WO2019088765A1

WO2019088765A1 - Pharmaceutical composition for anti-inflammation, or promotion of bone tissue formation or cartilage tissue formation, comprising, as an active ingredient, stauntonia hexaphylla leaf-derived extract or fractional purified product or novel flavonoid compound and caffeic acid compound separated therefrom

Info

Publication number: WO2019088765A1
Application number: PCT/KR2018/013264
Authority: WO
Inventors: 이용남; 이동구; 김새롬; 유지석; 최철웅; 반상오; 강후원; 김재용; 이규옥; 김재갑
Original assignee: 영진약품 주식회사; 재단법인 전남생물산업진흥원
Priority date: 2017-11-03
Filing date: 2018-11-02
Publication date: 2019-05-09
Also published as: KR102126575B1; KR20190050674A

Abstract

The present invention relates to a production method of a Stauntonia hexaphylla leaf-derived extract or a fractional purified product; a Stauntonia hexaphylla leaf-derived extract or a fractional purified product produced by the production method; and a pharmaceutical composition for anti-inflammation, or promotion of bone tissue formation or cartilage tissue formation, comprising, as an active ingredient, one or more selected from the group consisting of the Stauntonia hexaphylla leaf-derived extract or the fractional purified product, a caffeic acid compound and a novel flavonoid compound separated from the extract or the fractional purified product.

Description

DESCRIPTION OF THE INVENTION

Title of the Invention

A pharmaceutical composition for enhancing bone formation or cartilage formation, comprising extracts of fractions of alfalfa or fraction, or a novel flavonoid compound and a caffeic acid compound separated therefrom as an active ingredient;

TECHNICAL FIELD

Cross-reference with related application (s)

This application claims priority from Korean Patent Application No. 10-2017-01463, filed on November 3, 2017, the entire contents of which are incorporated herein by reference.

The present invention relates to a method for producing a Stauntoni a hexaphylla leaf-derived or fractionally purified product, a mulberry leaf-derived or fractionally purified product prepared by the above-mentioned preparation method, and a mulberry leaf-derived or fractionally purified product, A caffeic acid compound separated from a purified fraction, and a novel flavonoid compound as an active ingredient. The present invention also relates to a pharmaceutical composition for promoting bone formation, bone formation or cartilage tissue formation.

TECHNICAL BACKGROUND OF THE INVENTION

Bone tissue usually consists of a hard bone with a hard surface, and both ends of the center or long bone are associated with a spongy bone quality like bone. Most bones initially develop as cartilage in connective tissue, which later translates into bone tissue, some of which are made directly from connective tissue. At the epiphysis, there is a joint surface in contact with the neighboring bone, and its surface is covered with articular cartilage, which is a supernatural bone. The marine spoil (sponge small pillar) in the spongy is characterized by a constant arrangement. The large intestine of the caudal trunk is continuous with a small lumen of spongy soju and is filled with bone marrow. The hematopoietic bone marrow is reddish and red marrow. The bone structure is composed of five to 12 thick corrugated layers of dense quality and spongy matter. In the dense layer, the corrugated layered laminae (laminae) are arranged in several directions. There is a bus tube, and blood vessels pass. The osteocytes are arranged between the stratum corneum and have an irregular star shape It is a thin protoplasm and is connected to other adjacent bone cells. There is a rigid connective tissue periosteum on the surface of the bones, and nerve and blood vessels are distributed to protect and nourish the bones. If the periosteum is lost, it is difficult to survive, start and regenerate the bone. The bone is composed of 20% moisture, 35% organic and 35% inorganic, because the elasticity of the bone is organic. As age increases, minerals (mainly phosphate follicles) increase and bone hardness increases. Although the precise cause and mechanism of rheumatoid arthritis, a chronic inflammation, is not well known yet, one of the possible causes of the disease has been osteoblast that produces bone and osteoclast that destroys bone ) Is known to be caused by the collapse of the homeostasis of the bones due to the numerical imbalance. In normal state, osteoclasts are differentiated from monocytes and macrophages, destroying old bone or removing damaged bone. However, osteoclast patterns found in rheumatoid patients are caused by osteoclastogenesis, Survival and excessive activity are identified, leading to a pathologic form in which bone is severely impaired. Osteoclasts produce a variety of enzymes to break down bone, including tartrate-resistant acid phosphatase (TRAP), an acid phosphatase that oxidizes and degrades bone, and matrix metal (MMP-9), which degrades bone and cartilage cell- lopept idase-9), and collagen-degrading cathepsin K. These enzymes are used as special markers for mature osteoclasts. When RANKL is administered to macrophages, which are precursors of osteoclasts, activation of NF-κB and MAPK kinase is shown, producing inflammatory mediators.

Recently, it has been known that the progression of inflammatory reaction in the body is related to COX (cyclooxygenase) enzyme activity. The C0X enzyme is a major enzyme involved in the biosynthesis of prostaglandin present in vivo (Smith et al., J. Biol. Chem., 271, 33157 (1996)), two kinds of isozyme enzymes C0X-1 and C0X -2 is known to exist. The C0X-1 is constantly present in tissues such as the stomach or kidney and is involved in maintaining normal homeostasis, while the C0X-2 is involved in inflammation or other immune responses to mitogens or cytokines Is a transient and rapidly expressed enzyme in the cell. Nitric oxide (NO), another potent inflammatory mediator, is produced by L-arginine by NO synthase (NO) and is involved in external stresses such as UV, It is produced in many kinds of cells by substances such as cytokines. These inflammatory stimuli increase the expression of inducible NOS (iNOS) in the cells, thereby inducing NO production in the cells and activating macrophages to cause inflammatory reaction. Therefore, in order to effectively alleviate inflammation, researches on a substance capable of inhibiting the production of NO are under way.

However, some side effects of anti-inflammatory substances developed by these studies are problematic. For example, non-steroidal 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 C0X-2 enzyme as well as inhibiting C0X-1 enzyme. Recently, the mechanism of treatment for gastritis is mainly H2 inhibitors (H2-Blockers), which inhibit the second histamine receptor (H2 receptor) and decrease the secretion of gastric acid in the gastric wall cells. Reduced gastric acid prevents further damage to already damaged gastric cells (eg, gastric ulcer). Because these H2 inhibitors interfere with the metabolism of other drugs in the liver (potent inhibi tors of P-450), taking care with other medicines requires caution and has anti-androgen effects, (gynecomast ia), erectile dysfunction (impotence) and decreased sexual desire. In addition, it may pass the placenta and cerebrovascular barriers, which may be more dangerous for pregnant women and the elderly, and can cause headaches, wobbles, wobbles, and dizziness.

Therefore, it is possible to effectively inhibit the production of NO, inhibit the expression of iNOS and TNF-?, Effectively inhibit the activity of C0X-2 enzyme, It is required to develop a material which is excellent in osteoclast differentiation promotion and bone or cartilage tissue formation, and which has no risk of such side effects or cytotoxicity as a natural substance-derived substance, or which has little restriction on its use amount.

Mullen (Stauntonia hexaphyl la) is an evergreen vine plant with the native habitat of the southern coastal region and grows up to 15 m tall. White flowers bloom from mid April to mid May, It is a plant with fruit hanging (Ikuta, 1989; Lee, 1996). The fruit is known as a delicious fruit, which is derived from the name 'sweet as honey', but the coarse seed There are many fruits and few fruit. It has not been commercialized. Leaves can be wintered in the southern region, and the flesh is thick. The high value of ornamental value has led to an increase in use for gardening, plowing, etc., and consequently plant production is increasing.

Staphylococcus aphidophyll a) The caffeic acid contained in leaf plants Because of the very small amount of flavonoid compounds, there was a very inadequate aspect to be used in industry.

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 leaves of Staphylococcus aureus, and found that a more active ingredient, particularly a high content of flavonoid compound And extracts derived from Stauntoni a hexaphylla containing a large amount of caffeic acid phosphate and fractions thereof, have excellent antitumor activity or activity for promoting bone formation or promoting cartilage formation, thereby completing the present invention.

[Prior Art Literature]

. (Patent Document 1) Korean Patent Publication No. 1992-0005995

(Patent Document 2) Korean Patent Publication No. 1994-0006596

(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 10) Korean Patent Publication No. 10-2011-0021252

(Patent Document 11) Korean Patent Laid-Open No. 10-2010-0079786

(Patent Document 12) Korean Patent Publication No. 10-2016-0182923

DISCLOSURE OF THE INVENTION

[Problem to be solved]

Accordingly, one object of the present invention is

((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3 - (((2,3,4,5,6) 4, 5-trihydroxytetrahydro-2H-pyran-2- Yl) oxy) -2- (3,4-dihydroxyphenyl) -5-hydroxy-4H-chromen- (2,3,4,5,6) -4,5-dihydroxy-6-methyl-3 - (((2,3,4,5) -3,4,5-trihydroxy etr ahydr o_2H eu py r an-2-y 1 ) oxy) tetr ahydro- 2H-pyran ~ 2-yl) oxy) eu 2, eu (3, 4-di hydr oxypheny 1) -5-hydr oxy-4H ~ chr omen-4-one) in an amount of 0.1 to 10 parts by weight, chlorogenic acid (0.5 to 30 parts by weight) and cryptochlorogenic acid (Cryptochlorogenic acid) in an amount of 0.5 to 30 parts by weight Characterized in that it comprises a mulberry leaf extract and a fraction thereof. ^■

It is still another object of the present invention to provide a method for preparing extract of alfalfa leaf, which comprises repeating the step of adding a solvent to a dry mulberry leaf (Stauntoni a hexaphylla a) and repeating the step for 1 to 20 times. It is another object of the present invention to provide a method for preparing a fractionally purified product of mulberry leaf extract, which comprises treating the mulberry leaf extract with chromatography.

Another object of the present invention is to provide a composition containing at least one selected from the group consisting of mulberry leaf-derived extract or fractionally purified product, or a caffeic acid compound and a novel flavonoid compound separated from the extract or fractional purified product, Or a pharmaceutical composition for promoting bone formation or cartilage tissue production.

It is still another object of the present invention to provide a composition containing at least one selected from the group consisting of extracts derived from mulberry leaf or fractionally purified product, caffeic acid compounds separated from the extract or fractional purified product, and novel flavonoid compounds as an active ingredient , Anti-inflammatory, or bone-forming or cartilage tissue formation promoting composition.

Another object of the present invention is to provide a composition containing at least one selected from the group consisting of mulberry leaf-derived extract or fractionally purified product, or a caffeic acid compound and a novel flavonoid compound separated from the extract or fractional purified product, Or anti-inflammatory, or bone health, or a functional food for promoting bone formation or cartilage tissue production.

Another object of the present invention is to provide a health supplement containing as a main component at least one selected from the group consisting of extracts derived from mulberry leaf or fractionally purified, or caffeic acid compounds and novel flavonoid compounds separated from the extract or fractional purified product . Another object of the present invention is to provide a food additive containing at least one selected from the group consisting of extracts derived from mulberry leaves or fractionated tablets, or caffeic acid compounds and flavonoid compounds separated from said extracts or fractionated tablets .

Another object of the present invention is to provide a composition containing at least one compound selected from the group consisting of mulberry leaf-derived extract, fractional purified product thereof, caffeic acid compound and novel flavonoid compound isolated from the above extract or fractional purified product, And a pharmaceutical composition for preventing or treating rheumatoid arthritis or degenerative arthritis.

MEANS FOR SOLVING THE PROBLEMS

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

In one embodiment, the present invention relates to a novel extract of alfalfa leaf or fractionally purified product, and a salt of Alfalfa or extract of fraction, or a new flavonoid compound or caffeic acid compound separated therefrom as an active ingredient, or To a pharmaceutical composition for promoting bone formation or promoting cartilage tissue formation.

The Stauntonia hexaphylla leaves used in the present invention may be commercially available, purchased or naturally harvested or cultivated, but are not limited to, properly dried before extraction and purification, It can be pretreated so as to pass through the purification process. The novel extract or fractionated tablet as defined herein is prepared by extracting Stauntonia hexaphylla leaves with a solvent selected from 1 to 4 carbon atoms lower alcohols such as water, methane, ethane, butane, or the like, And the extraction method can be made into a fractionated product using a purification method known to a person skilled in the art to which the present invention belongs.

In addition, the extract or fractionally purified product of mulberry (Stauntonia hexaphylla) leaf of the present invention may be further subjected to a concentration and drying process such as drying such as concentration under reduced pressure, freeze drying or hot air drying, and further purification.

The extract or the fractionated preparation of the present invention is a novel flavonoid compound, (7 - (((2,3,4,5,6) -4,5-dihydroxy-6-methyl- 2H-pyran-2-yl) oxy) tetrahydro-2H-pyran-2-yl) oxy) - 4-one, 2- (3,4-dihydroxypheny 1) -5-hydoxy-4H-chromen-4-one, and caffeic acid compounds such as chlorogenic acid and cryptochlorogenic acid Within the above range.

Specifically, the above-mentioned extract or fractionally purified product is prepared by mixing (w / w), (7- (((2,3,4,5,6) -4,5-dihydr oxy -6-met hy 1 -3- (( (2, 3, 4, 5) -3, 4, 5-tri hydr oxy tetr ahydr o ~ 2H-pyran-2-yl) oxy) tetr ahydr o- 2H- pyran-2-yl) oxy) -2,3-dihydroxypheny 1 -5-hydoxy-4H-chr omen-4-one, 0.1 to 10% by weight of chlorogenic acid, 30% by weight and Cryptochlorogenic acid in an amount of 0.5 to 30% by weight,

(W / w) of 100% by weight of the total extract or fractionated tablet, the novel compound (7 - (((2,3,4,5,6) -4,5- dihydroxy- 6- IIletllyl- 3- (((2,3,4,5) -

2-yl) oxy) -2- (3,4-dimethoxyphenyl) -1H-pyrazolo [3,4-d] pyrimidin- 0.1 to 5% by weight of chloroplatinic acid, 0.5 to 20% by weight of chlorogenic acid, 0.5 to 20% by weight of cryptochlorogenic acid, %. &Lt; / RTI >

In one specific embodiment of the present invention, the crude extract of Allium cepacii according to the present invention contains a novel flavonoid compound (7 - (((2,3,4,5,6) -4,5-dihydr oxy-6 2H-pyran-2-yl) oxy) tetr ohydr 2HPyr (2, 3, 4,5) -3,4,5-trihydroxy- 0.56% by weight of chlorogenic acid, 1.61% by weight of chlorogenic acid, and 0.25% by weight of cryptochloric acid. The fractionated product of this crude extract contains 1.07% by weight of the acid, which is a novel flavonoid compound (7 - (((S) - 2H-pyran-2-yl) oxy] -2- (3,4-dimethoxyphenyl) di hydr oxypheny 1) _5 ᅳ

hydr oxy-4H-chr omen-4-one) 1.14 wt%, chlorogenic acid 5.34%, and cryptochlorogenic acid 3.85 wt%.

In yet another embodiment, the present invention relates to a method for producing extract of alfalfa leaf, which comprises repeating the step of extracting a mollusk (Stauntonia hexaphyll) leaf with a solvent by repeating 1 to 20 times.

In the above production method ^and, meolkkul (Stauntonia hexaphylla) leaves are suitably It may be pretreated by washing, drying, cutting or pulverizing according to a known method. Preferably, the mulberry leaves which are in a dry form can be used. These extracts of mulberry leaves are extracted using an appropriate extraction solvent. As the extraction solvent, there can be used a solvent selected from lower alcohol having 1 to 4 carbon atoms, such as water, methanol, ethanol, butane, or a co-solvent thereof, And preferably water can be used. The extraction temperature may be specifically from 10 ^° C to 100 ^° C, preferably from 20 ^° C to 10 ^° C, and the extraction time may be from 30 minutes to 72 hours, preferably from 6 hours to 48 hours However, this is not the case. The extraction method can be selected from a method selected from a needle extraction method, an ultrasonic extraction method, a reflux angle extraction method, a hot water extraction method, a room temperature extraction method, a reflux extraction method, or a combination of at least one of these extraction methods. . More preferably from 40 ^° C to 120 ^° C after extraction of the droplet for 30 minutes to 72 hours, preferably 6 hours to 48 hours at a silver concentration of io ° c to 6 rc, preferably 2 rc to 50 ^° C C, specifically at a temperature of 60 ^° C to 100 ^° C for 30 minutes to 72 hours, preferably for 6 hours to 48 hours, is carried out repeatedly about 1 to 20 times, preferably 2 to 10 times Lt; / RTI >

Preferably, the mulberry leaf extract may further be subjected to further concentration under reduced pressure after the extraction step. In another aspect, the present invention relates to a method for preparing a fractionally purified product of alfalfa leaf extract, which comprises treating the alfalfa leaf extract with chromatography. '

In a specific embodiment, the fractionated product of mulberry leaf extract can be prepared by first preparing an extract using mulberry leaves and then subjecting the extract to fractionation and purification by chromatography, wherein the mulberry leaf extract is prepared by a method comprising the steps of: The same method can be applied to the method for producing the extract.

Chromatography is used in the purification process _(a) reverse-phase partition chromatography method (Reverse phase part it ion chromatography) , (b) normal-phase top ⁱ tinted chromatography (Normal phase adsorpt ion chromatography), (c) Ion-exchange chromatography Ion exchange chromatography, (d) si ze exclusion chromatography, or one of them The above combination, preferably reverse phase partition chromatography, or chromatographic method using all resins characterized by polar materials first eluting and non-polar compounds being retained on stationary, more preferably by Sephadex, , Sephadex LH20, Sephadex G-25, Sephadex G-10, Sepharose, Superdex, methyl acrylate resin, carboxymethyl salicylose, sulfopropylcellulose, and carboxymethyl sephadex, A Sephadex series resin including propylsepadex, sulfopropylsephaose and carboxymethylseparos; Reversed-phase polymer resins using a family of styrene-divinylbenzene co-polymers such as Polymer X, HP20, and PRP-hl Polymers, and a family of methacrylate supports; Normal phase silica gels such as the adsorbed BPC (Bonded phase Chromatography) product line of Merck, the Si lica product line of YMC, the Si lica product line of DAISO, the Si lica product line of ASAHI, and the Si lica product line of C0SM0SYL; A resin selected from the reverse phase silica gel group such as 0DS product group of YMC, 0DS product group of DAIS0, 0DS product group of ASAHI, 0DS product group of CHEMC0, 0DS product group of Merck, 0DS product group of C0SM0SYL, 0DS product group of FUJI company, And a method of performing a chromatography method using a solvent.

Specifically, in (a) reversed-phase partition chromatography, the stationary phase is a reversed-phase column material, in which polar substances are first eluted and non-polar compounds are retained on stationary, C12, C12, C14, C16, or C18, preferably C12, C4, C6, C8, C16 or C18 or a polymer-based fixed bed such as a silica gel (Si lica Gel) based fixed bed or polystyrene, (C18 (IV) -D), (0DS-A / 0DS-AQ product family of YMC, C 0 -DDS-AQ product group, SP-C-0DS product line, SP-0DS-RPS product line of DAIS0 product, 5C18 product line of C0SM0SYL product, Chromatorex product line of FUJI company). The mobile phase may be prepared by dissolving distilled water, acetonitrile, and methane in an alcohol solvent such as ethanol, butanol, tetrahydrofuran or a mixed solvent thereof, preferably distilled water and a solvent for alcohol solvents such as methanol, ethanol, Is characterized by eluting a polar material by adjusting the ratio of water: methanol starting from 90:10 (v / v) to 60:40. Specifically, in the above-mentioned (b) normal phase chromatography, the stationary phase is a normal phase column material, and all the stationary phase, in which the nonpolar substances are first eluted and the polar compound is retained on the stationary phase, A reversed phase silica gel or a silica gel derivative thereof, more preferably a reversed phase silica gel (C18 (IV) silica gel, Fluorosyl, alumina based stationary phase, ) -D), (the 0DS-A / 0DS-AQ family of YMC, the SP-C-0DS family of CHMEC0, the SP-0DS-RPS family of DAIS0, the 5C18 family of C0SM0SYL and the Chromatorex family of FUJI) do.

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

Specifically, in (c) ion exchange resin column chromatography, "ion exchange chromatography material" uses all the floating high molecular weight solid features having covalently bonded charged groups as chromatographic functional groups Available; Preferably, a chromatography method using a cation exchange resin, an anion exchange resin, or a synthetic adsorbent, more preferably AG 50W-x8, Amberlite I-120, Dowex 50W-x8 and SK1B Strongly acidic cation exchange resins such as < RTI ID = 0.0 > 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; AMBERLITE CR1310 Na, AMBERLITE CR1310 Na, AMBERLITE CR1310 Na, AMBERLITE CR1310 Na, AMBERLITE CR1310 Na, AMBERLITE CR1310 Na, AMBERLITE CR1310 Na, AMBERLITE CR1310 Na, AMBERLITE CR1310 Na, AMBERLITE CR120, DIAI0N SK1B, DIAI0N PK216, DIAI0N CR11, DIAI0N CR20, DIAI0N UBK555 (Mitsubishi Chemical co.), TRILITE SPC 160H, TRILITE SPC 400H Lewatit VP 0C 1800, Lewatit VP 0C 1812, Lewatit MDS1368 Na, Lewatit K1221 (Bayer co.), PU 0LITE PCR833CA, PUR0LITE C145 (Purolite co.), AMBERLYST 131 WET, AMBERLYST 232 WET , MFG 210, and MFG 250 (Finex co.); Preferably a strongly acidic cation exchange resin, SK1B type exchange resin; Anion exchange resins include strong 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; 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 (e.g., Sephadex G-25) or polyacrylamide gel, For example, Sephacryl (e.g. Sephacryl_S400), agarose-based gels such as Superose or Sepharose (e.g. Sepharose CL-4B), and composite gels made from two kinds of gels, , Superdex 200 combination Dextran (Sephadex ( ^TM )) and crosslinked Aga 1 rose (Superose ^TM ) gels,

One

Is selected from the group of polymeric gels that are not limited; In a preferred embodiment, the buffer is selected from the group consisting of sodium phosphate, ammonium acetate, MES (2- (N-morpholino) ethanesulfonic acid), Bis-Tris (2- Methyl) -1,3-propanediol), ADA (N- (2-acetamido) iminodiacetic acid), PIPES (piperazine-N, N'- Aminoethanesulfonic acid), BES (N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid), MOPS (3- (N-morpholino) Propylsulfonic acid), TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid), HEPES (N-2-hydroxyethyl-piperazine- Is selected from the group consisting of sodium phosphate or ammonium acetate, more preferably ammonium acetate.

In addition, the present invention further relates to a process for preparing a compound represented by the above (a) reverse phase partition chromatography, (b) normal phase part chromatography, (c) km exchange chromatography (e) gel permeation or gel permeation using Sephadex LH-20 or Toyopearl HW-40, which can use organic solvents in addition to (d) size exclusion chromatography or a combination of one or more of these. Additional filtration chromatography may be used.

The synthetic adsorbent may be, but is not limited to, 0.5 to 20 times volume (v / v) HP20 of the suspended solvent volume. In another embodiment, the present invention relates to a composition comprising at least one compound selected from the group consisting of extracts derived from mulberry leaves or fractionated tablets, caffeic acid compounds separated from the extracts or fractionated tablets, and novel flavonoid compounds, , Or a pharmaceutical composition for promoting bone formation or cartilage tissue formation.

In the pharmaceutical composition according to the present invention, the content of the above-mentioned extract or fractionally purified product can be directly applied to the extracts derived from the mulberry leaves or the fractionated products.

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

The flavonoid compound can be prepared by reacting (7 - (((2,3,4,5,6) -4,5-dihydroxy-6-methyl- -tr i hydr o t t i r t a r h i r 2

2H-pyran-2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5-hydroxy-4H-chromen-4-

In one example of the present invention, it was confirmed that the extract of alfalfa leaf or fraction purified by the above-described method inhibits the osteoclast differentiation inhibitory activity (Example 3).

From the above results, it has been found that the inclusion of at least one selected from the group consisting of caffeic acid compounds and novel flavonoid compounds isolated from mulberry leaf-derived extract or fractionally purified product, The secretion amount and expression of inflammation-related substances of synovial cells of the present invention are reduced, and thus the anti-inflammatory therapeutic effect is exhibited.

Also, from the above results, it was confirmed that the extract containing at least one member selected from the group consisting of caffeic acid compounds and novel flavonoid compounds isolated from the extract of mulberry leaf produced by the above-mentioned production method or the fractionally purified product thereof, or the extract or fractional purified product The activity of inhibiting the loss of joints and bones caused by arthritis and the loss of function of joints, and thus, the extract of alfalfa leaf or fraction purified by the above-mentioned production method has an effect of inhibiting osteoclast activation, It can be seen that there is a promoting effect.

The present invention also relates to a pharmaceutical composition for preventing or treating a disease that destroys or damages bone tissue or cartilage tissue. The bone tissue or Diseases that destroy or damage cartilage tissue include, but are not limited to, arthritis, joint destruction caused by autoimmune disease, multiple myelitis, ankylosing spondylitis, systemic lupus erythematosus, multiple myelitis, rheumatoid myalgia polymyalgi a rheumat i ca), osteoporosis, bone cancer or Paget's disease (Paget's di sease). The arthritis may also include, but is not limited to, various forms of arthritis, for example, osteoarthritis, rheumatoid arthritis, degenerative arthritis, psoriatic arthritis, and react and arthritis. Preferably rheumatoid arthritis. The pharmaceutical composition containing the extract or the fractionated product or the compound according to the present invention may be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, And sterile injectable solutions. Examples of carriers, excipients and diluents that can be contained in the composition containing the extract include lactose, dextrose, sucrose, sorbic, mannitol, xyli, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, celluloses, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil . In the case of formulation, it is prepared by using a commonly used layering agent, a weighting agent, a binder, a wetting agent, a releasing agent, a surfactant or the like. Solid preparations for oral administration include tablets, pills, powders, Granules, capsules and the like. These solid preparations can be prepared by adding to the extract and fractionated product at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. . In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.

Examples of the suspending agent include propyleneglycol, polyethylene glycol, vegetable oil such as olive oil, And injectable esters such as ethyl oleate may be used. Examples of the suppository base include wi tepsol, macrogol, tween 61, cacao paper, laurin, and glycerin gelatin.

The preferred dosage of the pharmaceutical composition comprising the extract or fractionally purified compound or compound of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, have. However, for the desired effect, the pharmaceutical composition containing the extract or fractionated product of the present invention is preferably administered at 0.0001 to 1000 mg / kg, preferably 0.001 to 500 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The extract or fractionally purified product or the compound of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injection. In another embodiment, the present invention includes an extract containing at least one compound selected from the group consisting of extracts derived from mulberry leaf or fractionally purified product, caffeic acid compounds separated from the extract or fractional purified product, and novel flavonoid compounds Or anti-inflammatory, or a composition for promoting bone formation or cartilage tissue production.

The pharmaceutical composition for antiinflammatory or osteogenesis or promotion of cartilage tissue formation includes the following: a food composition for promoting bone formation or promoting bone formation or cartilage formation; a health functional food for promoting bone formation or promoting bone formation or cartilage formation; , Or health food supplements for promoting bone formation or promoting cartilage tissue formation, anti-inflammation, or food additives for promoting bone formation or cartilage tissue production.

In another embodiment, the present invention relates to a composition comprising at least one compound selected from the group consisting of extracts derived from mulberry leaves or fractionated tablets, caffeic acid compounds separated from the extracts or fractionated tablets, and novel flavonoid compounds as an active ingredient Inflammatory, or osteogenic or cartilage tissue formation promoting health functional food.

In another aspect, the present invention relates to a method for producing a caffeic acid compound (hereinafter, referred to as " caffeic acid compound ") isolated from a mulberry leaf-derived extract or fractionally purified product, And a novel flavonoid compound. The present invention also relates to a health supplement for promoting bone formation or cartilage tissue growth.

In another embodiment, the present invention relates to a method for the treatment and / or prophylaxis of an inflammatory disease, which comprises at least one selected from the group consisting of extracts derived from mulberry leaves or fractionally purified water, or caffeic acid compounds and novel flavonoid compounds , Or a food additive for promoting bone formation or cartilage tissue production.

&Quot; Health functional food " as defined herein means food prepared and processed using raw materials or ingredients having functionality useful to the human body in accordance with Law No. 6727 on Health Functional Foods. &Quot; Functional " Structure and function of the nutrient to control or physiological effects, such as to obtain a beneficial effect for health is intended to eat.

The health functional food of the present invention contains 0.01 to 95%, preferably 1 to 80%, of the above-mentioned extract or fractional purified product, based on the total weight of the composition.

In addition, for the purpose of anti-inflammation or inhibition of osteoclast activity, a pharmaceutical dosage form such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups or the like which is in the form of a tea bag, It can be manufactured and processed as functional food.

In addition, the above health functional foods may further include food additives, and whether or not they are suitable as "food additives" may be added to the relevant items in accordance with the general provisions of the Food Additives Ordinance approved by the Food and Drug Administration Shall be determined according to the relevant standards and standards.

Examples of the products listed in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, persimmon extracts, licorice extracts, Sodium glutamate preparations, noodle-added alkalis, preservative preparations, and tar coloring preparations.

Examples of the functional food containing the extract or fractionated product or compound of the present invention include confectionery ice cream such as bread, rice cakes, dried fruits, candy, chocolate, chewing gum and confectionery, milk of ice cream such as ice cream, ice cream powder, Milk, lactose decomposed milk, processed oil, goat milk, fermented milk, Dairy products such as butter oil, concentrated oil, yogurt cream, butter oil, natural cheese, processed cheese, powdered milk, milk products and processed meat products such as hamburger, ham, sausage and bacon The same fish products include lactic acid bacteria beverages such as ramen noodles, dried noodles, fresh noodles, noodle noodles, luxury noodles, improved sleep noodles, noodles, noodles such as pasta, vegetable beverages, carbonated beverages, But are not limited to, beverage such as beverage soy sauce, miso, kochujang, spring roll, chonggukjang, shrimp, vinegar, sauces, seasoned food margarine such as ketchup, curry, dressing, shortening and pizza.

The health functional beverage composition of the present invention is not particularly limited to the ingredients other than those containing the extract or the fractionated product or the compound as an essential ingredient in the indicated ratios and may contain various flavoring agents or natural carbohydrates, . Examples of the above-mentioned natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.); Disaccharides (e.g., maltose, sucrose, etc.); And polysaccharides (for example, dextrin, cyclodextrin and the like), and sugar alcohols such as xyli, sorbic acid, and erythritol. (Flavorings such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavorings other than the above have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additive is not so increased 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, the extract or fractionated product of the present invention, or the novel flavonoid and caffeic acid compound separated therefrom, may be added to food or beverage for the purpose of preventing or improving the objective disease. At this time, food or beverage The amount of the extract or fractionally purified product or compound in the whole food

0.01 to 15% by weight, and the health beverage composition may be added at a ratio of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100%.

The extracts, fractionated tablets or compounds according to the present invention, which are added to foods containing beverages in the process of manufacturing the health functional food, can be appropriately added or decreased as needed.

In another embodiment, the present invention provides a pharmaceutical composition comprising 100% (w / w), (7 - (((2 _> 3,4,5,6) -4,5-dihydroxy-6 2H-pyran-2-yl) oxy) tetr ahydr 2H-pyran-2 (1 H) -quinolinone 0.1 to 10% by weight of chlorogenic acid, 0.5 to 30% by weight of chlorogenic acid, 0.1 to 10% by weight of chlorogenic acid, And 0.5 to 30% by weight of cryptochlorogenic acid. The present invention also relates to a pharmaceutical composition for preventing or treating rheumatoid arthritis or degenerative arthritis.

In another aspect, the present invention relates to a method for promoting anti-inflammation, bone formation or cartilage formation, or prevention or treatment of rheumatoid arthritis or degenerative arthritis, comprising the step of administering the composition.

All of the methods of administration, routes, and contents of compositions applicable to the above methods can be applied as described in the above-mentioned composition.

【Effects of the Invention】

The present invention relates to a method for producing extracts or fractionated tablets derived from Sturtuntonia hexaphylla leaves, extracts or fractionated tablets from mulberry leaves prepared by the above-mentioned preparation method, and extracts or fractionated tablets from mulberry leaves, A caffeic acid compound separated from water, and a novel flavonoid compound as an active ingredient. The present invention also relates to a pharmaceutical composition for promoting bone formation or promoting bone formation, comprising New extracts or fractional preparations isolated from mulberry leaf-derived extracts or novel flavonoid compounds and caffeic acid compounds isolated therefrom are not only cytotoxic, but also cytokines related to inflammation, that is, IL-Ιβ, IL- 6, TNF-α, MCP-1, CXCL10, IL-8, VEGF, Kimyeo, the activation of NF-κΒ And the inhibitory activity of C0X-2 enzyme which is a causative factor of inflammation can be effectively inhibited by confirming the secretion activity of NO and the preventive, ameliorating or treating agent for various diseases related to destruction of bone tissue or cartilage tissue such as rheumatoid arthritis, or It is very useful as a functional food.

BRIEF DESCRIPTION OF THE DRAWINGS [

FIG. 1 shows the results of HPLC analysis of crude extract of mulberry leaf (ATC1) according to an embodiment of the present invention.

FIG. 2 shows the results of HPLC analysis of a mulberry leaf fraction (ATC2) according to an embodiment of the present invention.

FIG. 3 is a graph showing the results of TRAP activity measurement of osteoclasts of alfalfa leaf extract (ATC1) and its fractionated product (ATC2) according to an embodiment of the present invention as an osteoclast without treatment with RNAKL and ATC1 or ATC2 as a negative control (5 uM, or 20 uM) treated with C0X2 inhibitor after RANKL treatment as a positive control group and differentiated into osteoclasts as a positive control group.

4 is a photomicrograph showing the results of inhibition of osteoclast differentiation by aliquot (ATC1) and its fractionally purified product (ATC2) according to an embodiment of the present invention.

FIG. 5 is a bar graph showing the results of experiments confirming the apoptosis of rheumatoid arthritis synovial cells of fractionally purified (ATC2) according to an embodiment of the present invention.

FIG. 6 is a photograph and a bar graph showing the results of an experiment in which the mobility of rheumatoid arthritis synovial cells was examined during the treatment of the fractionated tablet (ATC2) according to an embodiment of the present invention.

FIGS. 7A and 7B are experimental results showing changes in secretion factors of inflammatory secretion factors (cytokines, chemokines, MMPs) in the treatment of fractionated tablets (ATC2) according to an embodiment of the present invention. 7B is a graph showing changes in secretion of TNF- ?, IL-6, IL-17A, MCP-1, CXCL10, IL-8 and VEGF, 2 and MMP-9.

FIG. 8 is a photograph showing the results of an experiment in which the expression of NF-kB in the cells was treated using fractional fluorescent staining for the treatment of fractionally purified (ATC2) according to an embodiment of the present invention.

FIG. 9 is a graph showing the results of the fractionated tablet (ATC2) treatment according to an embodiment of the present invention The results of confirming the activation of NF-kB in the inhibitory region and confirming the activation level showed that the reduction of NF-kB two-residue phosphorylation (reduction of NF-kB activation) was observed in the case of ATC2 treatment will be.

FIG. 10 is a graph showing the results of experiments in which inhibition of NO production was confirmed when 10 g / mL and 100 g / mL of fractionally purified (ATC2) according to an embodiment of the present invention was treated.

11 is the experimental results confirming the degree of change in enzyme activity, and expression of the purified fraction (ATC2) processed with gelatinase dehydratase _(e g _a l ti _nase) in accordance with one embodiment of the present invention eu

FIG. 12 is a graph showing changes in the expression level and the degree of inflammation and cytoskeleton-related proteins (COX-2, MMP-2, IL-1beta, vimentin, and fibronetin) in the treatment of fractionated tablets (ATC2) according to an embodiment of the present invention Experimental results.

FIG. 13 is a graph showing the result of screening of an inflammation-related factor that shows a difference in the amount of secretion when treating fractionated tablets (ATC2) according to an embodiment of the present invention using an antibody array kit (abl34003; Abeam).

FIG. 14 is a result of Western blot analysis of the signal transmission pathway inhibited during the treatment of the fractionated product (ATC2) according to an embodiment of the present invention. FIG. 15 is a graph showing the effect of ATC2 on the osteoclast differentiation according to one embodiment of the present invention. After 5 days of osteoclast differentiation, It is a photograph and a bar graph that I confirm.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided to further understand the present invention, and the present invention is not limited by the examples. Reference Example 1: Preparation of mulberry leaf crude extract and identification of indicator components

1.0 kg of Stauntonia hexaphylla leaf (GAP cultivation, 565-2, Busan-myeon, Busan-myeon, Jeollanam-do, Korea) was dried and cut, and 10 L of water was added to the mulberry leaf sample and then cold-extracted at room temperature for 24 hours. Thereafter, reflux extraction was carried out 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. The extract was concentrated under reduced pressure using a vacuum concentrator (EYELA, N-2100, JAPAN) to obtain 300 g of a mulberry leaf water crude extract (hereinafter referred to as ATC1). High performance liquid chromatography (HPLC, Agilent 1260, USA) was performed under the same conditions as the following Tables 1 and 2 (mobile phase conditions) in order to identify the surface components contained in the crude extract of mulberry leaves obtained above, 1.

In Table 2, mobile phase A is 0.025 M phosphate buffer (pH = 3.0) and mobile phase B is 9 (w / v) ACN (Acetonitrile).

[Table 1]

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

Based on the pattern (retention time) of the HPLC shown in FIG. 1, the content (%) of the indicator components was analyzed according to the following equation (1).

[Equation 1]

'¾ φ 靡^' )

(Mg / mU A;

A _T : Area of surface component in the test solution

As: Area of surface component in standard solution

It is used when the amount taken for the indicator components of the test solution and the standard solution are the same. As a result, crude extracts of mulberry leaves prepared by the method of Reference Example 1 contained 1.61 (w / w) of chlorogenic acid, 1.071% (w / w) of cryptochlorogenic acid, 0.56% / w). < / RTI >

[Table 3]

Example 1. Preparation of purified fraction of organic solvent-soluble fraction of mulberry leaf extract and identification of indicator components

700 mL of reversed phase silica gel (HP20, manufactured by Mitsubishi Co., Ltd. JAPAN) was layered on a column tube, and then the above 23 The brix mellower leaf extract (700 mL) was passed through to isolate the mutilla leaf blade fraction (ATC2). The mobile phase composition for separation was separated by controlling 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: methane (30:70) eluent was separated under the same conditions. Then 2-3 times of distilled water eluate (1.4 L) (v / v) methane was added to 2.8 L of the eluant 1-4 times, and the solvent was removed through reduced-pressure concentration. Then, 55 g of the finally purified mullin fraction or fraction was purified. (Hereinafter referred to as ATC2).

High performance liquid chromatography (HPLC) was carried out in the same manner as in Reference Example 1 to confirm the surface components contained in the Muller HP20 column extract or the fractionated product, and the results are shown in FIG.

As a result, as shown in Fig. 2, it was confirmed that a new compound was detected in chlorogenic acid at 8.67 min, cryptochlorogenic acid at 9.15 min, and 26.89 min. Based on the HPLC pattern (retention 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 mullen fraction extract or the fractionated product prepared by the preparation method of Example 1 contained 5.34% (w / w) of chlorogenic acid, 3.85% (w / w) of cryptochlorogenic acid, It was confirmed that the flavonoid compound contained 1.14% (w / w).

[Table 4]

Example 2. Isolation and structural analysis of caffeic acid 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.

2-1. Chlorogenic acid; Chlorogenic acid

D, J = 16 Hz, H-7 '), 7.03 (1H, d, J =

(1H, d, J = 8 Hz, H-2 '), 6.98 (1H, dd, J = 2 and 8 Hz, H- ), 6.15 (1H, d, J = 16 Hz, H-8 '), 5.08 (1H, br d, J = 5 Hz, H- 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).

^{1 3 C NMR (125 MHz,} DMS0-Q¾) δ: d 174.9 (C-7 '), 165.7 (C-9 1),

(C-4 '), 145.5 (03'), 144.8 (C-7 '), 125.6 2), 114.3 (C-8), 73.6 (C-1), 70.9 (C-3), 70.6 (C-4), 68.3 C-2). 2-2. Cryptochlorogenic acid; Cryptochlorogenic acid

(1H, d, J = 3, 4, 14 Hz, H-2eq), 2.06 (1H, ddd, J = (1H, ddd, J = 4, 9, 9), 2.17 (1H, dd, J = 4,14 Hz, H- 11 Hz, H-5), 4.28 (1H, ddd, J = 3,3,4 Hz, H-3), 4.79 (1H, dd, J = 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 1), 7.06 (1H, d, J = 2 Hz, H-2 '), 7.65 (1H, d, J = 16 Hz, H-7').

^{13 C-NMR (100 MHz,} DMS0- / 6) δ: 38.4 (C-2), 42.7 (C-6), 65.5 (C-5), 69.6 (03), 76.6 (C- 1), 79.3 (C-4), 115.1 (C- 2 '), 115.4 (08'), 116.5 (C-5 1), 123.0 (C-6 '), 127.8 (01 '), 146.8 (C 3'), 147.1 (C-7 '), 149.6 CC-4'), 169.0 (C-9 '), 177.3 (C-7'). 2-3. New flavonoid compounds

(1H, s, 5-OH), 7.46 (1H, d, J = 2.0 Hz, H-6 ') _. (1H, d, J = 8.5, 2.0 Hz, 2 '), 6.89 (1H, d, J = 8.5 Hz, H-3' J = 2.0 Hz, H-8), 6.35 (1H, d, J = 2.0 Hz, H-6), 5.20 4.5 Hz, 1H), 4.29 (1H, H-5 ''').

¹³ C-NMR (100 MHz, DMSO)?: 175.8 (C-4), 164.0 (C-7), 161.0 (C-4 '), 145.0 (02), 135.7 (C-3), 121.9 (C-?), 103.0 (C-10), 100.5 (01 "), 98.2 (C-6), 93.3 (08), 77.6 71.8 (C-4 "), 70.4 (C- 3 '', C-4 '11), 68.3 (C- 2 1'), 66.0 (C-5 ''), 18.0 (C-6"). exemplary Example 3. Confirmation of osteoclast differentiation inhibitory physiological activity of mulberry extract and fraction ^'

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

Tibia of 4-5 week-old ICR mice were excised and the both ends were cut. Bone marrow cells were collected by passing them through a-MEM essential medium and cultured with 50 ng / mL macrophage-co 1 ony stimulation factor And cultured for 24 hours. Untreated cells were washed with a -MEM, and the cells were subcultured to 48-wells at ⁵ × 10 ⁵ cells / well and cultured in α-ΜΕμ medium supplemented with 50 ng / mL of M-CSF for 3 days. Then, 50 ng / mL of MCSF and 100 ng / mL of RANKL (Receptor Activator for Nuclear Factor κB Ligand) were treated together and the samples were cultured for 4 days to differentiate into osteoclasts. 3-2. (ATC1), fractionated (ATC2) and osteoclast

Measurement of TRAP activity change

The osteoclasts are characterized by acid-phosphatase TRAP (tartrate-resistant), which is resistant to tartrate acid phosphatase, which is used as a cytochemical labeling enzyme for osteoclasts that can be distinguished from other bone tissue cells (Minkin, C, Calcif. Tissue Int., 34: 285-290, 1982).

In order to confirm the change of TRAP activity of osteoclasts by the aliquot (ATC1) and its fractionated product (ATC2) of the present invention, the following experiment was conducted. Specifically, in the same manner as in Example 3-1, cells were inoculated to 48-wells at 5 ^× 10 ⁵ cells / well, treated with differentiation factors (RANKL, MCSF) and samples, and cultured for 4 days. Were measured.

. For TRAP activity, 30 μl / well of culture supernatant was added to a new 96-well plate using the TRAP acivity assay kit (AK04F, COSMO BIO CO., LTD) for 4 days. Chromogenic substrate / Tart rate-containing buffer (170 μl / well), and the absorbance at 540 nm was measured at 37 ° C for 30 minutes to 3 hours.

The control group was a cell without bone marrow cells differentiated into osteoclasts and not treated with RNAKL and ATC1 or ATC2. The positive control group was treated with RANKL to differentiate into osteoclasts, and then treated with C0X2 inhibitor Celecoxib (5 uM, or 20 uM) and incubated for 4 days.

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

(ATC1), the second fraction extract or the fraction purified (ATC2) obtained in Example 3-1 were added to the extracts of mulberry leaf ATC1 (1, 10, 50, 100 ug / ml) The osteoclast was treated with the second fraction extract or fractionally purified ATC2 (0.1, 1, 10, and 50 ug / ml) and cultured for 4 days. The TRAP activity of the osteoclast (the TRAP activity exhibited resistance to tartrate Acid phosphatase activity), and the results are shown graphically in Fig.

3, it was confirmed that the activity of osteoclast was inhibited depending on the concentration of the aliquot (ATC1) extract, the second fraction extract or the fraction purified (ATC2) of the present invention.

More specifically, in the case of ATC2 extracts extracted from the fractionation process, 71.7% (0.1 ug / ml) of ATC2 (0.1, 1, 10, and 50 yg / ml) ml), 36.4% (1 ug / ml), 21.9% (10 UAP / ml) and 221.1% (50 yg / ml) TRAP activity, and 2 to 3 times more inhibitory effect on TRAP activity than the untreated crude extract (ATC1) .

In particular, when treated with 1 g / ml of ATC2 fraction, the inhibition rate of TRAP activity against the control group was 63.6% (inhibition rate: 100% -36.4 = 63.6%), (Inhibition rate: 100% -33.4% = 66.4%), which is the inhibition rate of JRAP activity when the concentration of ATC1 was 50 yg / ml. That is, the extract of fractionated mulberry leaf extract (ATC2) showed a 50-fold increase in activity compared to extract of mulberry leaf (ATC1).

3-3. Inhibitory effect of aliquot (ATC1) or fractionally purified (ATC2) on osteoclast differentiation

In the same manner as in Example 3-1, cells were inoculated to 48-wells at ⁵ × 10 ⁵ eel / well and treated with the fractionation product (ATC2) of the differentiation factor (RANKL, MCSF) and mullen leaf extract (ATC1) And cultured for 4 days. A wash to remove the culture medium with PBS, and then 1M (w / v) substrates in cells fixed with formaldehyde solution (1.36 mg / mL 4-nitrophenyl phosphate disodium salt, 10 mM tartrate, 50 mM citrate buffer P H 4.6) per 100 uL It was dispensed and stirred at 37 ^° C for 30 minutes. The medium was removed, washed with PBS, fixed with 10% (w / v) formaldehyde for 15 minutes, and washed three times with PBS. TRAP staining was ^{50 mM tartrate acid '50 mM sodium} acetate buffer 10 mL 1 mg / mL naphtol for _containing. AS-MX phosphate and Ν, N-dimethyl formamide 100 were added to each well, and 45 uL of the staining solution was added to the cells fixed with 10% (w / v) formaldehyde. After staining at 37 ^° C for 30 minutes, And the results are shown in Fig.

As shown in Fig. 4, TRAP-positive polynuclear osteoclasts were formed in the negative control cells, but the mullen leaf extract (ATC1) or. It was confirmed that osteoclast differentiation was inhibited in the concentration - dependent manner when the fraction purified (ATC2) was treated.

More specifically, in the case of the ATC2 extract extracted through the first extraction and the second fraction purification process, the extract (ATC1) that is 1/10 lower But also showed remarkable osteoclast differentiation inhibition effect of 2 to 3 times or more. Example 4. In vitro prophylactic and therapeutic activity of rheumatoid arthritis

4-1. Isolation and primary culture of synovial fibroblasts

Among the rheumatoid arthritis patients who meet the criteria of the American Rheumatology Society, synovial tissues obtained from patients requiring surgical treatment of the knee joints were washed with PBS, finely chopped to 2-3 mm, dissolved in serum-free DMEM The cells were treated with DMEM (10% (v / v) FBS) for 2 hrs. The cells were treated with collagenase (type I) , 1% (w / v) P / S) to obtain rheumatoid arthritis synovial fibroblasts.

4-2. Cell viability measurement of rheumatoid arthritis synovial cells

The ATC2 used in Example 3 was treated at a concentration of 0 to 1000 yg / mL for 1 hour in rheumatoid arthritic synovial cells obtained in the same manner as in Example 4-1, and then treated with LPS or 1 mg / mL of LPS And cultured at 37 ° C for 24 hours. Cell viability was measured by the CCK-8 assay method, and the results are shown in FIG.

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, it was confirmed that the cell number was increased by 20% when LPS was treated, and the cell survival rate was gradually decreased with increasing ATC2 treatment concentration. In the case of LPS treatment, it was confirmed that cell survival rate of ATC2 was more than 80% up to the treatment concentration of 100 ug / mL.

4-3. Measurement of movement inhibition using rheumatoid arthritis synovial cells

ATC2 used in Example 3 was treated at a concentration of 100 ug / mL for 1 hour in rheumatoid arthritic synovial cells obtained in the same manner as in Example 4-1, treated with 1 yg / mL of LPS and cultured at 37 ° C for 24 hours The cells were cultured for the time, and after removing the culture solution, the cells were stained with crystal violet to measure the ability to inhibit synovial cell migration, and a graph showing the number of cells migrated and the staining was shown in FIG.

As a result, as shown in Fig. 6, in the case of LPS alone treatment, The migration of synovial cells at the onset of arthritis was increased, but the migration of cells was not observed at the time of ATC2 treatment, and the degree of mobility was similar to that of the control (not treated with both LPS and ATC2), and the number of migrated cells was quantified. The mobility trends were similar in the graph on the right (*; p <0.05).

4-4. Rheumatoid arthritis synovial cell-mediated inhibitory capacity of inflammatory substances

ATC2 used in Example 3 was treated at a concentration of 10 < RTI ID = 0.0 > yg / mL < / RTI > and 100 pg / mL for 1 hour in rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1, IL-1β, IL-8, VEGF, MCP-1, CXCL10, MMP-1, 2, 3, and 4 were incubated for 24 hours at 37 ^° C and the cytokines and chemokines 9, 13) was measured by ELISA and the results are shown in Figs. 7a and b.

As a result, IL-6, IL-17A, MCP-1, CXCL10, IL-8, VEGF, and MMPs were detected in LPS-treated rheumatoid arthritis synoviocytes 1, MMP-2, MMP-3, MMP-9, and MMP-13). In addition, the ATC2-treated cells showed that the secretion of inflammatory substances related to rheumatism was reduced by ATC2 concentration even when LPS was simultaneously treated.

4-5. Expression and location of p65 NF-kB in rheumatoid arthritis synovial cells

ATC2 used in Example 3 was treated at a concentration of 100 pg / mL for 1 hour, treated with 1 yg / mL of LPS and incubated at 37 ^° C for 1 hour in rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 After the culture solution was removed, immunofluorescence staining was performed to observe the presence and expression of p65 NF-kB, and the result was shown in FIG.

As a result, as shown in FIG. 8, the expression of p65 NF-kB, which was present only in the cytoplasm when LPS was treated in rheumatoid arthritis synovial cells, was observed in the nucleus, indicating that the cytoplasmic p65 NF-kB migrated to the nucleus it means. When ATC2 treatment, migration of p65 NF-kB into the nucleus was not observed. Since NF-kB plays a major role in the expression of rheumatoid arthritis, based on the above results, ATC2 inhibits nuclear migration of NF-kB, suggesting that ATC2 has the preventive and therapeutic effect of rheumatoid arthritis. 4-6. Identification of NF-kB activation site using rheumatoid arthritis synovial cells

ATC2 used in Example 3 was treated at a concentration of 10 ii g / mL and 100 yg / mL for 1 hour and treated with 1 yg / mL of LPS to obtain rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1. Cells were fixed with 4% (w / v) formaldehyde after 15 min at 37 ^° C and Cel l-based ELISA was performed, and the results are shown in FIG.

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

From the above results, it can be seen that ATC2 is effective for the treatment of rheumatoid arthritis. 4-7. Test for confirming N0-producing ability using rheumatoid arthritis synovial cells The ATC2 used in Example 3 was treated for 1 hour at a concentration of 10 yg / mL and 100 yg / mL in rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1 The cells were incubated at 37 ^° C for 24 hours. The concentration of NO (Ni tr icoxide, Ni trate) in the culture medium was measured by Gr iess assay, and the results are shown in FIG.

As a result, as shown in FIG. 10, when LPS treatment was applied to rheumatoid arthritis synovial cells, N0 production was increased by about 2 times as compared with that of the normal group, whereas the concentration of ATC2 10 g / mL and 100 yg / mL after LPS treatment , It was confirmed that N0 production was remarkably suppressed in both conditions.

These results indicate that ATC2 has excellent anti-inflammatory effects.

4-8. Enzymatic Activity of Gelatinase Using Rheumatoid Arthritis Synovial Cells Identify changes and manifestations

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

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

As a result, as shown in FIG. 11, when the synovial cells of patients with rheumatoid arthritis were treated with LPS, the activated form of matrix metal loproteinase-2 (MMP-2) was increased whereas when ATC2 was treated, the activation of MMP-2 disappeared The expression level of MMP-2 was increased by treatment with LPS, but the expression of MMP-2 was decreased by treatment with LPS and ATC2, and the expression of MMP- 9, the expression of LPS and ATC2 completely disappeared.

4-9. Rheumatoid arthritis Synovial cell-mediated inflammatory and cytoskeletal protein expression and presence /

ATC2 used in Example 3 was treated at a concentration of 100 yg / mL for 1 hour in rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1, treated with 1 μg / mL of LPS, and cultured at 37 ^° C for 24 hours After incubation, the culture medium was removed and the cells were fixed with acetone. Immunofluorescence staining was used to confirm the presence and expression of inflammatory proteins (COX-2, MMP-2, IL-lbeta, Vimentin and fibronectin) .

As a result, as shown in FIG. 12, it was confirmed that the expression of C0X-2, MMP-2, IL-lbeta, vimentin, and fibronetin which were increased during LPS treatment was significantly reduced by ATC2 treatment, No change in the location of the inflammation-related protein expression was observed.

4-10. Identification of inflammatory factors using rheumatoid arthritis synovial cells

The rheumatoid arthritis synoviocytes obtained in the same manner as in Example 4-1 ATC2 used in Example 3 was treated at a concentration of 100 ug / mL for 1 hour. 1 ug / mL of LPS was cultured at 37 ^° C for 24 hours. Antibody array ki t (abl34003; Abeam) was used for screening inflammatory factors in culture. The results are shown in FIG.

As a result, as shown in FIG. 13, it was confirmed that the secretion of IL-6, CXCL8, CCL2 and CCL5 was increased in the synovial cells after Ant ibody array treatment. When LPS and ATC2 were simultaneously treated with LPS, And CCL5, an increased rheumatoid-related inflammatory factor, was again reduced to a statistically significant level. (*; P < 0.05)

4-11. Experimental Expression of Inflammatory Proteins Using Rheumatoid Arthritis Synovial Cells

ATC2 used in Example 3 was treated at a concentration of 100 yg / niL for 1 hour in the rheumatoid arthritis synovial cells obtained in the same manner as in Example 4-1. 1 yg / 5 minutes of LPS mL, 15 minutes, 30 minutes and after each treatment for 60 minutes obtained proteins of the cell Western blot analysi s method with _{p-Erk, p- P 38,} p-JNR and P-Akt The amount of protein expression was measured and shown in Fig.

As a result, as shown in FIG. 14, the expression of p38, JNK and Akt in rheumatoid arthritis increased at 60 minutes after LPS stimulation, but the signaling of p38, JNK and Akt related to rheumatoid arthritis .

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

CD14 ⁺ cells obtained by MACS method were separated from PBMC in normal human buf fy coat blood and fractionated at a concentration of 1 × 10 ⁵ cells / well in a 96-well plate and incubated with 20 ng / mL M -CSF treatment and cultured for 3 days. On day 4, 40 ng / mL RANKL and ATC2 used in Example 3 were treated at different concentrations to induce osteoclast differentiation for 5 days, and the number of osteoclasts after TRAP staining was measured and shown in FIG.

As a result, as shown in FIG. 15, when ATC2 was treated at different concentrations, it was confirmed that osteoclast differentiation was remarkably decreased from a concentration of 50 ug / ml or more. From the above results, it can be seen that ATC2 used in the present invention has an osteoclast differentiation-inhibiting action in a concentration-dependent manner.

4-13. In vitro rheumatoid arthritis prevention and treatment activity of ATC2

When the results of the above-mentioned studies are summarized, it can be seen that ATC2 used in the present invention reduces the secretion amount and expression of inflammation-related substances of synovial cells in patients with rheumatoid arthritis, thereby exhibiting an anti-inflammatory therapeutic effect.

In addition, the ATC2 decreased the secretion, expression and activity of MMPs which may cause cartilage and bone destruction, and exhibited osteoclast differentiation inhibition ability, which resulted in delayed joint and bone damage and joint loss due to arthritis The activity of ATC2 is similar to that of DMARD (DMARD), which is known to be active, and thus ATC2 has activity to delay joint and bone damage and joint loss by arthritis .

Claims

Claims:

[Claim 1]

Compared to 100% (w / w) total extract,

(0 (7 - (((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3 - (((2,3,4,5) 2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5-hydroxy-4H-pyrrolyl-2-yl) oxy) tetrahydro- (7 - (((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3 - (((2,3,4,5) - 2H-pyrrol-2-yl) oxy) -2- (3,4-dihydroxypheny l) -2,3-dihydroxy- 1) -5-hydoxy-4H-chromen-4-one) 0.1 to 10% by weight, (ii) chlorogenic acid 0.5 to 30%

(iii) 0.5 to 30% by weight of a cryptochlorogenic acid.

[Claim 2]

(% W / w) of the total fraction purified water 100,

(i) (((((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3 - (((2,3,4,5)

2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5 (4-hydroxyphenyl) -Hydroxy-4H-chromen-4-yl) 0.1 to 10 weight

(ii) 0.5 to 30% by weight of chlorogenic acid and

(iii) 0.5 to 30% by weight of a cryptochlorogenic acid.

[3]

Wherein the step of adding a solvent to leaves of Stemontania hexaphylla is repeated 1 to 20 times.

Claim 4

4. The method according to claim 3, wherein the solvent comprises at least one selected from the group consisting of water, methanol, ethanol, butanol, and a mixture thereof.

[Claim 5]

The method according to claim 3, wherein the extraction is carried out in a group consisting of 10 ^° C to 100 ^° C of haze and 30 minutes to 72 hours of isopropanol, a cold extraction method, an ultrasonic extraction method, a reflux cooling extraction method, a hot water extraction method, Choose one or more methods

32 Correction paper (Rule 91) ISA / KR &Lt; / RTI > wherein the method comprises the steps of:

[Claim 6]

(a) extracting a mulberry (Stauntonia hexaphylla) with a solvent by repeating the step of 1 to 20 times to obtain a mulberry leaf extract; And

(b) purifying the mullite extract obtained in step (a)

A method for producing a fractionally purified product of a mulberry leaf extract.

[Claim 7]

[7] The method of claim 6, wherein the step of purifying the mulberry extract comprises (a) reverse phase part chromatography, (b) normal-phase topcoat chromatography _. Wherein the purification is carried out by a method selected from the group consisting of normal phase adsorption chromatography, (c) ion exchange chromatography, (d) size exclusion chromatography or a combination of one or more thereof. A method for preparing a fractionally purified product of an extract.

8.

Wherein the composition contains at least one member selected from the group consisting of alfalfa leaf extract, fractional purified product thereof, or caffeic acid compound and flavonoid compound separated from the extract or fractional purified product thereof as an active ingredient. A pharmaceutical composition for promoting tissue formation.

[Claim 9]

The extract according to claim 18, wherein the extract from the mulberry leaves or the fraction thereof is prepared by mixing 100% (w / w)

(i) (((((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3 - (((2,3,4,5)

2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5 (4-hydroxyphenyl) -Hydroxy-4H-chromen-4-one) in an amount of 0.1 to 10%

(ii) 0.5 to 30% by weight of chlorogenic acid, and

(iii) 0.5 to 30% by weight of a cryptochlorogenic acid. < Desc / Clms Page number 24 >

[Claim 10] Wherein the composition comprises at least one selected from the group consisting of mulberry leaf-derived extract, fractional purified product thereof, caffeic acid compound and flavonoid compound isolated from the extract or fractional purified product thereof as an active ingredient. Food composition for promoting the production.

Claim 11

[Claim 11] The method according to claim 10, wherein the mulberry leaf-derived extract or fractionally purified product is prepared by mixing 100% (w / w)

(i) (7 - (((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3 - (((2,3,4,5) 2-yl) oxy) -2- (3,4-dihydroxyphenyl) -5-hydroxy-4H-pyrrolo [ -Chromen-4-one) in an amount of 0.1 to 10% by weight,

(ii) 0.5 to 30% by weight of chlorogenic acid, and

(iii) 0.5 to 30% by weight of a cryptochlorogenic acid (Cryptochlorogenic acid).

Claim 12

Wherein the composition contains at least one member selected from the group consisting of alfalfa leaf extract, fractional purified product thereof, or caffeic acid compound and flavonoid compound separated from the extract or fractional purified product thereof as an active ingredient. Health functional foods for promoting tissue formation.

[13]

The method of claim 12, wherein the meolkkul leaf extracts from or ^"purified fraction thereof Bull

Relative to 100% (w / w) of the total extract or fractionated tablet,

(i) (7 - (((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3 - (((2,3,4,5)

(ii) 0.5 to 30% by weight of chlorogenic acid, and

(iii) 0.5 to 30% by weight of a cryptochlorogenic acid.

[14] Or bone tissue or cartilage tissue, which contains at least one selected from the group consisting of mulberry leaf-derived extract, fractional purified product thereof, or a caffeic acid compound and a flavonoid compound separated from the extract or fractional purified product thereof Food additives for accelerating.

[Claim 15]

15. The method according to claim 14, wherein the extract of the mulberry leaf or the fraction thereof is

Relative to 100% (w / w) of the total extract or fractionated tablet,

(i) (7 - (((2,3,4,5,6) -4,5-dihydroxy-6-methyl-3 - (((2,3,4,5) 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) tetrahydro-

0.1 to 10% by weight, based on the total weight of the composition, of at least one compound selected from the group consisting of 2-hydroxy-2- (3,4-dihydroxyphenyl)

(ii) 0.5 to 30% by weight of chlorogenic acid, and

(iii) 0.5-30% by weight of a cryptochlorogenic acid. The food additive for promoting bone formation, bone formation or cartilage tissue formation.

16. The method of claim 16,

Relative to 100% (w / w) of the total extract or fractionated tablet,

Hydroxy-4H-chromen-4-one) in an amount of 0.1 to 10 parts by weight,

(ii) 0.5 to 30% by weight of chlorogenic acid, and

(iii) 0.5-30% by weight of a cryptochlorogenic acid. < RTI ID = 0.0 > 18. < / RTI >

17.

15. A method for promoting bone formation, bone formation or cartilage formation, comprising administering a composition according to any one of claims 8 to 15, a health functional food or a food additive.

Claim 18

18. A method for the prevention or treatment of rheumatoid arthritis or degenerative arthritis comprising administering a composition according to claim 16.