KR20100013436A - A composition comprising thteb compound for preventing or treating oxidation related disease - Google Patents

Abstract

PURPOSE: A 3,4,5-trihydroxy-N-[2-p-tolylethyl]-benzamide compound is provided to ensure excellent removal activity of free radical, superoxide radical, lipid peroxide, or hydroxyl radical and strong antioxidant effect on DNA damage inhibitory activity induced by hydrogen peroxide or ultraviolet rays. CONSTITUTION: A 3,4,5-trihydroxy-N-[2-p-tolylethyl]-benzamide compound and its pharmaceutically acceptable salt have a structure represented by chemical formula (I). A pharmaceutical composition for the prevention and treatment of oxidation-related diseases comprises the 3,4,5-trihydroxy-N-[2-p-tolylethyl]-benzamide compound and its salt as an active ingredient. The oxidation-related diseases are aging, cancer, diabetes, hepatitis, gastritis, rheumatoid arthritis, stroma, neurodegenerative disease or artery scleroma.

Description

A composition comprising THTEB compound for preventing or treating oxidation related disease}

The present invention relates to a composition for preventing and treating oxidation-related diseases containing 3,4,5-trihydroxy-N- [2- p- tolylethyl] -benzamide (THTEB) compound as an active ingredient.

1 Beckman J S., et al., Proc . Natl . Acad . Sci . USA, 87 , pp 1620-1624, 1990

2 Sagar S., et al., Mol . Cell Biochem ., 111 , pp 103-108, 1992

3 Ames B N., et al., Proc . Natl . Acad , USA, 90 , pp 7915-7922, 1993

Griffiths H R., et al., FEBS Lett ., 388 , pp 161-164, 1996

Squadrito G L., et al., Free Radic . Biol . Med ., 25 , pp 392-493, 1998

6 J Choi S., Phytochem . Res ., 16 , pp 232-235, 2002

7 Dreher D, et al., Eur . J. Cancer , 32A (1) , pp 30-38, 1996

8 Sohal RS, et al., Free Radic . Biol . Med ., 33 (1) , pp 37-44, 2002

9 Pietta P G., J. Nat . Prod . 63 , pp 1035-1042, 2000

Document 10 Burdon R H., Free Radic . Biol . Med . 18 , pp 775-794, 1995

11 Russo A et al., Cell Biol . Toxicol . 16 , pp 91-98, 2000

Document 12 Sanchez-Moreno C., Food Res . Int . 32 , pp 407-412, 1999

Document 13 Pratt D E., American Chemical Society , pp 54-71, 1994

[14] Larson R A., Phytochem ., 27 , pp 969-978, 1988

15 Decker E A., Nutr . Rev. 53 , pp 49-58, 1995

16. Haefner B., Drug Discov . Today , 8 (12) , pp 536-544, 2003

17. Blunt J W., et al., J. Nat . Prod . Rep . 20 , p 1, 2003

18 Faulkner D., J. Nat . Prod . Rep . 19 , pp 1-48, 2002

[19] Keum Y S., et al., Cancer Lett . 150 , pp 41-48, 2000

Kirby A J., et al., J. Ethnopharmacol ., 56 (2) , pp 103-108, 1997

[21] Eun M J., et al, life science, 74 , pp 1013-1026, 2004

Re 22 R., et al., Free Radic . Biol . Med . 26 , pp 1231-1237, 1999

Oxidative activity refers to an activity that prevents the production of free radicals in vivo and prevents oxidative phenomena that cause irreparable damage to cells.

Steady state oxygen (triplet oxygen) is a superoxide radical, hydroxyl radical, hydrogen peroxide (H ₂ O ₂ ) due to environmental and biochemical factors such as enzymes, reduction metabolism, chemicals, pollutants, photochemical reactions, etc. It is known to convert into reactive oxygen species (ROS), such as ROS, to oxidize various cell components, such as lipids, proteins, and DNA, causing inflammation or damaging various organs (Beckman J S., et al. , Proc . Natl . Acad . Sci . USA, 87 , pp 1620-1624, 1990; Sagar S., et al., Mol . Cell . Biochem ., 111 , pp 103-108, 1992; Ames B N., et al., Proc . Natl . Acad , USA, 90 , pp 7915-7922, 1993), examples include superoxide anion (O ₂ ), hydrogen peroxide, hydroxyl radicals, singlet oxygen (1O ₂ ), lipid peroxidation Alkoxyl radical (RO *), peroxyl radical (ROO), and nitrogen peroxide ion (ONOO-) produced | generated by the above are mentioned.

The action of these free radicals affects the action of antioxidant enzymes such as superoxide dismutase (SOD), catalase, and peroxidase, and vitamin C, E, and glutathione. If the biodefense is abnormal or exposed to excessive free radicals, the balance is broken and the free radicals irreversibly destroy substrates, proteins, DNA, and the like. ), Various diseases such as cancer, multiple atherosclerosis, arthritis and parkinson's disease have been reported (Griffiths H R., et al., FEBS Lett ., 388 , pp 161-164). , 1996; Squadrito G L., et al., Free Radic . Biol . Med ., 25 , pp 392-493, 1998; Choi J S., Phytochem . Res., 16 , pp 232-235, 2002; Dreher D, et al., Eur . J. Cancer , 32A (1) , pp 30-38, 1996; Sohal R S., et al., Free Radic . Biol . Med ., 33 (1) , pp 37-44, 2002). The mechanism of action of antioxidants includes the following three. (1) inhibits the production of free radicals by chelating enzymes or inhibitors associated with free radicals (2) removing free radicals (3) up-regulating antioxidant defenses active oxygen, including upregulating or promoting, superoxide anion, hydroxy radicals, singlet oxygen, hydrogen peroxide, all mammals from mitochondrial oxygen breathing or exposure to toxic substances Produced in cells. Some of these free radicals in vivo play a positive role in the production of energy and in the presentation of intracellular signaling pathways for apoptosis, stress, and proliferation or in the synthesis of biologically important components (Pietta P G., J. Nat . Prod . 63 , pp 1035-1042, 2000). Intracellular defense mechanisms from free radicals include correction systems, detoxifying enzymes such as superoxide dismutases (SOD), and scavengers such as glutathione. When the generation of free radicals and their protection mechanisms are not balanced, it is suggested that the production of unnecessary free radicals is strongly associated with the processes of aging and degenerative diseases such as cardiovascular disease and cancer. DNA can be attacked by inducing cell membrane damage such as peroxidation of cell lipids and decreased fluidity of cell membranes, resulting in DNA degeneration leading to cancer (Burdon R H., Free Radic . Biol . Med . 18 , pp 775-794, 1995; Russo A et al., Cell Biol . Toxicol . 16 , pp 91-98, 2000; Sanchez-Moreno C., Food Res . Int . 32 , pp 407-412, 1999), and these observations suggest that free radicals may be an important target of preventive chemotherapy for diseases associated with aging.

In order to prevent diseases caused by oxidative stress and to prevent oxidative deterioration of foods, many studies are being conducted to isolate, identify and develop powerful and harmless natural antioxidants from edible or medicinal plants. Natural compounds are said to retard oxidation by free radicals and scavengers or reducing agents of reactive oxygen species (Pratt D E., American). Chemical Society , pp 54-71, 1994: Larson R A., Phytochem ., 27 , pp 969-978, 1988).

Synthetic antioxidants developed so far include butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT) and nordihydro-guaiaretic acid (NDGA). Antioxidant enzymes such as oxidase and non-enzymatic antioxidants such as tocopherol (vitamin E), vitamin C (vitamin C, ascorbic acid), carotenoids and glutathione.

Phenolic compounds, widely distributed in nature and considered the most representative antioxidants, are secondary metabolites with one or more hydroxyl groups that can be substituted in the ring structure. These compounds act as inhibitors and promoters for a variety of mammalian enzyme systems and as scavengers and metal chelators of oxygen radicals. Since these antioxidant activities are related to conjugated rings and hydroxyl groups, it is not surprising that most phenolic compounds have antioxidant activities (Sanchez-Moreno C et al., Food Res. Int . 32 , pp 407-412, 1999; Decker E A., Nutr . Rev. 53 , pp 49-58, 1995).

Significantly physically and chemically different from the terrestrial environment, the marine environment is an infinite report of biologically active and uniquely structured natural products, which has recently revealed an infinite number of resources with remarkable activities such as anticancer, anti-inflammatory, analgesia, allergy, etc. Byostatin-1, with the exception of cephalosporins, cytarabine, and vidarabine, three medicines that have been proven and proven and are widely available from the ocean to date. , Squalamine, and ET743 have been recognized as rare drugs in the last two years (Haefner B., Drug Discov . Today 8 (12) , pp 536-544, 2003). In addition, more than twelve marine compounds or derivatives thereof are under recent clinical research and marine naturals will lead to a promising future in medicine development. Marine organisms have proven to be a rich source of structurally new and biologically active secondary metabolites, and these secondary metabolites have emerged as groundbreaking chemical resources for pharmaceutical development (Blunt J W., et al., J. Nat . Prod . Rep . 20 , p 1, 2003; Faulkner D., J. Nat . Prod . Rep . 19 , pp 1-48, 2002).

Therefore, the present inventors completed the present invention by confirming the antioxidant activity of THTEB using DNA damage measurement of hydroxyl radicals generated by DPPH, NBT, TEAC assay, hydrogen peroxide and ultraviolet rays as part of research and development efforts on antioxidant. It was.

In order to achieve the above object, 3,4,5-trihydroxy-N- [2- p- tolylethyl] -benzamide (hereinafter referred to as 'THTEB') compound represented by the following structural formula (I) and a pharmaceutically acceptable thereof salt:

(Ⅰ)

(Ⅰ)

The compounds of the present invention represented by the above formula (I) may be prepared with pharmaceutically acceptable salts and solvates according to conventional methods in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, and hydroiodic acid.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the invention include salts of acidic or basic groups which may be present in the compounds of the invention, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, Hydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts. It can be prepared through the process.

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

The THTEB compound of the present invention can be obtained as follows.

THTEB is a p-tyrosol and gallic acid (gallic acid) is bonded by an amide bond, the synthesis of THTEB is carried out in the following two steps.

3,4,5, -trimethoxybenzoyl chloride, 4-Methoxyphenethyl amine, potassium carbonate, distilled water and ethyl acetate After mixing in a solution containing 1-5: 1-15 (H ₂ O / EtOAc 1-5: 1-15), storing the reaction solution at room temperature for 20-40 hours, and then filtering the resultant Recrystallization from ethyl acetate (ethylacetate) and then purified to make 3,4,5-Trimethoxy-N- [2- (4-methoxyphenyl) ethyl] -benzamide compound;

Dissolve the first step of 3,4,5-Trimethoxy-N- [2- (4-methoxyphenyl) ethyl] -benzamide in 20 ml of freshly distilled dichloroethane (CH ₂ Cl ₂ ), Slowly add boron (BBr ₃ ) to the solution, stir the compound at room temperature for 20 to 40 hours, add purified water, stir again for 20 to 40 minutes, filter the resulting material, and recrystallize the material from dichloromethane. 3,4,5-trihydroxy-N- [2-p-tolylethyl] -benzamide (THTEB) compound can be obtained through two steps of separation and purification.

The present invention provides a pharmaceutical composition for the prevention and treatment of oxidation-related diseases containing THTEB compound obtained by the above production method as an active ingredient.

The oxidation-related diseases include aging, cancer, diabetes, hepatitis, gastritis, rheumatoid arthritis, epilepsy, neurodegenerative diseases or arteriosclerosis, preferably aging, cancer, diabetes or atherosclerosis, more preferably aging or atherosclerosis .

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

The pharmaceutical composition comprising the compound of the present invention may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Carriers, excipients and diluents that may be included in the compositions of the present invention 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.

Pharmaceutical dosage forms of the 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.

Compositions comprising the compounds of the invention are each formulated in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, according to conventional methods Can be used.

Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like form at least one excipient such as starch, calcium carbonate, sucrose in the compound. ), Lactose, gelatin and the like can be mixed. In addition to the simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solvents, emulsions and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances and preservatives, in addition to the commonly used simple diluents, water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter and glycerogelatin may be used.

Preferred dosages of the compounds of the present invention depend 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, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably in an amount of 0.001 to 100 mg / kg once to several times daily. The compound of the present invention in the composition may be formulated in an amount of 0.0001 to 50% by weight based on the total weight of the total composition.

The pharmaceutical composition 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 and intracerebroventricular injections.

The present invention also provides a health functional food for the prevention and improvement of oxidation-related diseases containing THTEB compound as an active ingredient.

The compounds of the present invention can be used in various ways, such as drugs, foods and beverages for the prevention and treatment of oxidation-related diseases. Foods to which the compound of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes and dietary supplements, and may be used in the form of powders, granules, tablets and capsules or beverages. have.

Since the compound of the present invention has little toxicity and side effects, it is a drug that can be used safely even for long-term administration for the purpose of prevention.

The compound of the present invention may be added to food or beverage for the purpose of preventing and treating oxidation related diseases. At this time, the amount of the compound in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 30 g based on 100 ml, preferably Can be added in a ratio of 0.3 to 10 g.

The health beverage composition of the present invention, in addition to containing the compound as an essential ingredient in the indicated proportions, has no particular limitation on the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, for example polysaccharides such as maltose and sucrose, and conventional sugars such as dextrin and cyclodextrin. Sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., 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 compounds of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the compounds 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 from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

As mentioned above, the THTEB compounds of the present invention are free radicals, superoxide radicals, lipid peroxidantion, or scavenging activity of hydroxyl radicals and are induced by hydrogen peroxide or ultraviolet light. By showing a strong antioxidant effect in relation to DNA damage inhibitory activity it can be usefully used as a pharmaceutical composition or health functional food for the prevention and treatment of oxidation-related diseases.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, the contents of the present invention is not limited by the following Examples, Reference Examples and Experimental Examples.

Example 1 Preparation of 3,4,5-Trimethoxy-N- [2- (4-methoxyphenyl) ethyl] -benzamide Compound (1)

3,4,5, -trimethoxybenzoyl chloride (0.01 mol), 4-Methoxyphenethyl amine (0.011 mol), potassium carbonate Was mixed with distilled water and ethyl acetate in a solution containing 1: 6 (H ₂ O / EtOAc 1: 6), the reaction solution was stored at room temperature for 24 hours, and the resultant was filtered and the material was ethylacetate. Purified by recrystallization).

The compound name of this first step is 3,4,5-Trimethoxy-N- [2- (4-methoxyphenyl) ethyl] -benzamide. The structure was confirmed by IR and ¹ H-NMR. It is as follows.

IR (KBr, cm ⁻¹ ): 3294, 2839, 1630, 1582, 1505, 1462, 1414, 1342, 1235, 1178, 1131, 1030, 993, 830.

¹ H-NMR (300 MHz, acetone- d ₆ ): δ 2.8 (t, 2H), 3.5 (q, 2H), 3.75 (d, 6H), 3.84 (s, 6H), 6.8 (d, 2H), 7.16 (d, 2 H), 7.19 (s, 2 H), 7.8 (s, 1 H).

Example 2 Preparation of 3,4,5-trihydroxy-N- [2-p-tolylethyl] -benzamide (THTEB) Compound (2)

The 3,4,5-Trimethoxy-N- [2- (4-methoxyphenyl) ethyl] -benzamide compound (0.003 mol) of Example 1 was dissolved in 20 ml of freshly distilled dichloroethane (CH ₂ Cl ₂ ) and cooled. After adding 0.015 mol of tribromoboron (BBr ₃ ) to the solution slowly, the compound was stirred at room temperature for 24 hours, and then purified water was added and stirred again for a few minutes, and the resultant water was filtered. A 3,4,5-trihydroxy-N- [2-p-tolylethyl] -benzamide (THTEB) compound can be obtained through a second step of recrystallization from methane and purification.

This THTEB structure was proved by IR, ¹ H-NMR and ¹³ C-NMR, and its physical properties are as follows.

IR (KBr, cm ⁻¹ ): 3346, 3237, 3035, 2965, 1586, 1554, 1445, 1354, 1308, 1236, 1033, 826.

¹ H-NMR (300 MHz, acetone- d ₆ ): δ 2.7 (t, 2H), 3.5 (q, 2H), 6.7 (d, 2H), 6.9 (s, 2H), 7.06 (d, 2H), 7.45 (s, 1 H), 8.03 (s, 1 H).

¹³ C-NMR (75 MHz, acetone- d ₆ ): δ 35.64, 42.40, 107.54, 116.00, 126.91, 130.5, 131.20, 136.70, 146.02, 156.60, 167.39.

Anal. Calcd .: C 62.22, H 5.18, N 4.84, O 27.65; Found: C 62.69, H 4.64, N 4.69, O 28.00

Experimental Example  One. DPPH  freedom Radical  Scavenging activity measurement

In order to measure the free radical scavenging activity, the following experiment was applied by applying the method described in the existing literature. (Keum Y S., et al., Cancer Lett . 150 , pp 41-48, 2000)

The DPPH free radical scavenging experiment is one of the methods used to identify which substance can act as a free radical trap, which measures the conversion of DPPH free radicals to 2,2-diphenyl-1-picryl hydrazine by antioxidants. The results show the values obtained by performing three experiments.

THTEB at various concentrations was reacted for 30 minutes at room temperature in a reaction mixture containing 0.4 mM DPPH-ethanol solution and 50 mM pH 7.4 Tris-HCl buffer 2: 1. By measuring the absorbance at 517 nm after the reaction, the degree of reduction of DPPH free radicals can be measured. In this experiment, vitamin C was used as a positive control group and the inhibition rate (%) was calculated by Equation 1 below.

% Inhibition = [(absorbance of control-absorbance of sample) / absorbance of control] x 100

As a result, as shown in Figure 1, THTEB showed a strong radical scavenging activity compared to vitamin C in the concentration range of 1.56μM to 25μM, compared with the concentration (IC ₅₀ ) showing 50% scavenging activity Vitamin C was 55.7 μM, while THTEB showed a lower IC ₅₀ of 22.8 μM, indicating that THTEB had a distinct DPPH free radical scavenging activity (see FIG. 1).

Experimental Example  2. Measurement of inhibitory activity of peroxide anion formation

In order to determine the peroxide anion formation inhibitory activity, the following experiment was conducted by applying the method described in the existing literature (Kirby A J., et al., J. Ethnopharmacol ., 56 (2) , pp 103-108, 1997).

Peroxide ions and hydroxyl radicals are the most representative free radicals, and the effects of these peroxide ions can be enhanced because peroxide ions are usually formed first in intracellular oxidation reactions, which produce other harmful free radicals and oxides. Xanthine oxidase is one of the main enzymatic causes of these free radicals in vivo. In the NBT method, hypoxanthine-xanthine oxidase produces peroxide ions and peroxide ions reduce NBT Forms a forazan of color. Observations of the absorbance change of NBT over time were expressed as percent inhibition relative to the rate value of the absorbance change obtained from the sample-free control, which is indicated by the relative peroxide anion scavenging ability of these samples. Vitamin C was used as a positive control, and all reagents for the experiment were prepared in an average state of 25 ° C. in 50 mM KH ₂ PO ₄ / KOH buffer (pH 7.4), and the reaction mixture was 20 μl of 15 mM Na ₂ EDTA. 50 μl of 0.6 mM NBT, 30 μl of 3 mM hypoxanthine, 3 μl of sample, and after the reaction was initiated by 50 μl of xanthine oxidase (1 unit / 10 ml buffer) at 25 ° C. Absorbance was measured at 570 nm for 10 minutes at 30 second intervals using a Microplate Reader (synergy HT, BIO-TEK). The control group uses ethanol, the same amount of sample solvent instead of the sample, and the result is expressed as a relative inhibition rate (%) relative to the control group and was calculated by Equation 2 below.

% Inhibition = [(control response-sample response) / control response] x 100

As a result of the experiment, it was confirmed that THTEB has a strong peroxide anion scavenging activity as compared to vitamin C in a concentration-dependent manner (see FIG. 2).

Experimental Example  3. Lipid Peroxide ( Lipid peroxide ) Measurement

In order to measure the lipid peroxide inhibitory activity, the following experiment was applied by applying the method described in the existing literature (Eun M J., et al, life science, 74 , pp 1013-1026, 2004).

First, 10 g of the liver of the rat was put in a homogeneous solution by adding HEPES-KCl solution, followed by centrifugation to obtain a supernatant, which was used after confirming the protein concentration. Liver microsomes, 10 vitamin C, 10FeSO ₄ and 220 calcium chloride were added with BHT or THTEB, respectively, and the concentration was measured to be 3.125 to 50 μM. After the experiment was finished using a centrifuge, the absorbance was measured at 532 nm. .

As a result of the experiment, as shown in FIG. 3, it can be seen that the absorbance increases with the concentration of THTEB, and it was confirmed that the activity was stronger than that of BHT, which is a well-known antioxidant compound (see FIG. 3).

Experimental Example  4. ABTS Radical  Scavenging activity measurement

In order to determine the value of trolox equivalent antioxidant capacities (THAC) for THTEB, the following experiments were carried out by applying the method described in the existing literature (Re R., et al., Free Radic . Biol . Med . 26 , pp 1231-1237, 1999).

The TEAC method by the generation of ABTS radicals is one of the spectrophotometric methods that have been used to measure the total antioxidant activity of a sample, and the ABTS radical (2,2 -azinobis (3-ethylbenzo-thiazoline-) of the Trolox antioxidant, a standard in the same experimental state. The antioxidative reactivity of 6-sulfonic acid in% compared to the antioxidative response of trolox, and ABTS · + (ABTS radical cation) was 2.45 mM potassium persulfate and 7 mM concentration. It was prepared by reaction of ABTS solution dissolved in water and prepared by placing it in the dark at room temperature 12 hours before use.The ABTS. + Solution was diluted to 0.70 ± 0.02 at 734 nm using 5 mM PBS. 10 μl of sample or trolox, a standard substance, was added to 1 ml of this ABTS · + solution and reacted at 30 ° C. for 6 minutes, and then the absorbance was measured at 734 nm. The antioxidant activities of THTEB and vitamin C were expressed as TEAC values using standard curves calculated by different concentrations of trolox.

As a result, as shown in FIG. 4, THTEB and vitamin C showed ABTS radical scavenging activity in a concentration-dependent manner. The TEAC values of THTEB and vitamin C were 0.623 and 0.130, respectively, which indicates that THTEB has antioxidant activity (see FIG. 4 and Table 1).

TABLE 1

TEAC value THTEB 0.623 Vt. C 0.130

Experimental Example  5. Induced by hydrogen peroxide or ultraviolet DNA  For chain cutting THTEB  Protective effect of compound

In order to measure the protective effect activity of THTEB on DNA chain cleavage induced by hydrogen peroxide or ultraviolet rays, the following experiment was applied by applying the method described in the existing literature (Keum Y S., et al., Cancer Lett ., 150) . , pp 41-48, 2000).

To 30 μL of reaction mixture (0.15 μg pBR322 plasmid DNA, 30 mM H ₂ O ₂ , 10 mM Tris-EDTA buffer at pH 8.0), 5 μL of various concentrations of THTEB compound were added to a final concentration of 2, 4, 8 μM, but the reaction mixture was The THTEB compound is mixed into the reaction solution before the hydrogen peroxide is added. After irradiating the reaction solution for 30 minutes at room temperature of 20 cm using a 12 W ultraviolet lamp to generate hydroxyl radicals, it contained 0.25% bromophenol blue tracking dye and 50% sucrose. The reaction is terminated by adding loading buffer. It was analyzed by 0.8% submarine agarose gel electrophoresis, the gel was stained with ethidium bromide (EtBr), washed with water and analyzed by UV transilluminator (TEX-20-M, VILBER LOURMAT).

As a result, as shown in Fig. 5, the DNA chain cleavage did not occur by hydrogen peroxide or UV irradiation alone, but the DNA was converted to 90% or more in the unfolded spiral form by treating supercoiled DNA with hydrogen peroxide and ultraviolet rays. It was confirmed that THTEB compounds could reduce the cleavage of DNA chains in a concentration dependent manner. When DNA was treated with 4 μM of THTEB compounds, DNA chain cleavage was protected by at least 50%, indicating that THTEB compounds effectively reduce the oxidative stress caused by hydroxyl radicals, a product of hydrogen peroxide and ultraviolet light (FIG. 5). Reference).

Examples of the pharmaceutical compositions containing the compounds of the present invention will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation example  One. Powder  Produce

THTEB 300 mg

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

THTEB 300 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

THTEB 300 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

THTEB 300 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

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

THTEB 300 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 to the purified water to dissolve, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

THTEB 1000 mg

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

Folate 50 ㎍

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. Manufacture of health drinks

THTEB 300 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B ₁

0.3 g of vitamin B ₂

Water quantification

After mixing the above components in accordance with the conventional healthy beverage manufacturing 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 refrigerated It is used to prepare a healthy beverage composition of the invention.

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.

1 is a diagram showing DPPH free radical scavenging activity against 3,4,5-trihydroxy-N- [2-p-tolylethyl] -benzamide (THTEB) and vitamin C.

2 is a diagram showing peroxide ion scavenging activity against 3,4,5-trihydroxy-N- [2-p-tolylethyl] -benzamide (THTEB) and vitamin C.

3 is a diagram showing the activity of lipid peroxidant against 3,4,5-trihydroxy-N- [2-p-tolylethyl] -benzamide (THTEB) and vitamin C,

4 is a diagram showing the antioxidant activity of 3,4,5-trihydroxy-N- [2-p-tolylethyl] -benzamide (THTEB) and vitamin C by TEAC assay,

FIG. 5 shows the effect of THTEB compounds on DNA chain cleavage induced by hydrogen peroxide and ultraviolet light.

Claims (7)

3,4,5-trihydroxy-N- [2- p- tolylethyl] -benzamide (hereinafter referred to as 'THTEB') compound represented by the following structural formula (I) and a pharmaceutically acceptable salt thereof:

(Ⅰ) A pharmaceutical composition for the prevention and treatment of oxidation-related diseases containing THTEB compounds and salts thereof as active ingredients. The pharmaceutical composition according to claim 2, wherein the oxidation-related disease is aging, cancer, diabetes, hepatitis, gastritis, rheumatoid arthritis, epilepsy, neurodegenerative diseases or arteriosclerosis. The method of claim 2, wherein the THTEB compound is 3,4,5-trimethoxybenzoyl chloride (3,4,5-Trimethoxybenzoyl chloride), 4-Methoxyphenethyl amine (4-Methoxyphenethyl amine), potassium carbonate (potassium carbonate) was mixed in a solution containing distilled water and ethyl acetate in 1-5: 1-15 (H ₂ O / EtOAc 1-5: 1-15), and then the reaction solution was stored at room temperature for 20-40 hours. Then, the resultant is filtered and the material is recrystallized from ethylacetate (ethylacetate) and purified to make a 3,4,5-Trimethoxy-N- [2- (4-methoxyphenyl) ethyl] -benzamide compound; The 3,4,5-Trimethoxy-N- [2- (4-methoxyphenyl) ethyl] -benzamide of step 1 was dissolved in 20 ml of freshly distilled dichloroethane (CH ₂ Cl ₂ ) and tribromoboron in a cool state. (BBr ₃ ) was slowly added to the solution, and the compound was stirred at room temperature for 20 to 40 hours, and then purified water was added and stirred again for 20 to 40 minutes. The resultant was filtered, and the material was recrystallized from dichloromethane. Pharmaceutical composition, characterized in that to obtain a 3,4,5-trihydroxy-N- [2-p-tolylethyl] -benzamide (THTEB) compound through two steps of purification. The pharmaceutical composition of claim 2, wherein the compound comprises 0.1 to 50 wt% of the THTEB compound based on the total weight of the composition. Health functional food for the prevention and improvement of oxidation-related diseases containing THTEB compound and its salt as an active ingredient. 7. The dietary supplement of claim 6 which is a powder, granule, tablet, capsule or beverage.

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