KR102336520B1 - Compositon for preventing or treating attention deficit hyperactivity disorder comprising luteolin - Google Patents

Abstract

The present invention is a signal transmission of the compounds with antagonistic activity on the dopamine D ₄ receptors and dopamine D ₄ receptor relates to a composition for preventing or treating attention deficit hyperactivity disorder, including luteolin (luteolin), as an active ingredient By inhibiting it, it can be usefully used for the prevention or treatment of attention deficit hyperactivity disorder (ADHD).

Description

A composition for preventing or treating attention deficit hyperactivity disorder comprising luteolin TECHNICAL FIELD

The present invention relates to a composition for preventing or treating attention deficit hyperactivity disorder comprising, as an active ingredient, a milk thistle extract, a fraction thereof, or a compound isolated therefrom, luteolin.

Attention Deficit Hyperactivity Disorder (hereinafter referred to as "ADHD") is a well-known neurodevelopmental disorder that exhibits attention deficit, hyperactivity, impulsivity, cognitive developmental disorders, language development disorders, memory and executive functions, etc. It is a kind of disability. ADHD has the highest prevalence among neurodevelopmental disorders, with about 5% of school-age children worldwide showing ADHD-related symptoms. If the symptoms of ADHD appearing in childhood are left untreated, various problems continue to occur during childhood. In addition, ADHD persists into adulthood with a rate of 40-60%, which not only causes difficulties in maintaining a job and marriage, but also increases the likelihood of alcohol and other substance abuse and dependence, antisocial behavior, delinquency, and crime, as well as problems with school violence and bullying. Problems related to suicide in adolescents and adolescents often coexist with ADHD.

The causes of ADHD include genetic factors, biochemical factors viewed as a reaction to lead levels and food additives in instant foods, the view that there is a problem in properly selecting the stimulus to be transmitted to the brain, and environmental factors, i.e. the relationship between parents and children I mentioned the social status of my parents and the mother's smoking and alcohol abuse during pregnancy, but the cause is still divided. However, neurobiological factors are considered to be more important than sociopsychological factors, and studies on drug treatment and biological factors for this disease are being actively conducted. In particular, as a biological factor, rather than a single nervous system developmental abnormality, the possibility of a non-uniform disease group caused by an abnormality in the correlation of various brain regions responsible for high-level cognitive functions is suggested. According to structural and functional brain imaging studies of children with ADHD until recently, the pathophysiology of ADHD is generally associated with dysfunction of the fronto-striatal tract, and methylphenidate (MPH) ) is known to be related to changes in dopaminergic function in this area. In particular, in the ADHD cause, and is reported as dopaminergic excessive neurotransmission caused by the D ₄ receptor in a variety of dopamine receptors, D ₄ difference in dopamine neurotransmission receptors individual D ₄ receptor gene (dopamine D4 receptor gene) was reported to be due to polymorphic mutation. In addition to dopaminergic neurons, ADHD symptoms are related to the regulation of noradrenergic neurons that control neural circuits in the frontal lobe. It has studied the behaviors of ADHD that cause an imbalance between dopamine and dopamine neuronal regulation of the acceptance by the Norwegian adrenal nerve shot and D ₄ receptor have been reported.

KR 10-2011-0055771 A

An object of the present invention is to provide a pharmaceutical composition for preventing or treating attention deficit hyperactivity disorder comprising luteolin as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving attention deficit hyperactivity disorder comprising luteolin as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating attention deficit hyperactivity disorder comprising a milk thistle extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving attention deficit hyperactivity disorder, comprising a milk thistle extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a method for inhibiting signal transduction by _{dopamine D 4} receptor, comprising the step of treating a sample with luteolin in vitro.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating attention deficit hyperactivity disorder comprising luteolin as an active ingredient.

In one embodiment of the present invention, the luteolin may be isolated from a milk thistle ( Cirsium japonicum var. maackii (Maxim.) Matsum) extract or a fraction thereof, but is not limited thereto, and is known in the art. Chemically synthesized or commercially available materials can be used in the prescribed method.

In one embodiment of the present invention, the luteolin may be a dopamine D ₄ receptor antagonist (antagonist).

In addition, the present invention provides a health functional food for preventing or improving attention deficit hyperactivity disorder comprising luteolin as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating attention deficit hyperactivity disorder comprising a milk thistle extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving attention deficit hyperactivity disorder comprising a milk thistle extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a method of inhibiting signal transduction by _{dopamine D 4} receptor, comprising the step of treating a sample with luteolin in vitro.

Luteolin derived from thistle ( Cirsium maackii ( Cirsium japonicum var. maackii (Maxim.) Matsum)) of the present invention was confirmed to act antagonistically on _{dopamine D 4} receptor (D _{4 R) in an in vitro experiment,} It has been confirmed that it is effective in attention deficit hyperactivity disorder (ADHD), a disease related to dopamine D _{4 , and can be usefully used as an effective treatment for ADHD.}

1 shows the chemical structural formula of luteolin or luteolin glycosides.
Figure 2a will showing the binding patterns of luteolin (in yellow), a square or a Pride (black) and clozapine (purple) to the human D ₄ R, 2b will showing the binding patterns of luteolin to human D ₄ R , 2c is a schematic of the 2D binding interaction between _{human D 4 R and luteolin.}

The present invention provides a pharmaceutical composition for preventing or treating attention deficit hyperactivity disorder comprising luteolin as an active ingredient.

The compound of the present invention, luteolin, refers to a compound represented by the following formula (1). The compound of the present invention may be isolated from a milk thistle ( Cirsium maackii ( Cirsium japonicum var. maackii (Maxim.) Matsum)) extract or a fraction thereof, but is not limited thereto, and is chemically synthesized or commercially available by a method known in the art. material can be used.

[Formula 1]

In the above formula, R ₁ and R ₂ are hydroxy groups (OH).

As used herein, the term "agonist (agonist)" refers to a substance that binds to a receptor and activates a receptor to produce a biological response, and "antagonist" binds to a receptor like the agonist, but as opposed to an agonist. A substance that inhibits a receptor in order to inhibit a biological response.

As used herein, the term "dopamine receptors" is a superfamily of G-protein-coupled receptors (GPCRs), which promotes the signal transduction of dopamine, a catecholamine-based neurotransmitter, and based on the similarity in signal transduction It _{is classified into D 1} -like (D ₁ R, D ₅ R) and D ₂ -like (D ₂ R, D ₃ R, D ₄ R) subtypes (Butini et al., 2016).

The compounds of the invention may be the antagonist (antagonist) of the dopamine D ₄ receptor, it is effective to inhibit the signal transduction by the dopamine D ₄ receptor, it can be used in the treatment of attention deficit hyperactivity disorder (ADHD).

In addition, the present invention provides a composition for preventing or treating attention deficit hyperactivity disorder comprising a milk thistle extract or a fraction thereof as an active ingredient.

In the present invention, the milk thistle extract may be extracted with water, an organic solvent, or a mixture thereof, and the organic solvent is methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride , may be any one or more selected from the group consisting of hexane and cyclohexane, preferably methanol, but is not limited thereto.

As the extraction method, any one of methods such as hot water extraction, cold extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process, and may be purified using a conventional purification method. For example, the primary extract is further purified using various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. fractions may be obtained. There is no limitation on the method for preparing the extract or fraction of the present invention, and any known method may be used.

In the present invention, the milk thistle fraction may be an ethyl acetate fraction, an n-butanol fraction, a dichloromethane fraction, or a water fraction, preferably an ethyl acetate fraction of a methanol extract, but is not limited thereto.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable” refers to exhibiting non-toxic properties to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art, such as buffers, preservatives, pain relievers, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, and the like.

In addition, the pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can Furthermore, it may be used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel, or external preparation for skin in the form of a gel. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the Cycotria rubra extract, for example, starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, internal solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance to an individual by an appropriate method, and the administration route of the composition may be administered through any general route as long as it can reach a target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, may be administered intrarectally, but is not limited thereto.

As used herein, the term “individual” refers to all animals including humans, rats, mice, and livestock. Preferably, it may be a mammal including a human.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is dependent on the patient's sex, age, body weight, health condition, disease type, severity, drug activity, sensitivity to drug, administration method, administration time, administration route, and excretion rate, duration of treatment, factors including drugs used in combination or concomitantly, and other fields of medicine It can be readily determined by one of ordinary skill in the art according to well-known factors. For administration, the recommended dosage may be administered once a day, or divided into several administrations.

The food composition of the present invention may include all foods in a conventional sense, and may be used interchangeably with terms known in the art, such as functional food and health functional food.

As used herein, the term "functional food" refers to food manufactured and processed using raw materials or ingredients useful for the human body in accordance with Health Functional Food Act No. 6727. It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients with respect to structure and function or physiological effects.

As used herein, the term "health functional food" refers to a food manufactured and processed using a specific ingredient as a raw material for the purpose of health supplementation or by extracting, concentrating, refining, or mixing a specific ingredient contained in a food raw material, It refers to food designed and processed to sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc. Functions related to it can be performed.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition of the present invention can be prepared by mixing known additives with other suitable auxiliary ingredients that may be included in food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the extract according to the present invention and a fraction thereof as a main component to juice, tea, jelly, juice, and the like.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (eg, formula milk, infant food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), health supplements , Seasoned foods (eg soy sauce, soybean paste, red pepper paste, mixed soy sauce, etc.), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.) ), beverages (eg, fruit, vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweetening agents, flavoring agents, or natural carbohydrates as additional ingredients, as in a conventional beverage. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin , alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Experimental method

1.1. Plant samples and their extraction, fractionation and isolation

Thistle ( Cirsium maackii ( Cirsium japonicum var. maackii (Maxim.) Matsum)) was collected in Ulsan, Korea in August 2001. Plant species identification was verified by Dr. Y. Kadota, a Cirsium taxonomy expert, and Professor Jaesu Choi, Professor of Fisheries Science, Pukyong National University, together with a certificate of specimen (2001-08) in the specimen room of the Botany Department of the National Museum of Natural Sciences, Tsukuba City, Japan.

Milk thistle ( Cirsium maackii ( Cirsium japonicum var. maackii (Maxim.) Matsum)) was fractionated and isolated in the same way as in previous studies (Jung et al., 2009; Wagle et al., 2018). Specifically, from the methanol extract of milk thistle and its ethyl acetate fraction, the separated compounds are analyzed by ^{spectroscopic methods such as 1} H and ¹³ C nuclear resonance magnetic analysis (NMR) or published spectroscopic data (Agrawal, 2013; Choi- et al., 1990). ) to reveal the structure of the compound through comparative analysis. The structure of the isolated compound (luteolin and its glycosides) is shown in FIG. 1 .

1.2. Functional GPCR (G-protein-coupled receptor) analysis

Cell-based functional GPCR analysis was performed according to the protocol used in Eurofins Cerep (Le Bois I'Eveque, France) using cells expressing human recombinant receptor, and the specific experimental conditions are the same as previously reported (Paudel et al. , 2019a; Paudel et al., 2019c; and Seong et al., 2019). A stable cell line expressing the recombinant GPCR was used in the experiment.

1.3. Measurement of cAMP levels

Dopamine D ₁ , D ₂ , D ₃ or Functional activity at D ₄ receptors (D ₁ R, D ₂ R, D ₃ R or D ₄ R) was analyzed as a cAMP modulating effect. Briefly, a plasmid containing the target GPCR gene (D ₁ R, D _2L R, D ₃ R or D ₄ R) was transfected into CHO cells (chinese hamster ovary cells). The transfected cell line (CHO-GPCR) was fixed with HBSS/20 mM HEPES buffer (pH 7.4) and 500 μM IBMX solution, and after luteolin was added at room temperature, cells were lysed by incubation for 30 minutes. Thereafter, a fluorescence acceptor (D ₂ -labeled cAMP) and a fluorescence donor (anti-cAMP antibody with europium cryptate) were added, and the mixture was further incubated for 1 hour. Thereafter, fluorescence transfer was measured at λex=337 nm, λem=620 and 665 nm using an Envision™ microplate reader (Perkin Elmer, Waltham, MA, USA).

1.4. intracellular Ca ²⁺ concentration measurement

Cells expressing other receptors were infected with an expression vector encoding a receptor polypeptide and proliferated until the corresponding receptors were expressed. A fluorescent probe (Fluo8 Direct; Invitrogen, Carlsbad, CA, USA) treated with HBSS/20 mM HEPES buffer (pH 7.4) with probenecid was added to each well and the cells were stored at 37 °C for 1 hour. balanced with Then, the degree of luminescence was measured using a CellLux™ microplate reader (PerkinElmer, Waltham, MA, USA) to the plate to which luteolin or a positive control (agonist or antagonist) was added. The agonist or antagonist effect was calculated as a percentage of the control or inhibition of the control antagonist corresponding to the target.

1.5. in the virtual environment ( in-silico ) docking simulation

Automated single docking simulations were performed with AutoDock 4.2 (Goodsell et al., 1996). The X-ray crystal structure of the D ₄ receptor was obtained at 5 WIU (Chien et al., 2010; De Colibus et al., 2005; Son et al., 2008; and Wang et al., 2017). Water and ligand molecules except for FAD (flavin adenine dinucleotide) were removed using Discovery Studio (v17.2, Accelrys, San Diego, CA, USA). The structure of luteolin was created and converted to a 3D structure using Marvin Sketch (v17.1.30, ChemAxon, Budapest, Hungary). The final docking result was determined when it was located at the lowest energy (kcal/mol) in the densest cluster. The results were analyzed and visualized using Discovery Studio and the UCSF Chimera tool (http://www.cgl.ucsf.edu/chimera/).

1.6. Absorption, distribution, metabolism and dissolution (ADME) prediction

The pharmacokinetic parameters of luteolin were predicted using the web-based software PreADMET (v2.0, YONSEI University, Seoul, Korea) (https://preadmet.bmdrc.kr/adme/).

1.7. statistical analysis

Statistical analysis was performed using Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA), and was analyzed using Student's t- test, and ^* p < 0.05 and ^** p < 0.001 were considered statistically significant. All experiments were performed as three independent trials and were expressed as mean ± standard deviation (SD) (n=3).

Example 2. Functional GPCR analysis results

The present inventors reported that luteolin for dopamine D ₁ , D _2L , D ₃ and D ₄ receptors (D ₁ R, D _2L R, D ₃ R and D ₄ R) and serotonin 1A receptor (5HT _1A ) GPCR analysis was performed to confirm the effect as an agonist or antagonist. A % stimulation or % inhibition higher than 50% was judged to be an agonist or antagonist effect, respectively. As a result, as shown in Table 1, luteolin did not act as an agonist against all of _{D 1} R, D _2L R, D ₃ R, D ₄ R and 5HT _1A. On the other hand, luteolin was effective as an antagonist by showing an inhibition rate of 61.30 ± 1.70% _{for D 4 R at a concentration of 50} μM, and it was confirmed that the IC 50 value was 39.59 ± 1.46 μM. However, the antagonist effect on the _{remaining D 1} R, D _2L R, D ₃ R and 5HT _{1A was not confirmed.}

Example 3. Docking simulation results for dopamine receptors

The present inventors confirmed the interaction of luteolin with dopamine receptors through docking simulation. As a result, as shown in Table 2 and Figure 2, luteolin showed a low binding energy of -8.21 kcal/mol _{, formed 7 hydrogen bonds with human D 4} R, and 5, 7, 3 of luteolin It was confirmed that the hydrogen groups at the 'and 4' positions and the ketone group (4th position) affect hydrogen bonding. The luteolin- h D ₄ R conjugate was stabilized through π-alkyl with Cys185 and Leu111, π-anion with Glu95, and amide-π stacked interactions with Leu90. In contrast, the positive controls nemonapride and clozapine _{formed 4 and 1 hydrogen bonds with D 4} R, respectively, and acted as an agonist and antagonist, and the binding energy was -11.82 kcal/mol and It was found to be -9.49 kcal/mol.

From the above results, the present inventors confirmed that luteolin has an antagonistic effect on _{dopamine D 4} receptor (D _{4 R).} Accordingly, it was confirmed that luteolin can be usefully used as a therapeutic agent for neurological diseases, in particular, a therapeutic agent for ADHD.

Example 4. ADME prediction results

The present inventors performed an experiment to predict pharmacokinetic parameters with PreADMET. As a result, the lipophilicity index (Log Po/w) of luteolin was confirmed to be 2.20, and the plasma protein binding rate was 99.72%, indicating that it was strongly bound to plasma proteins, and the intestinal absorption rate (human intestinal absorption) was 79.43%, confirming good intestinal absorption. In addition, the blood brain barrier permeability was 0.37, which was expected to be absorbed at a normal value.

From the above results, the present inventors confirmed that luteolin has a structure suitable for drug development.

Therefore, it was confirmed that the milk thistle-derived luteolin of the present invention _{acts antagonistically on the dopamine D 4} receptor (D ₄ R) in an in vitro experiment, and attention deficit hyperactivity disorder, a disease related to dopamine D4, It can be used as an effective treatment for ADHD by confirming that it is effective for hyperactivity disorder (ADHD).

Claims (7)

delete delete delete delete delete delete A method of inhibiting signal transduction by _{dopamine D 4} receptor, comprising treating a sample with luteolin in vitro.

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