KR20060033071A - Composition comprising ferulic acid as an effective component using as an anti-oxidant, anti-aging or anti-inflammatory agent - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory with ferulic acid as an active ingredient, the composition of the present invention removes free radical species and protects cells from oxidative damage, aging Since it has an excellent inhibitory effect on gene expression regulated by NF-κB and NF-κB, which are known as indicators, it can be used as an effective antioxidant, anti-aging or anti-inflammatory drug and health functional food.

Antioxidant, anti-aging, anti-inflammatory, free radicals, NF-κB, ferulic acid

Description

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

1 is a diagram showing the protective effect of ferulic acid 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 Is a graph showing the protective effect of vascular endothelial cytotoxicity induced by t-BHP according to the concentration of ferulic acid,

Figure 2 is a diagram showing the effect of inhibiting the generation of total reactive oxygen species in vascular endothelial cells induced by ferulic acid t-BHP, Figure 2a shows the total active oxygen species of the cell according to the administration of the concentration of ferulic acid It is a graph showing the production inhibitory effect, Figure 2b is a fluorescence microscope picture thereof,

3 is a graph illustrating whether ferulic acid inhibits NF-κB binding by overexpressing κB sites by vascular endothelial cells by t-BHP.

FIG. 4 is a graph of the scavenging activity of total reactive oxygen species (FIG. 4A) and the inhibition of lipid peroxidation (FIG. 4B) in the kidneys of aged white strains of ferulic acid,

5 is a graph showing the total SH production capacity (Fig. 5a) and glutathione inhibition of ferulic acid in the kidney of aged white mice (Fig. 5b),

6 is a graph showing the inhibitory effect of NF-κB levels in the nucleus of ferulic acid in aged kidney rats,

FIG. 7 is a graph showing decreased IκBα / β in the cytoplasm and increased phospho-IκBα in the kidneys of aged white rats of ferulic acid.

8 is a graph showing the inhibitory effect of NF-κB DNA binding activity in the kidneys of aged white rats of ferulic acid,

9 is a graph showing the inhibitory effect on NF-κB-regulated gene expression in ferulic acid aged white rat kidney,

Figure 10 is a diagram of the mechanism of action of NF-κB of ferulic acid, Figure 10a is a graph showing the pathway through which COX-2 is regulated by NF-κB in vascular endothelial cells, Figure 10b is a vascular endothelial It is a graph showing NF-κB inhibitory effect and ERK inhibitory effect through IKK / ERK / p38 / JNK in cells,

11 is a graph illustrating the effect of NF-κB inhibition of ferulic acid in vascular endothelial cells.

The present invention relates to an antioxidant, anti-aging or anti-inflammatory composition containing ferulic acid 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 consumption theory (Chung. HY et al., Kor. J. Gerontol). 2 : pp 1-11, 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. like gene set (pleiotropic gene theo r y), telomere theory (telomere theory), mutation accumulation set (mutation accumulation theory) it is typical.

On the other hand, consumption theory explains that constituent cells of an organism deteriorate with time (wear & tear 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 by human metabolism, radiation exposure, viruses, heavy metals and air pollution form toxic substances that not only promote aging but also cause various diseases related to aging This is 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.

Oxygen-related toxic substances in humans are called reactive oxygen species (ROS). These types of ROS include superoxide, hydroxyl, peroxyl, alkoxyl, Free radicals such as hydroperoxyl and hydrogen peroxide, hypochlorous acid, ozone, singlet oxygen, and peroxynit There are non free radicals, such as peroxinitrite. The most studied and important role of oxygen toxicity is superoxide free radical (active oxygen or harmful oxygen) is known (Fridovich L., Science , 201 : pp175-180,1978).

In particular, in vivo in the old trick oxide (NO: Nitric Oxide) and superoxide (O ₂ ^-: superoxide) ^- non-free radical peroxy nitrite (Peroxynitrite, ONOO ^-) by radical reaction is a radical from the conditions that occur at the same time is easy to produce do. Peroxynitrite has a half-life in milliseconds (milliseconds) compared to other radicals for a long time and its reactivity is also very high, which is the biggest cause of poisoning derived from active nitrogen species. The toxic effects of peroxynitrite have been reported to be associated with various diseases and age-related diseases, including Alzheimer's, cancer, arthritis, arteriosclerosis, and skin inflammation (Ray G, Husain SA, Indian J. Exp. Biol. , Nov; 40 (11) : pp 1213-1232, 2002; Oliveira GV et al., Shock., Sep; 22 (3) : pp278-282, 2004; Lancel S et al., J. Am. Coll. Cardiol., 16; 43 (12) : pp2348-2358, 2004).

The resulting peroxynitrite is sufficient time for its half-life to pass through the cell in the millisecond range, reacting with intracellular targets, causing oxidation, nitration and disrupting cellular signaling. Peroxy nitrite is nitric oxide (NO) and superoxide (O ₂ ^-), and more toxicity is known to be more resistant, lipid, protein, and through oxidation and nitration process of DNA vasodilator of smooth muscle cells, platelet aggregation It is involved in the inhibition and modification of amino acids and cytotoxicity by induction of lipid peroxidation. It is reported that such peroxynitrite plays an important role in the aging process as well as cancer, arthritis, and arteriosclerosis (Ray G, Husain SA, Indian J. Exp. Biol. , Nov; 40 (11) : pp1213- 1232, 2002; Oliveira GV et al., Shock., Sep; 22 (3) : pp278-282, 2004; Lancel S. et al., J. Am. Coll. Cardiol., 16; 43 (12) : pp2348 -2358, 2004)

Various efforts have been made to prevent oxidative damage caused by peroxynitrite, and many antioxidants and synthetic oxidants derived from natural products have been developed and used in medicine and food fields. In particular, since there is no enzyme for removing peroxynitrite in the living body, studies to prevent toxicity against peroxynitrite using natural products have been studied by several researchers, and flavonoids and vitamin E have been reported. (Stevens JF et al., Chem. Res. Toxicol., 16 (10) : pp 1277-1286, 2003; Beharka AA et al., Free Radic. Biol. Med., 15; 32 (6) : pp503-511, 2002).

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 has 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, and COX (cyclooxygenase), an enzyme involved in synthesizing prostaglandins from arachidonic acid, are the main targets in blocking inflammation.

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 is inactivated by binding to I-κB, but it 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).

Ferulic acid is named after extraction from the resin of plants in 1866 (H. Hlasiwetz, et al., Ann., 138 , p61, 1866) and has a mild antioxidant activity against various active oxygen species derived from oxygen molecules, It has been reported to exhibit potent antioxidant effects on oxidative transition metals such as iron and copper ions (MG Smart, et al., Aust. J. Plant Physiol. 6 , p485, 1979). It has also been reported to be used to prevent the automatic oxidation of natural oils such as linseed oil. However, since ferulic acid has only a simple antioxidant function, its main use is only as an additive to prevent oxidation of other active ingredients contained in the product by transition metals or other introduced active acid species (T. Tsukiya, et. al., Jpn. Kokai, 75 , p18621, 1975).

However, in all of the above documents, ferulic acid removes peroxitrite, which is harmful to the human body, and regulates the mechanism of action of NF-κB, which is known as an indicator of aging or inflammation. COX-2), inducible nitric oxide (inducible nitric oxide (iNOS), etc. It is not taught or disclosed that there is activity to inhibit the expression.

Therefore, the present inventors studied to find a new compound that can inhibit the damage caused by reactive oxygen species and the aging index NF-κB. As a result, ferulic acid removes free radicals in cells or in vivo to inhibit oxidative damage of cells. -An enzyme that protects the SH phase, inhibits the binding of NF-κB, inhibits gene expression associated with inflammation or aging regulated by NF-κB, and produces reactive oxygen species that are involved in the inflammatory response as a major cause of the aging process. The present invention was completed by confirming that it can be used as a pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory by inhibiting them.

The present invention relates to a composition containing ferulic acid, and more particularly, to remove the active oxygen species in the cell or in vivo, to protect the -SH group which is an antioxidant regulating system that inhibits oxidative damage of cells, and to prevent inflammation or aging. It inhibits the binding of NF-κB, which is an indicator, inhibits gene expression associated with inflammation or aging regulated by NF-κB, and by inhibiting enzymes that produce active species involved in the inflammatory response as a major cause of aging and antioxidation, It is an object to provide a pharmaceutical composition for aging or anti-inflammatory.

In order to achieve the above object, the present invention provides a pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory containing ferulic acid (ferulic acid) represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

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 It is not only due to aging inhibitory effects by inhibiting gene expression associated with aging, but also as an important cause of the aging process, it exhibits antioxidant, anti-aging or anti-inflammatory activity by inhibiting enzymes that produce active species involved in inflammatory reactions. It is done.

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. 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 above ferulic acid include salts of acidic or basic groups which may be present in ferulic acid, 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.

Ferulic acid of the present invention It can be obtained or commercially available by synthetic methods well known in the art, and can also be synthesized through the synthesis and fractionation of conventional 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 the ferulic acid as an active ingredient.

In order to examine the efficacy of the antioxidant, anti-inflammatory, and anti-aging of the composition containing the ferulic acid of the present invention, the effect of inhibiting the cytotoxicity caused by t-BHP was observed when the ferulic acid of the present invention was administered It was confirmed that the inhibitory effect of cytotoxicity was excellent, and the ability of ferulic acid to enhance -SH group, which is an anti-oxidant and antioxidant regulator in cells and in vivo, was found to enhance -SH group not only in cells but also in vivo. As a result of observing the aging marker NF-κB inhibitory ability, it was confirmed that the ability to inhibit NF-κB that regulates aging-related genes, the activity that is involved in the inflammatory response as a major cause of the aging process By inhibiting the enzymes that produce species, the antioxidant, anti-aging and anti-inflammatory effects of ferulic acid were confirmed.

The pharmaceutical composition containing ferulic acid 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 health functional food comprising the ferulic acid and a food acceptable food supplement additive exhibiting an antioxidant, anti-aging or anti-inflammatory effect. The health functional food of the present invention includes the form of tablets, capsules, pills or liquids, and the food to which the compound of the present invention can be added, for example, various foods, beverages, gums, teas, vitamin complexes, etc. 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 Cytotoxic Inhibitory Effect of Ferulic Acid by t-BHP

1-1. reagent

Ferulic acid (ferulic acid) and t-BHP ( tert- butylhydroperoxide) were purchased from Sigma Chemical Co., B-2633-St. Louis, MO, USA. DCFDA (2 ', 7'-dichlorodihydrofluorescein diacetate) was purchased from Molecular probes (Eugene, OR, USA) and polyvinyridine fluoride membranes (USA) were Milfoasa (USA), Chemilumino 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

Tecan's microplate fluorescence Genious (Tecan, Austria) and fluorescence microscope (Axiovert 100, Zeiss Co., Germany) 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. Inhibitory effect of ferulic acid on t-BHP

1-1. Determination of scavenging effect on peroxynitrite

To determine whether the ferulic acid of the present invention has an scavenging action against peroxynitrite, Kooy et al. (Kooy, NW et al. Free. Rad. Biol. Med., 16 (2) , pp149-156) By measuring the oxidation of dihydrodamine123. Fluorescence intensity of oxidized dihydrodamine 123 was determined by adding a sample and peroxynitrite solution to a buffer consisting of 90 mM sodium chloride, 50 mM sodium phosphate, 5 mM calcium chloride (pH 7.4) and 100 mM diethylenetriamipentaacetic acid. It was measured in a microplate fluorescence reader having a wavelength of 480 nm and an emission wavelength of 535 nm. At this time, IC ₅₀ is shown in Table 1 as the amount of ferulic acid (μM) required to reduce the fluorescence value added only perrosinite by 50%.

Peroxynitrite Scavenging Power by Ferulic Acid compound ONOO ^- Elimination Force IC ₅₀ (μM) Ferulic acid 2.44 ± 0.33 Penicillamine 2.38 ± 0.72

1-2. Determination of scavenging effect on free radicals

After the addition of non-fluorescent 2 ', 7'-DCFDA (2', 7'-dichlorodihydrofluorescein diacetate) to the kidney lysate, the change in fluorescence with the addition of ferulic acid was measured. This is because the non-fluorescent substance DCFDA is oxidized to the fluorescent substance DCF by the active oxygen generated by itself in the kidney crushed liquid. It was measured with a microplate fluorescence reader for 30 minutes at an excitation wavelength of 484 nm and an emission wavelength of 535 nm at 5 minute intervals. At this time, IC ₅₀ is the amount of ferulic acid (μM) required to reduce the fluorescence value added only ferulic acid to the kidney crushing liquid by 50%, and the scavenging power of active oxygen and hydroxy radical by ferulic acid are shown in the following table. 2 and Table 3.

Free radical scavenging ability by ferulic acid compound ROS scavenging power IC ₅₀ (μM) Ferulic acid 9.46 ± 0.41 Trolox 10.23 ± 0.15

Scavenging power of hydroxy radicals by ferulic acid compound Hydroxy Radical Scavenging IC ₅₀ (μM) Ferulic acid 6.38 ± 0.68 Trolox 5.98 ± 0.33

1-3. Cytotoxic Effects of t-BHP

In order to examine the cytotoxicity induced in vascular endothelial cells by t-BHP treatment of the present invention, after culturing vascular endothelial cells at 1.5x10 ⁴ / ml in 96-well plate and growing for one day, t-BHP 5, 10 After 20, 30, 40, 60 μM treatment for 30 minutes, the cytotoxic effect was observed using a microculture tetrazolium assay (USA).

As a result, as shown in Figure 1a it was confirmed that the cytotoxicity caused by t-BHP decreases depending on the concentration of ferulic acid of the present invention.

1-4. Cytoprotective effect of ferulic acid on the toxic effects of t-BHP

In order to examine the cytoprotective effect of the ferulic acid of the present invention from cytotoxicity induced in vascular endothelial cells by t-BHP treatment of the present invention, the vascular endothelial cells were cultured in 96-well plates at 1.5x10 ⁴ / ml After growing for one day, ferulic acid was pretreated with 50 and 200 μM for 1 hour, followed by 15 μM of t-BHP for 6 hours. Thereafter, the cytoprotective effect of ferulic acid was observed using a microculture tetrazolium assay.

As a result, as shown in Figure 1b it was confirmed that the number of cells protected in concentration dependent on the ferulic acid treatment of the present invention.

Experimental Example 2 Measurement of Intracellular Antioxidant Capacity of Ferulic Acid

2-1. Determination of Intracellular Reactive Oxygen Species of Ferulic Acid

In order to examine the ability of ferulic acid of the present invention to inhibit the production of free radicals induced in vascular endothelial cells by t-BHP treatment, after culturing vascular endothelial cells at 96 × well plate at 1.5 × 10 ⁴ / ml and growing for 5 days 1 hour treatment with 10, 50, 100, 200 μM ferulic acid followed by 15 μM t-BHP for 30 minutes, followed by addition of DCFDA to observe the change in fluorescence, excitation wavelength 485 nm and emission wavelength 535 nm It was measured with a microplate fluorescent reader at 5 minute intervals for 30 minutes at.

As a result, as shown in FIG. 2A, free radical generation was increased in cells treated with t-BHP compared to cells not treated with t-BHP, and in the group treated with ferulic acid of the present invention, the production of free radicals was determined by ferulic acid. It was confirmed that the concentration was inhibited depending on the treatment.

2-2. Fluorescence Microscopy Measurement of Reactive Oxygen Scavenging Capacity of Ferulic Acid

In order to examine the ability of the ferulic acid of the present invention to inhibit the production of free radicals induced in vascular endothelial cells by t-BHP treatment, vascular endothelial cells were cultured in 6 well plates at 20 × 10 ⁴ / ml and grown for one day, followed by 200 μM. After pretreatment with ferulic acid for 1 hour, 15 μM of t-BHP was treated for 30 minutes, and the change of fluorescence was measured by fluorescence microscopy by adding DCFDA.

As a result, as shown in FIG. 2B, free radical generation was increased in cells treated with t-BHP compared to cells not treated with t-BHP, and it was confirmed that free radical production was inhibited by the addition of ferulic acid.

Experimental Example 3. Measurement of ferulic acid's ability to enhance -SH group, an in vivo antioxidant and antioxidant regulator

3-1. In Vivo Antioxidant Activity of Ferulic Acid

In order to examine whether the ferulic acid of the present invention has antioxidant capacity in vivo, 24 months old aged rats were orally administered with ferulic acid at 2, 4 mg / kg weight, and then dissected 10 days later. The kidneys were excised and crushed, and the change in fluorescence was measured with a microfluorescence reader at 5 minute intervals for 30 minutes at an excitation wavelength of 484 nm and an emission wavelength of 535 nm using DCFDA. In FIG. 4A, 6 months old young rats (Young), 24 months old white rats (Old), and 24 months old aged rats were ferulic acid 2, 4 mg / kg / day, respectively. It is administered in the amount of.

As a result, the generation of free radicals was increased in the aged white rats compared to the young white rats, it was confirmed that the increase in free radicals of the aged white rats concentration-dependently reduced according to the administration of the ferulic acid of the present invention.

3-2. In Vivo Antilipid Peroxidation Activity of Ferulic Acid

In order to examine whether the ferulic acid of the present invention has antioxidant capacity in vivo, 24 months old aged rats were orally administered with ferulic acid at 2, 4 mg / kg weight, and then dissected 10 days later. Kidneys were excised and crushed to determine the degree of lipid peroxidation. The degree of lipid peroxidation was determined by measuring malondialdehyde production using thiobarbituric acid (TBA). Specifically, 0.5 ml of the reaction solution (1.2% TBA solution: 8.1% SDS solution: 20% acetic acid = 20: 4: 30) was added to the kidney crushed fraction, and heated at 94 ° C for 30 minutes. After cooling, n-butanol was added and centrifuged at 2,000 rpm for 10 minutes, and then the supernatant was measured for fluorescence at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. In FIG. 4b, 6 months old young rats (Young), 24 months old white rats (Old), the rest 24 months old aged rats 2, 4 mg / kg / day It is administered in the amount of.

As a result, it was confirmed that the production of lipid peroxide was increased in the aged white rats compared to the young white rats, and the increase in lipid peroxidation in the aged white rats was reduced in a concentration-dependent manner according to the administration of ferulic acid of the present invention.

3-3. Determination of -SH Phase Protective Capacity, an In Vivo Antioxidant Regulator of Ferulic Acid

In order to examine whether the ferulic acid of the present invention protects the -SH group, which is an antioxidant regulating system in vivo, oral administration of ferulic acid at 2, 4 mg / kg weight and oral administration to aged rats of 24 months of age, followed by 10 days After the dissection, the dissection was performed to excise and crush the kidneys to measure changes. In FIG. 5, 6 months old control rats (Young), 24 months old aging rats (Old), the rest 24 months old aging white rats 2, 4 mg / kg / day It is administered in the amount of.

As a result, the total -SH phase was decreased in the aged white rats compared to the young white rats, and the administration of the ferulic acid of the present invention decreased the total -SH phase in the aged rats. Increased concentration dependent (FIG. 5A). In addition, in the aged white rats, glutathione production ability was reduced compared to young white rats, and it was confirmed that administration of the ferulic acid of the present invention increased the concentration-dependent decrease in glutathione production capacity in aged rats (FIG. 5B).

Experimental Example 4. Measurement of NF-κB Inhibitory Activity of Ferulic Acid

4-1. Intracellular NF-κB Inhibitory Activity of Ferulic Acid

In order to determine whether ferulic acid of the present invention inhibits NF-κB in cells, plasmids having four κB sites were inserted into vascular endothelial cells using a reagent called FuGENE 6 (USA). In addition, stimulation with t-BHP was performed to determine the extent to which NF-κB binds to the κB site and whether ferulic acid inhibits the binding.

As a result, as shown in FIG. 3, the overexpressed cells (T-CON) increased NF-κB activity more than the overexpressed cells (CON), and the t-BHP treated group increased significantly. When the ferulic acid of the present invention was treated, it was confirmed that NF-κB activity was inhibited in a concentration-dependent manner.

4-2. Measurement of NF-κB Mechanism of Ferulic Acid

In order to examine whether ferulic acid of the present invention inhibits NF-κB, ferulic acid was orally administered at 2, 4 mg / kg / day to 24 months old aged rats, and then dissected and fractured after 10 days. Was used. Renal nuclear protein lysate was homogenously mixed with bromophenol blue stain and electrophoresed on 10% SDS-PAGE. After electrophoresis, the protein was transferred to the polyvinyridine fluoride membrane, and the nonspecific reaction was performed by immersing in TBS-Tween solution (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween 20, pH 7.5) containing 5% skim milk. Blocked. Thereafter, the antibodies against p50 and p65 (Santacruz) 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-Tween solution, reacted with ECL detection reagent for 30 minutes, and then exposed to X-ray film at room temperature.

As a result, as shown in FIG. 6, the expression levels of p50 and p65 genes were increased in aged rats aged 24 months compared to young rats aged 6 months, and the group administered ferulic acid compared to aged rats aged 24 months. It was confirmed that the reduction effect.

4-3. Mechanism of IκBα / β Action of Ferulic Acid

The mechanism of IκBα / β action of ferulic acid of the present invention was examined. As aging progresses, IκBα / β is released by phosphorylation when NF-κB is activated, and the amount of IκBα / β is reduced in the cytoplasm. In order to measure the protein level of IκBα / β, ferulic acid was orally administered at 2, 4 mg / kg / day to 24 months old aged rats, and then dissected and shredded after 10 days. 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 polyvinyridine fluoride membrane, and the nonspecific reaction was performed by immersing in TBS-Tween 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) 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-Tween solution, reacted with ECL detection reagent for 30 minutes, and then exposed to X-ray film at room temperature.

As a result, as shown in FIG. 7, the amount of protein of IκBα / β was decreased in the aged rats of 24 months of age compared with the young rats of 6 months of age, and fed ferulic acid in comparison with the aged rats of 24 months of age. The group showed an increasing effect. On the contrary, in FIG. 7, the amount of protein was increased in aging rats of 24 months of age compared with young white rats of 6 months of p-IκBα, and in the group fed ferulic acid compared to the same age of 24 months of aging rats. It was.

4-4. Measurement of NF-κB Binding Inhibition of Ferulic Acid

In order to examine whether ferulic acid of the present invention inhibits NF-κB in vivo, oral administration of ferulic acid at 2, 4 mg / kg / day to 24 months old aged rats was dissected 10 days later to excise kidney and nuclear protein. Was separated and measured using an electrophoretic mobility shift assay (EMSA). Renal nuclear proteins were labeled with ³² P isotopes and electrophoresed on 7% SDS-PAGE. After electrophoresis, the gel was exposed to X-ray film at room temperature.

As a result, as shown in Figure 8 it can be seen that compared to the young white rats of 6 months of age in the group administered ferulic acid to the aged white rats of 24 months of age showed an excellent inhibitory effect of NF-κB binding activity.

4-5. Determination of Inflammatory Gene Expression Inhibition Regulated by NF-κB of Ferulic Acid

In order to examine whether ferulic acid of the present invention inhibits NF-κB in vivo, 10 months of ferulic acid was orally administered to 24 months old aging white rats. . Kidney crushed solution was mixed with bromophenol blue dye in the same amount and electrophoresed on SDS-PAGE. After electrophoresis, the protein was transferred to the polyvinylidene fluoride membrane and 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) containing 0.5% skim milk. It was. After that, the solution of goat's antibody (Upstate) to COX-2 diluted 1/1000, the Levit's antibody to iNOS (Upstate) diluted 1/10000, Goat for VCAM-1, ICAM-1 The antibody (Santacruz) was reacted with a solution diluted to 1/500 for 3 hours at room temperature, and then reacted with an anti goot and Levit IgG antibody as a secondary antibody. After completion of the reaction, the membrane was washed four times with TBS-Tween solution, reacted with ECL detection reagent for 30 minutes, and then exposed to X-ray film at room temperature.

As a result, as shown in FIG. 10, the expression levels of COX-2, iNOS, VCAM-1, and ICAM-1 in 24 months old white rats were increased compared to 6 months old young white rats. Compared with the month-old aging white rats, the expression of COX-2, iNOS, VCAM-1, and ICAM-1 was reduced in the group administered ferulic acid.

4-6. Mechanism of NF-κB action of ferulic acid

In order to examine the mechanism of NF-κB action of ferulic acid of the present invention was examined using vascular endothelial cells. First, vascular endothelial cells were pretreated with ferulic acid and NF-κB, p38, JNK, and ERK inhibitors for 1 hour and t-BHP 15μM for 6 hours, followed by the expression of COX-2 expressed by NF-κB. Recognized sheep. As a result, as shown in FIG. 10A, it was found that each kinase inhibitor was inhibited.

Secondly, based on the results of FIG. 10A, the vascular endothelial cells were pretreated with ferulic acid for 1 hour and t-BHP was treated for 20 minutes to confirm p-JNK, p-p38, pERK, and p-IKK. As shown in 10b, it was confirmed that the activity of ERK is significantly reduced by ferulic acid.

Third, as a result of confirming p50 and p65 by pretreating ferulic acid to vascular endothelial cells for 1 hour and t-BHP for 30 minutes, as shown in FIG. 11, it was confirmed that p50 and p65 were inhibited by ferulic acid. there was.

Although a formulation example of a pharmaceutical composition comprising a compound of the present invention will be described, the present invention is not intended to be limiting but merely illustrative.

Formulation Example 1 Preparation of Powder

Ferulic Acid 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

Ferulic Acid 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

Ferulic Acid 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

Ferulic Acid 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

Ferulic Acid 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

Ferulic acid compound 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B_One                                                   0.13 mg

Vitamin B₂                                                   0.15 mg

Vitamin B₆                                                   0.5 mg

Vitamin B₁₂                                                   0.2 μg

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 vitamin and mineral mixture is a composition suitable for a relatively healthy food in a preferred embodiment, the composition 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

Ferulic acid 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.

The composition containing the ferulic acid 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 to inhibit NF-κB. Inhibits the expression of genes associated with inflammation or aging, and inhibits the enzymes that produce active species involved in the inflammatory response as a major cause of the aging process, leading to antioxidant, anti-aging or anti-inflammatory pharmaceutical compositions and health foods It can be usefully used.

Claims (5)

A pharmaceutical composition for antioxidant, anti-aging or anti-inflammatory, containing ferulic acid 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 ferulic acid of claim 1 and a food supplement acceptable food supplement. The dietary supplement of claim 4 which is a tablet, capsule, pill or liquid.

Images