KR20240100553A - Composition comprising extract of Polygonum aviculare for for preventing or treating brain diseases - Google Patents

Abstract

The present invention relates to a composition for preventing or treating brain diseases containing an extract of Polygonum aviculare . The extract activates brain neurons by inducing endocytosis and phosphorylation of TrkB (Tropomyosin receptor kinase B) and induces dendritic development of striatal neurons, thereby treating obsessive-compulsive disorder, autism spectrum disorder, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. It can be easily used as a pharmaceutical composition for the prevention, treatment, and improvement of various brain diseases, such as an effective health functional food for the development of brain cells.

Description

Composition comprising extract of Polygonum aviculare for for preventing or treating brain diseases}

The present invention relates to a composition for preventing or treating brain diseases containing an extract of Polygonum aviculare .

The actions of drugs related to brain function disorders include changes in long-term potentiation (LTP), increased expression of BDNF (Brain Derived Neurotropic Factor) in local areas such as the hippocampus and inferior temporal cortex, and its receptor, TrkB. It is related to the activation of (Tropomyosin receptor kinase B). TrkB is a Receptor Tyrosine Kinase-type receptor, which is highly expressed in the neocortex, hippocampus, striatum, and brain epistemum. It is one of the targets for neuroprotection in neurodegenerative diseases, and is important for neuron survival, differentiation, and function in the brain. It is one of the most widely distributed neurotrophic tyrosine kinases (NTRs). Binding of TrkB to its ligand, BDNF, results in activation of signaling pathways including MARK, PI3K, and PLC-γ through conformational changes and autophosphorylation of tyrosine residues in the intracellular domain. Therefore, TrkB agonists such as BDNF may have therapeutic potential in numerous neurological, mental, and metabolic disorders, making it possible to search for brain disease therapeutic agents using cell systems using TrkB.

Meanwhile, TrkB (Tropomyosin receptor kinase B), which is distributed in the plasma membrane, has the function of being phosphorylated and endocytosed into the plasma when it binds to a ligand, and induces the activation of various brain nerve cells through downstream signaling targets.

Therefore, while studying the activity of natural products using CLIP-TrkB HEK293 cells and striatal neurons prepared according to this principle, the inventors of the present invention confirmed that the extract of the knotweed acts as a TrkB agonist such as BDNF, thereby preventing novel brain diseases. By confirming that it can be used as a therapeutic agent, the present invention was completed.

Republic of Korea Patent No. 10-2013457 (Title of invention: Composition with AMPK activation effect, Applicant: R&S Co., Ltd., Registration date: 2012.08.13) Republic of Korea Patent Publication No. 10-2011-0083817 (Title of invention: Cosmetic composition for improving skin inflammation, Applicant: RNS Co., Ltd., Publication date: 2010.01.15) Chinese Patent No. 102901815 (Title of invention: A detection trkb receptor 816/817 the activity of tyrosine site elisa kit and method of use thereof, Applicant: Wuhan Yuanzheng Century Pharmaceutical Co., Ltd, Registration date: 2014.10.22) U.S. Patent No. 5622862 (Title of invention: Assay systems for trkb neurotrophin activity, Applicant: Regeneron Pharmaceuticals, Inc, Registration date: 1997.04.22)

The purpose of the present invention is to provide a composition for preventing or treating brain diseases containing an extract of Polygonum aviculare .

The present invention relates to a composition for preventing or treating brain diseases containing an extract of Polygonum aviculare .

The brain disease is characterized as being selected from the group consisting of obsessive-compulsive disorder, autism spectrum disorder, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.

The nodal plant can be used by selecting parts selected from flowers, stems, rhizomes, leaves, above-ground parts, or sentinels.

The extract can be extracted from the knotweed using water, C1 to C4 alcohol, or a mixed solution thereof as a solvent.

The present invention can also provide a health functional food for preventing or improving brain diseases containing the polygonum aviculare extract.

Hereinafter, the present invention will be described in detail.

The knotweed extract can be extracted from the knotweed using water, C1~C4 alcohol, or a mixed solution thereof as a solvent, and the C1~C4 alcohol may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and isobutanol. You can. The extract may also be a 30-90% (v/v) alcohol aqueous solution extract, preferably a 50-80% (v/v) alcohol aqueous solution extract.

Water, C1 to C4 alcohol, or a mixed solution thereof used in the production of the above joint extract can be used in an amount of 1 to 40 times the volume (1 to 40 liters per kg) based on the weight of the joint extract, and preferably 5 to 40 times the volume. You can use it. Extraction conditions for the joint extract may be 1 minute to 48 hours at 20 to 100°C. The above process can be repeated 1 to 4 times.

In addition, as a common method in the art, the extract of water, C1 to C4 alcohol or a mixture thereof of the nodule plant is dissolved in water and then selected from the group consisting of n-hexane, methylene chloride, acetone, chloroform, ethyl acetate and n-butanol. It can be prepared into fractions by additional fractionation using one or more selected solvents.

As a device for extracting the extract or its fractions, a conventional extraction device, an ultrasonic grinding extractor, or a fractionator can be used. The knotweed extract or fraction prepared in this way can be dried with hot air, dried under reduced pressure, or freeze-dried to remove the solvent. Additionally, the extract or fraction of the knotweed can be purified and used using column chromatography.

The chromatography includes silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, and medium pressure liquid chromatography. chromatography), thin layer chromatography (TLC), silica gel vacuum liquid chromatography, and high performance liquid chromatography.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of brain diseases containing an extract of the knotweed. The extract may be added to the pharmaceutical composition of the present invention in an amount of 0.001 to 100% by weight.

The pharmaceutical composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogenatin, etc. can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebrovascular injection. The extract of the present invention has almost no toxicity and side effects, so it is a drug that can be safely used even when taken for a long period of time for preventive purposes.

In addition, the present invention provides a health functional food for preventing or improving brain diseases containing a joint extract and a foodologically acceptable food supplement. The knotweed extract can be added to the health functional food of the present invention in an amount of 0.001 to 100% by weight. The health functional food of the present invention includes the form of tablets, capsules, pills, or liquid, and foods to which the extract of the present invention can be added include, for example, various drinks, meat, sausages, bread, candies, These include snacks, noodles, ice cream, dairy products, soups, electrolyte beverages, beverages, alcoholic beverages, gum, tea, and vitamin complexes.

The present invention relates to a composition for preventing or treating brain diseases containing an extract of Polygonum aviculare . The extract activates brain neurons by inducing endocytosis and phosphorylation of TrkB (Tropomyosin receptor kinase B) and induces dendritic development of striatal neurons, thereby treating obsessive-compulsive disorder, autism spectrum disorder, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. It can be easily used as a pharmaceutical composition for the prevention, treatment, and improvement of various brain diseases, such as an effective health functional food for the development of brain cells.

Figure 1 is a schematic diagram showing the structure of the pCCL-Hass-HA-CLIP-rTrkB vector of the present invention.
Figure 2 shows the protein expression results of HA in CLIP-TrkB HEK293 cells transformed with the pCCL-Hass-HA-CLIP-rTrkB vector.
Figure 3 is a schematic diagram showing the endocytosis process of CLIP-TrkB protein bound to the ligand BDNF in CLIP-TrkB HEK293 cells.
Figure 4 shows the results confirming that the node extract causes endocytosis of CLIP-TrkB protein like BDNF in CLIP-TrkB HEK293 cells.
Figure 5 shows the results confirming that the joint extract causes phosphorylation of CLIP-TrkB protein like BDNF in CLIP-TrkB HEK293 cells.
Figure 6 shows the results confirming that the leaf extract extract induces dendritic development of striatal neurons.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to ensure that the content introduced here is thorough and complete, and to sufficiently convey the spirit of the present invention to those skilled in the art.

<Example 1. Preparation of knotweed extract>

The aerial part of the nodal plant was taken and dried, then 10L of 70 (v/v)% ethanol aqueous solution was added to 100 g of the nodal plant, extracted under reflux, concentrated under reduced pressure to remove ethanol, and freeze-dried to obtain a powder sample.

<Example 2. Preparation of CLIP-TrkB HEK293 cell line>

In order to establish a system that allows easy high-throughput screening of brain disease treatments through fluorescence imaging, a cell line was prepared targeting TrkB, a receptor tyrosine kinase type receptor.

This uses the principle that TrkB, which is located in the plasma membrane, is endocytized into the plasma when it binds to a ligand. To this end, a CLIP tag that expresses fluorescence was combined with TrkB to enable subcellular localization in living cells.

This CLIP tag is a modified version of the SNAP tag that reacts with benzylguanine derivatives and is a self-labeling protein derived from O6-alkylguanine-DNA-alkyltransferase. CLIP tags react with benzylcytosine derivatives within cells and can produce fluorescence in living cells, so they are mainly used as protein tags.

In the present invention, CLIP-tagged TrkB recombinant DNA (SEQ ID NO: 1) was first produced so that the TrkB receptor can display its subcellular localization in living cells.

For this purpose, the CLIP tag was first introduced into the 5' position of rat TrkB cDNA through overlapping PCR*.

*primer

sense: CTA GCT AGC GGA TCC GAC AAG GAT TGT G (DG1; NheI-CLIP-TrkB-Forward) 　　

antisense: CCG CTC GAG CTA GCC TAG GAT GTC CAG G (DG2; XhoI-TrkB-Reverse)

The pCCL vector was created by inserting the CLIP TrkB sequence (SEQ ID NO: 1) into the subcloning site of the pCCL-HAss vector using NheI and Through sequencing, it was also confirmed whether the CLIP TrkB sequence (SEQ ID NO: 1) was properly inserted.

The structure of the pCCL-Hass-HA-CLIP-rTrkB vector prepared in this way and used for cell transformation is shown in Figure 1.

Afterwards, lentivirus was created in HEK293 using the prepared pCCL-Hass-HA-CLIP-rTrkB. This was soon transduced into HEK293 cells, and well-transformed cells were obtained. During transduction, the number of HEK293 cells and the amount of lentivirus were adjusted to ensure that most cells expressed HA-CLIP-TrkB, and this was confirmed through HA staining and CLIP staining.

In addition, the fact that the cells prepared in this way were transformed by inserting the vector was confirmed again through a biochemical method using Western blotting using an anti-HA antibody, as shown in Figure 2.

<Example 3. Confirmation of the mechanism of action of a cell line as a tester for a treatment for brain diseases and testing the efficacy of the extract of the knotweed>

In the cell line established in Example 2, as shown in FIG. 3, CLIP-TrkB in a fluorescent state on the surface of the cell's plasma membrane binds to a ligand (eg, BDNF) and moves into the plasma.

Therefore, it was verified through immunocytochemistry (ICC) whether the cell line directly reacted with BDNF (Brain Derived Neurotropic Factor) and the fluorescence moved from the plasma membrane into the plasma through endocytosis as follows.

For this purpose, a washed glass coverslip was placed in a 24-well plate after treatment with nitric acid for 2 days, and then coated with PDL (poly D lysin). Afterwards, 1x10 ⁵ CLIP-TrkB HEK293 cells were dispensed into each well and cultured in DMEM containing 10% FBS under humidified 5% CO ₂ conditions at 37°C for 24 hours, and the reactivity of the cell line to BDNF was tested. To optimize this, the CLIP-TrkB HEK293 cells were replaced with serum free media and left in the same conditions again for 24 hours.

Next, for fluorescent staining of the CLIP tag, 1 μM Surface 547 dye was used for 1 hour, and BDNF (25 ng/ml) was treated for 15 minutes (the control group was treated with only DMSO, a solvent in which BDNF was dissolved).

At the same time, the test sample, the node plant extract, was treated with BDNF at 10 μg/ml for the same time.

To confirm fluorescence, the stained cells were washed with PBS, fixed with 4% paraformaldehyde solution, transferred to a glass slide, and confirmed under a fluorescence microscope.

The results of fluorescence microscopy are shown in Figure 4. As in the control result on the left, fluorescent staining was confirmed on the plasma membrane surface in CLIP-TrkB HEK293 cells that were not treated with BDNF, while fluorescence staining was observed in the BDNF-treated CLIP-TrkB HEK293 cells in the center. It can be seen that it is expressed scattered throughout the protoplasm of these cells. This same phenomenon was also observed in the group treated with the node extract shown in the right photo of Figure 4, with fluorescence moving into the protoplasm.

Accordingly, it was confirmed that CLIP-TrkB HEK293 cells can function as useful cells that can search for active substances that function to activate brain cells, such as BDNF.

In addition, it can be confirmed that the extract of the knotweed of the present invention can be used as a treatment for brain diseases that can activate brain cells through TrkB.

<Example 4. Confirmation of phosphorylation of CLIP-TrkB>

TrkB, a receptor, is autophosphorylated by stimulation of a ligand such as BDNF, and must then be internalized (endocytosis) into the cell protoplasm for subsequent activation of brain cells.

Accordingly, in order to confirm that TrkB is not simply internalized into cells through a non-specific reaction without a phosphorylation process in the CLIP-TrkB HEK293 cell line, BDNF and the extract of the plant of the present invention were used for direct brain cell activation or as a treatment for brain diseases. To prove that it can be used, the degree of phosphorylation of TrkB was confirmed through immunoprecipitation (IP) and Western blot (WB).

For this, a 6 well plate was coated with PDL as in Example 3, ² Cultured. As a control group, DMSO, BDNF (25ng/ml) for cell line establishment confirmation, and 10-200㎍/ml of knotweed extract were treated for 15 minutes.

Afterwards, cells were lysed using RIPA (Radioimmunoprecipitation assay) buffer, and phosphorylated CLIP-TrkB was precipitated using an anti-phosphotyrosine antibody. After washing the sediment three times with RIPA buffer, IPed TrkB was obtained using SDS sample buffer.

For each IP sample obtained in this way, Western blotting using an anti-HA antibody was performed in the same manner as in Example 2 to confirm the amount of phosphorylated protein.

The experimental results are shown in Figure 5. Referring to Figure 5, it can be seen that tyrosine phosphorylation of TrkB in CLIP-TrkB HEK293 cells appears through BDNF, which means that the CLIP-TrkB HEK293 cells activate brain cells such as BDNF. This result can be said to prove that it is useful in the search for materials.

In addition, it can be confirmed that, like BDNF, the extract of the plant can also play a role as a treatment for brain diseases by causing tyrosine phosphorylation of TrkB at 10-200㎍/㎖.

<Example 4. Confirmation of dendritic development in striatal neurons>

In order to further verify the efficacy of the extract of the extract confirmed through CLIP-TrkB HEK293 cells, it was demonstrated whether dendritic growth occurred in the striatal neurons treated with the extract of the extract of the extract of the extract of brain cells.

First, fetuses were isolated from pregnant mice on embryonic day 16, and primary striatal neurons were extracted. These cells were cultured in Neurobasal media containing B-27 supplements, dendritic development was induced, and used in experiments. Dendritic development was confirmed through the expression of its marker protein, MAP2.

For this purpose, primary striatal neurons were cultured by treating them with 10 ㎍/㎖ of node plant extract for 5 days. Afterwards, the cultured striatal neurons were fixed with 4% paraformaldehyde solution, permeablized with 0.2% Triton The image was secured. After randomly selecting about 40 images obtained in this way, the development of primary and secondary dendrites was analyzed, and this was quantified and shown in Figure 6.

Referring to the results in Figure 6, it can be seen that the primary dendritic development in striatal neurons was significantly increased by about 15% in the node plant extract-treated group compared to the untreated group.

The above results demonstrate that the extract of the knotweed of the present invention is a material that can be applied in various ways to activation, treatment, regeneration, and differentiation of nerve cells through CLIP-TrkB HEK293 cell line and striatal nerve cells.

<Formulation Example 1. Pharmaceutical formulation - Preparation of tablets>

200 g of the knotweed extract of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. A 10% gelatin solution was added to this mixture, then pulverized and passed through a 14 mesh sieve. This was dried and 160g of potato starch, 50g of talc, and 5g of magnesium stearate were added to make the resulting mixture into tablets.

<Formulation example 2. Food production>

Formulation Example 2-1. Manufacturing of cooking seasoning

A cooking seasoning for health promotion was prepared by adding 1% by weight of the joint extract of the present invention to the cooking seasoning.

Formulation Example 2-2. Manufacturing of flour foods

The knotweed extract of the present invention was added at 0.1% by weight to flour, and this mixture was used to prepare bread, cakes, cookies, crackers, and noodles to prepare health-promoting foods.

Formulation Example 2-3. Preparation of soups and gravies

Soup and gravy for health promotion were prepared by adding 0.1% by weight of the joint extract of the present invention to soup and gravy.

Formulation Example 2-4. Manufacturing of dairy products

The knotweed extract of the present invention was added to milk at 0.1% by weight, and various dairy products such as butter and ice cream were manufactured using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

Vegetable juice for health promotion was prepared by adding 0.5 g of the knotweed extract of the present invention to 1,000 ml of tomato juice or carrot juice.

Formulation Example 2-6. Fruit juice manufacturing

Health-promoting fruit juice was prepared by adding 0.1 g of the joint extract of the present invention to 1,000 ml of apple juice or grape juice.

Sequence list electronic file attached

Claims (8)

A composition for preventing or treating brain diseases, characterized in that it contains an extract of Polygonum aviculare . According to paragraph 1,
A composition for preventing or treating brain diseases, wherein the brain diseases are selected from the group consisting of obsessive-compulsive disorder, autism spectrum disorder, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. According to paragraph 1,
A composition for preventing or treating brain diseases, characterized in that the nodal plant is used by selecting parts selected from flowers, stems, rhizomes, leaves, aerial parts, or sentinels. According to paragraph 1,
The extract is a composition for preventing or treating brain diseases, characterized in that the extract is extracted from the nodal plant using water, C1 to C4 alcohol, or a mixed solution thereof as a solvent. A health functional food for preventing or improving brain disease, characterized by containing extract of Polygonum aviculare . According to clause 5,
A health functional food for preventing or improving brain diseases, wherein the brain diseases are selected from the group consisting of obsessive-compulsive disorder, autism spectrum disorder, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. According to clause 5,
The joint plant is a health functional food for preventing or improving brain disease, characterized in that the part selected from the flower, stem, rhizome, leaf, aerial part, or sentinel is used. According to clause 5,
The extract is a health functional food for preventing or improving brain diseases, characterized in that the extract is extracted from the nodal plant using water, C1 to C4 alcohol, or a mixed solution thereof as a solvent.