KR101841604B1 - Composition for preventing or treating of depression and anxiety comprising purpurin - Google Patents

Abstract

The present invention relates to a composition for preventing or treating depression and anxiety comprising purpurin as an active ingredient. The purpurin selectively, reversibly, and competitively inhibits an activity of monoamine oxidase A type (MAO-A), which is associated with onset of depression and anxiety. In addition, the purpurin has little cytotoxicity so that the purpurin can be safely used in pharmaceutical or food compositions for prevention or treatment of depression and anxiety.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating depression and anxiety,

The present invention relates to a pharmaceutical composition or food composition for the prevention or treatment of depression and anxiety, which comprises purpurin as an active ingredient.

Korea has undergone rapid development over the last fifty years in such a way that it is hard to find any analogy in the world as it has experienced the industrialization, urbanization, westernization, nuclear family, advanced education, aging and informationization process in a short period of time. These rapid developments have led to high economic growth, but they have been associated with aging, excessive disparities in wealth, family dismemberment, alienation, neurological diseases such as Parkinson's disease due to psychological stress from intense competition, dementia, , Depression, and anxiety disorder (neuropsychiatric disorder) are increasing.

The onset of this neuropsychiatric disorder plays an important role in the metabolism of monoamines in vivo, and monoamine oxidase (MAO, E.C. 1.4.3.4) is known to be involved as an enzyme.

Specifically, monoamine oxidase (MAO) is an enzyme that is widely distributed in the central nervous system and peripheral tissues. It catalyzes the oxidative deamination of amine compounds and is derived from monoamine compounds and intestinal bacteria Which break down hormonal amines. That is, the neurotransmitter is decomposed by the monoamine oxidase, resulting in the deficiency of the monoamine compound in vivo, resulting in the neuropsychiatric disorder.

This monoamine oxidase (MAO) is mainly used as an endogenous substrate such as dopamine, tyramine, epinephrine or norepinephrine, and serotonin is selectively eliminated by substrate specificity The amino acid can be divided into two types: A-type (type A, MAO-A), B-type (type B, MAO-B) that selectively deaminates phenylethylamine and benzylamine (Youdim MB et al. , 2006, Nat. Rev. Neurosci., 7: 295-309). Among these, MAO-A is associated with the onset of neuropsychiatric disorders such as depression and anxiety, and MAO-B is associated with the onset of neurological diseases such as Alzheimer's diseases and Parkinson's diseases.

Thus, MAO inhibitors have been used to prevent or treat neuropsychiatric disorders domestically and internationally, and the MAO inhibitors include MAO-A selective inhibitors, MAO-B selective inhibitors, and MAO-A / And irreversible inhibitors [Malcomson T et al. , 2015, FEBS J., DOI: 10.111 / feeb.13260 (Epub ahead of print); Mostert S et al. , 2015, Chem. Med. Chem., DOI: 10-.1002 / cmdc.201500059 (Epub ahead of print)].

(1990, Br. J. Clin. Pharmacol., 30: 805-816), Fowler et al. (1990), and the like in connection with MAO-A selective inhibitors that can prevent or treat depression and anxiety, Neuropsychopharmacology, 35: 623-631), Gentili F et al (2006, J. Med. Chem., 49: 5578-5586), and Lotufo-Neto F et al. (1999, Neuropsychopharmacology, 20: 226-247 ) Reports that moclobemide, brofaromine, toloxatone, amifuraline, and CX157 can be used as reversible inhibitors of MAO-A. (1997, Arch. Biochem. Biophys., 337: 137-142) and Mattsson C et al. (2014, Eur J. Med. Chem., 73: 177-186) have discovered novel beta-carbolines derivatives and coumarin derivatives, which have been reported to be available as selective inhibitors of MAO-A . However, the MAO-A reversible and selective inhibitor is a chemically synthesized substance that causes hepatotoxicity and somatic hypotension, and has the problem that side effects such as insomnia, excitement, and convulsions occur when overdose.

Therefore, there is a demand for the development of medicines or health functional foods which are safe for long-term use and can treat or ameliorate depression and anxiety.

On the other hand, purpurin is anthraquinone found in the roots of madder (Rubia tinctorum or Rubia akane) containing anthraquinone alizarin. The perforin inhibits xanthine oxidase, protects against DNA damage, has antifungal activity and antioxidant and cytotoxic activity, and spermidine-induced autoactivity of plasease plasma hyaluronan-binding protein, O-acetylpeptido Glycans esterase, DNA topoisomerase VI, adipocyte-derived leucine aminopeptidase, and angiogenesis.

The effects of some anthraquinones on MAO isomers have been studied, but emodin has been reported to selectively inhibit mixed morphology for MAO-B. However, there is no report on the inhibitory activity of perforin on MAO enzyme.

Accordingly, the present inventors have found that purpurin contained in a madder is used as a preventive or therapeutic agent for neuropsychiatric disorders such as depression and anxiety while searching for a substance that is safe for long-term use and can prevent or treat depression and anxiety. And the present invention has been completed.

Accordingly, a technical problem to be solved by the present invention is to provide a composition for preventing or treating depression and anxiety.

In order to solve the above technical problems, the present invention provides a pharmaceutical composition for preventing or treating depression and anxiety, which comprises perrin or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a food composition for preventing or ameliorating depression and anxiety, which comprises perrin or a pharmaceutically acceptable salt thereof as an active ingredient.

The term "depression" as used in the present invention is a feeling of feeling and feeling of sadness, tastelessness, loss of emotion, lack of pleasure (lack of pleasure), grief, agitation or delay, guilt and worthlessness, , ≪ / RTI > and delusions.

As used herein, the term "anxiety" refers to a psychological disorder in which the physical or behavioral symptoms such as headache, increased heart rate, increased respiratory rate, abnormal gastrointestinal symptoms, panic disorder, sweating, It means disorder.

The term "prevention ", as used herein, refers to inhibiting the occurrence of a disease or disease in an individual who has never been diagnosed as having a disease or disease, but has a tendency to become susceptible to such disease or disease.

The term "treatment ", as used herein, refers to (a) inhibiting the development of a disease or disorder, (b) relieving the disease or disorder and (c) eliminating the disease or disorder.

The term "individual" as used herein means a mammal such as a monkey, a cow, a horse, a pig, a sheep, a dog, a cat, a rat, a mouse, a chimpanzee, etc. including a human having a disease whose symptoms may be improved by administering the composition of the present invention Of mammals.

Purpurin, an active ingredient of the composition of the present invention, is a compound having a structure represented by the following general formula (1).

The perrin can be isolated or chemically synthesized from the madder and the purpurin is found in the roots of the madder (Rubia tinctorum or Rubia akane) containing anthraquinone alizarin Anthraquinone. The perforin inhibits xanthine oxidase, protects against DNA damage, has antifungal activity and antioxidant and cytotoxic activity, and spermidine-induced autoactivity of plasease plasma hyaluronan-binding protein, O-acetylpeptido Glycans esterase, DNA topoisomerase VI, adipocyte-derived leucine aminopeptidase, and angiogenesis.

On the other hand, in one specific embodiment of the present invention, alizarin is used to compare the effect of perforin, and alizarin has a structure similar to perforin with the structure of the following formula (2). Such alizarin is used as a probe dye and an osteoporosis drug for detecting micro-damage of a skeleton and is known to have antioxidant activity, antiviral activity, antifungal activity, larva and insecticidal activity, antibiotic membrane and anti-hemolytic activity.

According to one embodiment of the present invention, perforin has the effect of selectively, reversibly, and competitively inhibiting the activity of monoamine oxidase A (MAO-A), which is associated with the onset of depression and anxiety, Alizarin, which has a similar structure to perforin, has little effect on inhibiting the activity of MAO-A.

Accordingly, the composition containing the perforin of the present invention as an active ingredient can be usefully used for preventing or treating depression and anxiety.

The perforin represented by the formula (1) of the present invention can be used in the form of a pharmaceutically acceptable salt or a pharmaceutically acceptable salt, and the salt includes an acid addition salt formed by a pharmaceutically acceptable free acid Is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be prepared by dissolving the perpurine in an excess amount of an aqueous acid solution by a conventional method such as precipitation using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile . It is also possible to prepare the mixture by evaporating a solvent or an excess acid in the mixture, or by suction filtration of the precipitated salt.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

The perrin contained as an active ingredient in the composition of the present invention may be contained in an amount of 0.001 to 50% by weight, preferably 0.01 to 30% by weight, more preferably 0.01 to 10% by weight, based on the total weight of the total composition have. When the content of perforin is less than 0.001% by weight, the MAO-A inhibitory activity is insufficient. When the content of perforin is more than 50% by weight, the effect of increasing the content is not proportionally increased, There is no problem.

As one specific use of the present invention, the composition of the present invention can be used as a pharmaceutical composition for preventing or treating depression and anxiety.

When the composition of the present invention is used as a pharmaceutical composition, it may further comprise a pharmaceutically acceptable carrier other than perrin as an active ingredient. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include carbohydrate-type compounds (e.g., lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, Corn oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, cottonseed oil, But are not limited to, polyethylene glycol, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as aerosols, external preparations, suppositories, and sterilized injection solutions according to a conventional method.

The pharmaceutical composition may prevent or treat depression and anxiety. The method of treatment includes administering the pharmaceutical composition in a pharmaceutically effective amount by various routes in a mammal such as a mouse, a mouse, a domestic animal, or a human. The administration method may be carried out in any manner, for example, by oral, rectal or intravenous infusion, intraperitoneal administration, intramuscular administration, or intracerebral administration.

The dosage of the pharmaceutical composition may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, and the like of the patient, and may be appropriately selected by those skilled in the art. In general, the pharmaceutical composition of the present invention may be administered in an amount of 0.001-100 mg / kg on an adult basis, once to several times a day. However, the dose is not limited in any way to the scope of the present invention.

As another specific use of the present invention, the composition of the present invention can be used as a food composition for preventing or ameliorating depression and anxiety.

The term "improvement" as used in the present invention means all actions that improve symptoms of a disease or disease in an individual.

When the composition of the present invention is used as a food composition, in addition to perforin as an active ingredient, it further includes components commonly added in the manufacture of food such as proteins, carbohydrates, fats, nutrients, seasonings and flavors can do.

Examples of such carbohydrates are monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavors (saccharin, aspartame, etc.) may be used as flavorings.

The food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juices, beverages and vegetable drinks. These components may be used independently or in combination.

There is no particular limitation on the kind of the food. Examples of the food to which the perpurin of the present invention can be added include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, , A drink, an alcoholic beverage, and a vitamin complex, all of which include health foods in a conventional sense.

When the food composition of the present invention is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and the like may be further added in addition to the perforin of the present invention.

In addition, the food composition of the present invention may further contain food additives, and the suitability of the food additives as a food additive may be determined according to the General Rules and General Test Methods approved by the Food and Drug Administration (KFDA) It shall be judged according to standards and standards for items.

Examples of the foodstuffs that have been used in the above food additives include chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; Natural additives such as crystalline cellulose and guar gum; Sodium L-glutamate preparation; Noodle-added alkaline agent; Preservative formulation; And mixed preparations such as tar coloring agents.

On the other hand, the perforin of the present invention is a harmless substance having no cytotoxicity. Therefore, the perforin of the present invention can be safely used even in long-term use, and can be safely used especially in pharmaceutical or food compositions as described above.

The perforin of the present invention exhibits an effect of selectively, reversibly, and competitively inhibiting the activity of monoamine oxidase A (MAO-A), which is associated with the onset of depression and anxiety, and has little cytotoxicity, Can be safely used in pharmaceutical or food compositions for the prevention or treatment of anxiety.

Figure 1 shows the time-dependence of MAO-A inhibition by perforin.
Figure 2 shows the recovery of MAO-A by perforin.
Figure 3 shows the residual activity of MAO-A by perforin under dilute conditions.
Figure 4 shows the line weighed-Burk plot (A) of inhibition of MAO-A by perforin and the second plot (B) of the slope versus inhibitor concentration.
Figure 5 shows a docking simulation between MAO-A (A) or MAO-B (B) and perforin.

Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings. It is to be understood that the scope of the present invention is not limited by these reference examples, examples and the like in accordance with the gist of the present invention. It will be obvious to those who have.

Material preparation

Benzylamine, quinolamine, and recombinant human MAO-A and MAO-B were purchased from Sigma-Aldrich (St. Louis, Mo.); MAO-A and MAO-B were stored at -70 C in 100 mM potassium phosphate, pH 7.4, 0.25 M sucrose, 0.1 mM EDTA, and 5% glycerol. Chloroglirin is a component of the monoamine oxidase kit obtained by BioAssay Systems (Hayward, Ca. USA).

Example 1 Measurement of MAO-A and MAO-B Inhibitory Activity on Purpurin

Perforin and alizarin were analyzed for inhibition of MAO-A and MAO-B; IC ₅₀ values are shown in Table 1 below.

The initial oxidation rate of MAO was measured at 25 DEG C in a 1 ml cuvette containing 50 mM sodium phosphate (pH 7.2). The activity of MAO-A was assayed at 316 nm for 30 minutes using 0.2 mM quinolamine as substrate and the activity of MAO-B was analyzed at 250 nm for 10 minutes using 0.2 mM benzylamine. The reaction was initiated by adding a substrate to the enzyme mixture. The reaction rate was expressed as the change in absorbance per minute. When using this method, K _m values for the Kinugawa suramin is 0.029mM was substrate concentration was 6.9 X Km, K _m value for a benzylamine is 0.26mM was the substrate concentration was 7.7 X Km.

The MAO-A selectivity index was calculated using IC ₅₀ (MAO-B) / IC ₅₀ (MAO-A).

In Table 1, superscript C shows a suppression rate of 23.2% at 40 μM, and superscript d shows a suppression rate of 16.5% at 60 μM. As seen here, perforin effectively inhibited MAO-A with an IC ₅₀ value of 2.50 μM, but MAO-B did not effectively suppress selectivity index (SI) values greater than 16.0. On the other hand, alizarin was less effective than perforin for MAO-A with an IC ₅₀ value of 30.1 μM, and was not effective against MAO-B (IC ₅₀ > 60 μM).

<Example 2> Measurement of time-dependent inhibitory activity of MAO-A according to pre-incubation time of perforin

The time-dependent inhibitory activity of MAO-A was determined by pre-incubation time of perforin. Specifically, the residual activity was incubated at 316 nm and 25 캜 for 30 minutes using 0.2 mM quinolamine as a substrate.

As a control, the reaction was carried out in the presence of 0.2 mM substrate without inhibitor. Clorgyline was used as a reference irreversible inhibitor.

Figure 1 shows the time-dependence of MAO-A inhibition by perforin. As shown here, with respect to the time-dependence of inhibition, MAO-A activity in the presence of perforin showed little change during pre-incubation up to 30 min. This result implies that perforin does not inhibit human MAO-A in a time-dependent manner.

In addition, the reversibility of the MAO-A inhibition of perrin by addition of the substrate was evaluated and shown in Fig. Enzyme activity was measured for 10 minutes in the presence of 0.2 mM of quinamine and 5.0 μM of perrin, and the same amount of substrate was added and enzyme activity was measured after an additional 10 minutes. Fig. 2 shows the recovery of MAO-A by perforin due to substrate addition. In this case, 0.2 is the reaction (2.0 x IC ₅₀₎ of the buffer purine of the substrate and 5.0 μM of 0.2 mM, 0.2 + 0.2 is buffer purine of 5.0 μM and additionally adding a substrate of 0.2 mM at the end of the reaction (2.0 x IC ₅₀ ), and 0.4 is the reaction with 0.4 mM substrate at the start of the reaction in the presence of the inhibitor. As shown in FIG. 2, when the MAO-A and perforin were mixed for the first time, the residual activity was 36.6% and the addition of the additional substrate restored to 55.2%. This recovery value was similar to the residual activity of 54.4% observed at an initial substrate concentration of 0.4 mM.

The recovery of the enzyme activity was diluted as described above and analyzed with some modifications. An excess of purin (100 X IC ₅₀ ) was incubated with MAO-A for 10 minutes and then diluted 100-fold. The residual activity was compared to the residual activity in the undiluted state (i.e. 1.0 X IC ₅₀ ). Chloroglirin was used as an irreversible reference MAOA inhibitor.

Figure 3 shows the residual activity of MAO-A by perforin under dilute conditions. Here, I + D is the reaction with a 100-fold diluted enzyme mixture (final concentration of inhibitor = 1.0 X IC ₅₀ ). As shown in Fig. 3, the recovery activity under the dilution conditions of perforin was almost the same (67.2-63.9%) as in the undiluted condition. However, the activity of chloroglycan was about half of that of 47.9% to 24.9%. These results show that perforin is not an irreversible inhibitor of MAO-A.

Example 3 Measurement of MAO-A Inhibitory Activity by Perfuran Concentration or Substrate Concentration

MAO-A and MAO-B inhibition modes by perforin were tested using different concentrations of substrate and inhibitor. The inhibition pattern and K _i values were measured using a Lineweaver-Burk plot.

Figure 4 shows the line weighed-Burk plot (A) of inhibition of MAO-A by perforin and the second plot (B) of the slope versus inhibitor concentration. Promoting ratios of MAO-A and MAO-B were measured at six different concentrations 0.01-0.5 mM using quinolamine as substrate in the absence or presence of perlin.

As shown in FIG. 4, the plot for MAO-A inhibition of MAO-A by perforin was linear and crossed the y-axis. This means that perforin is a competitive inhibitor of MAO-A. The K _i value for inhibition of MAO-A by perforin in the second plot of slope versus inhibitor concentration was 0.422 ± 0.035 μM.

Example 4: MAO docking simulation of perforin

To simulate perforin for MAO docking, we used the DOCK 6.7 program with an automated docking facility and AutoDock Vina. To confirm the docking pockets of MAO-A and MAO-B, a complex of MAO-A and 7-methoxy-1-methyl-9H-beta-carboline (PDB ID: 2Z5X) and a complex of MAO-B and pioglitazone (PDB ID: 4A79) was used. The docking simulation of MAO and perforin was performed using the following steps: (1) conversion of the 2D structure to a 3D structure, (2) energy minimization using the ChemOffice program, (3) calculation of the charge, and (4) Addition of hydrogen atoms using.

Figure 5 shows a docking simulation between MAO-A (A) or MAO-B (B) and perforin.

As a result of docking simulation, perforin was found to bind to 7-methoxy-1-methyl-9H-beta-canolin binding site (PDB: 2Z5X) complexed with MAO-A and phyoglinazone pioglitazone) (PDB: 4A79).

The docking affinity (-40.0 kcal / mol) of perforin for MAO-A was similar to the docking affinity for MAO-B (-33.9 kcal / mol) by AutoDock Vina. The perforin was found to form hydrogen bonds through the Ile207 and Gly443 residues and bind to MAO-A and MAO-B.

The oxygen in the carbonyl group of each residue was expected to act as an H-coupled receptor. This involvement of the carbonyl group in the interaction between MAO-A and derivatives of 2-methoxy-4- (5-phenyl-4,5-dihydro-1H-pyrazol-3-yl) phenol has been reported have. Alizarin, on the other hand, was predicted to interact with MAO-A by hydrogen bonding through Gly443 alone. Porphin has three hydroxyl groups at positions 1, 2 and 4 of the anthraquinone skeleton, and alizarin has two hydroxyl groups at positions 1 and 2. On the basis of the measured IC ₅₀ values, the hydroxyl value of perforin was found to play an important role in the inhibition of MAO-A.

In addition, perforin did not show cytotoxicity to L929 cells and was found to significantly increase their proliferation at 100 μM concentration. The Ki value of perforin for MAO-A was determined using other natural compounds, for example, 2-methylnorbornane (1.43 [mu] M, b-carboline derivative) and aniotactin A (1.84 [mu] M).

Based on these results, perforin can be regarded as a potent and selective reversible inhibitor of MAO-A.

The perforin of the present invention exhibits an effect of selectively, reversibly, and competitively inhibiting the activity of monoamine oxidase A (MAO-A), which is associated with the onset of depression and anxiety, and has little cytotoxicity, Can be safely used in pharmaceutical or food compositions for the prevention or treatment of anxiety.

Claims (2)

A pharmaceutical composition for preventing or treating depression and anxiety comprising purpurin or a pharmaceutically acceptable salt thereof as an active ingredient. A food composition for preventing or ameliorating depression and anxiety comprising perforin or a pharmaceutically acceptable salt thereof as an active ingredient.

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