KR20220107430A - Composition for preventing or treating parkinson's disease comprising evernic acid - Google Patents

Abstract

The present invention relates to a composition for preventing or treating Parkinson's disease containing evernic acid as an active ingredient. In the present invention, evernic acid, one of candidate substances having a neuroprotective effect, was discovered through library screening of lichen metabolites, and evernic acid's effect on Parkinson's disease was confirmed. In the present invention, in an animal model of Parkinson's disease induced by MPTP, it was confirmed that evernic acid stabilizes the mitochondria of neurons to have a neuroprotective effect, and has a dual effect of suppressing the neuroinflammatory response occurring in astrocytes such that the composition suggests the possibility that evernic acid can be used as an effective treatment for neurodegenerative diseases comprising Parkinson's disease.

Description

A composition for preventing or treating parkinson's disease comprising evernic acid as an active ingredient TECHNICAL FIELD

The present invention relates to a composition for preventing or treating Parkinson's disease containing evernic acid as an active ingredient.

Parkinson's disease is a neurodegenerative disease that gradually increases with aging, and is caused by a decrease in dopaminergic neurons present in the midbrain dopamine pathway responsible for motor functions. Therefore, movement disorders such as muscle stiffness and tremor of the extremities appear as the main symptoms. Parkinson's disease is believed to be caused by oxidative stress, loss of mitochondrial function, aging, chronic neuroinflammation, etc., but the pathological cause is still unknown.

Lichens are symbionts of fungi and algae, and there are over 20,000 types of lichens around the world. Therefore, numerous metabolites produced by lichens exist, and various biological activities such as anticancer, antimicrobial, antioxidant, and anti-inflammatory effects have been reported. However, little is known about its action in neurodegenerative diseases including Parkinson's disease.

PCT International Patent Publication WO 2008/109521 A2 (published on September 12, 2008)

Accordingly, an object of the present invention is to provide a use for preventing, improving or treating Parkinson's disease of evernic acid.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease containing evernic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving Parkinson's disease containing evernic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention relates to a composition for preventing or treating Parkinson's disease containing evernic acid as an active ingredient. acid) was discovered and confirmed what effect it had on Parkinson's disease. In the present invention, it was confirmed that evernic acid has a neuroprotective effect by stabilizing the mitochondria of nerve cells in an animal model of MPTP-induced Parkinson's disease, and has a dual effect of inhibiting the neuroinflammatory response occurring in astrocytes. , suggesting the possibility that evernic acid can be used as an effective treatment for neurodegenerative diseases including Parkinson's disease.

1 shows a schematic diagram of an animal model of Parkinson's disease induction and drug administration by MPTP.
Figure 2 shows a behavioral pattern measurement test (Rota-rod test) process.
3 shows the neuroprotective effect of evernic acid in primary cultured neurons.
4 shows the mitochondrial function improvement effect of evernic acid in primary cultured neurons.
5 shows the anti-inflammatory effect of evernic acid in primary cultured astrocytes.
6 shows the effect of improving exercise capacity of evernic acid in a Parkinson's disease model.
7 shows the neuroprotective effect of evernic acid in a Parkinson's disease model.
8 shows the inhibitory effect of evernic acid on astrocyte activation in a Parkinson's disease model.

The present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease, comprising as an active ingredient evernic acid represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

Preferably, the composition may have a neuroprotective effect through mitochondrial stabilization of neurons and an inhibitory effect on neuroinflammatory responses occurring in astrocytes.

Preferably, the composition may have an effect of improving athletic performance and a protective effect of dopaminergic neurons.

Preferably, the pharmaceutically acceptable salt is hydrochloride, bromate, sulfate, phosphate, nitrate, citrate, acetate, lactate, tartrate, maleate, gluconate, succinate, formate. , trifluoroacetate, oxalate, fumarate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, camphorsulfonate, sodium salt, potassium salt, lithium salt, calcium salt and magnesium salt However, the present invention is not limited thereto.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, and a lubricant. Alternatively, a solubilizing agent such as a flavoring agent may be used. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

The pharmaceutical formulation of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained-release formulations of the active compound. can The pharmaceutical composition of the present invention may be administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for preventing or treating a disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, weight, general health status, sex and diet of the patient, administration time, administration route and composition It can be adjusted according to various factors including secretion rate, duration of treatment, and drugs used at the same time.

In addition, the present invention provides a health functional food composition for preventing or improving Parkinson's disease, comprising as an active ingredient evernic acid represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

Preferably, the composition may have a neuroprotective effect through mitochondrial stabilization of neurons and an inhibitory effect on neuroinflammatory responses occurring in astrocytes.

Preferably, the composition may have an effect of improving athletic performance and a protective effect of dopaminergic neurons.

The health functional food composition of the present invention may be provided in the form of powder, granule, tablet, capsule, syrup or beverage, and the health functional food composition is used with other foods or food additives in addition to the active ingredient, and according to a conventional method can be used appropriately. The mixed amount of the active ingredient may be suitably determined according to the intended use thereof, for example, prophylactic, health or therapeutic treatment.

The effective dose of the active ingredient contained in the health functional food composition can be used according to the effective dose of the pharmaceutical composition, but in the case of long-term intake for health and hygiene or health control, it should be less than or equal to the above range. It is certain that the active ingredient can be used in an amount greater than the above range because there is no problem in terms of safety.

The type of health food is not particularly limited, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, and the like.

In the composition according to the present invention, the evernic acid represented by Formula 1 or a pharmaceutically acceptable salt thereof is contained in an amount of 0.001 to 30.0% by weight with respect to the total composition, and if it is out of the content, it is represented by Formula 1 The pharmacological effect of evernic acid or a pharmaceutically acceptable salt thereof, such as prevention, improvement or treatment of Parkinson's disease, does not appear, or the degree of increase in the effect of increasing the content is insignificant, there is a problem in safety and stability of the formulation, and economic not even an enemy

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

< Experimental example >

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

One. evernic acid purchase

Evernic acid was purchased from Santa Cruz Biotechnology (Cat No. sc-294581).

2. Neuron and glial cell primary culture

Neurons and astrocytes were isolated from the cortex tissues of embryos 17 to 18 days old and 1 to 2 days old rats, respectively, and in vitro as follows. Experiments were performed.

(1) Cell viability measurement

After 6 hours of pretreatment with evernic acid in primary cultured neurons, the neurotoxic substance MPP ⁺ was treated for 24 hours to observe the protective effect of evernic acid. Cell viability was measured by measuring the absorbance of the degree of reaction using the MTT reagent.

(2) Mitochondrial functional evaluation (membrane potential measurement, metabolism analysis)

MPP ⁺ , a neurotoxic substance, inhibits complex I of the mitochondrial electron transport system, leading to apoptosis. Therefore, it was observed whether evernic acid could prevent mitochondrial damage caused by MPP ⁺ . After treating primary cultured neurons with evernic acid and MPP ⁺ for 6 hours, the mitochondrial membrane potential was observed by staining using MitoTracker, and cellular respiration ability was measured with a metabolic gas analyzer (Seahorse XF).

(3) Measurement of active oxygen

It is well known that reactive oxygen species can be excessively generated by mitochondrial damage, and oxidative stress can act as one of the causes of neurodegenerative diseases. Therefore, DCF-DA dye was used to determine whether treatment with evernic acid in primary cultured neurons could lower the reactive oxygen species produced by MPP ⁺ .

(4) Observation of astrocyte activation and inflammatory response through immunostaining

In primary cultured astrocytes Treatment with MPP ⁺ activates astrocytes, which can be observed by changing the expression level of GFAP, a well-known marker for astrocytes. Therefore, it was observed whether evernic acid could lower the activation of astrocytes and inhibit the inflammatory marker p65 through immunostaining.

(5) Western blot analysis

Primary cultured neurons and astrocytes were sampled after the experiment, and proteins related to apoptosis and inflammation were observed through biochemical analysis.

3. to MPTP Parkinson's disease animal model induction and drug administration

MPTP is not toxic by itself when administered intraperitoneally, but when it passes through the blood-brain barrier and enters the central nervous system, it is metabolized to MPP ⁺ by an enzyme called MAO-B in astrocytes. At this time, MPP ⁺ is selectively uptaken by the transporter of dopaminergic neurons, thereby specifically killing dopaminergic neurons. Therefore, it is a widely used substance to induce Parkinson's disease. In the present invention, in order to investigate the preventive effect of evernic acid on Parkinson's disease, Parkinson's disease was acutely induced by intraperitoneal administration of evernic acid for 10 days first, and then, 24 hours after the last administration, by intraperitoneal administration of MPTP 4 times at 2 hour intervals. After that, the behavioral test was performed for 3 days and sacrificed, and histological analysis was performed (FIG. 1).

(1) animal model

C57BL/6 mice and 6-week-old male mice were purchased and stabilized in the animal room of Pusan National University for 1 week, followed by behavioral testing and evernic acid administration at 7 weeks.

(2) dosing concentration

Evernic acid was administered orally at a concentration of 5 and 80 mg/kg for 10 days, and was administered after dissolving corn oil + 5% DMSO as a solvent.

(3) Population

A control group (control and vehicle group inducing Parkinson's disease) and a group by concentration of evernic acid were tested with a total of 4 populations, and 5 animals were used in each group.

4. Behavior measurement (Rota-rod test)

Rota-rod is a method of measuring the locomotion ability of a mouse without falling for 180 seconds on a cylinder rotating at a constant speed. In the case of a normal mouse, it walks straight without falling off the machine for 180 seconds, but if there is an abnormality in motor ability, it cannot walk straight and fall off the machine. If MPTP, which induces Parkinson's disease, is administered intraperitoneally to mice 4 times a day at an interval of 2 hours, their motor ability deteriorates and they cannot walk. It is a method of measuring whether exercise ability is improved when a drug that protects Parkinson's disease is administered (FIG. 2).

5. Tissue Immunostaining

- In order to study the degree of damage to dopaminergic neurons in the striatum (STR) and substantia nigra (SN) of the midbrain, the lesion site of Parkinson's disease, an appropriate brain tissue section is prepared.

- Experimental animals are euthanized after administration, and brains are fixed through cardiac perfusion. The extracted brain is sectioned in a microtome and subjected to immunohistochemistry with antibodies to various target proteins. The processed brain sections are stored in a cryoprotectant solution.

- In the immunostaining method, the prepared brain sections are blocked and then reacted overnight at 4°C after treatment with an appropriate primary antibody. After reacting with biotinylated secondary antibody at room temperature for 3 hours, after washing, react with ABC solution and room temperature for 1 hour, stain with DAB kit, and check the degree of staining under a microscope. When analyzing co-localization, etc., fluorescent immunostaining using a fluorescently labeled secondary antibody is used.

- Using TH (tyrosine hydroxylase) antibody specific for dopaminergic neurons, neuronal damage caused by MPTP can be confirmed, and microglia and astrocytes can be detected by immunostaining using an antibody specific for glial cells. activity can be checked.

The types of antibodies used in the present invention are shown in Table 1.

primary antibody target dilution concentration (species) manufacture company TH dopaminergic neurons 1:500 (ms) Chemicon GFAP astrocyte 1:500 (ms) Cell
signaling Iba-1 microglia 1:500 (Rb) Wako

< Example 1> In primary cultured neurons Evernic acid neuroprotective effect

Primary cultured neurons were pre-treated with evernic acid for 6 hours and then treated with MPP ⁺ for 24 hours to observe the effect. As a result, neuronal cell death by MPP ⁺ and protection by evernic acid could be observed under a microscope (FIG. 3A), which was also confirmed in the cell viability measurement experiment using the MTT assay (FIG. 3B). In addition, it was confirmed that the form of cell death by MPP ⁺ was apoptosis through western blot, and it was found that evernic acid prevented apoptosis ( FIGS. 3C and 3D ). . Therefore, it was confirmed that evernic acid has the effect of directly protecting nerve cells in the Parkinson's disease model.

< Example 2> In primary cultured neurons Evernic acid The effect of improving mitochondrial function

The primary cultured neurons were treated with evernic acid and MPP ⁺ for 6 hours, and mitochondrial functionality was observed. As a result, it was confirmed that the mitochondrial membrane potential was damaged by MPP ⁺ , and it was found that it was protected by evernic acid ( FIG. 4A ). In addition, when the mitochondrial respiratory function was checked using a metabolic gas analyzer, it was found that the mitochondrial function, such as the basic respiratory level, proton leak, and ATP production, was restored by evernic acid by MPP ⁺ (Fig. 4B and 4C). In addition, damage to mitochondria can cause excessive production of free radicals. When the active oxygen in the cells was measured using DCF-DA, it was confirmed that the generation of active oxygen was inhibited in the evernic acid-treated group ( FIG. 4D ). Therefore, evernic acid is It was confirmed that the mitochondrial function of primary cultured neurons was improved and oxidative stress was lowered.

< Example 3> In primary cultured astrocytes Evernic acid Activation inhibitory effect through anti-inflammatory action

Various experiments were performed by culturing primary cultured astrocytes, pre-treated with evernic acid for 6 hours and treated with MPP ⁺ for 24 hours. As a result of immunostaining and western blotting, it was confirmed that astrocytes were activated by MPP ⁺ , and it was confirmed that the activation was inhibited by treatment with evernic acid ( FIG. 5A ). It was found that evernic acid lowered the activation of astrocytes through anti-inflammatory action through co-labeling experiments with the representative inflammatory marker NF-κB (p65) and Western blot ( FIGS. 5C and 5D ). Therefore, it was confirmed that evernic acid has an anti-inflammatory effect by inhibiting the NF-κB pathway in astrocytes, and has a dual effect of directly protecting neurons and lowering the activity of glia.

< Example 4> to MPTP induced Parkinson's disease in animal models on Mount Evernic improvement of athletic performance by

In order to confirm the efficacy of evernic acid verified in vitro also in vivo , an MPTP-induced Parkinson's disease mouse model was used. First, the motor ability was measured through the Rota-rod test. Two hours after the last MPTP is administered, the MPTP group has a marked decrease in exercise capacity compared to the control group. After intraperitoneal administration of MPTP, it can be seen that exercise ability gradually recovers over time. 6).

< Example 5> to MPTP induced Parkinson's disease in animal models Evernic acid Observation of neuroprotective effect

After sectioning the striatum (STR) and substatia nigra (SN) areas of the nigra-striatal dopamine pathway in Parkinson's disease lesion, DAB staining was used to express Tyrosine hydroxylase (TH), a marker of dopaminergic neurons. was observed. As a result of observation, it can be seen that a lot of dopaminergic neurons were lost by MPTP, as the TH staining became pale in the MPTP group. However, it can be seen that the group administered with MPTP and evernic acid 80 mg/kg has effective protection of dopaminergic neurons compared to the MPTP group ( FIG. 7 ).

< Example 6> to MPTP induced Parkinson's disease in animal models Evernic acid Observation of anti-inflammatory effect

In order to observe the degree of glial cell activation in the STR (FIG. 8A) and SN (FIG. 8B) regions, GFAP, an astrocyte marker, and Iba-1, a microglia marker, were double fluorescently stained to observe the expression level. As a result, the expression of both GFAP and Iba-1 was increased by MPTP, indicating that glial cells were activated. However, in the group administered with MPTP and evernic acid 80 mg/kg, the expression of astrocytes was significantly reduced compared to the MPTP group, and the inhibitory effect on microglia did not appear ( FIG. 8 ). Therefore, it was found that evernic acid has the effect of inhibiting neuroinflammation mediated by astrocytes in the Parkinson's disease model.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

A pharmaceutical composition for preventing or treating Parkinson's disease, comprising as an active ingredient evernic acid represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.
[Formula 1]

According to claim 1, wherein the composition is a pharmaceutical composition for preventing or treating Parkinson's disease, characterized in that it has a neuroprotective effect through mitochondrial stabilization of neurons and an inhibitory effect on a neuroinflammatory response occurring in astrocytes. According to claim 1, wherein the composition is a pharmaceutical composition for preventing or treating Parkinson's disease, characterized in that it has a motor ability improvement effect and a dopaminergic neuron protective effect. The pharmaceutically acceptable salt of claim 1, wherein the pharmaceutically acceptable salt is hydrochloride, bromate, sulfate, phosphate, nitrate, citrate, acetate, lactate, tartrate, maleate, gluconate, succinate, formate, trifluoroacetic acid salt, oxalate, fumarate, methanesulfonate, benzenesulfonate, para-toluenesulfonate, camphorsulfonate, sodium salt, potassium salt, lithium salt, calcium salt and magnesium salt, characterized in that at least one salt selected from the group consisting of A pharmaceutical composition for preventing or treating Parkinson's disease. A health functional food composition for preventing or improving Parkinson's disease, comprising as an active ingredient evernic acid represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.
[Formula 1]

[Claim 6] The health functional food composition for preventing or improving Parkinson's disease according to claim 5, wherein the composition has a neuroprotective effect through mitochondrial stabilization of nerve cells and an inhibitory effect on neuroinflammatory responses occurring in astrocytes. [Claim 6] The health functional food composition for preventing or improving Parkinson's disease according to claim 5, wherein the composition has an athletic ability improvement effect and a dopaminergic neuron protective effect.

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