KR20060025693A - Composition comprising betaine as an effective component using as an anti-oxidant, anti-aging or anti-inflammatory agent - Google Patents

Abstract

The present invention relates to an antioxidant, anti-aging or anti-inflammatory pharmaceutical composition comprising a betaine compound isolated from goji berry extract, specifically, the betaine compound of the present invention by removing the active oxygen in the cell or in vivo of the cell It protects -SH group which inhibits oxidative damage, inhibits NF-κB binding, inhibits gene expression related to inflammation or aging regulated by NF-κB, and is involved in inflammatory response as a major cause of aging process By inhibiting the enzymes that produce species, it can be used as an antioxidant, anti-aging or anti-inflammatory medicine and health food.

Betaine, -SH protection, NF-κB, anti-aging, antioxidant, anti-inflammatory, medicines, health food

Description

Composition comprising Betaine as an effective component using as an anti-oxidant, anti-aging or anti-inflammatory agent}             

1 is a diagram showing the protective effect of betaine on cytotoxicity by t-BHP ( tert -butylhydroperoxide), Figure 1a is a graph of vascular endothelial cytotoxicity according to the administration of t-BHP concentration, Figure 1b It is a graph showing the protective effect of vascular endothelial cytotoxicity induced by t-BHP according to the concentration of betaine,

Figure 2 is a graph showing the inhibitory effect (%) of the generation of total reactive oxygen species in the vascular endothelial cells induced by t-BHP, Figure 2a is a graph showing the effect of the administration according to the concentration of betaine, 2b is its fluorescence micrograph,

Figure 3 is a diagram showing the effect of betaine on the cytotoxicity induced by homocysteine, Figure 3a is a graph of vascular endothelial cytotoxicity according to the administration of homocysteine concentration, Figure 3b is a homocysteine according to the administration of the concentration of betaine A graph showing the protective effect of vascular endothelial cytotoxicity induced by

FIG. 4 is a graph showing the scavenging ability of total reactive oxygen species (FIG. 4A), glutathione generating ability (FIG. 4B), and total SH generating ability (FIG. 4C) occurring in kidneys of aged white rats of betaine. FIG.

5 is a diagram showing the inhibitory ability of betaine NF-κB, Figure 5a is a graph showing the inhibitory effect of the level of NF-κB in the nucleus in the kidney of betaine aged white rats, Figure 5b is the aged white rat of betaine Figure 5c is a graph showing the inhibitory effect of NF-κB DNA binding activity in the kidney of, overexpressing the κB site by t-BHP to vascular endothelial cells, whether NF-κB binds a lot or betaine Is a graph showing whether or not

FIG. 6 is a graph showing the mechanism of action of betaine on IκBα / β. FIG. 6A is a graph showing the increase in cytoplasmic IκBα / β in the kidney of betained aged rats. A graph showing a decrease in phospho-IκBα in the kidney,

7 is a graph showing the inhibitory effect of betaine on gene expression regulated by NF-κB in the kidneys of aged white rats,

8 is a diagram illustrating the mechanism of action of NF-κB of betaine, FIG. 8A is a graph illustrating the pathway through which COX-2 expressed by NF-κB is inhibited in vascular endothelial cells, and FIG. 8B is a vessel of betaine It is a graph showing the effect of NF-κB inhibition through IKK / ERK / p38 / JNK in endothelial cells, Figure 8c is a graph showing that betaine inhibits NF-κB in vascular endothelial cells.

The present invention relates to a pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory containing betain as an active ingredient.

Aging is a functional, structural, and biochemical process that occurs continuously from birth to death, and occurs throughout the cells and tissues that make up the human body. It shows metabolic rate, increased disease, and decreased adaptability. This is a phenomenon that leads to the death of the whole body. Theories explaining the aging process and causes are largely genetic (Chung. HY et al., Kor. J. Gerontol . 2 : pp1-11, 1992) and conservative theory (Chung. HY et al., Kor. J. Gerontol). 2 : pp1-11, 1992). The theory of genes is the theory that aging clock theory and pleiotropicity are genes that have genes that have been programmed to determine lifespan from the birth of an organism, and that aging proceeds in a predetermined order by the activity of genes related to aging. Pleiotropic gene theoty, telomere theory, and mutation accumulation theory are typical.

On the other hand, consumption theory explains that constituent cells of living organisms deteriorate with time (wear & teat theory) as if the machine is consumed for a long time, or it is explained by accumulation of harmful substances. Free radical theory that free radicals generated through human metabolic processes, radiation exposure, viruses, heavy metals and air pollution form toxic substances that not only promote aging but also cause various diseases related to aging It is accepted as the most influential theory.

Free radicals collectively refer to a material having an electron that is not paired with the outermost electron orbit, and its structure is very unstable and highly reactive due to its nature of obtaining and stabilizing electrons. In particular, free radicals derived from oxygen are called free radicals, and they react with proteins, lipids, and carbohydrates, causing lipid peroxidation, DNA damage, and oxidation of proteins, resulting in damage to intracellular structures, resulting in cell death. In particular, it is involved in the inflammatory process as a cause of vascular aging and increases homocysteine in arteriosclerosis, Alzheimer's, and blood. Reactive oxygen species such as, for example, superoxide (O ₂ −), hydrogen peroxide (H ₂ O ₂ ), hydroxy radicals (OH) and the like are representative.

Therefore, in order to prevent the accumulation of free radicals and harmful oxidative action which promotes aging, the free radical removal system is operated in the cell. The component containing the -SH group has a reducing power, thereby removing the oxidizing ability of the active oxygen by providing electrons to the active oxygen. In addition, catalase, ham oxygenase, and quinone reductase also play a role in protecting cells from oxidative stress (Chung. HY et al., Kor. J.). Gerontol . 2 : pp1-11, 1992; Chung. HY et al., Kor. J. Gerontol ., 10 : pp46-59, 2000).

Recently, inflammatory reactions and their associated proinflammation processes have been reviewed as major causes of the aging process. In this inflammatory response, not only reactive oxygen species (ROS) but also enzymes that produce active species such as COX-2 (Cyclooxygenase-2), iNOS (indused nitric oxide synthase), XOD (Xanthine oxidase), and NADPH oxidase play an important role. I am in charge. It proposed a molecular inflammation hypothesis on aging to identify new molecular perspectives linking aging mechanisms and related senile diseases at the molecular level (Chung. HY et al., Rev. Clin. Gerontol . 10 : pp207-). 222, 2000)

NF-κB is a redox-sensitive transcript known to respond to various stimuli to treat aging and the complex relationship between NF-κB, inflammatory and oxidative stress is dependent on NF-κB regulation of reactive oxygen species (ROS). It is finely adjusted according to the state of redox. The NF-κB family has been identified in a wide variety of organisms, from Drosophila to mammals, and has been known to play a pivotal role in many responses related to defense through rapid induction of gene expression. In particular, NF-κB regulates the expression of iNOS, COX-2, acute-phase proteins, immunoreceptors, cell adhesion molecules, and various inflammatory cytokines. The activation of NF-κB and its gene expression are associated with a number of pathologies, including aging, cancer, atherosclerosis, radioactive tissue damage, viral replication, acute inflammation, tissue transplant host reactions, and toxic / corrosive tissue damage. It is related to the state.

It is now clear that the inflammatory response is closely related to the aging process and senile disease. It is well known that Alzheimer's disease and vascular disease are improved by administration of anti-inflammatory drugs. NF-κB is a key regulator of inflammatory processes because aging and senile disease are closely related to reactive oxygen species (ROS) and reactive nitrogen species (RNS) reactions that lead to the activation of NF-κB. (Key regulator). The recent research suggests that NF-κB plays a major role in the molecular inflammation hypothesis of aging. Therefore, recently, factors that can regulate the activation of NF-κB are likely to be therapeutic indicators, and many studies using natural and synthetic materials have been conducted. Examples include flavonoids, phenolic, and hydroxycinnamate. (Kim. HJ et al., Free Radic. Biol. Med. , 32 : pp991-1005, 2000; Muraoka. K et al., Transplant Proc. , 34 : pp1335-1340, 2002)

Wolfberry is the mature fruit of Lycium chinense MILL. It contains 3.39 mg% carotene, 0.23 mg% of vitamin B ₁ , 1.7 mg% of nicotinic acid, and ₃ mg% of vitamin C. It contains β-sitosterol, linoleic acid, and beta. It contains betaine. Goji has the effect of lowering blood sugar and cholesterol, and inhibits the sedimentation of light fat in hepatocytes, and promotes the development of hepatocytes. It has the effect of increasing the phospholipids in the serum and liver, and it acts to excite the central and peripheral parasympathetic nerves and lowers blood pressure by inhibiting the heart. Hwanhyeol function has the effect of having a promoting action enhances the phagocytic function of the tonic endothelial system (information boseop and new mingyo, hyangyak Dictionary, Younglim four, pp826-828, 1998)

Betaine, a component of the wolfberry, refers to the amphoteric electrolyte containing quaternary ammonium as a generic term for trialkylamino acids. Trimethylglycine is sometimes called betaine. Gojija contains 6 to 11 mg / g of betaine, and choline is oxidized to betaine aldehyde, which, in turn, becomes betaine, a source of methyl groups with important physiological functions to the human body. Goji betaine is effective in promoting the function of the liver and stomach, preventing atherosclerosis and hypertension, and preventing musculature and anemia.

The present invention relates to betaine, one component of goji berry, and has been reported to have effects such as anti-interstitial action, blood pressure strengthening, and anti-glycemic action.

However, none of these documents teaches or discloses that betaine has the activity of protecting the -SH group, which removes free radicals from cells, and inhibiting the binding of NF-κB, thereby inhibiting the expression level of genes regulated by it. .

Therefore, the present inventors protect the -SH group by inhibiting the oxidative damage of the cell by removing the active oxygen in the cell or in vivo by the betaine compound isolated from the wolfberry, regulated by NF-κB by inhibiting the binding of NF-κB By inhibiting the expression of genes associated with inflammation or aging, and inhibiting enzymes that produce active species involved in the inflammatory response as a major cause of the aging process, by confirming that it can be used as a pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory The present invention has been completed.

The present invention relates to betaine, removes free radicals in cells or in vivo, protects -SH, an antioxidant regulatory system that inhibits oxidative damage of cells, and inhibits binding of NF-κB, which is an indicator of inflammation or aging. Pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory by inhibiting gene expression associated with inflammation or aging regulated by NF-κB and inhibiting enzymes that produce active species involved in the inflammatory response as a major cause of the aging process. The purpose is to provide.

In order to achieve the above object, the present invention provides a pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory containing a betaine compound of formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient that can be isolated or synthesized from the Goji berry extract do.

The pharmaceutical composition removes active oxygen species in cells or in vivo, protects -SH group, which is an antioxidant regulatory system that inhibits oxidative damage of cells, and inhibits the binding of NF-κB to regulate inflammation by NF-κB or By inhibiting the gene expression associated with aging not only due to the aging inhibitory effect, but also as a major cause of the aging process by inhibiting the enzymes that produce active species involved in the inflammatory response to provide a pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory For the purpose of

The betaine compound of Formula 1 of the present invention may be isolated from known synthetic methods or wolfberry extracts well known in the art.

The compounds may be prepared with pharmaceutically acceptable salts and solvates according to methods conventional 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. An equimolar amount of the compound and an acid or alcohol (eg, glycol monomethylether) in water can be heated and then the mixture is 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, hydroiodic acid, and the like.

Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are 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 above betaine compounds include salts of acidic or basic groups which may be present in the betaine compound, 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, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, and methods or processes for preparing salts known in the art It can be prepared through.

Hereinafter, the present invention will be described in detail.

Betaine of the present invention Obtainable from goji berry extract, obtainable by synthetic methods well known in the art or commercially available.

For example, the separation process for obtaining betaine from the extract of goji berry in detail,

After washing and drying the wolfberry, about 1 to 15 times the dry weight, preferably about 10 to 15 times the volume of water, a lower alcohol such as methanol, ethanol or a mixed solvent thereof, preferably about 1: 0.1 to Extraction temperature of 20 to 100 ° C., preferably 50 to 90 ° C. in mixed solvent or water, 50%, 70%, 95% ethanol with a mixing ratio of 1:10, more preferably 1: 0.2 to 1: 3 At about 0.5 hours to 2 days, preferably 1 hour to 1 day by repeated extraction method, such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction, 1 to 5 times, preferably 2 to 3 times After obtaining, the resultant was filtered under reduced pressure and concentrated under reduced pressure, and then the residue was dried with a vacuum freeze dryer to obtain crude alcohol extracts available in lower alcohols such as water, ethanol and methanol or mixed solvents thereof. Poured into this crude extract Through the purification process using the conventional process and the column fractions in the art it is possible to obtain the betaine compound of the present invention.

The betaine compounds of the present invention can also be synthesized through conventional synthesis and fractionation of substituents (Herbert O. House: Modern Synthetic Reactions , 2nd Ed ., The Benjamin / Cummings Publishing Co., 1972).

The present invention provides a pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory containing a betaine compound isolated from the Goji berry extract obtained by the above method as an active ingredient.

In order to determine the efficacy of the antioxidant, anti-inflammatory, and anti-aging of the betaine compound isolated from the Gojija extract obtained by the above method, the effect of inhibiting the cytotoxicity induced by t-BHP as a result of the present invention When betaine was administered, it was confirmed that the inhibitory effect of cytotoxicity was excellent, and the ability of betaine to enhance the -SH group, which is an antioxidant and antioxidant regulation system in cells and in vivo, was observed not only in cells but also in vivo. It was confirmed that the ability to enhance the SH group was excellent, and NF-κB inhibitory activity of betaine was observed, and it was confirmed that the ability to inhibit NF-κB that regulates aging-related genes was the main cause of the aging process. As a result, the antioxidant, anti-aging and anti-inflammatory effects of betaine were confirmed by inhibiting enzymes that produce active species involved in the inflammatory response.

The pharmaceutical composition containing the betaine compound of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.

Pharmaceutical compositions comprising the compounds of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

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.

The pharmaceutical compositions comprising the compounds of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be used. Carriers, excipients and diluents that may be included in the composition comprising the compound 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 in the compound. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. 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 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 is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 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 compounds of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like 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 dietary supplement comprising the betaine and a food acceptable food supplement additive exhibiting an antioxidant, anti-aging or anti-inflammatory effect. Examples of the food to which the 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 antioxidant, anti-aging or anti-inflammatory effects. At this time, the amount of the 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 may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.

Food supplement additives as defined herein include food additives customary in the art, such as flavorings, flavors, 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 compound as an essential ingredient in the indicated ratios, 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 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 said 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 usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the 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 only illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples.

Example 1 Preparation of Experiment

1-1. reagent

Betaine, t-BHP ( tert- butylhydroperoxide) was purchased from Sigma Chemical Co., B-2633-St. Louis, MO, USA.

DCFDA (2 ′, 7′-dichlorodihydrofluorescein diacetate) was purchased from Molecular probes (Eugene, OR, USA). Sense assay reagent (USA) was purchased from Amersham life science and NF-κB antibody was purchased from Santa Cruz Biotechnology-Santa Cruz CA.USA.

1-2. Experiment apparatus

Microplate fluorescence Genious (Tecan, Austria), a fluorescence microscope (Axiovert 100, Zeiss Co., Germany), and Western Blot System-3 kit (Mini Protean 3 Electrophoresis, Bio Rad) were used.

1-3. Preparation of Experimental Animals

Fischer 344 male rats of around 400 g, 6 months and 24 months of age, were purchased from Samtaco (Osan, Korea), and adapted to the experimental environment for 1 week with sufficient feed and water. It was.

Experimental Example 1. t-BHP of betaine ( tert -butylhydroperoxide) Inhibitory Effects of Cytotoxicity

1-1. Cytotoxic Effects of t-BHP

In order to test the cytotoxicity induced in vascular endothelial cells by t-BHP treatment of the present invention, after culturing vascular endothelial cells in a 96-well plate at 1.5 × 10 ⁴ cells / ml and growing for one day t-BHP After treatment with 5, 10, 20, 30, 40, 60 μM for 30 minutes, the cytotoxic effect was observed using a microculture tetrazolium assay (USA).

As a result of performing the experiment, t-BHP concentration-dependently of the present invention as shown in Figure 1a It was confirmed that the cytotoxicity caused by.

1-2. Cytoprotective effect of betaine on toxic effects of t-BHP

T-BHP of the present invention To test whether the betaine cells of the present invention are protected from cytotoxicity induced by vascular endothelial cells by treatment, the vascular endothelial cells were incubated at 1.5 × 10 ⁴ cells / ml in 96-well plates and grown for one day before betaine. Was pretreated to 50, 200 μM for 1 hour and then 15 μM of t-BHP Was treated for 6 hours. Thereafter, the cytoprotective effect of betaine was observed using a microculture tetrazolium assay.

As a result of the experiment, as shown in Figure 1b it can be seen that the number of cells protected betaine concentration-dependent of the present invention increases.

Experimental Example 2 Measurement of Intracellular Antioxidant Capacity of Betaine

2-1. Intracellular Reactive Oxygen Species Removal of Betaine

In order to examine the ability of betaine of the present invention to inhibit the production of free radicals induced in vascular endothelial cells by t-BHP treatment, vascular endothelial cells were grown in 96-well plates at 1.5 × 10 ⁴ cells / ml and grown for one day. After 1 hour treatment with 10, 50, 100 and 200 μM of betaine, and then treated with 15 μM of t-BHP for 30 minutes, and then observe the change in fluorescence by adding DCFDA at excitation wavelength 485 nm and emission wavelength 535 nm. It was measured with a microplate fluorescent reader at 5 minute intervals for 30 minutes.

As a result of the experiment, as shown in FIG. 2A, free radical generation in the cells treated with t-BHP was increased as compared to cells not treated with t-BHP, and in the group treated with betaine, free radical production was It was confirmed that the concentration of betaine is inhibited.

2-2. Fluorescence Microscopy Measurement of Betaine's Reactive Oxygen Scavenging Ability

In order to examine the ability of betaine of the present invention to inhibit the production of free radicals induced in vascular endothelial cells by t-BHP treatment, the endothelial cells were cultured in 6 well plates at 20 × 10 ⁴ cells / ml, and then grown for one day. After pretreatment with 32, 75, 150 μM of betaine for 1 hour, 15 μM of t-BHP for 30 minutes, DCFDA was added, and the change in fluorescence was measured by fluorescence microscope.

As a result of performing the experiment, as shown in FIG. 2B (A), free radical generation was increased in cells treated with t-BHP as in FIG. 2B (B) compared to cells not treated with t-BHP, and FIG. 2B (C). As shown in E) it was confirmed that free radicals were inhibited by betaine concentration dependently.

Experimental Example 3. Measurement of the ability to enhance -SH group, which is an intracellular antioxidant regulator of betaine

3-1. Intracellular -SH Phase Measurement by Homocysteine

To test the ability of the -SH phase in vascular endothelial cells to be treated by homocysteine, vascular endothelial cells were cultured at 1.5 × 10 ⁴ cells / ml in 96-well plates and grown for one day, 20, 50, 100, 200 μM. In order to observe the change in fluorescence by adding FM (Fluorescein-5-Maleimide, USA) after treating homocysteine, the microplate fluorescence reader was measured at an interval of 2 minutes at an excitation wavelength of 485 nm and an emission wavelength of 535 nm for 10 minutes.

As a result of performing the experiment, as shown in FIG. 3a, it was confirmed that the -SH group production of the cells treated with homocysteine was reduced in a concentration-dependent manner compared to the cells not treated with the homocysteine.

3-2. Measurement of Intracellular -SH Phase Protective Effect of Betaine Reduced by Homocysteine

In order to test the protective ability of the -SH group in vascular endothelial cells by homocysteine treatment, betaine of the present invention was incubated at 1.5 × 10 ⁴ cells / ml in 96 well plates, and then grown for 15 days, 15, 75 , 150, 250μM of betaine was pretreated for 1 hour, 200μM of homocysteine, and then FM was added to change the fluorescence was measured by a microplate fluorescence reader at an excitation wavelength of 485 nm and emission wavelength of 535 nm for 2 minutes at 10 minutes intervals. .

As a result of the experiment, it was confirmed that -SH group production of betaine-treated cells was increased in a betaine concentration-dependent manner as compared to the betaine-treated cells of the present invention as shown in Figure 3b.

Experimental Example 4 Measurement of Betaine's Antioxidant Activity and Ability to Enhance -SH Group, an Antioxidant Regulator

4-1. In vivo antioxidant activity of betaine

In order to examine whether the betaine of the present invention has an antioxidant activity in vivo, 24 months old aged white rats prepared in Examples 1-3 were mixed orally with beta at a weight of 2, 4, 8 mg / kg, and fed orally. After dissection, the kidneys were excised and crushed to measure the change in fluorescence using DCFDA, and measured with a microplate fluorescence reader at an excitation wavelength of 484 nm and an emission wavelength of 535 nm for 5 minutes at 30 minutes intervals.

As a result of performing the experiment, the control group (Young) of young white rats of 6 months old, the control group of aged white rats (Old) of 24 months old, the rest of the betaine 2, 4 in 24 months old white rats , 8 mg / kg / day. In the aged white rats, free radical production was increased in comparison with the young white rats, and the increase in free radicals in the aged white rats was found to decrease in a concentration-dependent manner when the betaine of the present invention was administered.

4-2. Determination of -SH Phase Protective Activity, an In Vivo Antioxidant Regulator of Betaine

In order to examine whether the betaine of the present invention protects -SH, an antioxidant regulator in vivo, 24-month-old aging white rats prepared in Examples 1-3 were fed with betaine at 2, 4 and 8 mg / kg weight. After 10 days of mixing and oral administration, it was dissected, dissected, and broken down to measure changes in fluorescence using DCFDA, and measured once with a microplate fluorescence reader at an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

As a result of performing the experiment, as shown in Figure 4b, the control group (Young) of young white rats of 6 months old, the control group of aged white mice (Old) of 24 months old, the rest of the betaine in aged white mice of 24 months It is administered in amounts of 2, 4 and 8 mg / kg / day each. Aged white rats were found to have reduced glutathione production ability compared to young white rats, and the decrease in glutathione production ability of aged white rats was increased in a concentration-dependent manner of betaine. In Figure 4c, the total -SH group was measured as a measure of the total -SH group was reduced compared to the young white rats total -SH group production capacity, the decrease in the total -SH group production capacity of the aged white rats of the betaine of the present invention It was confirmed that the increase in concentration dependent.

Experimental Example 5 Measurement of NF-κB Inhibitory Activity of Betaine

5-1. Measurement of NF-κB Inhibitory Effect of Betaine

To test whether betaine of the present invention inhibits NF-κB, 24 months old aged rats of Example 1-3 were orally administered at 2, 4, 8 mg / kg / day, and dissected 10 days later. Was used by ablation and crushing. The crushed kidney nuclear protein was mixed with bromophenol blue dye in the same amount and electrophoresed on 10% SDS-PAGE (SDS-polyscrylamide gel). After electrophoresis, the protein was transferred to the polyvinyridine fluoride membrane, and the TBS-Twin (Tris Buffered Saline-Tween) solution containing 5% skim milk (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween 20, pH 7.5). ) To block nonspecific reactions. Then, the antibodies against p50 and p65 (Santacruz, USA) were reacted with a solution diluted to 1/500 for 3 hours at room temperature, and then reacted with an anti-rabbit IgG antibody as a secondary antibody. After completion of the reaction, the membrane was washed four times with TBS-twin solution, reacted with an ECL (enhanced chemiluminescence, USA) detection reagent for 30 minutes, and then exposed to an X-ray film at room temperature.

As a result of the experiment, as shown in FIG. 5A, the gene expression levels of p50 and p65 in aged white rats (Old) of 24 months old were increased as compared with 6 months old young white rats (Young). Compared with the 24-month-old aging rats, the betaine fed group of the present invention was able to confirm the effect of decreasing the gene expression of p50, p65.

5-2. Determination of NF-κB Binding Inhibition of Betaine

In order to test whether betaine of the present invention inhibits NF-κB in vivo, it was labeled with ³² P isotope after separation using centrifugation, and subjected to 7% SDS-PAGE in aged white rats of Example 1-3 in Example 1-3. Betaine was orally administered at 2, 4, 8 mg / kg / day, dissected 10 days later, and resected, and nuclear proteins were separated and measured using an electrophoretic mobility shift assay (EMSA). After electrophoresis of the nuclear protein of the kidney, the gel was photosensitive to the X-ray film at room temperature.

As a result of the above experiment, as shown in FIG. 5B, NF-κB is produced more in the case of aged white rats than in young white rats compared with young white rats of 6 months and 24 months of age. In the same aged white rats, it was confirmed that NF-κB was inhibited in the betaine-administered group of the present invention.

5-3. Measurement of Intracellular NF-κB Inhibitory Activity of Betaine

In order to test whether betaine of the present invention inhibits NF-κB in the cells, plasmids having four κB sites are inserted into vascular endothelial cells using a reagent called FuGENE 6 (USA) for overexpression. T-BHP was stimulated to determine the extent to which NF-κB binds to the κB site and whether betaine inhibits the binding.

As a result of the experiment, as shown in FIG. 5C, the overexpressed cells (T-CON) increased more than the cells without overexpressing NF-κB activity (CON), and the group treated with t-BHP was about the same. Although significantly increased, it was confirmed that NF-κB activity was inhibited in a concentration-dependent manner when treated with betaine of the present invention.

5-4. Measurement of the mechanism of action of IκBα / β by betaine

This experiment was carried out to test the mechanism of action of IκBα / β of betaine. As senescence ages, IκBα / β is separated by phosphopolylation when NF-κB is activated, and the amount of IκBα / β is reduced in the cytoplasm. To determine the protein level of IκBα / β, 24 months old aged rats of Example 1-3 were orally administered with betaine at 2, 4, and 8 mg / kg / day after 10 days to dissect and disrupt the kidney. Was used. Renal cytoplasmic protein lysate was mixed with bromophenol blue staining solution and electrophoresed on 10% SDS-PAGE. After electrophoresis, the protein was transferred to the polyvinylidene fluoride membrane, and the nonspecific reaction was performed by immersing in TBS-Twin solution (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween 20, pH 7.5) containing 5% skim milk. Blocked. Thereafter, the antibody against IκBα / β (Santacruz, USA) was reacted with a solution diluted to 1/500 for 3 hours at room temperature, and then reacted with an anti-mouse IgG antibody as a secondary antibody. After completion of the reaction, the membrane was washed four times with TBS-Twin solution and ECL (enhanced chemiluminescence, USA) After reacting for 30 minutes with the detection reagent, the X-ray film was exposed to light at room temperature.

As a result of the experiment, as shown in FIG. 6A, the amount of protein of IκBα / β in aged white rats (Old) of 24 months of age was decreased compared to 6 months of young white rats. Compared with the aged white rats of the betaine fed group of the present invention was confirmed that the increase in the protein amount of IκBα / β. Conversely, in FIG. 6B, the protein amount of the p-IκBα / β protein increased in aged white rats (Old) at 24 months of age, compared to 6 months of young white rats. Compared with the white rat aged months, the betaine-treated group of the present invention was found to reduce the amount of p-IκBα / β protein.

5-5. Determination of Gene Expression Inhibition Regulated by NF-κB of Betaine

In order to test whether betaine of the present invention inhibits NF-κB in vivo, oral administration of betaine 2, 4, 8 mg / kg / day to 24 months old aging rats was dissected 10 days after dissection and disintegration Used. Kidney crushed solution was mixed with bromophenol blue dye in the same amount and electrophoresed on SDS-PAGE. After electrophoresis, the protein is transferred to the polyvinylidene fluoride membrane, and 0.5% skim milk is included. The non-specific reaction was blocked by soaking in TBS-Tween solution (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween 20, pH 7.5). Since the solution of goat antibodies (Upstate, USA) to COX-2 diluted 1/1000, the rabbit antibody (Upstate, USA) to iNOS diluted 1/10000, VCAM-1, ICAM-1 The goat antibody (Santacruz, USA) to the reaction diluted to 1/500 for 3 hours at room temperature, and then reacted with anti- goat, rabbit IgG antibody as a secondary antibody. After completion of the reaction, the membrane was washed four times with TBS-Twin solution, reacted with ECL detection reagent for 30 minutes, and then exposed to X-ray film at room temperature.

As a result of the experiment, as shown in Figure 7, the genes of COX-2, iNOS, VCAM-1, ICAM-1 of aged white rats (Old) of 24 months old compared to the young white rats (Young) of 6 months old The expression level was increased, and compared with the same aged 24 months old white rats, the betaine group of the present invention was found to reduce the gene expression of COX-2, iNOS, VCAM-1, ICAM-1.

5-6. Measurement of the mechanism of action of NF-κB by betaine

In order to test the mechanism of NF-κB action of betaine of the present invention, it was examined using vascular endothelial cells. First, the amount of COX-2 expressed by NF-κB after pretreatment with betaine, NF-κB, p38, JNK, and ERK inhibitors for 1 hour and t-BHP for 30 minutes in vascular endothelial cells. Learned.

As a result of the experiment was confirmed that it is inhibited by each inhibitor as shown in Figure 8a.

Secondly, on the basis of the results of FIG. 8A, p-JNK, p-p38, pERK, and p-IKK were confirmed by pretreatment of betaine to vascular endothelial cells for 1 hour and t-BHP for 30 minutes.

As a result, as shown in Figure 8b, it was confirmed that significantly reduced by betaine.

Third, as a result of confirming p50 and p65 by pretreatment with betaine for 1 hour and t-BHP for 30 minutes on vascular endothelial cells, as shown in FIG. 8C, p50 and p65 were inhibited by the betaine of the present invention. there was.

Examples of the formulation of a pharmaceutical composition comprising the compound of the present invention will be described, but the present invention is not intended to be limited thereto but merely to be described in detail.

Formulation Example 1 Preparation of Powder

Betaine Compound 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

Betaine 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

Betaine Compound 50 mg

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

Betaine 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

Betaine Compound 100 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

Betaine Compound 1000 mg

Vitamin Mixture

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 may be mixed according to a conventional health food manufacturing method. Next, 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

Betaine Compound 1000 mg

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 betaine compound of the present invention removes active oxygen species in cells or in vivo, protects -SH group, which is an antioxidant regulation system that inhibits oxidative damage of cells, and inhibits binding of NF-κB. Pharmaceutical composition and health for antioxidant, anti-aging or anti-inflammatory by inhibiting gene expression associated with inflammation or aging regulated by NF-κB and inhibiting enzymes that produce active species involved in the inflammatory response as a major cause of the aging process It can be usefully used as a functional food.

Claims (5)

A pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory, containing a betaine compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. (Formula 1)

The pharmaceutical composition according to claim 1, wherein the anti-aging pharmaceutical composition is due to inhibiting reactive oxygen species or inhibiting NF-κB, which is an aging indicator. The pharmaceutical composition of claim 1, wherein the anti-inflammatory pharmaceutical composition is a major cause of the aging process due to inhibition of enzymes that produce active species involved in the inflammatory response. A dietary supplement containing the betaine compound of claim 1 and a food supplement acceptable food additive. The health functional food of Claim 4 which is a health drink.

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