KR102671319B1 - Composition for improving mental health containing a novel compound - Google Patents

Abstract

The present invention relates to a composition for improving mental health containing a novel compound, which can improve and prevent psychotic symptoms by improving impaired pre-stimulus inhibition and impaired social interaction, which are schizophrenia-like behaviors.
In addition, it has an excellent effect of inhibiting the activity of monoamine oxidase and inhibiting amyloid beta self-aggregation, and can prevent the occurrence of side effects.

Description

Composition for improving mental health containing a novel compound}

The present invention relates to a composition for improving mental health containing a novel compound.

As society develops and diversifies rapidly, modern people are surrounded by various types of stress. Failure to successfully cope with stress results in psychological reactions such as depression, anxiety, fatigue, anger, and mood changes, as well as physiological reactions such as decreased α-waves in brain waves and increased blood pressure and pulse rate, and these reactions continue to repeat. When this happens, diseases such as depression and anxiety appear, cause personal, family and social losses, and lower an individual's quality of life.

Recently, mental illness in adolescents is also on the rise due to causes such as excessive enthusiasm for learning. According to the 2016 Epidemiological Survey on Mental Illnesses published by the Ministry of Health and Welfare, the ‘one-year prevalence rate of mental disorders’, which is the percentage of people experiencing at least one mental illness in one year, is about 1 in 8 adults between the ages of 18 and 64. It is 12.1%, and the 'lifetime prevalence rate of mental illness', which is the proportion of the population experiencing at least one mental illness during their lifetime, was also found to be 26.6%, about 1 in 4 adults.

The incidence rate is increasing worldwide, and the World Health Organization predicts that depression will become the most common disease among all ages by 2020.

Despite this, only 18.2% of people diagnosed with mental illness have any experience with counseling or treatment for mental problems.

Among mental illnesses caused by stress, depression refers to a psychological state of depressed mood characterized by feelings of sadness, hopelessness, and frustration. This refers to a condition that progresses from the normal emotion of “depression” to dysthymic disorder to major depressive disorder.

Symptoms of depression include a major depressive episode, which includes changes in appetite and weight patterns, psychomotor agitation or retardation, decreased ability to think, concentrate or make decisions, lack of energy, fatigue and feelings of worthlessness. These include feelings of guilt, remorse or guilt, frequent thoughts of death or suicide, plans or attempts to commit suicide, low mood in daily life, or loss of interest or pleasure in most activities.

There are various hypotheses about the biological cause of depression, but the prevailing view is that it is a disorder of the brain neurotransmitter system such as adrenaline, dopamine, or serotonin.

Currently, tricyclic antidepressants (TCAs) or selective serotonin re-uptake inhibitors (SSRIs) are mainly used as treatments for depression, but these serotonin reuptake inhibitors have the problem of being ineffective and taking a long time. .

According to the American Psychiatric Association (APA) recommendations, the serotonin reuptake inhibitor should be taken for at least 4 months, and depending on the patient, this may extend to 2 to 3 years.

In addition, it is known that there is a risk that most depression symptoms will recur within 6 to 12 months when medication is stopped.

Most drugs used for mental illness are classified as psychotropic drugs. In particular, benzodiazepine drugs act directly on gamma-aminobutyric acid (GABA) receptors in the central nervous system, causing excessive sedation and psychological or There is a high risk of drug abuse due to high physical dependence.

Schizophrenia is characterized by delusions, hallucinations, disorganized speech (positive symptoms), flat affect, avolition, speech and language deficits, and social withdrawal (negative symptoms). , a widespread mental disorder characterized by deficits in attention, planning, abstract thinking, and short- and long-term memory deficits (cognitive dysfunction).

To date, treatments for schizophrenia have been developed with positive symptoms as their target symptoms. However, since negative symptoms or cognitive impairment are closely related to the chronicity of schizophrenia and the difficulty of returning to society, there is a strong demand for drugs that improve these symptoms.

Therefore, improving not only positive symptoms but also negative symptoms or cognitive impairment is believed to be a useful treatment method for schizophrenia.

In recent years, drugs that improve not only positive symptoms but also negative symptoms or cognitive impairment have been developed, but the number is still small and the effectiveness is not sufficient.

There is a need to develop materials that are effective in relieving tension, preventing or treating anti-depression, or mental illness, but do not cause the above side effects even when taken repeatedly.

KR 10-1776143 B1

The purpose of the present invention is to provide a composition for improving mental health containing a novel compound.

Another object of the present invention is to provide a composition that contains a novel compound as an active ingredient and can improve and prevent psychotic symptoms by improving impaired pre-stimulus inhibition and impaired social interaction, which are schizophrenia-like behaviors.

Another object of the present invention is to include a novel compound as an active ingredient, which has an excellent effect of inhibiting the activity of monoamine oxidase and inhibiting amyloid beta self-aggregation, and has a tension-relieving or anti-inflammatory effect that can prevent the occurrence of side effects. To provide a composition for depression.

In order to achieve the above object, the present invention relates to a composition for improving mental health, which may include a compound represented by the following formula (1) as an active ingredient:

[Formula 1]

here,

n and m are the same or different from each other and are each independently an integer from 0 to 4,

R ₁ and R ₂ are the same or different from each other, and are each independently hydrogen, deuterium, cyano group, nitro group, halogen group, hydroxy group, substituted or unsubstituted alkylthio group having 1 to 4 carbon atoms, substituted or unsubstituted carbon number Alkyl group of 1 to 30 carbon atoms, substituted or unsubstituted cycloalkyl group of 3 to 20 carbon atoms, substituted or unsubstituted alkenyl group of 2 to 30 carbon atoms, substituted or unsubstituted alkynyl group of 2 to 24 carbon atoms, substituted or unsubstituted Aralkyl group with 7 to 30 carbon atoms, substituted or unsubstituted aryl group with 6 to 30 carbon atoms, substituted or unsubstituted heteroaryl group with 1 to 60 carbon atoms, substituted or unsubstituted heteroarylalkyl group with 2 to 30 carbon atoms, substituted or an unsubstituted alkoxy group having 1 to 30 carbon atoms, a substituted or unsubstituted alkylamino group having 1 to 30 carbon atoms, a substituted or unsubstituted arylamino group having 6 to 30 carbon atoms, or a substituted or unsubstituted aralkyl having 7 to 30 carbon atoms. Amino group, substituted or unsubstituted heteroarylamino group having 1 to 24 carbon atoms, substituted or unsubstituted alkylsilyl group having 1 to 30 carbon atoms, substituted or unsubstituted arylsilyl group having 6 to 30 carbon atoms, and substituted or unsubstituted carbon atoms. It is selected from the group consisting of 6 to 30 aryloxy groups.

The mental health can show excellent improvement effects on schizophrenia, schizophreniform disorder, schizoaffective disorder, delusional disorder, brief psychotic disorder, shared psychotic disorder, social isolation, atonia or depression.

The compound represented by Formula 1 has an excellent effect of inhibiting the activity of monoamine oxidase and inhibiting amyloid beta self-aggregation.

The compound represented by Formula 1 can improve pre-stimulus inhibition impairment and social interaction impairment.

The composition for improving mental health can pass through the blood-brain barrier and be absorbed into the central nervous system (CNS).

The composition for improving mental health exhibits excellent human intestinal absorption (HIA) and can be taken by oral administration.

A functional food composition according to another embodiment of the present invention may include the composition for improving mental health.

A pharmaceutical composition according to another embodiment of the present invention may include the composition for improving mental health.

In the present invention, “hydrogen” refers to hydrogen, light hydrogen, deuterium, or tritium, unless otherwise specified.

In the present invention, the “halogen group” is fluorine, chlorine, bromine, or iodine.

In the present invention, “alkyl” refers to a monovalent substituent derived from a straight-chain or branched-chain saturated hydrocarbon having 1 to 40 carbon atoms. Examples thereof include methyl, ethyl, propyl, isobutyl, sec-butyl, pentyl, iso-amyl, hexyl, etc., but are not limited thereto.

In the present invention, “alkenyl” refers to a monovalent substituent derived from a straight-chain or branched-chain unsaturated hydrocarbon having 2 to 40 carbon atoms and having one or more carbon-carbon double bonds. Examples thereof include vinyl, allyl, isopropenyl, 2-butenyl, etc., but are not limited thereto.

In the present invention, “alkynyl” refers to a monovalent substituent derived from a straight or branched chain unsaturated hydrocarbon having 2 to 40 carbon atoms and having at least one carbon-carbon triple bond. Examples thereof include ethynyl, 2-propynyl, etc., but are not limited thereto.

In the present invention, “alkylthio” refers to the alkyl group described above bonded through a sulfur linkage (-S-).

In the present invention, “aryl” refers to a monovalent substituent derived from an aromatic hydrocarbon having 6 to 60 carbon atoms, either a single ring or a combination of two or more rings. In addition, forms in which two or more rings are simply pendant or condensed with each other may also be included, specifically naphthyl group, anthracenyl group, phenanthryl group, triphenyl group, pyrenyl group, phenalenyl group, perylenyl group, cryo group. It may be a cenyl group, a fluorenyl group, etc., but is not limited thereto. The fluorenyl group may be substituted, and adjacent groups may combine with each other to form a ring.

In the present invention, “heteroaryl” refers to a monovalent substituent derived from a monoheterocyclic or polyheterocyclic aromatic hydrocarbon having 6 to 30 carbon atoms. At this time, at least one carbon, preferably 1 to 3 carbons, of the ring is replaced with a heteroatom such as N, O, S or Se. In addition, a form in which two or more rings are simply pendant or condensed with each other may be included, and a condensed form with an aryl group may also be included. Examples of such heteroaryls include 6-membered monocyclic rings such as pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl, phenoxathienyl, indolizinyl, and indolyl ( Polycyclic rings such as indolyl, purinyl, quinolyl, benzothiazole, carbazolyl, and 2-furanyl, N-imidazolyl, 2-isoxazolyl , 2-pyridinyl, 2-pyrimidinyl, etc., but are not limited thereto.

In the present invention, “aryloxy” is a monovalent substituent represented by RO-, where R refers to aryl having 6 to 60 carbon atoms. Examples of such aryloxy include phenyloxy, naphthyloxy, diphenyloxy, etc., but are not limited thereto.

In the present invention, “alkyloxy” is a monovalent substituent represented by R'O-, where R' refers to alkyl having 1 to 40 carbon atoms and has a linear, branched, or cyclic structure. may include. Examples of alkyloxy include, but are not limited to, methoxy, ethoxy, n-propoxy, 1-propoxy, t-butoxy, n-butoxy, and pentoxy.

In the present invention, “alkoxy” may be straight chain, branched chain, or ring chain. The number of carbon atoms of alkoxy is not particularly limited, but is preferably 1 to 20 carbon atoms. Specifically, methoxy, ethoxy, n-propoxy, isopropoxy, i-propyloxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentyloxy, neopentyloxy, Isopentyloxy, n-hexyloxy, 3,3-dimethylbutyloxy, 2-ethylbutyloxy, n-octyloxy, n-nonyloxy, n-decyloxy, benzyloxy, p-methylbenzyloxy, etc. It may be possible, but it is not limited to this.

As used herein, “aralkyl” refers to an aryl-alkyl group where aryl and alkyl are defined above. Preferred aralkyl contains lower alkyl groups. Non-limiting examples of suitable aralkyl groups include benzyl, 2-phenethyl, and naphthalenylmethyl. Bonding to the parent moiety is via the alkyl.

In the present invention, “arylamino group” refers to an amine substituted with an aryl group having 6 to 30 carbon atoms.

In the present invention, “alkylamino group” means an amine substituted with an alkyl group having 1 to 30 carbon atoms.

In the present invention, “aralkyl amino group” refers to an amine substituted with an aryl-alkyl group having 6 to 30 carbon atoms.

In the present invention, “heteroarylamino group” refers to an amine group substituted with an aryl group or heterocyclic group having 6 to 30 carbon atoms.

In the present invention, “heteroaralkyl group” refers to an aryl-alkyl group substituted with a heterocyclic group.

In the present invention, “cycloalkyl” refers to a monovalent substituent derived from a monocyclic or polycyclic non-aromatic hydrocarbon having 3 to 40 carbon atoms. Examples of such cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and adamantine.

In the present invention, “heterocycloalkyl” refers to a monovalent substituent derived from a non-aromatic hydrocarbon having 3 to 40 carbon atoms, and at least one carbon in the ring, preferably 1 to 3 carbons, is N, O, S or Se. It is substituted with a hetero atom such as Examples of such heterocycloalkyl include, but are not limited to, morpholine and piperazine.

In the present invention, “alkylsilyl” refers to silyl substituted with alkyl having 1 to 40 carbon atoms, and “arylsilyl” refers to silyl substituted with aryl having 6 to 60 carbon atoms.

In the present invention, “condensed ring” means a condensed aliphatic ring, a condensed aromatic ring, a condensed heteroaliphatic ring, a condensed heteroaromatic ring, or a combination thereof.

In the present invention, "forming a ring by bonding with adjacent groups" means a substituted or unsubstituted aliphatic hydrocarbon ring by bonding with adjacent groups; Substituted or unsubstituted aromatic hydrocarbon ring; Substituted or unsubstituted aliphatic heterocycle; Substituted or unsubstituted aromatic heterocycle; Or it means forming a condensation ring thereof.

In the present invention, examples of “aromatic hydrocarbon rings” include phenyl groups, naphthyl groups, and anthracenyl groups, but are not limited to these.

In the present invention, “aliphatic heterocycle” refers to an aliphatic ring containing one or more heteroatoms.

In the present invention, “aromatic heterocycle” refers to an aromatic ring containing one or more heteroatoms.

In the present invention, "substitution" means changing a hydrogen atom bonded to a carbon atom of a compound to another substituent. The position to be substituted is not limited as long as it is the position where the hydrogen atom is substituted, that is, a position where the substituent can be substituted, and if two or more substituents are substituted. , two or more substituents may be the same or different from each other. The substituents include hydrogen, cyano group, nitro group, halogen group, hydroxy group, alkyl group with 1 to 30 carbon atoms, alkenyl group with 2 to 30 carbon atoms, alkynyl group with 2 to 24 carbon atoms, heteroalkyl group with 2 to 30 carbon atoms, and 6 to 6 carbon atoms. Aralkyl group of 30, aryl group of 5 to 30 carbon atoms, heteroaryl group of 2 to 30 carbon atoms, heteroarylalkyl group of 3 to 30 carbon atoms, alkoxy group of 1 to 30 carbon atoms, alkylamino group of 1 to 30 carbon atoms, 6 to 6 carbon atoms Arylamino group of 30, aralkylamino group of 6 to 30 carbon atoms, heteroarylamino group of 2 to 24 carbon atoms, substituted or unsubstituted alkylsilyl group of 1 to 30 carbon atoms, substituted or unsubstituted arylsilyl group of 6 to 30 carbon atoms and substituted Alternatively, it may be substituted with one or more substituents selected from the group consisting of unsubstituted aryloxy groups having 6 to 30 carbon atoms, but is not limited to the above examples.

The present invention can improve and prevent psychotic symptoms by improving impaired pre-stimulus inhibition and impaired social interaction, which are schizophrenia-like behaviors.

In addition, it has an excellent effect of inhibiting the activity of monoamine oxidase and inhibiting self-aggregation of amyloid beta, so it has excellent tension relieving or antidepressant effects and can prevent the occurrence of side effects.

Figure 1 is an image of the combination of coumestrol and hMAO-A according to an embodiment of the present invention.
Figure 2 shows the results of an experiment on whether Aβ25-35 self-aggregates with plant chemicals according to an embodiment of the present invention.
Figure 3 shows the results of an experiment on whether Aβ25-35 self-aggregates with phytochemicals according to an embodiment of the present invention.
Figure 4 shows the results of an experiment on the pre-stimulus inhibition ability of the composition of the present invention according to an embodiment of the present invention on an animal model of pre-stimulus inhibition impairment induced by MK-801.
Figure 5 shows the results of an experiment on the recovery of social cognition in an animal model with social cognitive impairment induced by MK-801 using the composition of the present invention according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement it. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.

In this specification, “active ingredient” refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

Schizophrenia is a mental disorder that affects less than 1% of the general population and is a widespread endogenous disease that involves changes in the thinking, perception, and behavior of those affected.

The symptoms of schizophrenia include negative symptoms characterized by lack of voluntary behavior, severe maladaptation, monotonous emotions, lack of interpersonal relationships, and abnormal thinking, and positive symptoms such as psychomotor excitement, delusions, auditory hallucinations and hallucinations, and cognitive impairment. It is classified as

The cause of schizophrenia is related to an imbalance of dopamine, a neurotransmitter in the brain, and damage to the frontal lobe, and is known to be a result of genetic causes, prenatal environment, social causes, or drug abuse.

The treatment of schizophrenia involves drug treatment centered on tranquilizers, separate from psychopsychological treatment. Existing typical antipsychotic drugs focus on treating positive symptoms such as hallucinations, delusions, and confusion.

These drugs are known to be useful as dopamine antagonists, and most require long-term administration of these drugs over a year, specifically haloperidol, chlorpromazine, and fluphenazine. It is being used.

However, these drugs not only have many side effects such as movement disorders, involuntary muscle spasms, emotional blunting, fatigue, lack of drive, and weight gain, but also have limitations in that they cannot treat vocal symptoms and cognitive impairment.

Recent atypical antipsychotics target the limbic system and block dopamine D2 receptors. They have fewer side effects than traditional antipsychotics and treat both positive and negative symptoms.

Examples of these atypical antipsychotics include clopazine, risperidone, asenapine, olanzapine, and iloperidone (Kay et al., 1987; Lieberman et al., 2005; Molteni et al., 2009; Howes and Kapur, 2009).

However, in the case of olazapine, diabetes has recently been reported as a side effect. This confirmed that olazanpine can cause atypical diabetes by causing structural abnormalities in proinsulin in the endoplasmic reticulum.

As mentioned above, although atypical antipsychotics are known to have fewer side effects than typical existing antipsychotics, their continued use may be problematic because they do not have any side effects.

Monoamine oxidase (MAO) catalyzes the oxidation of biological and heterogeneous amines, including primary (dopamine, serotonin, norepinephrine, etc.), secondary (adrenaline), and tertiary amines to form H ₂ O ₂ and aldehydes. A group of flavoenzyme enzymes that produce Therefore, the monoamine oxidase plays an important role in regulating monoamine levels in the central nervous system (CNS).

Among the two isomers (types A and B) of monoamine oxidase, MAO-A shows substrate selectivity for serotonin, melatonin, norepinephrine, and epinephrine, and MAO-B represents substrate selectivity for phenylethylamine and benzylamine. However, the two isomers are known to non-selectively catalyze dopamine and tyramine.

Therefore, MAO-A is widely used as a drug target for anxiety disorders such as depression, and MAO-B is widely used as a drug target for neurodegenerative diseases such as Parkinson's disease (PD).

However, MAO inhibitors (MAOIs) are not popular as CNS drug candidates due to side effects such as high blood pressure due to the 'cheese effect' caused by non-selective and non-selective MAOIs. However, a selectively reversible MAOI (moclobemide) that does not cause the ‘cheese effect’ has also been developed and does not require dietary restrictions.

MAO-B levels were found to be increased in the brains of Alzheimer's patients, and MAO-B activation increased amyloid beta (Aβ) production through gamma-secretase activation.

Aggregated Aβ is a major component of senile plaques, the main cause of Alzheimer's disease, and is being widely studied as a biomarker for developing treatments for Alzheimer's disease. Therefore, it is essential to develop compounds that affect MAO activation and Aβ production/aggregation.

The present invention relates to a novel compound that can be used as an antipsychotic, has no side effects even when taken for a long time, and can affect MAO activation and Aβ production/aggregation.

Additionally, the novel compounds can be found in natural materials such as arrowroot, soybeans, alfalfa sprouts, clover, and bean sprouts.

In other words, since it is a compound derived from natural materials, it not only has no side effects, unlike synthetic drugs previously taken to improve mental health, but can also have the same or improved mental health improvement effect as synthetic drugs.

Specifically, the composition for improving mental health according to an embodiment of the present invention may include a compound represented by the following Chemical Formula 1 as an active ingredient:

[Formula 1]

here,

n and m are the same or different from each other and are each independently an integer from 0 to 4,

R ₁ and R ₂ are the same or different from each other, and are each independently hydrogen, deuterium, cyano group, nitro group, halogen group, hydroxy group, substituted or unsubstituted alkylthio group having 1 to 4 carbon atoms, substituted or unsubstituted carbon number Alkyl group of 1 to 30 carbon atoms, substituted or unsubstituted cycloalkyl group of 3 to 20 carbon atoms, substituted or unsubstituted alkenyl group of 2 to 30 carbon atoms, substituted or unsubstituted alkynyl group of 2 to 24 carbon atoms, substituted or unsubstituted Aralkyl group with 7 to 30 carbon atoms, substituted or unsubstituted aryl group with 6 to 30 carbon atoms, substituted or unsubstituted heteroaryl group with 1 to 60 carbon atoms, substituted or unsubstituted heteroarylalkyl group with 2 to 30 carbon atoms, substituted or an unsubstituted alkoxy group having 1 to 30 carbon atoms, a substituted or unsubstituted alkylamino group having 1 to 30 carbon atoms, a substituted or unsubstituted arylamino group having 6 to 30 carbon atoms, or a substituted or unsubstituted aralkyl having 7 to 30 carbon atoms. Amino group, substituted or unsubstituted heteroarylamino group having 1 to 24 carbon atoms, substituted or unsubstituted alkylsilyl group having 1 to 30 carbon atoms, substituted or unsubstituted arylsilyl group having 6 to 30 carbon atoms, and substituted or unsubstituted carbon atoms. It is selected from the group consisting of 6 to 30 aryloxy groups.

Where n and m are 1, R ₁ and R ₂ may be hydroxy groups.

Specifically, the compound represented by Formula 1 may be a compound represented by Formula 2 below:

[Formula 2]

The compound represented by Formula 2 may specifically be coumestrol.

As will be described later, the coumestrol extract has an excellent effect of inhibiting the activity of monoamine oxidase and inhibiting amyloid beta self-aggregation, so it can exhibit excellent effects in alleviating tension or improving depression.

Additionally, in animal models treated with NMDA receptor antagonists, excellent effects on impaired pre-stimulus inhibition and impaired social interaction were confirmed.

“Functional food” as defined herein refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Functional Food. “Functional” refers to the structure of the human body and It means ingestion for the purpose of controlling nutrients for function or obtaining useful health effects such as physiological effects.

A pharmaceutical composition according to another embodiment of the present invention may include the composition for improving mental health.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, for a desirable effect, the pharmaceutical composition is preferably administered at 0.01 mg/kg to 10 g/kg per day, preferably at 1 mg/kg to 1 g/kg. Administration may be administered once a day, or may be administered in several divided doses. Therefore, the above dosage does not limit the scope of the present invention in any way.

Experiment example

Materials and Methods

chemical substance

Human monoamine oxidase (hMAO) isozymes, robinin, rutin, nicotiflorin, daidzin, genistin, Coumestrol, daidzein, genistein, curcumin, amyloid _ peptide 25-35, A_25-35, hexafluoroisopropanol, HFIP) and selegiline hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA).

In vitro hMAO inhibition assay

The inhibitory potential of phytochemicals against MAO was evaluated using luminescence method. Various doses of phytoestrogens and 5% 1,2,3-propanetriol for MAO-A and 5% 1,2,3-propanetriol for MAO-B in 0.1 M HEPES buffer (pH 7.5). Propanetriol and 10% dimethyl sulfoxide were mixed with substrate (160 μM for MAO-A, 16 μM for MAO-B) and 0.5 g of enzyme and incubated at 25°C. After 1 hour of incubation, reconstituted luciferin-detection reagent was added to the reaction mixture and incubated at 25°C for 20 minutes. Afterwards, the luminescence signal was monitored using a microplate reader (Biotech, Winoski, VA, USA). Selegiline hydrochloride (final concentration: 6.25 to 25 μM) was used as a positive control.

Kinetic analysis for hMAO-A

The type of inhibition exhibited by coumestrol was analyzed based on changes in enzyme activity according to various concentrations of substrate and inhibitor. The experimental method was the same as that used in the hMAO inhibition assay, using 0.5 μg of enzyme, 40-160 μM of substrate, and 0, 2.5, 10, and 20 μM of coumestrol. Based on the results of the kinetic analysis, a Lineweaver-Burk plot (LB plot) was created using Sigmaplot 12.0, and an auxiliary plot of the LB plot (LB-secondary plot) was created using the EK macro module for exploration.

The equation for the LB plot is:

The equation for the LB-2th plot is:

Aβ25-35 autoaggregation assay

Aβ25-35 self-aggregation assay was performed by pretreating the peptide with HFIP for 1 day at 22°C to obtain the non-amyloidogenic form. Various doses of phytoestrogens were mixed with 0.1mM monomeric Aβ25-35 solution at a 1:29 ratio (v/v) in 34.5mM phosphate buffer (pH 7.4) containing 17.5% MeCN and incubated overnight at 4°C. After incubation, the reaction mixture was supplemented with 0.025mM thioflavin-T in 50mM glycine-NaOH buffer (pH 8.5).

Fluorescence emission was monitored using Gemini XPS (Molecular Devices, Sunnyvale, CA, USA) with excitation at 490 nm to 446 nm. Curcumin was used as a standard material.

In Silico Docking Simulation

The X-ray crystallographic structure of hMAO-A was obtained from PDB (ID 2z5x). Water and co-ligands were removed from the crystallographic structure using Discovery Studio (v17.2, Accelrys, San Diego, CA, USA). The 3D structure of coumestrol was created using Marvin Sketch (v17.1.30, ChemAxon, Budapest, Hungary). Fifteen docking postures were created with the same parameters using AutoDock 4.2. Results were scrutinized using Discovery Studio v17.2.

Pharmacokinetic parameter prediction

The pharmacokinetic properties of coumestrol were calculated using Marvin Sketch (v17.1.30) and PreADMET server v1.0 (https://preadmet.bmdrc.kr/, accessed on June 1, 2022).

Selection of test animals

Male ICR mice 5 weeks old (26-28 g) were supplied from Orient Co., Ltd. (Sangdaewon-dong, Jungwon-gu, Seongnam-si, Gyeonggi-do). The experimental animals were acclimated for one week, and during the acclimation period, the temperature in the animal room was maintained at a constant temperature of 23 ± 1°C, humidity around 60 ± 10%, and a 12-hour light/dark cycle. The experimental animals were provided with unlimited food and water during the adaptation period, and this experiment was performed with animal approval (approval number: KHUASP-22-530) from the Institutional Animal Care and Use Committees (IACUC).

MK-801 induction model and dosing

MK-801 is a non-competitive NMDA receptor antagonist (Foster and Fagg, 1987; Huettner and Bean, 1988) and acts on GABAergic interneurons, and due to its action and specificity for NMDA receptors, it causes hyperactive locomotor activity and reduced social interaction in experimental animals. and can induce impairments in cognitive flexibility, latent learning, long-term spatial memory, working memory, and sensorimotor gating (Rung et al., 2005; Avecawa et al, 2007; Manahan-Vaughan et al., 2008; Zou et al. ., 2008; Wiescholleck and Manahan-Vaughan, 2012). From the results of the previous study, MK-801 was administered intraperitoneally 30 minutes before the start of the behavioral test at a dose of 0.2 mg/kg, and samples 1 to 4 (samples 1 to 3 were coumestrol 1 mg/kg, 3 mg/kg, and 10 mg/kg, For sample 4, aripiprazole (1 mg/kg) was administered orally 1 hour before the behavioral test.

Acoustic startle response test (ASR)

It is known that in the sound startle response test, prepulse inhibition (PPI) is not achieved in a schizophrenia-like animal model administered MK-801, an NMDA receptor antagonist.

Based on this fact, it is possible to determine whether a test drug is effective in an animal model of schizophrenia by providing sound stimulation to experimental animals in a startle box and then measuring the response.

The startle box consists of a box in which the rat can be lightly detained, a system that can produce sound stimulation consistently, equipment that can measure the animal's movements, a program that can digitize the measured stimulus, a source that can provide sound, and It is composed of a speaker, and for each test, the rat was acclimatized to the startle box for 5 minutes, and a sound stimulus of 80, 90, 100, 110, or 120 dB was provided to measure the animal's response to each stimulus.

Then, in the prestimulus suppression test, a prestimulus of 73, 76, 82, or 86 dB [stimulus of 3, 6, 12, or 16 dB and white noise (70 dB)] was given for 20 ms, followed by 80 ms for 40 ms. A sound stimulus of 120 dB was provided. After measuring the animal's startle response to each stimulus, the degree of pre-stimulus inhibition (PPI) was determined by measuring the response ratio between a situation in which only 120 dB was stimulated (SS) and a situation in which 120 dB was stimulated after pre-stimulation (PP). was calculated, and the calculation formula is as follows:

% prestimulus inhibition (PPI) = (SS - PP)/SS × 100

As a positive control, 1 mg/kg of Aripiprazole, which is known to be effective in treating mental illness, namely schizophrenia, was administered.

Sociability test (SI)

The social interaction test using three chambers is a representative behavioral experiment that measures general sociability and social novelty. Rodents prefer to live with other animals and new animals rather than being isolated alone. Based on their preferred habits, they were allowed to freely explore a 3-chamber space for 5 minutes.

Afterwards, an unfamiliar rat was placed in one space, the time spent exploring the unfamiliar rat was measured, and a new rat was placed in the opposite empty space for 10 minutes to evaluate social interaction (SI).

As a positive control, 1 mg/kg of Aripiprazole, which is known to be effective in treating mental illness, namely schizophrenia, was administered.

statistical analysis

The 50% inhibitory concentration (IC50) calculated from the dose-inhibition curve was expressed as the mean_standard deviation (SD) of three independent experiments. The statistical significance of the group treated with the test compound compared to the control group for the Aβ25-35 self-aggregation assay was calculated using Student's t-test (Microsoft Excel 2019, Microsoft Corporation, Seattle, WA, USA).

Experiment result

Inhibitory activity of phytochemicals against human MAO isozymes.

The MAO assay protocol was validated using selegiline hydrochloride as a positive control. Additionally, because factors such as organic solvents, metal ions (e.g. Cu ²⁺ , Cd ²⁺ , Al ³⁺ ), pH, and temperature affect MAO activity, the analysis was performed in a controlled laboratory environment. The optimal conditions for the enzyme reaction, including reaction time, temperature, pH, and concentration of enzyme and substrate, were established based on previous studies. All test samples were diluted in reaction buffer containing up to 10% DMSO, and solvent controls were tested together for each assay to minimize false positive/false negative effects. The MAO inhibition efficacy of components in phytochemicals was evaluated. Among the compounds tested, coumestrol was the most effective MAO-A inhibitor with an IC50 of 1.99 μM.

However, the coumestrol showed a moderate inhibitory effect on MAO-B with an IC50 of 77.79 (selective index (SI) = 0.02). As shown in Table 1 below, genistein showed excellent inhibitory activity against both MAO isoenzymes, with IC50 values of 4.77 and 3.42 μM for MAO-A and MAO-B, respectively.

On the other hand, daidzein, which has a structure similar to genistein without a hydroxyl group at the C5 position, showed a weak inhibitory effect on both MAO isoenzymes (IC50 values of 304.05 and 356.87 μM for MAO-A and MAO-B, respectively). It has been proven through conventional enzyme kinetics studies and docking simulations that genistein exhibits excellent inhibitory activity against MAO, and these results were consistent with the above experimental results. Additionally, rutin showed weak MAO-A inhibitory activity, while the other substances showed no activity against both MAO isoenzymes at the concentrations tested.

Table 3 below shows the results of experiments on the inhibitory activity of phytochemicals against hMAO.

a. Selectivity index (SI) = hMAO-A IC ₅₀ /hMAO-B IC ₅₀ .

b. Positive control.

Competitive inhibition of hMAO-A by coumestrol

Through the experiments of the present invention, the strong and selective inhibitory activity of coumestrol against MAO-A was revealed. Therefore, enzyme kinetics analysis was performed to identify the mode of inhibition of MAO-A by coumestrol. Similar to previous studies, it was confirmed that the lower the concentration of substrate, the lower the enzyme reaction rate in the control group (treated with buffer solution only).

As shown in Figure 1A, the y-intercept of each linear regression did not change even when the substrate and coumestrol concentrations were changed, and the above kinetic pattern is a typical kinetic pattern of a competitive inhibitor.

According to the above quadratic plots (Figures 1B and 1C), the inhibition constant of coumestrol on MAO-A was calculated to be 1.32 μM (Table 2).

In addition, in silico docking simulations were performed to predict the binding sites of coumestrol and MAO-A and key residues that affect binding.

As shown in Figures 1D and 1E, the coumestrol was stably docked at a site known to be the main active site of MAO-A. The ketone moiety of coumestrol is hydrogen bonded (2.16 ) interacted with the nitrogen at position 5 of the isoalloxazine ring of FAD, and the hydroxyl moiety of the B ring of coumestrol and the Tyr444 residue of MAO-A formed a hydrogen bond (1.85 ) was formed. Additionally, coumestrol formed pi-pi bonds with Phe352 and Tyr407. Therefore, the strong hydrogen bond and pi-pi interaction as described above may have influenced the binding ability of coumestrol and MAO-A.

suppression mode
(Inhibition
Mode) suppression constant
(Inhibition Constant, K _i , μM) binding energy
(Binding energy, kcal/mol) ^a Hydrogen-bond interaction residues
(H-Bond Interaction Residues) Other interacting residues
(Other interaction residues) ^b Coumestrol Competitive 1.32 -9.63 FAD, Tyr444 FAD and Phe352 (π-π T shaped), Tyr407 and Tyr444 (π - π stacking), Ile335, and
Leu337 (π-alkyl) Harmine ^c N.D. N.D. -8.43 N.D. Tyr407 (π - π stacking, π -Alkyl),FAD (Van derWaals), Cys323
(π -sulfur), Ile335 (π -π,
π -alkyl), Tyr444, Ile180, and
Leu337 π_-alkyl)

a. Determined by Autodoc 4.2.b. Determined by Discovery Studio v17.2.

c. Co-ligand of human MAO-A (2z5x) obtained from the Protein Data Bank.

ND not detected.

Inhibitory activity of phytoestrogens on Aβ25-35 autoaggregation

To evaluate the inhibitory activity on Aβ25-35 self-aggregation based on the fluorescence of thioflavin-T and its inhibitory activity against MAO and curcumin (a well-known inhibitor of A_25-35/1-42 self-aggregation). Four types of phytoestrogens that showed were investigated.

Based on previous studies, optimal conditions for Aβ25-35 aggregation were established, including the ratio of Aβ25-35 to inhibitor, temperature, reaction time, and pH. Figure 5A shows the inhibitory activity (%) of the four phytoestrogens and curcumin on autologous Aβ25-35 aggregation after 24 hours of incubation.

When treated with 100 μM coumestrol, the degree of Aβ25-35 self-aggregation was reduced by 76.14% compared to the control group. Additionally, 100 μM rutin, daidzein, and genistein reduced Aβ25-35 self-aggregation by 49.31%, 35.54%, and 34.90%, respectively. As shown in Figure 2B, coumestrol inhibited Aβ25-35 aggregation in a dose-dependent manner with an IC50 value of 37.40 ± 1.70 μM, while curcumin showed an IC50 value of 10.57 ± 1.31 μM.

Pharmacokinetic parameters of coumestrol

Pharmacokinetic parameters were predicted based on the chemical structure of coumestrol. As a result of the pharmacokinetic analysis of coumestrol, the blood-brain barrier (BBB) permeability score of coumestrol was 0.76, confirming that coumestrol can pass through the BBB and be appropriately absorbed into the CNS.

Additionally, coumestrol showed a human intestinal absorption (HIA) of 93.51%, which means that coumestrol can be easily absorbed into the human intestine and is suitable for oral delivery.

According to the CMC-like rule, which is the lipophilicity index of a drug, the log P of a neurotropic drug should be between 1.3 and 4.1. As can be seen in Table 4 below, the Log P _o/w value of coumestrol is 2.94. Additionally, coumestrol was predicted to be a non-inhibitor of p glycoprotein. Analysis of mutagenic properties predicted that coumestrol was not carcinogenic to rats and mice. The predicted CNS MPO scores suggested that coumestrol has appropriate CNS drug-like properties based on several physicochemical properties (partitioning coefficient, number of hydrogen bond donors, pKa, molecular size, and topological polarity surface area values). Therefore, these predicted pharmacokinetic properties make coumestrol an attractive CNS drug candidate for MAO inhibition and self-assembly of Aβ peptides.

Table 3 below shows the pharmacokinetic properties of coumestrol:

predicted values Log P _o/w ^a 2.94 BBB penetration ^b 0.76 HIA ^c 93.51 P-glycoprotein Non-inhibition Carcino-rat/mouse Negative CNS MPO score ^d 5.24

a. The ratio of 1-octanol to water in the logarithm of the solvent partition coefficient.b. Absorption levels below 0.1 are considered low, between 0.1 and 2.0 are considered moderate, and above 2.0 are considered good.

c. Absorption levels of 0-20% are considered low, 20-70% moderate, and 70-100% good.

d. CNS multiparameter optimization (MPO), >4.0: desirable score.

In the present invention, the inhibitory effect of MAO by plant chemicals was confirmed. According to the above experimental results, coumestrol showed the best inhibitory effect on MAO-A, followed by genistein, daidzein, and rutin. For MAO-B, genistein showed the strongest inhibitory effect, followed by coumestrol and daidzein.

On the other hand, the remaining components did not show significant inhibitory effects on the two isozymes. Additionally, we discovered for the first time that coumestrol acts as a selective and competitive MAO-A inhibitor.

Cumestrol is a coumestan derivative found in soybeans, alfalfa (Medicago sativa), and Trifolium. The coumestrol is a phytoestrogen with antioxidant, anti-inflammatory and estrogenic activities, and is biosynthesized from daidzein, a representative isoflavone abundant in legumes. According to previous experimental results, psoralidin extracted from hazel seeds, which additionally has a prenyl moiety at the C2 position of coumestrol, does not show a significant inhibitory effect on MAO-A or MAO-B. In another experimental result, glycirol extracted from licorice root, in which a prenyl group is added at the C2 position and a methoxyl group is added at the C3 position of coumestrol, was confirmed to have an IC50 value of 29.5 μM for MAO-B (MAO No activity below 40 μM for -A). In the present invention, coumestrol, unlike psoralidin and glycirol, showed a strong and selective inhibitory effect on MAO-A at low concentration (SI = 0.02). This means that the functional groups attached to C2 and C3 of psoralidin and glycirol form unnecessary interactions with other amino acid residues of MAO-A, preventing the ligand from docking at the active site of the enzyme, thereby lowering the inhibitory activity of the enzyme. It is estimated.

However, in silico docking simulation analysis showed that coumestrol forms hydrophobic or hydrogen bonds with the active site residues (Tyr444, Tyr407, and Phe352) of MAO-A and the cofactor FAD, thereby stably docking at the substrate binding site.

In addition, it was confirmed that coumestrol interacts with Ile335, which represents the main structural difference between MAO isoforms, through a hydrophobic bond (p-alkyl), affecting the selectivity for MAO-A.

Additionally, through analysis of enzyme kinetics depending on coumestrol and substrate concentration, it was revealed that coumestrol acts as a competitive inhibitor. According to the linear Uber-Burk plot and its auxiliary plots, V remained constant despite changes in substrate or coumestrol concentration, which corresponds to a typical pattern of competitive inhibition. Therefore, taking coumestrol may not cause the side effects (e.g. high blood pressure) associated with the ‘cheese effect’ of non-selective MAOI intake. However, additional reversibility analysis is needed to confirm whether coumestrol is a reversible inhibitor.

Additionally, changes in thermal stability of the free enzyme and inhibitor-enzyme complex should be scanned to determine the thermodynamic effect of the inhibitor on the enzyme.

Thioflavin-T assay for inhibitory compounds against MAO (rutin, daidzein, genistein, coumestrol) was used to investigate the inhibitory effect on self-aggregation of Aβ, which is known to cause neuroinflammation and Alzheimer's disease. did. Cumestrol showed the strongest inhibitory effect with an IC50 of 37.40 μM. In a previous study, it was known that nanomolar concentrations of coumestrol showed a neuroprotective effect by significantly reducing Aβ-induced cytotoxicity and inflammatory cytokine levels in mouse astrocytes.

Additionally, coumestrol also showed an in vitro inhibitory effect on β-secretase, which influences Aβ formation. Therefore, the inhibitory action of coumestrol on Aβ self-aggregation and β-secretase may maintain the Aβ peptide in a soluble monomeric state, thereby promoting the clearance of the peptide from the brain through normal physiological mechanisms.

In the present invention, the potential of coumestrol as a central nervous system drug for the treatment of Alzheimer's disease and depression was confirmed through in vitro and in silico approaches, and its pharmacokinetic properties were also predicted to support its potential. Cumestrol has lipophilicity suitable for use as a nervous system drug and is predicted to have normal BBB permeability.

In addition, it was demonstrated that the structure of coumestrol is suitable for development as a CNS drug based on the CNS MPO score, considering several general physicochemical properties. Several studies have shown that Aβ1-40/1-42 reduces the expression of p-glycoprotein at the BBB, thereby reducing Aβ in the brain. prevented the removal of. Therefore, BBB-permeable coumestrol inhibits Aβ aggregation and inhibits Aβ in the brain. It may be a potential therapeutic drug that acts to improve clearance.

Efficacy of coumestrol in improving prestimulus inhibition impairment in psychiatric disease-induced animal model

An animal model with prestimulus inhibition impairment induced by MK-801 (0.2 mg/kg) was used in the experiment, and an acoustic startle response test was performed to evaluate the degree of prepulse inhibition. did.

The experimental results are shown in Figure 4.

According to FIG. 4, it was confirmed that pre-stimulus inhibition (P<0.001) was significantly decreased in the MK-801-induced pre-stimulus inhibition impaired animal model compared to the control group, and the deficiency in the group administered 10 mg/kg of kumetstol was confirmed. It was confirmed that the pre-stimulation inhibition ability was significantly improved, and the degree was similar to that of the positive control group.

Efficacy of coumestrol in improving social interaction in an animal model of induced mental illness

Figure 5 shows the results of an experiment on the efficacy of coumestrol to improve social interaction in an animal model of social cognitive impairment induced by MK-801. According to the above experiment results, general sociability was improved by social interaction in both the normal group and the administered group. It was confirmed to work.

In social novelty, it was confirmed that the preference for unfamiliar rats was decreased in the group administered MK-801 alone compared to the control group, and it was confirmed that social novelty was restored to the normal level at all doses due to the administration of coumestrol. It was similar to the positive control group.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims are also possible. falls within the scope of rights.

Claims (8)

It is a composition for improving mental health containing a compound represented by the following formula (2) as an active ingredient,
The above mental health refers to schizophrenia, schizophreniform disorder, schizoaffective disorder, delusional disorder, or shared psychotic disorder.
Compositions for improving mental health:
[Formula 2]
delete According to paragraph 1,
The compound represented by Formula 2 has an excellent effect of inhibiting the activity of monoamine oxidase and inhibiting amyloid beta self-aggregation.
Composition for improving mental health. According to paragraph 1,
The compound represented by Formula 2 can improve pre-stimulus inhibition impairment and social interaction impairment.
Composition for improving mental health. According to paragraph 1,
The composition for improving mental health passes through the blood-brain barrier and is absorbed into the central nervous system (CNS).
Composition for improving mental health. According to paragraph 1,
The composition for improving mental health exhibits excellent human intestinal absorption (HIA) and can be taken by oral administration.
Composition for improving mental health. Containing a composition for improving mental health according to any one of paragraphs 1, 3 to 6.
Functional food composition. Containing a composition for improving mental health according to any one of paragraphs 1, 3 to 6.
Pharmaceutical composition.