KR100694569B1 - Composition comprising the extract of Eurya emarginata or the compounds isolated therefrom having anti-inflammatory activity - Google Patents

Abstract

The present invention relates to a composition having an antioxidant and anti-inflammatory effect containing Eurya emarginata extract or a compound Utigoside B, C isolated therefrom, wherein the extract or compound of the present invention Utigosides B and C are antioxidants by inducing the inhibition of pro-inflammatory cytokine production, expression of iNOS and inhibition of COX-2 production, such as TNF-α, IL-6 and IL-1β. And anti-inflammatory effects.

Anti-inflammatory, Antioxidant, Cytokine, Umukasarepi, Utigoside B, Utigoside C

Description

Composition containing the extract of Eurya emarginata or the compounds isolated therefrom having anti-inflammatory activity}             

1 is a diagram showing the inhibitory effect of the production of each solvent fraction of NO in RAW 264.7 cells,

2 is a diagram showing the inhibitory effect of the components of Wumusaspirop on NO production in RAW 264.7 cells,

3 is a diagram showing the inhibitory effect of the concentration of utigoside B on NO production in RAW 264.7 cells.

4 is a diagram showing the inhibitory effect of the concentration of utigoside C on NO production in RAW 264.7 cells.

5 is a diagram showing the inhibitory effect of the production of each solvent fraction of TNF-α, IL-6 and IL-1β in RAW 264.7 cells,

6 is a diagram showing the inhibitory effect of utigosides B and C on TNF-α production in RAW 264.7 cells,

7 is a diagram showing the inhibitory effect of utigosides B and C on IL-6 production in RAW 264.7 cells,

FIG. 8 is a diagram showing the inhibitory effect of Wumusa respiratory extract on early-inflammatory cytokine mRNA expression in activated macrophages.

9 is a diagram showing the inhibitory effect of the lower mucosa respiratory fraction on the early-inflammatory cytokine mRNA expression in activated macrophages,

10 is a diagram showing the inhibitory effect of the lower mucosa respiratory fraction on iNOS, COX-2 and β-actin mRNA expression in activated macrophages,

FIG. 11 is a diagram showing the inhibitory effect of utigosides B and C on early-inflammatory cytokine mRNA expression in activated macrophages.

12 is a diagram showing the inhibitory effect of utigosides B and C on anti-inflammatory cytokine mRNA expression in activated macrophages,

FIG. 13 is a diagram showing the inhibitory effect of Umusaxerapi extract on iNOS and β-actin producing protein expression in activated macrophages.

14 is a diagram showing the inhibitory effect of the lower mucosa respiratory fraction on iNOS and β-actin-producing protein expression in activated macrophages,

15 is a diagram showing the inhibitory effect of utigosides B and C on iNOS producing protein expression in activated macrophages,

16 is a diagram showing the inhibitory effect of utigosides B and C on COX-2 protein expression in activated macrophages,

Figure 17 is a diagram showing the separation process of the bioactive components of cow mucosa respiratory,

FIG. 18 is a diagram illustrating spots on ethyl acetate / methanol (10: 1 / v / v) TLC during the separation of bioactive components of Umusa resperus;

19 is a diagram showing a spot on the TLC of the ethyl acetate fraction 5-4 during the separation process of the bioactive component of the mucosa respiratory tract.

20 is a diagram illustrating a process of separating Utigosides B and C from woomusa repi,

FIG. 21 shows ¹ H-NMR and ¹³ C-NMR spectra of Utigoside B. FIG.

FIG. 22 shows ¹ H-NMR and ¹³ C-NMR spectra of Utigoside C. FIG.

The present invention is a composition containing Utigoside B and C, which are extracts of Eurya emarginata having antioxidant and anti-inflammatory activity or compounds isolated therefrom, for the prevention and treatment of various inflammation-related diseases and health Relates to nutraceuticals.

Inflammatory reactions are associated with various mediators and immune cells of local blood vessels and body fluids when infected with tissues (cells) or external infectious agents (bacteria, fungi, viruses, and various types of allergens). It exhibits a series of complex physiological reactions including substance secretion, fluid infiltration, cell migration and tissue destruction, as well as external symptoms such as erythema, edema, fever and pain. In normal cases, the inflammatory response removes external infectious agents and regenerates damaged tissues to restore the function of life.However, if the inflammatory response is excessive or persistent due to the absence of antigens or internal substances, the mucosal damage is promoted. Some lead to diseases such as cancer.

In vivo, inflammation causes various biochemical phenomena. Especially, nitric oxide synthase (NOS) and prostaglandin, enzymes that generate nitric oxide (NO), are involved. Enzymes associated with biosynthesis are known to play an important role in mediating the inflammatory response. Therefore, NOS, an enzyme that produces NO from L-Arginine, or COX (cyclooxygenase), an enzyme involved in synthesizing prostaglandins from arachidonic acid, is a major target for blocking inflammation. have.

According to recent research, there are several types of NOS, such as bNOS (brain NOS) in the brain, neuronal NOS in the nervous system, and endothelial NOS in the vascular system. It is expressed at a level, and a small amount of NO produced by them plays an important role in maintaining normal homeostasis, such as inducing neurotransmission and vasodilation, whereas iNOS induced by various cytokines or external stimulants NO, which is rapidly over-produced by induced NOS, is known to cause cytotoxicity and various inflammatory reactions, and chronic inflammation has been associated with an increase in iNOS activity (Miller et al., Mediators of inflammation , 4 , pp 387-396, 1995: Appleton L. et al., Adv. Pharmacol. , 35 , pp 27-28, 1996).

In addition, there are two kinds of isoforms of cyclooxygenases. Cyclooxygenase-1 (COX-1) is always present in the cell, and is used to synthesize prostaglandins (PGs) necessary for cytoprotective action. In contrast, COX-2 is known to play an important role in causing an inflammatory response due to a rapid increase in cells during the inflammatory response (Weisz A., Biochem. J. , 316 , pp209-215, 1996). Transcriptional inflammatory factors, including iNOS and COX-2, which enhance the degree of NO and PGs, are chronic diseases including various sclerosis, parkinson's disease, Alzheimer's disease and colon cancer. Related to the etiology of.

NF-κB is a nuclear protein of the Rel gene family. In the cytoplasm, NF-κB binds to I-κB and exists in an inactive form. However, NF-κB is reactive oxygen, TNF-α (tumor necrosis factor-α), I-κB kinase is activated by various stimuli such as chemokines and lipopolysaccharide (LPS) such as IL-1 (Interleukin-1), and then I-κB is released through phosphorylation. . NF-κB, composed of p50 and p65 heterodimers, is known to promote gene expression that, after being activated, moves to the nucleus to induce an inflammatory response (Oh, GT et al., Atherosclerosis , 159 (1)). , pp 17-26, 2001: Epstein, FH et al., The New England Journal of Medicine , 336 (15), pp 1066-1071, 1997: Zhang WJ et al., FASEB J. , 15 (130), pp 2423-2432 , 2001: Denk, A. et al., J. Biol. Chem. , 276 (30), pp28451-28458, 2001: Sahnoun Z. et al., Physiology , 53 (4), pp315-339, 1998: Lindner V., Pathobiology , 66 (6), pp 311-320, 1998: Landry, DB et al., Am. J. Pathol. , 151 (4), pp 1085-1095, 1997: Gerritsen, ME et al., Am. J. Pathol. , 147 (2), pp 278-292, 1995).

Multifunctional cytokines such as TNF-α are not only expressed in normal tissues but also increased in the course of lesions, and play an important role in skin inflammation, particularly during cancer promotion. TNF-α has been reported to be associated with inflammatory skin diseases in humans (Verhoef, J. et al., J. Antimicrob. Chemother., 35 , 1996). The treatment of TNF-α with various inflammatory diseases and allergic symptoms was alleviated. TNF-α also acts as an endogenous cancer promoter by activating neutrophils and increasing hydrogen peroxide production. Therefore, proinflammatory molecules (proinflammatory) may be inhibited by inhibiting the expression of the cytokine TNF-α, which plays a pivotal role in the inflammatory stage closely related to the oncogenic stage, or by inhibiting the production of PGE ₂ due to COX-2 activity inhibition. It is likely that the process of lesions accompanied by an increase in molecules can be controlled. When treated with antibiotics under bacterial infection, LPS released from the extracellular membrane while killing bacteria causes endotoxin shock and, when severely destroyed by this process, does not neutralize the LPS that continues to be produced (Dunn, DL, Surg. Clin.North.Am ., 74 , 1994: Giroir, BP, Crit.Care Med., 21 , 1993: Yamaguchi, Y. et al., J. Reticuloendothel. Soc., 409 , 1982). Therefore, drug treatment may be effective by reducing the action of LPS. Reduction of inflammation due to the use of nonsteroidal anti-inflammatory drugs (NSAIDs) to date has often resulted in relief of pain for a considerable period of time, and most of the non-narcotic analgesics (such as aspirin) are also anti- It has an inflammatory effect and has been used properly for the treatment of acute and chronic inflammatory diseases. However, due to side effects, it is difficult to use in the long term, and as a result, the development of new or improved therapeutics is essential and urgent, because the seriousness of the side effects is too great for the present treatment. In connection with the problem of overcoming these side effects, efforts have recently been made to find the active ingredient in natural products that are used in the private sector.

Eurya emarginata ( Eurya emarginata ) of the present invention is an evergreen shrub of evergreen shrubs and grows in the coastal mountains of warm islands such as Jeju Island, and is used as a medicine for diuresis and phlegm removal in the private sector. Seoul National University Press, pp114-115, 1996). However, there is little report on the composition and physiological activity of the leaves. In addition, none of the above documents discloses or teaches that the mucosa respiratory extract and the compounds isolated therefrom have anti-inflammatory activity.

Accordingly, the present inventors completed the present invention by extracting the active ingredient of such natural products commonly used in folklore, observing the inflammatory cytokine inhibitory effect and its mechanism of action, and discovering the excellent anti-inflammatory effect of the mucosa respiratory extract.

It is an object of the present invention to provide a pharmaceutical composition for the treatment and prevention of inflammation-related diseases containing Umutasarepi extract or compounds isolated from the Utigoside B and C having an antioxidant and anti-inflammatory efficacy as an active ingredient. .

In order to achieve the above object, the present invention contains an extract of Wumuxarespi organic solvent having an antioxidant and anti-inflammatory effect as an active ingredient, and the treatment and prevention of inflammation-related diseases comprising a pharmaceutically acceptable carrier, excipient or diluent Provide a pharmaceutical composition for

In the present invention, the extract of the mutated mucosa respira can be used to extract the leaves, stems, leaves and roots of the mucosa sapire, preferably the mucosa sapire.

Examples of the organic solvent extract include polar solvents such as C ₁ to C ₄ lower alcohols such as methanol, ethanol and butanol, nonpolar solvents such as hexane, ethyl acetate, acetone, chloroform and methylene chloride and mixed solvents thereof. It includes. Preferably, the extract is extracted from methanol, butanol, hexane, ethyl acetate and a mixed solvent thereof.

In addition, the present invention is an inflammation containing a compound of the general formula (I) having the antioxidant and anti-inflammatory effect isolated from the extract of the mucosa respiratory extract as an active ingredient, including a pharmaceutically acceptable carrier, excipient or diluent It provides a pharmaceutical composition for the treatment and prevention of related diseases.

(Ⅰ)

(Ⅰ)

In the general formula (I), R is H or OH.

In the present invention, in the general formula (I), when R is an H group, it is Utigoside C, and when R is an OH group, it is named Utigoside B.

The inflammation-related diseases include rheumatoid arthritis, asthma, acute pain, chronic pain, neuropathic pain, postoperative pain, pain such as migraine and arthralgia, neuropathy, nerve damage, irritable bowel syndrome, shock by endotoxin, or inflammatory bowel disease This includes.

Hereinafter, the present invention will be described in detail.

Specifically, the oomu-sasiprei extract of the present invention is collected by drying the oomu-sasiprei leaves and then pulverized and pulverized, the volume of water and methanol, ethanol, butanol of about 2 to 20 times the weight of the oomu-sasipye sample A polar solvent of C ₁ to C ₄ lower alcohol such as a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably about 10 hours to 48 days at 20 to 50 ° C by adding methanol, Preferably, after extraction using an extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for 20 to 30 hours, it can be obtained by filtration, concentration and drying according to a conventional method.

In addition, the non-polar solvent soluble extract of the present invention, after suspending the crude extract in distilled water, the suspension is about 1 to 100 times, preferably about 1 to 5 times the volume of non-polar solvents such as hexane, ethyl acetate, chloroform It can be obtained by extracting and separating the non-polar solvent soluble layer 1 to 10 times, preferably 2 to 5 times, and may be further subjected to a conventional fractionation process (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis, 3rd Ed. , p6-7, 1998).

For example, the preferred organic solvent extract of the mucosa respiratory tract is ethanol, butanol, hexane, ethyl acetate, acetone, chloroform, methylene chloride, and a mixed solvent thereof, preferably hexane, butanol, and ethyl acetate. It can be obtained by a systematic extraction method, and in particular, the dried Wumusa leaf leaf extract with ethyl acetate and butanol to obtain an extract, and then fractionated by silica gel column chromatography to obtain an ethyl acetate and butanol fraction.

More specifically, by using reverse phase column chromatography using 20 to 100% methanol as an eluting solvent, the sol-mucosa nonpolar solvent soluble extract obtained by the above process is preferably fractionated into 5 to 8 fractions. After separation, the lower fraction 1 was mixed with dichloromethane: acetone: methanol in a ratio of 3: 2: 0, 1: 4: 0, 2: 7: 1, and subjected to Sephadex LH-20 column chromatography to obtain 30 to 30 Separation into 32 fractions can be performed by RP HPLC in double subfractions 26 and 30, respectively, to obtain the utigoside B and C compounds of the present invention, respectively.

The present invention provides a pharmaceutical composition for the treatment and prevention of inflammation-related diseases containing the woomusa respiratory extract obtained by the above method or a compound isolated therefrom Uticoside B and C as an active ingredient.

In addition, Wumusa repi is a drug that has been used for a long time as a herbal medicine and edible extracts of the present invention extracted from them or compounds isolated there are also problems such as toxicity and side effects.

The pharmaceutical composition for preventing and treating inflammation-related diseases of the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.

The pharmaceutical composition comprising the extract of the present invention or a compound separated therefrom may further comprise a suitable carrier, excipient or diluent commonly used in the preparation of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts or compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

The pharmaceutical composition comprising the extract or compound according to the present invention may be prepared according to a conventional method, respectively, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions. It can be formulated and used in the form. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the extract or compound. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts or compounds of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract or compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The extracts or compounds of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food comprising the extracts or compounds Utigosides B and C isolated therefrom and food acceptable food additives, which show a prophylactic effect of inflammation-related diseases. Examples of the food to which the extract or compound of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing the inflammation-related diseases. At this time, the amount of the extract or compound in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml Can be added.

Food supplement additives as defined herein include food additives customary in the art, such as flavorings, flavoring agents, colorants, fillers, stabilizers, and the like, and are exemplified below.

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

In addition to the above, the extracts or compounds of the present invention are various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract or compound of the present invention.

Below, The invention is illustrated in detail by the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1 Preparation of Umusa SAR Methanol Crude Extract

After collecting and drying the leaves of Umusa-shrapi native to Jeju Island, dry it with a grinding machine, and finely grind it. Then, 25.8 of 80% methanol was added to 175.8g of Umu-sas-lepi leaf fine powder sample and stirred at room temperature for 24 hours. The supernatant was recovered by. This process was repeated three times to collect the supernatant, and then concentrated under reduced pressure to give 58.21 g of crude mucosa extract of wulmusapi.

Example 2. Preparation of Wumulsasule fraction by solvent

58.21 g of the crude mucosa leaf leaf crude crude extract obtained in Example 1 was suspended in 1 L of water, and extracted with hexane (1 L x 3), ethyl acetate (1 L x 3) and butanol (1 L x 3), respectively. The fractions were dried in vacuo to give a hexane fraction (0.518 g), an ethyl acetate fraction (9.976 g) and a butanol fraction (8.09 g). Finally, the remaining aqueous layer was concentrated under reduced pressure to give an aqueous layer fraction (8.759 g). Among these extract fractions, 9.976 g of ethyl acetate extract fraction was dissolved in 10 ml of ethyl acetate, followed by column chromatography using silica gel (Kiesel gel 60) (column size: 3 × 5 cm, eluent: ethyl acetate / methanol (10: 1 / v / v), flow rate; 10 ml / min), and TLC was carried out with the same solvent at the same time, and the seven fractions were separated at a 0.1 interval of Rf of the spot on the TLC, and concentrated under reduced pressure. In the same manner as above, 8.09 g of butanol fraction was dissolved in 10 ml butanol, and four butanol fractions were separated and concentrated under reduced pressure. Fractions obtained here were used as experimental samples (see FIGS. 17-19).

Example 3 Isolation and Identification of Utigosides from Solvent-Resistant Fractions

A portion of the ethyl acetate fraction obtained in Example 2 (1.5 g) was taken and methanol was gradually increased to 20 to 100% to give five fractions (fr. 1 to fr. 5) through a reverse phase silicon dioxide (SiO ₂ ) column. Got. The polar fraction fr.1 (603 mg) was purified using Sephadex LH-20 column chromatography using dichloromethane: acetone: methanol in a ratio of 3: 2: 0, 1: 4: 0, 2: 7: 1 Fractions were obtained. In this fraction, fr.26 (13 mg) and fr.30 (30 mg) were further purified by C18 HPLC to give Utigoside C (5.3 mg) and Utigoside B (3.8 mg) having the following spectroscopic data. Was obtained (see Fig. 20).

Utigoside B: C ₂₃ H ₂₆ O _10.

¹ H-NMR (400 MHz, methanol-d4) δ ppm 7.63 (1H, d, 16.0, H7``), 7.47 (2H, d, 8.6, H2 '', H6 ''), 6.97 (2H, m, H2, H6), 6.81 (2H, br d, 8.6, H3``, H5 ''), 6.35 (1H, d, 16.0, H8 ''), 6.07 (2H, d, 10.0, H3, H5), 4.48 (1H, dd, 12.0, 2.2, H6 '), 4.29 (1H, dd, 12.0, 6.0, H6'), 4.24 (1H, d, 7.8, H1 '), 3.92 (1H, dt, 11.9, 6.1, H8 ), 3.64 (1H, dt, 11.9, 6.1, H8), 3.49 (1H, dd, 9.0, 6.0, H5 '), 3.31-3.39 (2H, m, H3', H4 '), 3.16 (1H, dd, 9.0, 7.8, H2 '), 2.04 (2H, t, 6.1, H7). (See Figure 21)

¹³ C-NMR (100 MHz, methanol-d4) δ ppm 187.8 (C4), 169.1 (C9 ''), 161.4 (C4 ''), 154.4 (C2), 154.3 (C6), 146.8 (C7 ''), 131.2 (C2 '', C6 ''), 128.0 (C3), 127.9 (C5), 116.9 (C3 '', C5 ''), 115.0 (C8 ''), 104.4 (C1 '), 77.9 (C3') , 75.5 (C5 '), 75.0 (C2'), 71.8 (C4 '), 69.2 (C1), 65.9 (C8), 74.6 (C6'), 41.0 (C7). (See Figure 21)

Utigoside C: C ₂₃ H ₂₆ O ₉ .

¹ H-NMR (400 MHz, methanol-d4) δ ppm 7.71 (1H, d, 16.0, H8``), 7.62 (2H, m, H2 '' and H6 ''), 7.40 (3H, m, H3 ''', H4 '', H5 ''), 6.97 (2H, m, H2, H6), 6.56 (1H, d, 16.0, H8 ''), 6.06 (2H, br d, 10.4, H3, H5), 4.50 (1H, dd, 11.8, 2.0, H6 '), 4.32 (1H, dd, 11.8, 2.0, H6'), 4.25 (1H, d, 7.8, H1 '), 3.92 (1H, dt, 10.2, 6.5, H8 ), 3.65 (1H, dt, 10.2, 6.5, H8), 3.51 (1H, dd, 9.0, 6.0, H5 '), 3.34 (2H, m, H3', H4 '), 3.16 (1H, dd, 9.0, 7.8, H2 '), 2.04 (2H, t, 6.5). (See Figure 22)

¹³ C-NMR (100 MHz, methanol-d4) δ ppm 187.8 (C4), 168.5 (C9 ''), 154.4 (C2), 154.3 (C6), 146.5 (C7 ''), 135.7 (C1 ''), 131.6 (C4 ''), 130.1 (C2 '', C6 ''), 129.3 (C3 '', C5 ''), 128.0 (C3), 127.9 (C5), 118.7 (C8 ''), 104.4 (C1 ' ), 77.9 (C3 '), 75.4 (C2'), 75.0 (C5 '), 71.7 (C4'), 69.2 (C1), 65.9 (C8), 64.8 (C6 ''), 41.0 (C7) (Figure 22 Reference)

Experimental Example 1. Cell Culture and Reagent

RAW 264.7 cells, a murine macrophage cell line, were distributed from the Korea Cell Line Bank (KCLB), using DMEM medium containing 100 units / ml penicillin-streptomycin and 10% FBS (fetal bovine serum). Incubated at 37 ° C., 5% CO ₂ incubator. Subcultures were performed once every 3 to 4 days, and lipopolysaccharide (LPS. E. coli serotype 0111: B4) was purchased from Sigma. Interferon-γ (mIFN-γ, recombinant E. coli) was purchased from Roche and used for the experiment.

Experimental Example 2. Screening of Antioxidant Activity and NO Production Inhibition of Umuksa Repy

2-1. Screening of Antioxidant Activity by DPPH Radical Scavenging Activity

Antioxidant activity was determined using the Blois method, which measures the radical scavenging effect of the sample using DPPH (1,1-Diphenyl-2-picrylhydrazyl, Aldrich) (Blois MS, Nature 181 , pp 1198-1200, 1958). About 2 mg of DPPH was dissolved in 15 ml of ethanol to prepare a DPPH solution. 6.25 ml of DMSO was added to 12 ml of this solution, followed by UV-Vis. The absorbance was diluted with ethanol to 0.94-0.97 and shaken for 10 seconds. In addition, 1 mg of the sample extracted as a powder was mixed with 1 ml of the solvent, and then sufficiently dissolved. 50 µl of the sample solution was added to 450 µl of the prepared DPPH, and the mixture was left at room temperature for 10 minutes, and then absorbance was measured at 517 nm. As a control group, BHA, vitamin C, vitamin E, and pycnogenol (pine extract) were used, and the antioxidant effect was expressed by the concentration of the sample (RC ₅₀ ) which appears when the absorbance of DPPH was reduced by 50%. The experiment was repeated three times to obtain an average value.

As a result of the above experiment, the ethyl acetate fraction, butanol fraction, EF 5-4-6, EF 5-4-7, BF 1 to the RC ₅₀ value of synthetic antioxidants BHA and vitamin C, vitamin E, pine extract Pycnogenol In comparison, they showed high antioxidant activities of 10.9 μg / ml, 12.7 μg / ml, 9.93 μg / ml, 10.09 μg / ml and 9.09 μg / ml, respectively (Table 1).

sample RC ₅₀ (μg / ml) DPPH radical scavenging effect Vitamin c 3.0 Vitamin E 17.0 Pycnogenol 6.3 BHA 9.04 80% Methanol Extract 32.3 Hexane fraction 89.0 Ethyl acetate fraction 10.9 Butanol fraction 12.7 Aqueous fraction 89.3 Butanol-20% Methanol Fraction (BF1) 9.93 Butanol-40% Methanol Fraction (BF2) 19.74 Butanol-80% Methanol Fraction (BF3) 97.94 Butanol-100% Methanol Fraction (BF4) ＞ 100 Ethyl Acetate Fraction 5-4-1 (EF 5-4-1) ＞ 100 Ethyl Acetate Fraction 5-4-2 (EF 5-4-2) ＞ 100 Ethyl Acetate Fraction 5-4-3 (EF 5-4-3) ＞ 100 Ethyl Acetate Fraction 5-4-4 (EF 5-4-4) ＞ 100 Ethyl Acetate Fraction 5-4-5 (EF 5-4-5) 78.9 Ethyl Acetate Fraction 5-4-6 (EF 5-4-6) 10.09 Ethyl Acetate Fraction 5-4-7 (EF 5-4-7) 9.09

2-2. NO generation inhibition rate search

2-2-1. Efficacy of Wumusa Respiratory Extract

RAW264.7 cells were adjusted to 1.5 × 10 ⁵ cells / ml using DMEM medium, and then inoculated into 24 well plates, incubated for 48 hours with treatment of test medium and fresh medium containing LPS (1 μg / ml) simultaneously. It was. The amount of generated NO was measured in the form of NO ₂ ⁻ present in the cell culture liquid using a grease reagent. Mix 100 μl of cell culture supernatant and 100 μl of grease reagent (1% (w / v) sulfanilamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid) in a 96-well plate for 10 minutes. After coagulation, the absorbance at 540 nm was measured. 540 nm was measured using an ELISA reader, and sodium nitrite (NaNO ₂ ) was compared as a standard.

As a result of the experiment, ethyl acetate fraction, butanol fraction, EF 5-4-6-3-2, BF 1 showed higher NO production inhibitory activity than the control LPS treatment (see Fig. 1; A: 80% methanol extract) , B: hexane extract, C: ethyl acetate extract, D: butanol extract, E: aqueous fraction, F: EF 5-4-6-3-2, G: EF 5-4-6-3-3, H: EF 5-4-7, I: BF 1).

2-2-2. Effect of Utigosides B and C

In order to examine the effect of uteigosides B and C on NO production induced by LPS, the same results as those of Experiment 2-2-1 using Utigosides B and C obtained in Example 3 as samples. An experimental procedure was performed.

As a result of performing the experiment, as shown in Figure 2, when the extract of Umuxasa lepi was administered more than the control LPS treatment, it showed a higher NO production inhibitory activity, Umusasapi methanol crude extract, ethanol fraction in the order of high NO inhibition The activity was confirmed, and when utigosides B and C were administered, higher NO production inhibitory activity was observed. In addition, in FIG. 3, the concentration-dependent concentration of utigoside B of the present invention, and in FIG. 4, it was confirmed that the NO production inhibitory activity was increased in concentration-dependent manner. Utigoside B has an IC ₅₀ value of 24.41 μM and Utigoside C has an IC ₅₀ value of 24.66 μM.

Experimental Example 3. Measurement of the effect on the production of pro-inflammatory molecules

3-1. In Vitro Cytokine Production and Quantitation

3-1-1. Efficacy of Wumusa Respiratory Extract

RAW 264.7 cells, a murine macrophage cell line, were adjusted to 1.0 × 10 6 cells / ml using DMEM medium, inoculated into 24 well plates, and pre-incubated for 18 hours in a 5% CO ₂ incubator. . Thereafter, the medium was removed, and 50 μl of test substance prepared at 10-fold concentration and fresh medium containing 450 μl final LPS concentration (1 μg / ml) were simultaneously treated and cultured under the same conditions as the pre-culture. After 6 hours TNF-α and IL-6 were centrifuged (12,000 rpm, 3 min) in the culture medium to obtain a supernatant. Quantification of IL-1β comprises 150 mM NaCl, 50 mM Tris HCl (pH7.6), 1.0 mM PMSF, and 0.25% Nonidet P-40 after treatment with the test drug in the same manner as TNF-α and after 6 hours of incubation. After 10 minutes at 4 ° C using 1 ml of lysis buffer, centrifugation (2 minutes at 12,000 rpm) to obtain a supernatant. All samples were stored at −20 ° C. or below until quantification.

TNF-α and other cytokines were quantified using a mouse ELISA kit, and the r ² value of the standard curve for the standard was 0.99 or more.

As a result of the above experiment, TNF-α production inhibitory was 24.84%, 16.77%, 13.39 in EF 5-4-6-3-2, EF 5-4-6-3-3, EF 4-7, BF 1, respectively. % And 19.35% showed inhibitory effects. In addition, the inhibition of IL-6 and IL-1β production was slightly different from that of TNF-α, whereas IL-6 production was inhibited by EF 5-4-6-3-2 and EF 5. -4-6-3-3, EF 5-4-7, and BF 1 showed 24.67%, 12.1%, 11.71%, and 46.14%, respectively, and EF 5-4-6-3 for IL-1β production inhibition -2, EF 5-4-6-3-3, EF 5-4-7, and BF 1 showed 24.65%, 15.21%, 14.82%, 44.32%, respectively. It was confirmed that EF 5-4-6-3-2 and butanol fraction BF 1 affects inflammatory cytokine inhibition expressed by LPS (see FIG. 5).

3-1-2. Effect of Utigosides B and C

In order to examine the effects on the production of TNF-α and IL-6 of utigosides B and C, the same experimental procedure as in 3-1-1 was performed.

As shown in Figure 6 and Figure 7, the results of the administration of the Utigoside B and C obtained in Example 3 was treated only LPS and TNF-α and IL- compared to the group without administration It was confirmed that the production of 6 significantly inhibited the production of anti-inflammatory cytokines.

3-2. RNA Isolation and RT-PCR

3-2-1. Efficacy of Wumusa Respiratory Extract

Total RNA extraction from RAW 264.7 cells (1.0 × 10 5 cells / ml) was performed using TRI-reagent (MRC) and under RNase-free conditions. 1 μg total RNA was added with oligo (dT) 18 primer, dNTP (0.5 μM), 1 unit RNase inhibitor and M-MuLV reverse transcriptase (2U) 5 min at 70 ° C., 5 min at 37 ° C. The reaction was stopped by heating at 37 ° C. for 60 minutes and at 70 ° C. for 10 minutes.

Polymerase Chain Reaction (PCR) was performed using 2 μl cDNA, 4 μM to amplify TNF-α, IL-6, IL-1β, iNOS, COX-2, β-Actin from the synthesized cDNA. 5 ′ and 3 ′ primers, 10 × buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 250 μM dNTP, 25 mM MgCl ₂ , 1 unit Taq polymerase, Promega Inc., USA) were mixed and distilled water was adjusted to 25 μl, followed by PCR using a Perkin-Elmer Thermal Cycler. At this time, the PCR conditions are 94 ℃ / 45 seconds, 55 ~ 60 ℃ / 45 seconds, 72 ℃ / 60 seconds, 35 times, the product produced by PCR is subjected to electrophoresis on 1.5% agarose gel and ethidium bromide ( Staining with ethidium bromide confirmed specific bands.

As a result of conducting the experiment, when the solvent fraction of mumussa rep to suppress the TNF-α, IL-6, IL-1β production by treatment with LPS stimulation at a concentration of 100 ㎍ / ㎖, TNF-α production inhibition is ethyl The acetate fraction showed an inhibitory effect, the inhibition of IL-6 production was the ethyl acetate fraction, the IL-1β production inhibition was the inhibitory effect in the butanol fraction (see FIG. 8; A: control (LPS +), B: 80% Methanol extract, C: hexane extract, D: ethyl acetate extract, E: butanol extract, F: aqueous fraction). In addition, EF 5-4-6-3-2 obtained from the ethyl acetate fraction and BF 1 obtained from the butanol fraction showed high inhibitory effects not only on TNF-α but also on IL-6 and IL-1β (FIG. 9). EF 5-4-6-3-2: 24.67%, EF 5-4-6-3-3: 12.1%, EF 5-4-7: 11.71%, BF 1: 46.14%). In addition, iNOS and COX-2, which showed the expression level of cytokines from RAW 264.7 cells, showed high inhibitory effects in ethyl acetate fraction and butanol fraction (see FIG. 10; A: EF 5-4-). 6-3-2, B: EF 5-4-6-3-3, C: EF 5-4-7, D: BF 1).

3-2-2. Effect of Utigosides B and C

Experimental experiments were performed in the same manner as in the RT-PCR experiment of the Umusa respiratory extract of Experimental Example 3-2-1, and the effects on the production of the initial inflammatory molecules of the utigosides B and C were examined. In this experiment, however, RAW264.7 cells were initially cultured for 18 hours, and the expression level of mRNA for early inflammatory cytokines was measured 6 hours after LPS (1 µg / ml) administration to these cells.

As a result of the experiment, as shown in FIG. 11, utigosides B and C were found to significantly inhibit the production of TNF-α and IL-6, but did not have any effect on IL-1β production. Could. However, as shown in FIG. 12, it was confirmed that the production of IL-10 was increased during the treatment of Utigoside B and C (100 µg / ml).

3-3. Western blot analysis

3-3-1. Efficacy of Wumusa Respiratory Extract

In the RAW 264.7 cells (1.0 × 105 cells / ml) Cells were collected after treatment of the extracts and fractions with a concentration of 100 μg / ml, respectively. After washing the cells 2-3 times with PBS (Phosphate Buffered Saline), 1 ml of lysis buffer was added, lysed for 30 minutes to 1 hour, and then centrifuged at 12,000 rpm for 20 minutes to form cell membrane components. Etc. were removed. Protein concentration was quantified using a Bio-Rad Protein Assay Kit by standardizing BSA (Bovine serum albumin). 30-50 μg of lysate was denatured by 8% mini gel Poly Acrylamide Gel Electrophoresis (SDS-PAGE) and transferred to PVDF cell membrane (BIO-RAD) at 200 mA for 2 hours. . Blocking of the cell membrane was performed for 2 hours at room temperature in TTBS (TBS + 0.1% Tween 20) solution containing 5% skim milk. Anti-mouse iNOS (1: 1000) (Santa-Cruz) was used as an antibody for examining the expression level of iNOS. Anti-rabbit COX-2 (1: 1000) was used as an antibody for examining the expression level of COX-2. (Santa-Cruz) was diluted in TTBS solution, reacted at room temperature for 2 hours, and washed three times with TTBS. As a secondary antibody, anti-mouse or anti-rabbit IgG (Amersham Co.) conjugated with HRP (Horse Radish Peroxidase) was diluted 1: 5000, reacted at room temperature for 30 minutes, washed three times with TTBS, and then ECL. After reacting with the substrate (Amersham Co.) for 1 to 3 minutes, the X-ray film was photosensitive.

As a result of the experiment, it was confirmed that iNOS and COX-2 protein expression is significantly inhibited in the ethyl acetate fraction and butanol fraction (see FIG. 13), and also EF 5-4-6-3- obtained in the ethyl acetate fraction. It was confirmed that iNOS protein expression was significantly inhibited in BF 1 obtained from 2 and butanol fraction (see FIG. 14; A: EF 5-4-6-3-2, B: EF 5-4-6-3-3). , C: EF 5-4-7, D: BF 1).

3-3-2. Effect of Utigosides B and C

i Experimental Example 3-3-1 using the uteigosides B and C obtained in Example 3 as a test sample to examine the effect of uteigosides B and C on NOS and COX-2 expression. The same experimental procedure was performed. In this experiment, RAW264.7 cells were initially cultured for 18 hours, and protein expression of iNOS was measured after 24 hours stimulation with LPS (1 µg / ml).

As a result of the experiment, it was confirmed that when the utigosides B and C of the present invention were administered, the expression level of iNOS (see FIG. 15) and the expression level of COX-2 (see FIG. 16) were significantly inhibited.

Experimental Example 4. Cytotoxicity Test

RAW 264.7 cells were placed in each well of a 96 well plate at a concentration of 1.0 × 0 ⁵ cells / ml, and cultured for 24 hours. Then, the samples obtained in Examples 2 and 3 were added at a concentration of 100 µg / ml. After incubation for 24 hours, 100 ㎍ of MTT sample (3- (4,5-dimethylthiazol) -2,5-diphenyl tetrazolium bromide, Sigma) was added and further incubated for 4 hours. The plate was centrifuged at 1000 rpm for 10 minutes and the medium was carefully removed, then 150 μl of DMSO (Sigma) was added to dissolve the formazan precipitate produced by the reduction of MTT, followed by absorbance at 540 nm using a microplate reader. Was measured (Carmichael, J., et al., Cancer Research , 47 , pp936-941, 1987). The average absorbance values for each sample group were obtained. As a result of examining the degree of growth inhibition compared with the absorbance values of the control group, no cytotoxicity was observed.

Although the preparation example of the pharmaceutical composition comprising the woomusa repi extract or utigoside compound of the present invention will be described, the present invention is not intended to limit it, only intended to explain in detail.

Formulation Example 1 Preparation of Powder

300 mg of dry extract of Example 1

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

Utigoside Compound 50 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

50 mg of dry extract of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Utigoside Compound 50 mg

Appropriate sterile distilled water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Utigoside Compound 100 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

1000 mg of dry extract of Example 1

Vitamin mixture proper amount

       70 μg of Vitamin A Acetate

       Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

       Vitamin C 10 mg

       10 μg biotin

       Nicotinic Acid 1.7 mg

       50 μg folic acid

       Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

1000 mg of dry extract of Example 1

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the mutigosaccharide extract of the present invention or Utigoside B and C isolated therefrom exhibits antioxidant and anti-inflammatory effects, and thus is used as a pharmaceutical composition and health functional food for the prevention and treatment of inflammation-related diseases. Can be.

Claims (7)

For the treatment and prevention of rheumatoid arthritis and inflammatory bowel disease containing the extract of Eurya emarginata organic solvent leaf having an antioxidant and anti-inflammatory effect as an active ingredient, and containing a pharmaceutically acceptable carrier, excipient or diluent Pharmaceutic composition. The pharmaceutical composition according to claim 1, wherein the organic solvent is a polar solvent of methanol, ethanol or butanol polar solvent, hexane, ethyl acetate, acetone, chloroform or methylene chloride, and a mixed solvent thereof. A pharmaceutical composition for the treatment and prophylaxis of rheumatoid arthritis and inflammatory bowel disease, which comprises a utigoside compound having the structure of formula (I) as an active ingredient, and includes a pharmaceutically acceptable carrier, excipient or diluent.

(Ⅰ) Wherein R is H or OH. delete A health functional food for the prevention and improvement of rheumatoid arthritis and inflammatory bowel disease, which contains as an active ingredient woosamsa repi leaf extract showing antioxidant and anti-inflammatory effects. delete A health functional food for the prevention and improvement of rheumatoid arthritis and inflammatory bowel disease, which contains the utigoside compound of the formula (I) structure of claim 3, which exhibits antioxidant and anti-inflammatory effects.

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