KR20130090578A - Composition comprising naphthazarin for preventing and treating parkinson's disease - Google Patents

Abstract

PURPOSE: A composition for treating and preventing Parkinson's disease containing naphthazarin as an active component is provided to be used as an effective pharmaceutical composition or a health functional food for treating and preventing Parkinson's disease by expressing tyrosine hydroxylase in the striatum and the substantia nigra and deterring the activation of astrocyte and microglia. CONSTITUTION: A pharmaceutical composition for treating and preventing Parkinson's disease contains naphthazarin as an active component. The naphthazarin is used in an effective amount of 0.1 to 2 mg/kg. A health functional food for treating and preventing Parkinson's disease contains naphthazarin as an active component. A food or a food additive for treating and preventing Parkinson's disease contains naphthazarin as an active component.

Description

Composition comprising naphthazarin for preventing and treating Parkinson's disease}

The present invention relates to a composition for the prevention and treatment of Parkinson's disease containing naphthazarin as an active ingredient.

Parkinson's disease (PD) is the second most prevalent cerebral degenerative disease after Alzheimer's disease, and about 1% of people over 50 have been reported to suffer from it [Polymeropoulos MH, Higgins JI, Golbe LI, Johnson WG, Ide SE, Di Iorio G, Sanges G, Stenroos ES.Mapping of a gene for Parkinson's disease to chromosome 4q21-4q23. Science 1996; 274: pp. 1197-1199.

Parkinson's disease is a degenerative neuropathy with progressive degeneration of dopamine-secreting neurons distributed from the vaginal stratum to the striatum. Clinically, 70-80% of the nerve cell ends of the striatum are reduced and 50 neutrophils in the striatum. It is known to decrease by -60% [Bernheimer H, Birkmayer W, Hornykiewicz O et al: Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J. Neurol. Sci, 1973: 20: pp. 415-55; Agrid Y: Parkinson's disease: pathophysiology, Lancet, 1991; 337: pp. 1321-54.

Parkinson's disease is distinguished from other degenerative disorders, in which normal cognitive function and normal dyskinesia are distinguished from other degenerative diseases. The main symptoms are 1) bradykinesia, and 2) tremor-atrest. 3) muscle rigidity, 4) loss of postural reflexes, 5) abnormally flexed posture, and 6) freezing. Is expressed, and one is diagnosed with Parkinson's disease when one of the symptoms is slow motion or steady progression [Fahn S, Przedborski S. Parkinsonism. In: Merritt's neurology (Rowland LP, ed). Philadelphia: Lippincott Williams & Wilkins, 2000, pp. 679-693). Familial form of Parkinson's disease often occurs early (around 50 years old), and its pathogenesis is caused by genetic abnormalities such as parkin, DJ-1, PINK1, alpha-synuclein, and LRRK2. While [Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, Yokochi M, Mizuno Y, Shimizu N. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 1998; 392: pp. 605-608 .; Bonifati V, Rizzu P. Squitieri F, Krieger E, Vanacore N, van Swieten JC, Brice A, van Dujin CM, Oostra B, Meco G, Heutink P. DJ-1, a novel gene for autosomal recessive, early onset parkinsonism. Neurol. Sci. 2003; 24: pp. 159-160 .; Valente EM, Salvi S, Ialongo T, Marongiu R, Elia AE, Caputo V, Romito L, Albanese A, Dallapiccola B, Bentivoglio AR. PINK1 mutations are associated with sporadic early-onset parkinsonism. Ann. Neurol. 2004; 56: pp. 336-341 .; Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root H, Rubenstein J, Boyer R, Stenroos ES, Chandrasekharappa S, Athanassiadou A, Papapetropoulos T, Johnson WG, Lazzarini AM, Duvoisin RC , Di Iorio G, Globe LI, Nussbaum RL. Mutation in the alpha-synuclein gene identified in famillies with Parkinson's disease. Science 1997; 276: pp. 2045-2047.], Idiopathic form of Parkinson's disease has been reported to be the major etiology of various genetic characteristics, environmental toxins and old age [Nagatsu T, Sawada M. Cellular and molecular mechanisms]. of Parkinson's disease: neurotoxins, causative genes, and inflammatory cytokines. Cell Mol. Neurobiol. 2006; 26: pp. 781-802.

Currently, the treatment of Parkinson's disease uses dopamine replacement therapy and dopamine receptor agonists such as L-DOPA, but since long-term administration causes serious side effects such as dyskinesia, In addition to improving symptoms, it is urgent to develop a drug having a neuroprotective effect with safety.

Rodent models commonly used to study the mechanism of Parkinson's disease and to evaluate the pharmacological efficacy of anti-Parkinson compounds include DJ-1 gene deficient mice, PINK1 deficient mice, and parkin in the case of familial form. Gene deficient mice, alpha-synuclein gene overexpressing mice, and the like, and in the idiopathic form, 1-methyl-4-phenyl-tetrahydropyridine (1-methyl-4-phenyl-tetrahydropyridine , MPTP), 6-hydroxydopamine, paraquat, rotenone, and the like. [Terzioglu M, Galter D. Parkinson 'disease: -genetic versus toxin-induced rodent models. FEBS J. 2008; 257: pp. 1384-1391.]. In addition, amphetamines such as methamphetamine (MA) are administered to rodents as a model of drug-induced Parkinsonism [Sonsalla PK, Jocnowitz MD, Zeevalk GD, Oostveen JA, Hall ED . Treatment of mice with methamphetamine produces cell loss in the substantia nigra. Brain Res. 1996; 738: pp. 172-175.

Naphthazarin is a 1,4-naphthoquinone derivative and is found in dental plants and is used in food colors, textiles, and cosmetics. Recently, naphthoquinone derivatives are known to act as anti-inflammatory, antioxidant, anti-viral, antipyretic, and so far in the literature, experimental models directly related to Parkinson's disease, for example, in vivo model MPTP model, etc. In application, no activity has been taught or described for naphthazarine's activity against Parkinson's disease.

Therefore, the present inventors evaluated the pharmacological efficacy in the animal model of Parkinson's disease induced by MPTP using naphthazarin. Parkinson's disease model was evaluated by rota-rod to evaluate ataxia symptoms, expression of tyrosine hydroxylase enzyme in striatum and black matter, striatum and black matter site. As a result of evaluating the activity of the astrocyte (microglia) and microglia (astgocyte), the excellent anti-Parkinson effect was confirmed to complete the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing and treating Parkinson's disease (PD) containing naphthazarin as an active ingredient.

In addition, the present invention has another object to provide a health functional food for preventing and improving Parkinson's disease (PD) containing naphthazarin as an active ingredient.

As a means for solving the above problems, the present invention provides a pharmaceutical composition for the prevention and treatment of Parkinson's disease (PD) containing naphthazarin as an active ingredient.

In addition, as another means for solving the above problems, the present invention provides a functional food for preventing and improving Parkinson's disease (PD) containing naphthazarin as an active ingredient.

The naphthazarin of the present invention has been found to have an anti-Parkinson effect in an animal model of MPTP-induced Parkinson's disease. By inhibiting astrocyte) and microglia activity, it can be used in pharmaceutical compositions and dietary supplements useful for the prevention and treatment of Parkinson's disease (PD).

Figure 1 shows the chemical formula of the Nachitazarin (naphthazarin) and the dental clinic plant is found.
2 shows a protocol for animal experiments.
3 is a photograph of measuring the motor capacity (Rota rod) of the mouse for 180 seconds at 30rpm.
Figure 4 shows the results of the exercise capacity measurement experiment through the Rota rod.
5 is a result of observing the degree of loss of dopamine neurons (Dopaminergic neuron) through the STR and SN sites by TH staining.
6 is a result of observing the degree of loss of dopamine neurons and the activity of GFAP through the TH and GFAP double fluorescence staining SN site.
7 is a result of observing the degree of loss of dopamine neurons and the activity of Iba-1 through SN and GFAP double fluorescence staining.
Figure 8 shows the activity of primary cultured astrocytes by naphthazarin [(A) immunostaining results; (B) Western blot results].

The present invention relates to a pharmaceutical composition for the prevention and treatment of Parkinson's disease containing naphthazarin as an active ingredient.

The present invention also provides a health functional food for the prevention and improvement of Parkinson's disease (PD) containing naphthazarin as an active ingredient.

The anti-Parkinson effect was confirmed in the animal model of MPTP-induced Parkinson's disease against naphthazaline of the present invention. Since it inhibited the activity of the cells (astrocyte) and microglia (microglia), it was confirmed that it is useful for the prevention and treatment of Parkinson's disease.

The pharmaceutical composition containing the naphthazarin of the present invention comprises 0.1 to 10% by weight of the naphthazarin relative to the total weight of the composition. However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents that may be included in the composition comprising naphthazaline 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid form may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the active ingredient of the present invention is preferably administered at 0.1 mg / kg to 2 mg / kg per day, preferably at 0.2 mg / kg to 1 mg / kg. The administration may be carried out once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention also provides a health functional food for the prevention and improvement of Parkinson's disease (PD) containing naphthazarin as an active ingredient.

Therefore, the present invention also provides a food and food additive containing the naphthazarin as an active ingredient having an effect of preventing and improving Parkinson's disease (PD).

The composition comprising the naphthazarin of the present invention can be used in a variety of drugs, food and beverages for the prevention and improvement of Parkinson's disease (PD). Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used as powders, granules, tablets, capsules or beverages have.

Naphthazarin of the present invention may be added to food or beverages for the purpose of preventing and ameliorating Parkinson's disease (PD). Accordingly, the present invention provides a food and / or food additive containing naphthazarin as an active ingredient having a prophylactic and improving effect of Parkinson's disease (PD).

At this time, the amount of naphthazarin in the food or beverage is generally the health food composition of the present invention can be added to 0.001 to 1% by weight of the total food weight, the health beverage composition is 0.1 to 10 mg, preferably based on 100 ml Can be added at a rate of 0.2 to 5 mg.

The health beverage composition of the present invention, in addition to containing the naphthazarin as an essential ingredient in the indicated ratio, there is no particular limitation on the liquid component, and the conventional beverage preparation may contain various flavors or natural carbohydrates as additional ingredients. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, such as conventional sugars such as maltose, sucrose and polysaccharides such as dextrin, cyclodextrin, and the like. Sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 mL of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like.

In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by examples and production examples.

However, the following Examples and Preparation Examples are merely illustrative of the present invention, the contents of the present invention is not limited by the following Examples.

Reference Example  One: MPTP Parkinson's Disease Animal Model Induction and Drug Administration

Seven-week-old C57BL / 6 male mice were purchased and stabilized in the animal breeding room at the Pusan National University Experimental Animal Center for one week, followed by pretraining for three days for the Rota rod test at week eight, followed by naptazaline for five days. MPTP was intraperitoneally administered 4 times at 2 hour intervals 24 hours after the last dose of naphthazarin. Rota rod test was then performed for 3 days, followed by biochemical analysis and histology.

Cardiac perfusion, 5 groups for behavior, 5 groups for biochemical analytical studies, and 10 individuals were used for histology for each group [see FIG. 2].

Example  1: Measure behavior ( Rota rod test )

Rota rod is a method for measuring the motor capacity of the mouse for 180 seconds at 30rpm [Fig. 3]. In normal rats, they walk straight for 180 seconds without falling on the machine, but if they have an abnormality in their motor skills, they do not walk straight and fall on the machine. Intraperitoneal administration of MPTP, which leads to Parkinson's disease, to mice every two hours, can not walk due to poor motor skills. It is a method to measure whether exercise ability is improved when administration of drugs to protect Parkinson's disease.

MPTP group is significantly less athletic ability than the control group (CON). After the MPTP intraperitoneal administration, it can be seen that the exercise ability significantly decreased up to 2 hours, 6 hours, 24 hours, and 48 hours. However, the group administered with 1 mg / kg of MPTP and naphthazarin was found to improve exercise performance at 2 hours, 6 hours, 24 hours, and 48 hours compared to the MPTP group [FIG. 4]. However, the 0.1 mg / kg group of naphthazarin did not show any significant improvement in motor performance.

Example  2: tissue staining and immunostaining

Appropriate brain tissue sections were prepared to study the dopamine neuron damage in melanoma and striatum.

The experimental animals were euthanized after the administration, and the brain was fixed through cardiac perfusion. The extracted brain was sliced in a microtome and subjected to immunohistochemistry with antibodies to various target proteins. Processed brain sections were stored in cryoprotectant solution.

In immunostaining, the prepared brain sections were blocked and then reacted overnight at 4 ° C. with an appropriate primary antibody. After reacting for 3 hours at room temperature with a biotinylated secondary antibody, and washing with an ABC (avidin-biotin complex, Vector Laboratories, Burlingame, CA) solution for 1 hour at room temperature, DAB (3,3) After staining with -diaminobenzidine) kit, the degree of staining was checked under a microscope. Fluorescence immunostaining was used to analyze colocalization.

The TH antibody specific for dopamine neurons can be used to identify neuronal damage caused by MPTP, and the activity of microglia and astrocytes can be confirmed through immunostaining using antibodies specific for glial cells.

Type of antibody used Primary antibody Target Dilution concentration (species) manufacture company TH Dopaminergic Neuron 1: 2000 (ms) Chemicon GFAP Astrocyte 1: 500 (ms) Cell signaling Iba-1 Microglia 1: 400 (Rb) Wako

One) MPTP Induced Parkinson's Disease in Animal Models Naphthazarin  Protection of Dopamine Neurons by

The expression of TH (Tyrosine hydroxylase), a marker of dopamine neurons, was observed through DAB staining of STR (Striatum) and SN (Substantia nigra) sites of the dopamine pathway.

As a result, it can be seen that the dopamine neurons in the MPTP group is much lost compared to the control (CON) (B, F of Figure 5). However, the group administered with both naphthazarin and MPTP can confirm that dopamine neurons were protected according to the concentration compared to the MPTP group (C, D, G, H of FIG. 5).

2) MPTP Induced Parkinson's Disease in Animal Models Naphthazarin  Inhibition of Astrocyte Activity

Double fluorescence staining of STR and SN sites was used to observe the expression of TH, a marker of dopamine neurons, and GFAP, a marker of astrocytes.

As a result, TH expression was the same as the DAB staining results, it was confirmed that the astrocytic cells of the MPTP group was activated compared to the control group (CON) (GF, B, F, J of Figure 6). However, the group administered with the MPTP and naphthazarin can be seen that the activity of the astrocytic cells is reduced according to the concentration compared to the MPTP group (C, D, G, H, K, L of Figure 6).

3) MPTP Induced Parkinson's Disease in Animal Models Naphthazarin  Inhibition of microglial activity

Double fluorescence staining of STR and SN sites was used to observe the expression of TH, a marker of dopamine neurons, and Iba-1, a marker of microglia.

As a result, TH expression was the same as the DAB staining results, and it was confirmed that the microglia of the MPTP group were activated in the case of Iba-1 compared to the control group (B, F, J of FIG. 7). However, the group administered with the MPTP and naphthazarin can be seen that the activity of the microglia decreased with concentration compared to the MPTP group (C, D, G, H, K, L of Figure 7).

4) in primary cultured astrocytes MPP ⁺ When processing Naphthazarin  Activity inhibition

After 24 hours treatment of 500 μM MPP ⁺ to the primary cultured astrocytes, the activation of astrocytes can be confirmed by glial fibrillary acidic protein (GFAP), an astrocyte activity marker.

When pretreated with naphthazarine for 6 hours and treated with 500 μM of MPP ⁺ for 24 hours, it was confirmed that the activity of astrocytes was reduced compared with the treatment with MPP ⁺ alone. These results show that naphthazarin reduces the activity of astrocytic cells [see FIG. 8].

Formulation Example 1: Preparation of powder

Naphthazarine 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation Example 2: Preparation of tablets

Naphthazarin 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3: Manufacture of capsule

Naphthazarin 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4: Preparation of injection

Naphthazarin 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ , 12H ₂ O 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5: Liquid  Produce

Naphthazarine 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, and then, lemon flavor is added, the above components are mixed, and then purified water is added to adjust the total amount to 100 ml. Manufacture.

Formulation example  6: Manufacture of health food

Naphthazarine 5 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7: Manufacture of health drinks

Naphthazarine 5 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (8)

A pharmaceutical composition for the prevention and treatment of Parkinson's disease (PD), containing naphthazarin as an active ingredient.
The pharmaceutical composition for preventing and treating Parkinson's disease (PD) according to claim 1, wherein the naphthazarin is used in an effective amount of 0.1 to 2 mg / kg.
The pharmaceutical composition for preventing and treating Parkinson's disease (PD) according to claim 1, wherein the naphthazarin is used in an effective amount of 0.2 to 1 mg / kg.
Health functional food for the prevention and improvement of Parkinson's disease (PD) containing naphthazarin as an active ingredient.
The dietary supplement for preventing and improving Parkinson's disease (PD) according to claim 4, wherein the naphthazarin is used in an effective amount of 0.1 to 2 mg / kg.
The dietary supplement for preventing and improving Parkinson's disease (PD) according to claim 4, wherein the naphthazarin is used in an effective amount of 0.2 to 1 mg / kg.
A food or food additive containing naphthazarin as an active ingredient having a prophylactic and improving effect of Parkinson's disease (PD).
The food or food additive of claim 7, wherein the food is a variety of foods, beverages, gums, teas, or vitamin complexes of candies.

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