WO2012161358A1 - Pharmaceutical composition for preventing or treating attention deficit hyperactivity disorder (adhd), containing ginkgo leaf extract with enhanced terpenlactone - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating attention deficit hyperactivity disorder (ADHD), and more specifically, to a pharmaceutical composition for preventing or treating ADHD, containing a ginkgo leaf extract with enhanced terpenlactone. In addition, the pharmaceutical composition of the present invention can further comprise a ginseng extract with enhanced prosapogenin such as ginsenoside Rg3 and the like. The pharmaceutical composition of the present invention is enhanced in terpenlactone compared with a conventional ginseng leaf extract or ginseng extract, and is thus effective for improving brain function disorders and enhancing cognition, and in particular, can remarkably prevent or treat ADHD.

Description

Pharmaceutical composition for the prevention or treatment of attention deficit hyperactivity disorder (ADHD) containing terpenactone-enriched ginkgo biloba extract

The present invention relates to a pharmaceutical composition containing terpenlactone or terpenlactone and ginsenoside Rg ₃ , and attention deficit hyperactivity disorder including ginkgo biloba extract and ginseng extract containing the terpenlactone and ginsenoside Rg ₃ as main components. (ADHD) relates to a pharmaceutical composition for the prevention and treatment.

Attention Deficit Hyperactivity Disorder (ADHD) is the most common psychiatric disorder in preschool and school-age children, with persistent attention, hyperactivity, impulsivity, etc. Is a disorder that causes serious problems in children's normal school life and family life. ADHD has been recognized as a medical disease for more than 100 years, and the history of medical treatment is over 60 years, but the diagnosis and treatment rate is still low compared to the history. In 1902, British pediatrician GF Still reported a patient's hyperactivity behavior and presumed a medical condition. ADHD was found to be a disease, and in 1937 doctors found that amphetamine reduced hyperactivity and impulsivity. Found effective. Also, in the early 1960s, Stella Chess defined the disease as a Hyperactivity Child Syndrome, and the spread of treatment with amphetamines (Methylphenidate) spread, and the American Psychiatric Association in the 1980s Attention Deficit Disorder was used as the official name, and in 1987, ADHD was renamed Attention Deficit Hyperactivity Disorder.

Various hypotheses have been raised about the causes of ADHD, but the interpretation that the most fundamental is due to neurological and chemical factors is dominant, and complex anatomical, genetic and environmental factors interact.

Concentration enhancer, the most commonly used drug for the treatment of ADHD, is a central nervous stimulant and has been proven to be effective and safe for treating ADHD in children. CNS is used as a primary treatment for ADHD because of its rapid onset of action and clear symptoms, and it acts by activating dopamine and norepinephrine, the brain neurotransmitters that control attention. have. The concentration-enhancing effect is verified through neuropsychological tests, and it improves attention, cognitive impulsivity, reaction time, short-term memory, and verbal and nonverbal learning. Widely used concentration enhancers include methylphenidate, d-Amphetamine, and pemoline. However, concentration-enhancing drugs are anti-regulatory drugs that have a mechanism of stimulating the central nervous system, which can cause many side effects, especially insomnia, rebound phenomena, decreased appetite, discomfort, dizziness, irritability, anxiety and restlessness Symptoms appear and this is a factor that amplifies the anxiety of parents involved in the medication, whether mild or severe, it is required to develop a drug that is secured.

On the other hand, ginseng is described in <Agreement Bogam>, 'Ginseng is governing, wise and reinforces the mind, spirit and boundaries, and enhances memory.' In Chinese Medicine Dictionary, it is described that ginseng plays an active role in brain function, enhances mental function, enhances eyesight, hearing, thinking and memory, and concentrates attention. In Chinese medicine, prescription for forgetfulness usually includes ginseng.

Standardized GinkgoBiloba Extract (German Pharmacopoeia DAB 2000) consists of 5-7% of terpentrilactone, 22-27% of gingoflavone glycosides, and less than 5 ppm of gingkolic acid, among which Gingoflavone glycosides And free radical scavenging activity, terpentrilactone has been shown to exhibit platelet activating factor (PAF) antagonism, platelet aggregation inhibition, nitric oxide production inhibition, neuronal cell protection (Curr Drug Targets, Jul: 1 ( 1), 25-58, 2000), ginkgolic acid is known to cause allergies and cancers. As described above, ginkgo biloba extract has been shown to have excellent neuronal protective effects and effects on vascular dementia, Alzheimer's dementia, and cognitive improvement, and have been used for treatment of organic brain dysfunction and peripheral arterial circulation disorder for a long time.

Despite all these attempts, however, ginseng, ginkgo biloba and its combined prophylactic and therapeutic agents, which are effective in strengthening brain dysfunction and cognition, in particular ADHD disease, are somewhat insufficient in fundamentally treating the cause of etiology. They found that certain components of ginseng and ginkgo biloba are effective for ADHD, and completed the present invention by presenting a novel complex herbal extract composition enhanced with these specific components.

It is an object of the present invention to provide a pharmaceutical composition containing terpenactone-enriched ginkgo biloba extracts useful for improving brain dysfunction and enhancing cognition, in particular for preventing and / or treating attention deficit hyperactivity disorder.

The present invention provides the following problem solving means to achieve the above object.

The present invention relates to a pharmaceutical composition for the prevention or treatment of attention deficit hyperactivity disorder (ADHD) containing terpenactone-enriched ginkgo biloba extract.

The present invention is characterized in that it further comprises ginseng extract enhanced by ginsenoside Rg ₃ .

In the present invention, the terpene lactone is characterized in that at least one selected from the group consisting of bilobaride, jinkoride A, jinkoride B and jinkoride C.

In the present invention, the ginkgo extract and ginseng extract is characterized in that it is included in a weight ratio of 1: 0.1 to 1:10.

In the present invention, the ginkgo biloba extract is characterized in that it contains 6 to 18% by weight terpenlactone and 20 to 30% by weight of ginkgo flavone glycosides.

In the present invention, the ginseng extract is characterized by containing 1% by weight or more of ginsenosite Rg ₃ .

Since the pharmaceutical composition of the present invention contains terpenlactone as a main component compared to the conventional ginkgo biloba extract, it can effectively prevent and / or treat brain dysfunction and cognition enhancement, especially attention deficit hyperactivity disorder. In addition, it is not only low toxicity and high stability, but also can show an excellent improvement effect compared to the efficacy with synthetic drugs.

The pharmaceutical composition of the present invention is a complex herbal composition comprising a ginseng extract containing ginsenoside Rg ₃ enriched through a vinegar treatment process and a ginkgo biloba extract containing a high concentration of terpenlactone and ginkgo flavone glycosides. It can exhibit excellent therapeutic effects on behavioral disorders (ADHD).

Figure 1 shows the expression of p-AMPK by drug administration in human neuroblastoma.

Figure 2 shows the expression change of TH (ser19) by drug administration in rat chromocytoma.

Figure 3 shows the expression changes of TH (ser40) by drug administration in rat chromocytoma.

Figure 4 shows the increase in motility by chronic administration of bisphenol A.

5 shows the time until the mouse finds water in the water finder test.

FIG. 6 measures the time taken from the first avoidance to the next avoidance in the passive avoidance test.

7 shows the time until the mouse finds a new object.

Figure 8 shows the increase in the number of times the mouse enters the open-arm by measuring the open-arm-entry in the high-priced maze puzzle.

Figure 9 shows the increase in downtime in percent by forced swimming test.

Figure 10 shows the glucose transport capacity change by drug administration in human neuroblastoma using a flow cytometer.

Figure 11 shows the change in glucose transport capacity by drug administration in human neuroblastoma using confocal microscopy.

12 shows the expression changes of p-AMPK and glucose transporter-3 by drug administration in neuroblastoma.

Figure 13 shows the expression changes of p-AKT and glucose transporter-4 by drug administration in neuroblastoma.

Figure 14 shows the expression change of p-TH (ser19) by drug administration in Chromocytoma.

Figure 15 shows the expression changes of p-CAMKII by drug administration in Chromocytoma.

Figure 16 shows the expression change of p-TH (ser40) by drug administration in Chromocytoma.

Figure 17 shows the expression change of p-PKA-c-α by drug administration in Chromocytoma.

The present invention relates to a pharmaceutical composition for preventing and treating attention deficit hyperactivity disorder (ADHD), and more particularly for the prevention or treatment of attention deficit hyperactivity disorder (ADHD) containing terpenactone-enhanced ginkgo biloba extract It relates to a pharmaceutical composition. In another aspect, the present invention may further comprise a ginseng extract with enhanced prosapogenin components such as ginsenoside Rg ₃ in the pharmaceutical composition. The pharmaceutical composition of the present invention is effective in improving brain dysfunction and cognitive reinforcement by enhancing terpenlactone as compared to conventional ginkgo biloba extract or ginseng extract, and in particular, can significantly prevent or treat attention deficit hyperactivity disorder.

Hereinafter, the present invention will be described in more detail.

Terpenlactone of the present invention is known as one of the active ingredients obtained from the extract of Ginkgo biloba, Terpenlactone obtained from the ginkgo biloba extract is bilobalide (Bilobalide), Ginkgolide A, Ginkgo Ginkgolide B and Ginkgolide C include, but are not limited to these.

Chemical formula of the bilobaride is the following [formula 1], the chemical formula of ginkgoride A, B and C is the same as [formula 2].

[Formula 1]

[Formula 2]

 Ginkgolide A: R1 = OH R2 = H R3 = H

 Ginkgolide B: R1 = OH R2 = OH R3 = H

 Ginkgolide C: R1 = OH R2 = OH R3 = OH

In addition, the pharmaceutical composition of the present invention may further include ginsenoside Rg ₃ (Ginsenoside Rg ₃ ) having the structure of [Formula 3].

When ginkgo biloba extract and ginseng extract are included together, the composition ratio is 1: 0.1 to 1:10, preferably 1: 0.2 to 1: 5, and most preferably 1: 0.6 to 1: 2.

[Formula 3]

The ginkgo biloba extract of the present invention contains 20 to 30% by weight of Gingko Flavonoids, terpenactone contains 6 to 18% by weight, preferably 8 to 16% by weight and most preferably 10 To 14 wt%. In addition, the ginseng extract preferably contains at least 1%, preferably at least 2% and most preferably at least 4% of ginsenoside Rg ₃ .

The pharmaceutical composition according to the invention is preferably from 10 mg to 1000 mg, in particular from 80 mg to 320 mg per unit dosage form. In addition, the daily dosage of the pharmaceutical composition of the present invention may vary depending on the administration method or treatment conditions, but when administered orally, it is preferable to administer 1-3 times a day for adults.

Dosage forms of the pharmaceutical compositions according to the invention may be used alone, in combination with other pharmaceutically active compounds, or in any suitable collection.

The pharmaceutical compositions of the present invention may be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. have.

The pharmaceutical composition according to the present invention may include one or more excipients, carriers, diluents, excipients, etc., and pharmaceutically acceptable auxiliaries described in Korean Pharmacopoeia (KP), US Pharmacopoeia (USP), Japanese Pharmacopoeia (JIS), and German Pharmacopoeia (DAB). can do. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the compound. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

The following examples are intended to specifically describe the contents of the present invention, whereby the scope of the present invention is never limited and can be interpreted.

Example 1 Preparation of Raw Material Extract and Complex Herbal Extract

Comparative Example 1

10 g of 50 v / v% ethanol was added to 500 g of dried ginseng, extracted twice from a water bath of 75 ° C., and the aqueous solution obtained by filtration was lyophilized to prepare 102 g of ginseng extract.

Comparative Example 2

Ginkgo biloba extract was prepared according to the method of preparing Ginkgo biloba extract of German Pharmacopoeia after adding 5 kg of dried ginkgo biloba powder to an extraction container and adding 10 times 60 v / v% aqueous acetone. The final amount of extract obtained was 125 g (GBE). As a result of the content analysis, it was confirmed that it contained 6% by weight of terpenlactone and 24% by weight of ginkgo flavone glycosides.

<Manufacture example 1>

The ginseng extract prepared in Comparative Example 1 was added to 10 times the amount of brine vinegar (pH2.2 7), extracted at 90 ° C. for 6 hours, and lyophilized to obtain an extract 9.8 g (YY-351). As a result of content analysis, it was confirmed that YY-351 contained 4% by weight of ginsenoside Rg ₃ .

<Manufacture example 2>

5 kg of ginkgo biloba dry powder is filled in an extraction container, and 50 L of 50 v / v% ethanol aqueous solution is leached. After 12 hours, the resultant was filtered under pressure and the filtrate was concentrated to 5 L under reduced pressure. Water was added to the concentrated solution to make 10 L, stirred vigorously, left to cool at 15 ° C. for 12 hours, and the supernatant was separated from the precipitate and filtered. After extraction three times with 5 L of heptane to remove the heptane soluble part, the remaining aqueous layer was extracted three times with 10 L of a butanol-toluene mixed solvent (1: 1, v / v). The extracted mixed solvent layer was left at 15 ° C. for 12 hours, filtered through filter paper, and the filtrate was concentrated under reduced pressure and dried to give 57.3 g of purified ginkgo biloba extract (YY-1224). As a result of content analysis, it was confirmed that it contained 12% by weight of terpenlactone and 24% by weight of ginkgo flavone glycosides.

<Manufacture example 3>

YY-351 of Preparation Example 1 and YY-1224 of Preparation Example 2 were combined at a ratio of 1: 0.2 to prepare a complex herbal extract.

<Manufacture example 4>

YY-351 of Preparation Example 1 and YY-1224 of Preparation Example 2 were combined at a ratio of 1: 0.6 to prepare a complex herbal extract (YY162A).

Production Example 5

YY-351 of Preparation Example 1 and YY-1224 of Preparation Example 2 were combined at a ratio of 1: 2 to prepare a complex herbal extract.

<Manufacture example 6>

YY-351 of Preparation Example 1 and YY-1224 of Preparation Example 2 were combined at a ratio of 1: 5 to prepare a complex herbal extract.

<Manufacture example 7>

YY-351 of Preparation Example 1 and GBE of Comparative Example 2 were combined at a ratio of 1.2 to prepare a complex herbal extract (YY-162B).

<Manufacture example 8>

YY-351 of Preparation Example 1 and YY-1224 of Preparation Example 2 were combined at a ratio of 1: 1.2 to prepare a complex herbal extract (YY-162C).

TABLE 1

Example 2 Formulation Example

(1) tablet formulation

Tablets are prepared by tableting and coating the components of the following [Table 2] according to a conventional manufacturing method.

TABLE 2

(2) Hard Capsule

The following Table 3 components are mixed and filled according to a conventional manufacturing method to prepare a hard capsule.

TABLE 3

(3) Soft Capsule

To prepare a soft capsule by mixing and filling the components of the following [Table 4] according to a conventional soft capsule manufacturing method.

TABLE 4

(4) Injection formulations

Injectables are prepared by preparing and filling the ingredients of Table 5 below according to a conventional method for preparing injectables.

TABLE 5

Example 3 In Vitro Activity Test of Example 2

Various hypotheses have been raised about the cause of ADHD, but the dominant interpretation is that it is based on neurochemical factors, and the complex characteristics of other anatomical, genetic and environmental factors interact. Positron emission tomography of brains of patients with ADHD recently revealed that the function of the frontal lobe is lower than that of normal individuals, which is attributed to a decrease in neurotransmitter activity and glucose utilization (Avis R). et al. Ann NY Acad Sci. 2008 . ; Zimmer L. neuropharmacology. 2009.)

Glucose transport is via insulin-independent / independent pathways, and insulin-independent pathways are regulated by p-AMPK expression. Depletion of sugar in the body and reduction of adenosine triphosphate (ATP) / adenosine diphosphate (ADP) ratio resulted in increased p-AMPK expression and finally induced glucose transporter (GLUT) expression. Increase the transport of glucose used as an energy source. Insulin-dependent pathways are regulated by AKT protein kianse (AKT) expression, which also induces glucose transporter expression.

On the other hand, catecholamines are used as neurotransmitters between brain neurons, and tyrosine introduced into cells is converted into dopamine and norepinephrine by enzymes. The enzyme that acts as a rate step in this mechanism is tyrosine hydroxylase, and the higher factors are protein kinase A (PKA), protein kinase C (PKC) and calcium / calmodulin dependent protein kinase II ( calcium-calmodulin-dependent protein kinase II (CAMKII).

Therefore, in Example 2, the in vitro activity according to the blending ratio of the preparation prepared in Example 1 was tested.

Experimental Example 1 Cell Culture and Drug Treatment

Human neuroblastoma SH-SY5Y (Korea Cell Line Bank) was dispensed on a confocal microscope slide (Millipore) and 6-well plate at 1 × 10 ⁶ cell / ml, and then in a cell culture medium (5% CO ₂ , 37 ° C.). Incubated for 24 hours. Test drug (Preparation Example 1 to Preparation Example 6) was dissolved in dimethyl sulfoxide to make 10 mg / ml, and diluted with culture to make 1%. 2 ml of the diluted test drug was incubated for 12 hours in a cell incubator, and then Western blot analysis was performed to determine the expression of p-AMPK.

PC12 (Korea Cell Line Bank), a rat chromophore cell species, was dispensed into 6-well plates at 1 × 10 ⁶ cells / ml, and then cultured in a cell culture medium (5% CO ₂ , 37 ° C.) for 24 hours. Test drug (Preparation Example 1 to Preparation Example 6) was dissolved in dimethyl sulfoxide to make 10 mg / ml, and diluted with culture to make 1%. 2 ml of diluted test drugs were incubated in cell culture cells for 24 hours and 48 hours, and then p-TH (ser19), p-TH (ser40), and pan-TH were used to confirm the neurotransmitter activity. Western blot was performed.

Experimental Example 2 Protein Extraction

Adenosine monophosphate kinase (AMPK) and phosphorylated adenosine kinase (p-AMPK), tyrosine hydroxylase (TH) TH- (ser40), TH- (ser19) and phosphorylated tyrosine hydroxylase In order to measure the protein expression levels of p-TH (ser19) and p-TH (ser40) by Western blotting, about 10 mg of cells were obtained from 6-well plates and homogenized with 0.1 ml of protein extract solution. After 30 minutes in a cold ice bath for cell lysis, the whole homogenate was centrifuged at 10,000 rpm for 10 minutes. The supernatants were collected and the protein concentration of the supernatants extracted from the total homogenate was determined by the Bradford method using Bio-Rad protein quantitative reagents. Fetal bovine albumin (Bradford) was used as a standard solution.

Experimental Example 3 Western Blot Method

To determine the contents of monophosphate adenosine kinase, phosphorylated adenosine kinase, tyrosine hydroxylase, phosphorylated tyrosine hydroxylase 19, 40, 20 μg of the water-soluble protein of the cell homogenate was used. Protein samples are mixed with 2x reduction buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% sodium dodecyl sulfate (SDS), 10% 2-mercetoethanol, 0.02% bromophenol blue), boiling water Heat treatment for 7 minutes. The denatured protein was separated by 10% SDS-PAGE, transferred to a polyvinylidenedifluoride membrane, and left at room temperature for 60 minutes in a buffer containing 5% dried skim milk powder to prevent nonspecific binding. Bands were immunologically detected using human p-AMPK, AMPK, p-TH (ser19), p-TH (ser40), polyclonal antibodies against pan-TH and monoclonal antibodies against β-actin. . Binding of all antibodies was detected with the ECL detection system (Millipore) according to the manufacturer's instrument. The intensity of the immune response bands was determined using a concentration assay program (ImageQuant LAS 4000, FUJI FILM Co., Japan).

Experimental Example 4 Statistical Processing

All experimental results were expressed as mean ± standard error, and data analysis was performed using one-way analysis of variance (ANOVA) to test statistical significance at P <0.05 level. Group comparisons used the Bonferroni method.

<Experiment Result>

(1) increased p-AMPK expression

Protein expression by aminoimidazole carboxamide ribonucleotide (AICAR) used as a positive control in human neuroblastoma and the test substance obtained in Preparation Examples 1 to 6 is shown in FIG. 1. As shown in FIG. 1, p-AMPK expression was significantly increased in the AICAR, Preparation Example 1, Preparation Example 3, and Preparation 4 treatment groups compared to the control group.

(2) increased p-TH (ser19) expression

Protein expression by the test substance obtained in Preparation Examples 1 to 6 in PC12 cells is as shown in FIG. As shown in FIG. 2, p-TH (ser19) expression was significantly increased in Preparation Example 2, Preparation Example 4, Preparation Example 5, and Preparation Example 6 treatment group compared to the control group.

(3) increased p-TH (ser40) expression

Protein expression by the test substance obtained in Preparation Examples 1 to 6 in PC12 cells is shown in FIG. As shown in FIG. 3, p-TH (ser40) expression was significantly increased in Preparation Examples 2 to 6 compared to the control group.

Example 4 Pharmacological Efficacy Test

Effects of the pharmaceutical composition according to the present invention on the improvement of organic brain dysfunction and cognition enhancement were tested.

Since ADHD rarely leads to death, which makes it difficult to identify the etiology, the establishment of the animal model also includes the face validity of ADHD patients and whether the drug applied to the ADHD patient is reproduced in the animal model. It has been developed with a focus on p redictive validity. Animal models of ADHD mainly applied to rodents include spontaneously hypertensive rats (SHR), dopamin e transporter knockout mice, peractive wheel running mice, Genetic models such as thyroid mutant mice, neonatal anoxia, taumatic models such as hippocampal irradiation, and neonatal 6-OHDA damage ( Impairment models such as neonatal 6-OHDA lesions, and environmental toxicant models treated with bisphenol A, polychlorinated biphenyls, and lead. Etc.

In the present pharmacological efficacy test, an in vivo ADHD model was established using bisphenol A, an environmental hormone substance, and Aroclor 1254, a polychlorinate biphenol complex. Drug administration was performed by setting the administration group as a single extract Preparation Example 1, Preparation Example 2 and Mixed Preparation Preparation Example 4, Preparation Example 8. Bisphenol A and Aroclor 1254 are both endocrine disruptors and have been reported to show ADHD-like symptoms during prenatal and childhood administration. As a control drug, methylphenidate (MP: methylphenidate) and GBE, a standard extract of Ginkgo biloba, which are clinically applied to ADHD, were applied.

<Experiment 1> Confirmation of improvement effect in attention deficit / hyperactivity disorder model Ⅰ

(1) experimental animal and drug treatment

Immature mice were used in the experiments because ADHD is a disease mainly seen in children. ICR mice of 14 days of gestation were purchased and adapted to the laboratory and treated with drugs from 1 week after the birth of the pups. Briefly, bisphenol A was orally administered to lactating mothers during pregnancy and up to 3 weeks of age at 500 ug / kg / day, and long-term oral administration to immature mice at 500 ug / kg / day for 3 to 4 weeks after 3 weeks of age. . Preparation Example 1 (YY-351), Preparation Example 2 (YY-1224) and Preparation Example 4 (YY-162A) (30 or 60 mg / kg, oral administration) were administered 1 hour before the bisphenol A, respectively, GBE (30 or 60 mg / kg, oral administration) was used as a control drug. When the pups are 5 weeks old, the behavioral and cognitive functions are assessed based on the following criteria. During the behavioral evaluation, the drugs were administered after the behavioral and cognitive functions were evaluated in order to prevent the drugs from directly affecting the behavior.

(2) Confirmation of the improvement effect on the excessive behavior of ADHD model

As shown in Figure 4, the increase in locomotor activity by chronic administration of bisphenol A was significantly inhibited by high dose administration (60 mg / kg, oral administration) of Preparation Example 2 (YY-1224) (P <0.05), administration of GBE and Preparation Example 1 (YY-351) showed no significant efficacy. Administration of Preparation Example 4 (YY-162A) also significantly inhibited the hyperactivity induced by bisphenol A with the greatest effect.

(3) Confirmation of improvement effect on attention concentration disorder of ADHD model

Attention concentration disorder in the ADHD model was measured using a water finding latency test. As a result, as shown in FIG. 5, the immature mice administered with bisphenol A for a long time increased significantly incubation period for finding the nozzle of the water bottle compared to the mice administered with solvent only. Significantly decreased the latency period. The administration of Preparation Example 1 showed a tendency to decrease the incubation period, but was not statistically significant, and especially in Preparation 4 administration, the increase in the incubation time induced by bisphenol A was most significantly reduced.

(4) Confirmation of improvement effect on learning disability of ADHD model

Learning disabilities in the ADHD model were measured using a passive avoidance test. As a result, as shown in FIG. 6, in the immature mice administered with bisphenol A for a long time, the step-through latency was significantly reduced compared to the mice administered with vehicle, leading to a decrease in learning memory ability. , Preparation Example 2, the fighting over the GBE significantly increased the time to the next avoidance. The administration of Preparation Example 1 tended to increase the time to the next avoidance, but showed no statistically significant effect, but Preparation Example 4 significantly increased the time to the next avoidance induced by bisphenol A. .

<Experiment 2> Confirmation of improvement effect in attention deficit / hyperactivity disorder model Ⅱ

(1) experimental animal and drug treatment

Immature mice were used in the experiments because ADHD is a disease mainly seen in children. ICR mice were purchased and adapted to the experimental animal room and mated. From 6 days of gestational day to 21 days of age, mothers were orally administered Aroclor 1254 at a dose of 18 mg / kg once a day, and only 21 males were weaned after weaning. From the 21st day after the end of the behavioral experiment to the weaning infants Preparation Example 1 (100 or 150 mg / kg, po), Preparation Example 2 (120 mg / kg, po), GBE (120 mg / kg, po) or Preparation Example 7 (110 or 220 mg / kg, po) was administered or Preparation Example 8 (110 or 220 mg / kg, po) was administered. From 35 days after birth, behavioral and cognitive evaluations were performed based on the following criteria. As the positive control group, methylphenidate, which is most widely prescribed for ADHD disease, was used.

(2) Confirmation of improvement effect on attention concentration disorder / lowering learning memory of ADHD model

In mice treated with Aroclor 1254, the time spent in the central area where the new object was located was significantly reduced to meet the face validity of attention attention disorders. In the new object exploration test, attention reduction by Aroclor 1254 was significantly suppressed in Preparation 2 dose group (P <0.01), Preparation 7 dose and Preparation 8 dose group as shown in FIG. In particular, Preparation Example 2, Preparation Example 7 and Preparation Example 8 showed an improvement effect than the methylphenidate of the positive control group.

(3) Confirmation of improvement effect on impulsive behavior of ADHD model

Elevated plus maze is an anxiety when the number of open arm entries is significantly reduced by measuring open arm entry, and an impulsiveness is significantly increased. impulsiveness) increased. As a result, as shown in FIG. 8, the mice treated with Aroclor 1254 significantly increased open-cancer access (P <0.01) to satisfy the target validity for impulsive behavior, and methylphenidate was added to this indicator. All showed significant efficacy. (P <0.01 in the open arm entry) The increase in open-arm-entry by Aroclor 1254 in the high-cost cruciform maze test resulted in GBE (P <0.05), preparation 2 administration group (P <0.01) and preparation 8 administration group ( P <0.01) significantly inhibited the effect was confirmed.

(4) Confirmation of improvement effect on anxiety / depression symptoms of ADHD model

The forced swimming test and the tail suspension test are methods for assessing depressive symptoms. Depressive symptoms are not the main symptoms of ADHD, but depression, anxiety, hostile opposition, and behavioral disorders in ADHD patients. Most cases were accompanied by depression, so depression was evaluated. As a result, as shown in FIG. 9, the mice treated with Aroclor 1254 significantly increased the stop time in the forced swimming test (P <0.01) and the tail suspension method, leading to depression. The increase in stop time increased by Aroclor 1254 in the forced swimming test was found in the GBE administration group (P <0.05), Preparation Example 2 administration group (P <0.05), Preparation Example 7 administration group (P <0.05) and Preparation Example 8 administration group (P <0.05) was significantly suppressed to confirm its efficacy.

Example 5 Pharmacological Mechanism Study

The pharmacological mechanism of the pharmaceutical composition according to the present invention was experimentally identified at the molecular biological level.

In vivo experiments with Arochlor1254 demonstrated that YY162A was effective in improving ADHD symptoms. Thus, in vitro experiments showed the effect of YY162A on neurotransmitter production and glucose utilization at the molecular biological level.

Experimental Example 1 Confirmation of Glucose Transport Capacity in Neuroblastoma

Human neuroblastoma SH-SY5Y (Korea Cell Line Bank) was dispensed on a confocal microscope slide (Millipore) and 6-well plate at 1 × 10 ⁶ cell / ml, and then in a cell culture medium (5% CO ₂ , 37 ° C.). Incubated for 24 hours. The test drug (Preparation Example 1, Preparation Example 2 and Preparation Example 4) was dissolved in dimethyl sulfoxide to make 10 mg / ml, and diluted with culture to make 1%. 2 ml of the diluted test drug was incubated for 12 hours in a cell incubator, and then glucose transportability was measured using a flow cytometer and confocal microscope. Western blot method (AMPK, p-AMPK, AKT, p-AKT, GLUT-3, GLUT-4) were used to confirm molecular biological changes.

(1) Fluorescent labeled deoxidized glucose derivative transport ability

After stabilizing SH-SY5Y cells dispensed on a 6-well plate and a confocal microscope slide (Millipore) for 24 hours, the FBS concentration of the culture solution was lowered to 0.1%, and the drug group (Preparation Example 1, Preparation Example 2, and Preparation Example 4). , 100 μg / ml) was incubated for 12 hours. After 12 hours, washed three times with Crab's-Ringer-N-2-hydroxyethylpiperadine-N'-2-ethanesulfonic acid (KRH buffer) buffer, followed by 2- (N- (7-nitrobenz-2 Treatment with -oxa-1,3-diazol-4-yl) amino) -2-deoxyglucose (2-NBDG, molecular probes, 10 μM) was further incubated for 30 minutes and the reaction was terminated by phosphate buffered saline wash. Cells of 6 well plates were desorbed with trypsin and fractionated (1500 rpm, 5 min) and measured by flow cytometry (BD oscience). .

As a result, it was found that 2-NBDG fluorescence was increased in Preparation Example 1, Preparation Example 2, and Preparation Example 4 significantly compared to the control shown in FIG. 10. In addition, as a result of observing the transport capacity of 2-NBDG using a confocal microscope as shown in Figure 11 it can be seen that the fluorescence increased in the Preparation Example 4 group (Fig. 11B) compared to the control (Fig. 11A).

(2) expression of protein

After stabilizing SH-SY5Y cells dispensed on a 6-well plate and a confocal microscope slide (Millipore) for 24 hours, the FBS concentration of the culture solution was lowered to 0.1%, and the drug group (Preparation Example 1, Preparation Example 2, and Preparation Example 4). , 100 μg / ml) was incubated for 12 hours. Thereafter, the expression of AMPK, p-AMPK, GLUT-3, and GLUT-4 was confirmed using the methods of Experimental Examples 2 and 3 of Example 3. As a result, as shown in FIG. 12, p-AMPK expression was significantly increased in AICAR, Preparation Example 1, Preparation Example 2, and Preparation Example 4 used as a positive control, and AICAR used as a positive control. It was found that the expression of GLUT-3 increased significantly in Example 1 and Preparation Example 4 groups. As a result of measuring the expression of AKT, p-AKT and GLUT-4, p-AKT expression was significantly increased in the Insulin group, which is a positive control group, as shown in FIG. 13, and positive control group, Preparation Example 1 , Preparation Example 2 and Preparation Example 4 It was found that the expression of GLUT-4 significantly increased.

Therefore, the pharmaceutical composition of the present invention is expected to improve the symptoms of ADHD through improved glucose utilization.

Experimental Example 2 Production of Neurotransmitters in Rat Chromium-Affinity Cell Tumors

PC12 (Korea Cell Line Bank), a rat chromophore cell species, was dispensed in 6 well plates at 1 × 10 ⁶ cells / ml, and then cultured in a cell culture (5% CO ₂ , 37 ° C.) for 24 hours. The test drug (Preparation Example 1, Preparation Example 2 and Preparation Example 4) was dissolved in dimethyl sulfoxide to make 10 mg / ml, and diluted with culture to make 1%. 2 ml of the diluted test drug was incubated for 24 hours and 48 hours in a cell incubator, and then p-TH (ser19), p-TH (ser40), and pan were prepared using the methods of Experimental Examples 2 and 3 of Example 3. Expression of -TH, p-CAMKII and p-PKA-c-α was measured. As a result, as shown in FIGS. 14 and 15, the expression of p-TH (ser19) was significantly increased in Preparation Example 2 and Preparation Example 4 groups compared to the control group, and Preparation Example 1 and Preparation Example 2 compared to the control group. And it was found that the expression of p-CAMKII which is a higher factor of p-TH (ser19) in the Preparation Example 4 group significantly increased. As shown in FIGS. 16 and 17, expression of p-TH (ser40) and its higher factor, p-PKA-c-α, was significantly higher in the Preparation Example 1, Preparation Example 2, and Preparation Example 4 groups than the control group. It was found to increase.

Drug YY162A used in this experiment is a combination of a new composition of ginkgo biloba extract (YY1224) and enhanced ginseng extract (YY351) is a natural combination new drug developed for the treatment of ADHD in the present invention. Through the above experiments, it is expected that the combination drug YY162A may improve the symptoms of ADHD through improving neurotransmitter activity and glucose utilization.

Claims (6)

1. A pharmaceutical composition for the prevention or treatment of attention deficit hyperactivity disorder (ADHD) containing terpenactone-enriched ginkgo biloba extract.
2. The pharmaceutical composition of claim 1, further comprising ginseng extract fortified with ginsenoside Rg ₃ .
3. The pharmaceutical composition of claim 1, wherein the terpenlactone is at least one selected from the group consisting of bilobaride, ginkride A, ginkride B and ginkride C.
4. The pharmaceutical composition of claim 1 or 2, wherein the ginkgo extract and ginseng extract are included in a weight ratio of 1: 0.1 to 1:10.
5. The pharmaceutical composition of claim 1, wherein the ginkgo biloba extract contains 6-18 wt% of terpenlactone and 20-30 wt% of ginkgoflavone glycosides.
6. The pharmaceutical composition of claim 2, wherein the ginseng extract contains 1% by weight or more of ginsenoside Rg ₃ .

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