ion exchange chromatography material" is a chromatographic functional group using all immobilized high molecular weight solids having covalently bonded charged groups. Possible; Preferably, a cation exchange resin, an anion exchange resin, or a chromatography method using a synthetic adsorbent, more preferably, AG 50W-x8, Amberlite IR-120, Dowex 50W-x8 and SK1B Strong acid cation exchange resins such as; Weakly acidic cation exchange resins such as Amberlite IRC-50, Bio-Rex 70, Duolite-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 CR13 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.), Strong basic cation exchange resins such as MFG 210 and MFG 250 (Finex co.); Preferably, a strong acid cation exchange resin SK1B type 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 having 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 can be used; The synthetic adsorbent is SP207, HP20SS, HP20 or the like, and preferably HP20 can be used.

Specifically in (d) size exclusion chromatography, the gel is typically a dextran-based gel, such as Sephadex (eg Sephadex G-25) or polyacrylamide gel, eg For example, Sephacryl (e.g. Sephacryl-S400), agarose-based gels such as Superose or Sepharose (e.g. Sepharose CL-4B), and complex gels prepared from two types of gels, For example, Superdex 200 combination Dextran (Sephadex™) and crosslinked Agarose (Superose™) gels are selected from the group of polymerized gels including, but not limited to; 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 is further (a) reverse phase partition chromatography (Reverse phase partition chromatography), (b) normal phase partition chromatography (Normal phase partition chromatography), (c) ion exchange chromatography (ion exchange chromatography), (d) Sephadex LH-20 or Toyopearl HW-40, which can use organic solvents other than size exclusion chromatography or a combination of one or more of them. (e) Gel permeation or gel filtration chromatography. Chromatography (Gel filtration chromatography) can be additionally used.

The synthetic adsorbent may be 0.5 to 20 times the volume of the suspended solvent (v/v) HP20, 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 extract or fraction purified from the honeysuckle leaf, or a caffeic acid compound and a novel flavonoid compound separated from the extract or fraction purified. It relates to a pharmaceutical composition comprising, as anti-inflammatory, or to promote bone tissue production or cartilage tissue production.

In the pharmaceutical composition according to the present invention, the matter related to the extract or fractional tablet derived from mulberry leaves may be applied as it is to the extract or fractional tablet.

The caffeic acid compound is preferably one or more selected from the group consisting of chlorogenic acid and Cryptochlorogenic 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 mulberry leaves prepared by the above manufacturing method inhibits the differentiation inhibitory activity of osteoclasts (Example 3).

Through the above results, the extract comprising the extract or fraction purified from mulberry leaves or the caffeic acid compound separated from the extract or fraction purified prepared by the above manufacturing method includes at least one selected from the group consisting of rheumatoid arthritis patients It can be seen that the anti-inflammatory treatment effect is shown by reducing the secretion amount and expression of inflammation-related substances in synovial cells.

In addition, through the results, the extract containing the extract or fractions derived from honey leaves prepared by the above method or caffeic acid compounds separated from the extracts or fractions purified and containing at least one selected from the group consisting of novel flavonoid compounds include arthritis It has the activity of delaying the damage of joints and bones and loss of function of joints, and through this, the extract or fractions derived from honey leaves prepared by the above manufacturing method has an inhibitory effect on osteoclast activity, and promotes bone tissue production or cartilage tissue production It can be seen that it works.

In addition, the present invention 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 is, but is not limited to, arthritis, joint destruction disease caused by autoimmune disease, multiple myositis, ankylosing spondylitis, systemic lupus erythematosus, multiple myositis, rheumatoid bundle And polymyalgia rheumatica, osteoporosis, bone metastasis, or Paget's disease. In addition, the arthritis may include various types of arthritis, for example, diseases such as osteoarthritis, rheumatoid arthritis, degenerative arthritis, psoriatic arthritis, reactive arthritis, but is not limited thereto. It is preferably rheumatoid arthritis.

Pharmaceutical compositions comprising extracts or fractional tablets or compounds according to the present invention, respectively, according to a conventional method, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and the like It can be formulated and used in the form of a sterile injectable solution. 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. In the case of formulation, it is usually prepared using diluents or excipients such as fillers, weights, binders, wetting agents, sealants and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the extract and fractional tablets, for example, starch, calcium carbonate, It is prepared by mixing sucrose, lactose, or gelatin. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.

Non-aqueous solvents, suspending agents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as the above-described preparation. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerol gelatin may be used.

The preferred dosage of the pharmaceutical composition comprising the extract or fractional tablet or compound of the present invention varies depending on the patient's condition and body weight, the degree of disease, the drug form, the route and duration of administration, but can 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 per day, preferably 0.001 to 500 mg/kg per day. The administration may be administered once a day, or may be divided into several times. The above dosage does not limit the scope of the present invention in any way.

The extract or fraction purified or compound of the present invention can be administered to various mammals such as rats, mice, livestock, and humans by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine epidural or intracerebroventricular injection.

As another aspect, the present invention includes as an active ingredient one or more selected from the group consisting of extracts or fractions purified from honeysuckle leaves, caffeic acid compounds separated from the extracts or fractions purified and novel flavonoid compounds The present invention relates to an anti-inflammatory, or food composition for promoting bone tissue production or cartilage tissue production.

The anti-inflammatory, or for the pharmaceutical composition for promoting bone tissue production or cartilage tissue production is the following anti-inflammatory, or 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 Alternatively, the same may be applied to a food supplement for promoting bone tissue production or cartilage tissue production, an anti-inflammatory, or bone tissue production or cartilage tissue production promotion.

As another aspect, the present invention is an active ingredient comprising at least one selected from the group consisting of mulberry leaf-derived extracts or fractional tablets, or caffeic acid compounds and novel flavonoid compounds isolated from the extracts or fractional tablets. Contains, anti-inflammatory, or relates to a health functional food for promoting bone tissue production or cartilage tissue production.

In another aspect, the present invention, the anti-inflammatory, containing at least one selected from the group consisting of extracts or fractions purified from mulberry leaves, or caffeic acid compounds and novel flavonoid compounds isolated from the extracts or fractions purified, Or, it relates to a dietary supplement for promoting bone tissue formation or cartilage tissue formation.

In another aspect, the present invention, the anti-inflammatory, containing at least one selected from the group consisting of extracts or fractions purified from mulberry leaves, or caffeic acid compounds and novel flavonoid compounds isolated from the extracts or fractions purified, Or, it relates to a food additive for promoting the formation of bone tissue or cartilage tissue.

"Health functional food" as defined herein refers to food manufactured and processed using ingredients or ingredients having useful functionality for the human body in accordance with Act 6727 on the Health Functional Food, and "functional" means It means to ingest for the purpose of obtaining useful effects for health purposes such as adjusting nutrients for structure and function or physiological action.

The health functional food of the present invention includes the extract or fractional tablets in an amount of 0.01 to 95%, preferably 1 to 80% by weight, based on the total weight of the composition.

In addition, for the purpose of suppressing anti-inflammatory or osteoclast activity, as a pharmaceutical functional form such as powder, granule, tablet, capsule, pill, suspension, emulsion, syrup, or health functional food in the form of tea bags, leaching tea, health drinks, etc. Manufacturing and processing are possible.

In addition, the health functional food may additionally include food additives, and whether or not it is suitable as a "food additive" is applied to the relevant item according to the General Regulations and General Test Methods of the Food Additives Code approved by the Ministry of Food and Drug Safety unless otherwise specified. It is judged according to related standards and standards.

Items listed in the "Food Additives Exhibition" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamonic acid, natural additives such as chromosomes, licorice extract, crystalline cellulose, guar gum, and sodium L-glutamate. And mixed preparations such as formulations, noodles-added alkalis, preservatives, and tar colorants.

Functional foods containing the extract or fractional tablets or compounds of the present invention include breads, rice cakes, dried fruits, candies, chocolates, chewing gums, confectionary ice creams, ice creams, ice cream powders, ice cream products, milks, and low fat foods. Dairy products such as milk, lactose-degraded milk, processed milk, goat milk, fermented milk, butter oil, condensed milk, milk cream, butter oil, natural cheese, processed cheese, powdered milk, whey, meat processed food, egg processed food, hamburger and Same meat products Fish products such as fish cakes, ham, sausage, bacon, etc. Fish products such as ramen, dried noodles, raw noodles, fried noodles, luxurious dried noodles, improved sleeping noodles, frozen noodles, noodles such as pasta, fruit drinks, vegetable drinks , Carbonated drinks, soy milk, lactic acid bacteria drinks such as yogurt, beverages such as mixed drinks soy sauce, miso, red pepper paste, chunjang, cheonggukjang, mixed soy sauce, vinegar, sauces, tomato ketchup, curry, seasoning foods such as margarine, shortening and pizza However, it is not limited thereto.

The health functional beverage composition of the present invention is an essential component in the indicated proportions, and there is no particular limitation on other components other than those containing the extract or fractional tablets or compounds, and additional components such as various flavors or natural carbohydrates, such as ordinary beverages It can contain as. Examples of natural carbohydrates described above include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); And polysaccharides, common sugars such as (eg, dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (such as taumatin and stevia extract (for example, rebaudioside A, glycyrrhizine)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The proportion of the natural carbohydrate is generally about 1 to 20 g per 100 ml of the composition of the present invention, preferably about 5 to 12 g.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and the like. It may contain salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain flesh for the preparation of natural fruit juices and fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. The proportion of these additives is not so critical, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

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

In the process of manufacturing the dietary supplement, the extract or fractional tablet or compound according to the present invention, which is added to foods including beverages, may appropriately adjust its content as necessary.

In another aspect, the present invention is 100% by weight of the whole 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 0.5 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 is a method for producing extracts or fractions of extracts derived from leaves of Stauntonia hexaphylla, extracts of fractions of extracts derived from leaves of leaves prepared by the above manufacturing method, and extracts or fractions purified from extracts of fractions of leaves, or extracts or fractions As an active ingredient, containing at least one selected from the group consisting of caffeic acid compounds and novel flavonoid compounds separated from water, relates to a pharmaceutical composition for promoting anti-inflammatory, or bone tissue production or cartilage tissue production, according to the present invention Novel extracts or fractional refinements isolated from extracts derived from mulberry leaves or novel flavonoid compounds and caffeic acid compounds isolated therefrom are not only cytotoxic, but also cytokines associated with inflammation, namely IL-1β, IL- 6, TNF-α, MCP-1, CXCL10, IL-8, VEGF, reducing the secretion of MMP, reducing the activation of NF-κB to confirm the inhibitory activity of COX-2 enzyme that causes NO secretion and inflammation It can effectively inhibit inflammation, and is very useful as a preventive, ameliorating or treating agent for various diseases related to bone tissue or cartilage tissue destruction, such as rheumatoid arthritis, or as a functional food.

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

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Reference example One: Honey leaf Crude extract Manufacturing and identification of indicator components

After drying and cutting 1.0 kg of mulberry (Stauntonia hexaphylla) leaves (GAP cultivation, 565-2, Bujun-ri, Pusan-myeon, Jangheung-gun, Jeollanam-do), 10 L of water was added to the mulberry leaf sample, followed by extracting with cold for 24 hours at room temperature. Thereafter, reflux extraction was performed while 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 concentrator (EYELA, N-2100, JAPAN) to obtain 300 g of crude honey leaf water extract (hereinafter referred to as ATC1).

High performance liquid chromatography (HPLC, Agilent 1260, USA) was performed under the same conditions as Table 1 and Table 2 (mobile phase conditions) to confirm the indicator components included in the obtained honeycomb leaf extract. It is shown in 1.

In Table 2, mobile phase A is 0.025 M phosphate buffer (pH = 3.0), and mobile phase B is 90% (w/v) ACN (Acetonitrile).

High-speed 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 Detection wavelength (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 40-45 60 40 45-50 30 70 50 ~ 56 30 70 56 ~ 70 89 11

As a result, as shown in Fig. 1, chlorogenic acid at 8.67 minutes, cryptochlorogenic acid at 9.15 minutes, and a new compound at 26.89 minutes were detected.

Based on the HPLC pattern shown in FIG. 1 (stay time), the content (%) of the index components was analyzed according to Equation 1 below.

[Equation 1]

A _T : Surface area of indicator component in sample solution

A _S : Area of indicator component in standard solution

However, it is used when the amount taken for the indicator component of the test solution and the standard solution is the same.

As a result, the crude extract of mulberry leaves prepared by the preparation method of Reference Example 1 was 1.61% (w/w) of chlorogenic acid, 1.071% (w/w) of cryptochlorogenic acid, 0.56% of new compound ( w/w).

Surface composition ATC1 Retention time (minutes) Content (w/w%) Chlorogenic acid 8.67 1.61 Cryptochlorogenic acid 9.15 1.07 New flavonoid compound 26.89 0.56 Sum 3.24

Example One. Honey Of leaf extract Organic solvent Soluble fraction Purified water Manufacturing and identification of indicator components

After dissolving the extract of mulberry leaf water prepared in Reference Example 1 in distilled water to adjust to 23 brix, 700 mL of reversed phase silica gel (HP20, Mitsubishin Co., Ltd. JAPAN) was filled in a column tube, and the 23 was added to the filled column. Secondly, the crude honey leaf fraction purified (ATC2) was separated by passing through 700 mL of the brix honey leaf crude extract. Mobile phase composition for separation was performed by controlling the ratio of distilled water:methanol (100:0 to 30:70). First, 2.1 L of distilled water: methanol (100:0) eluent was separated, then 5.6 L of distilled water: methanol (30:70) eluent was separated under the same conditions, and then distilled water eluent 2-3 times (1.4 L) and 70% ( v/v) After adding 2.8 L of methanol eluent 1-4 times and removing the solvent through a concentrated process under reduced pressure, 55 g of the finally purified mulberry fraction extract or fraction purified product was separated. (Hereinafter referred to as ATC2).

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

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

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

Surface composition ATC2 Retention time (minutes) content(%) Chlorogenic acid 8.67 5.34 Cryptochlorogenic acid 9.15 3.85 New flavonoid compound 26.89 1.14 Sum 10.33

Example 2. Separation and structural analysis of caffeic acid compounds and flavonoid compounds

The structure of the compound identified in Example 1 was identified by comparing the NMR data of the compound with physical properties described in the existing literature.

One. 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, br d, J =5 Hz, H-3), 3.94 (1H, br s, H-5), 3.55 (1H, br d, J =4 Hz, H-4), 1.7-2.1 (2H, m, H-6), 1.98 (2H, br d, 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. New 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. Honey Extracts and fractions Purified water Inhibition of osteoclast differentiation and confirmation of physiological activity

3-1. Isolation of bone marrow cells and osteoclast differentiation

The tibia of a 4-5 week old ICR mouse is extracted, the ends are cut, and α-MEM essential medium is passed through to collect bone marrow cells and treated with 50 ng/mL macrophage-colony stimulation factor (M-CSF). And cultured for 24 hours. After washing non-adherent cells with α-MEM, the cells were dispensed into 5×10 ⁵ cells/well in 48-wells, and cultured in α-MEM medium containing 50 ng/mL of M-CSF for 3 days. Thereafter, 50 ng/mL MCSF and 100 ng/mL RANKL (Receptor Activator for Nuclear Factor κB Ligand) were treated together, and samples were treated for each concentration to incubate for 4 days to differentiate into osteoclasts.

3-2. Honey Leaf extract( ATC1 ), fraction Purified (ATC2) Measurement of TRAP activity change of osteoclasts

Osteoclasts express tartrate-resistant acid phosphatase (TRAP), an acid phosphatase, which is characteristicly resistant to tartrate, and this feature is a cytochemical 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 in TRAP activity of osteoclasts by the honeycomb leaf extract (ATC1) of the present invention and its fraction purified product (ATC2), the following experiment was performed.

Specifically, in the same manner as in Example 3-1, 48-well was inoculated with cells to be 5×105 cells/well, treated with a differentiation factor (RANKL, MCSF) and a sample, and cultured for 4 days. Changes were measured.

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

Negative control is not treated with RNAKL and ATC1 or ATC2, and is a bone marrow cell that has not differentiated into osteoclasts, and a positive control is treated with RANKL and differentiated into osteoclasts, and after processing the sample, COX2 inhibitor Celecoxib, which is a COX2 inhibitor (5 uM, or 20 uM) and cultured for 4 days.

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

Each of the extracts obtained from Example 3-1 of the extract of leaves (ATC1), the second fraction extract or fractions (ATC2) by concentration, namely, extract of leaves of honey ATC1 (1, 10, 50, 100 ug/ml), leaves of honey The second fraction extract or fraction purified ATC2 (0.1, 1, 10, and 50 ug/ml) was treated to osteoclasts, cultured for 4 days, and then TRAP activity of osteoclasts (TRAP activity shows resistance to tartrate) The effect on acid phosphatase activity) was measured, and the results are graphically shown in FIG. 3.

Through the results shown in Figure 3, it was confirmed that the activity of osteoclasts was inhibited depending on the concentration of the honeycomb leaf extract (ATC1), the secondary fraction extract or the fraction purified (ATC2) of the present invention.

More specifically, in the case of the ATC2 extract extracted through the fractionation purification process, 71.7% (0.1 ug) of each of the fraction purified purified ATC2 (0.1, 1, 10, and 50 ug/ml) compared to the positive control (100%) /ml), 36.4% (1 ug/ml), 21.9% (10 ug/ml) and 221.1% (50 ug/ml) with TRAP activity, 1/10 of crude extract without processing (ATC1) Even at the lower concentrations, it showed a significant TRAP activity inhibition effect of 2-3 times or more.

Particularly, when treated with 1 ug/ml of the concentration of purified fraction (ATC2) of the extract of mulberry leaf, the inhibition rate of TRAP activity was 63.6% (inhibition rate: 100%-36.4% = 63.6%) compared to the control, which is mulberry leaf When the concentration of the extract (ATC1) was treated with 50 ug/ml, the result was equivalent to the TRAP activity inhibition rate of 66.4% (inhibition rate: 100%-33.4% = 66.4%). In other words, it was shown that the fraction extract of mulberry leaf extract (ATC2) had a 50-fold increase in activity compared to mulberry leaf extract (ATC1).

3-3. Honey Leaf extract( ATC1 ), or fraction Purified (ATC2) Inhibitory effect of osteoclast differentiation

In the same manner as in Example 3-1, 48-well was inoculated with the cells to be 5×10 ⁵ cells/well, and differentiation factors (RANKL, MCSF), honeydew leaf extract (ATC1), and fraction purified products (ATC2) thereof Treatment was performed and cultured 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 fixed to cells fixed with 10% (w/v) formaldehyde. It dispensed and reacted at 37 degreeC for 30 minutes. The medium was removed, washed with PBS, and then the cells were fixed with 10% (w/v) formaldehyde for 15 minutes, and the cells were washed 3 times with PBS. TRAP staining was performed by adding 1 μg/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, followed by 10% (w/v ) Dispense 45 μL of the staining solution into cells fixed with formaldehyde, stain for 30 minutes at 37° C., and observe with a microscope to show the results in FIG. 4.

As shown in FIG. 4, TRAP-positive multinuclear osteoclasts were formed in the negative control cells, but when the honeycomb leaf extract (ATC1) or the fraction purified (ATC2) was treated, the concentration-dependent differentiation of the osteoclasts was suppressed. Could.

More specifically, in the case of the ATC2 extract extracted through the primary extraction and the secondary fractionation purification process, significant osteoclast differentiation of 2-3 times or more despite the treatment of a concentration that is 1/10 lower than the extract without processing (ATC1) ) It was confirmed that it exhibits an inhibitory effect.

Example 4. In vitro Rheumatism Prevention and treatment of arthritis

4-1. Synovial fibroblasts Separation and primary culture

Among the rheumatoid arthritis patients meeting the American Rheumatology Society's diagnostic criteria, synovial tissue obtained from patients requiring surgical treatment of the knee joint was washed with PBS, chopped into 2-3 mm, and dissolved in DMEM without serum (2). After treating collagenase (type I) at mg/mL for 2 hours in a 37°C incubator, neutralized in DMEM containing FBS, filtered using a strainer, and separated cells were DMEM (10% (v/v)FBS, Cultured at 1% (w/v) P/S) to obtain rheumatoid arthritis synovial fibroblasts.

4-2. Rheumatism arthritis Synovial Cell viability measurement

After treating ATC2 used in Example 3 with rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 at a concentration of 0 to 1000 μg/mL for 1 hour, LPS was not treated or 1 μg/mL LPS was used. After treatment and incubation at 37° C. for 24 hours, the cell viability was measured by the CCK-8 assay method, and the results are shown in FIG. 5.

As a result, as shown in FIG. 5, it was confirmed that the cell viability decreased and the cytotoxicity tended to increase as the ATC2 was treated at a high concentration. In addition, it was confirmed that when the LPS was treated, the number of cells increased by about 20% compared to the case where the LPS was not treated, and the cell viability gradually decreased as the concentration of ATC2 treatment increased. In the case of LPS treatment, it was confirmed that the cell viability was 80% or more until 100 μg/mL treatment concentration of ATC2.

4-3. Rheumatism arthritis Synovial cells Used movement Restraint Measure

ATC2 used in Example 3 was treated with rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 at a concentration of 100 μg/mL for 1 hour, followed by treatment with 1 μg/mL LPS for 24 hours at 37°C. After incubation, the culture medium was removed and stained with crystal violet to measure the ability to inhibit synovial cell migration, and the stained picture and the graph of the number of migrated cells are shown in Fig. 6, respectively.

As a result, as shown in FIG. 6, when LPS alone was treated, the mobility of synovial cells that appeared during the onset of rheumatoid arthritis was increased, but cell migration was not observed when treated with ATC2, similar to that of the control group (both LPS and ATC2 were not treated). It showed the mobility, and the number of migrated cells was quantified. In the graph on the right of FIG. 6, the mobility tendency was similar (*; p<0.05).

4-4. Rheumatism arthritis Synovial cells Measure the ability to suppress the amount of inflammatory substances used

ATC2 used in Example 3 was treated with rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 at a concentration of 10 μg/mL and 100 μg/mL for 1 hour, followed by treatment with 1 μg/mL LPS. Incubated at 37° C. for 24 hours and cytokines and chemokines in culture (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 results are shown in FIG. 7.

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

4-5. Rheumatism arthritis Synovial cells Used p65 NF - kB Expression and position change investigation experiment

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 LPS for 1 hour at 37°C. Thereafter, the result of observing the presence or absence and expression of p65 NF-kB expression using immunofluorescence staining after removing the culture medium is shown in FIG. 8.

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

Since NF-kB is a major factor acting on rheumatoid arthritis expression, based on the above results, it can be seen that ATC2 inhibits the intranuclear movement of NF-kB, so that ATC2 is effective in preventing and treating rheumatoid arthritis.

4-6. Rheumatism arthritis Synovial cells Used NF - kB Active site Confirmation experiment

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 LPS at 37°C. After reacting for 15 minutes at 4% (w/v) formaldehyde, the cells were fixed and subjected to Cell-based ELISA, and the results are shown in FIG. 9.

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

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

4-7. Rheumatism arthritis Synovial cells Experiment to check the NO generation capacity

ATC2 used in Example 3 was treated with rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 at a concentration of 10 μg/mL and 100 μg/mL for 1 hour, followed by treatment with 1 μg/mL LPS 37 It was incubated for 24 hours at ℃. The NO (Nitric oxide, Nitrate) concentration in the culture was measured by the Griess assay method, and the results are shown in FIG. 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 concentrations of ATC2 10 μg/mL and 100 μg/mL after LPS treatment When treated with, it was confirmed that NO production was significantly suppressed in both conditions.

Through the above results, it was confirmed that ATC2 has an excellent anti-inflammatory effect.

4-8. Rheumatism arthritis Synovial cells Used gelatinase Enzyme activity change and expression level confirmation

ATC2 used in Example 3 was treated with rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 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 perform gelatin zymography assay to confirm the activation of MMP-2 in the supernatant, and the results are shown in FIG. 11.

The enzyme of gelatinase is known to have increased activity in patients with rheumatoid arthritis, and the enzymes of gelatinase include MMP-2 and MMP-9.

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

4-9. Rheumatism arthritis Synovial cells Used inflammation and Cytoskeleton Related protein Expression And expression position confirmation experiment

ATC2 used in Example 3 was treated with rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 at a concentration of 100 μg/mL for 1 hour, and then treated with 1 μg/mL LPS for 24 hours at 37° C. After removing the culture solution and fixing the cells with acetone, the presence and absence of expression of inflammation-related proteins (COX-2, MMP-2, IL-1beta, Vimentin and fibronectin) were confirmed by immunofluorescence staining, and the results 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. Thereby, no change in the expression position of the inflammation-related protein was observed.

4-10. Rheumatism arthritis Synovial cells Used Inflammatory factors Confirmation experiment

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. After treating with 1 ug/mL LPS, the cells were cultured at 37° C. for 24 hours and observed using an antibody array kit (ab134003; Abcam) for screening inflammation-related factors in the culture solution, and the results are shown in FIG. 13.

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

4-11. Rheumatism arthritis Synovial cells Inflammation-related protein expression experiment

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 processing 1 μg/mL LPS for 5 min, 15 min, 30 min, and 60 min, respectively, after obtaining the protein of the cells, p-Erk, p-p38, p-JNK and p-Akt protein expression by Western blot analysis The amount was measured and shown in FIG. 14.

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

4-12. Rheumatism arthritis Synovial cells Used osteoclast differentiation Restraint Confirm

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

As a result, as shown in FIG. 15, when ATC2 was treated by concentration, it was confirmed that osteoclast differentiation was significantly decreased from a concentration of 50 μg/ml or more.

From the above results, it can be seen that the ATC2 used in the present invention has an inhibitory effect on osteoclast differentiation depending on concentration.

4-13. ATC2 In vitro Rheumatism Prevention and treatment of arthritis

When synthesizing the results of the above study, it can be seen that ATC2 used in the present invention has an anti-inflammatory treatment effect by reducing the secretion and expression of inflammation-related substances in synovial cells of rheumatoid arthritis patients.

In addition, the ATC2 reduced the secretion amount, expression and activity of MMPs, which can cause cartilage and bone destruction, and exhibits osteoclast differentiation inhibitory ability, thereby delaying damage to the joints and bones due to arthritis and loss of function of the joints. It is similar to the properties of a drug-difyingase-modifying anti-rheumatic drug (DMARD) known to have activity, through which it can be seen that the ATC2 has an activity of retarding joint and bone damage and loss of function of the joint due to arthritis. .

Claims (15)

100% by weight of total 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- Fractional purification of honeycomb leaf extract, characterized by containing 1.14% by weight of 4H-chromen-4-one, 5.34% by weight of chlorogenic acid, and 3.85% by weight of cryptochlorogenic acid. Water-containing, anti-inflammatory, or arthritis, joint destruction by autoimmune diseases, polymyositis, ankylosing spondylitis, systemic lupus erythematosus, multiple myositis, rheumatoid polymyalgia rheumatica, osteoporosis, bone metastasis And Paget's disease (Paget's disease) pharmaceutical composition for the prevention or treatment of any one selected from the group consisting of. The method of claim 1, wherein the extract of mulberry leaves anti-inflammatory comprising fractional purification of mulberry leaf extract, characterized in that it is prepared by repeating the extraction step by adding a solvent to the leaves of mulberry (Stauntonia hexaphylla) 1 to 20 times , Or arthritis, autoimmune disorders of the joints, multiple myositis, ankylosing spondylitis, systemic lupus erythematosus, multiple myositis, rheumatoid polymyalgia rheumatica, osteoporosis, bone metastases, and Paget's disease (Paget's) disease) is a pharmaceutical composition for the prevention or treatment of any one selected from the group consisting of. According to claim 2, wherein the solvent is water, methanol, ethanol, butanol and one or more selected from the group consisting of mixtures, anti-inflammatory, or arthritis, autoimmune disease comprising the honeycomb leaf extract fraction tablets Destructive diseases of joints caused by, multiple myositis, ankylosing spondylitis, systemic lupus erythematosus, multiple myositis, polymyalgia rheumatica, osteoporosis, bone metastasis, and Paget's disease A pharmaceutical composition for the prevention or treatment of any one disease. According to claim 2, The extraction is 10 ℃ to 100 ℃ temperature and 30 minutes to 72 hours, cold extraction, ultrasonic extraction, reflux cooling extraction method, hot water extraction method, room temperature extraction method, and at least one selected from the group consisting of reflux extraction method Anti-inflammatory, or arthritis, fractional purification of honeydew leaf extract using the method, arthritis, autoimmune diseases, joint destruction, polymyositis, ankylosing spondylitis, systemic lupus erythematosus, polymyositis, rheumatism A pharmaceutical composition for the prevention or treatment of any one disease selected from the group consisting of polymyalgia rheumatica, osteoporosis, bone metastasis and Paget's disease. delete delete delete delete 100% by weight of total 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- Fractional purification of honeycomb leaf extract, characterized by containing 1.14% by weight of 4H-chromen-4-one, 5.34% by weight of chlorogenic acid, and 3.85% by weight of cryptochlorogenic acid. Water-containing, anti-inflammatory, arthritis, autoimmune diseases of joints, multiple myositis, ankylosing spondylitis, systemic lupus erythematosus, multiple myositis, rheumatoid polymyalgia rheumatica, osteoporosis, bone metastasis and A food composition for the prevention or amelioration of any one disease selected from the group consisting of Paget's disease. delete 100% by weight of total 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- Fractional purification of honeycomb leaf extract, characterized by containing 1.14% by weight of 4H-chromen-4-one, 5.34% by weight of chlorogenic acid, and 3.85% by weight of cryptochlorogenic acid. Water-containing, anti-inflammatory, arthritis, autoimmune diseases of joints, multiple myositis, ankylosing spondylitis, systemic lupus erythematosus, multiple myositis, rheumatoid polymyalgia rheumatica, osteoporosis, bone metastasis and Health functional food for prevention or improvement of any one disease selected from the group consisting of Paget's disease. delete 100% by weight of total 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- Fractional purification of honeycomb leaf extract, characterized by containing 1.14% by weight of 4H-chromen-4-one, 5.34% by weight of chlorogenic acid, and 3.85% by weight of cryptochlorogenic acid. Water-containing, anti-inflammatory, arthritis, autoimmune diseases of joints, multiple myositis, ankylosing spondylitis, systemic lupus erythematosus, multiple myositis, rheumatoid polymyalgia rheumatica, osteoporosis, bone metastasis and Food additives for the prevention or amelioration of any one of the diseases selected from the group consisting of Paget's disease. delete delete

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