KR102314268B1 - Mosichongmyeongcha for protecting brain neuroral cells - Google Patents

Abstract

The present invention relates to a Boehmeria nivea Chongmyung tea composition obtained by mixing and extracting Boehmeria nivea leaves, Salvia miltiorrhiza, Curcuma aromatica, and Polygala tenuifolia. The present invention has an effect of being able to be manufactured economically and safely without a fermentation process. In addition, the present invention possesses anti-inflammatory activity which suppresses inflammatory mediators secreted by activation as LPS in a BV2 cell line, which is mouse microglia, and antioxidant activity which suppresses oxidative stress induced by glutamate in a HT22 cell line, which is a mouse hippocampal cell line.

Description

Mosichongmyeongcha for protecting brain neuroral cells

The present invention relates to a composition extracted from ramie leaf, dandelion ginseng, ganghang and raw paper mixed extract. The present invention has the effect that it can be manufactured economically and safely without a fermentation process. In addition, the anti-inflammatory activity of inhibiting inflammatory mediators secreted by activation as LPS in the BV2 cell line, a mouse microglia, and the antioxidant activity of suppressing the oxidative stress induced by glutamate in the mouse hippocampal cell line, HT22 cell line. holds

The present invention is a result derived through the following research project.

[Project No.: RA201906-4-C4

Department name: Jeollabuk-do / Project management organization name: Jeonbuk Techno Park

Research project name: 2019 Jeollabuk-do innovative growth R&D+ business technology development project

Research project name: Development of extracts and health functional foods for cognitive function improvement using ramie grass and mechacin

Name of project performing organization: (Nong)SBS Biopharmaceuticals, Wonkwang University Industry-Academic Cooperation Foundation, Chonbuk National University Industry-Academic Cooperation Foundation

Research period: 2019.6.1~2020.5.31]

Rapeseed (Boehmeria nivea (L.) GAUDICH.) is a herbaceous perennial plant belonging to the nettle family, and is also called ramie or ramie. There are about 100 species in the tropics, and about 8 species are distributed in our country. , turtle tails and grass turtle tails are growing, and there is a rock ramie (non-sheeping tree) in the rock ramie.

Salvia miltiorrhiza (Salvia miltiorrhiza) is a herbaceous perennial plant belonging to the family Lamiaceae. The height is 40-80 cm, and the flowers are purple and bloom from May to June and run in layers. The roots are used as herbal medicine. It contains tansinnon and vitamin E, and it has been recognized that it dilates peripheral blood vessels and lowers blood pressure in animal experiments.

Ginseng contains tanshinone I, II (tanshinone I, II), dihydrotanshinone, cryptotanshinone, methyl tanshinone, methylene, tanshiquinone. and beta-sisoneterol (β-sitosterol), and is effective in cardiovascular diseases, hepatitis, gynecological diseases, diabetes and chronic kidney disease.

Turmeric (姜黃/薑黃, Curcuma longa Rhizoma) is an annual plant of the monocotyledonous Ginger family, and its rhizome is used as a herbal medicine. Turmeric contains curcumin, turmerone and arturmerone. It is a yellowish medicinal with a spicy and bitter taste, and is effective in relieving pain and menstrual irregularities.

Wonji (遠志, Polygala tenuifolia) is a perennial plant of the dicotyledonous arboraceae family, and the root is called Wonji in oriental medicine. The taste of raw paper is spicy and bitter, and the nature is warm. It is used when your heart beats while being surprised, when you cough with phlegm, and forgetfulness. It has effects such as sedation, hypnosis, strong heart, phlegm clearing, and hemolysis.

Looking at the prior art literature invented by the present inventor, Korean Patent Publication Nos. 10-1584513 and 10-1673204 disclose that the fermented product of ramie leaves with Seonokbacterium liquid was oxidative stress induced by glutamate against the mouse hippocampal cell HT22 cell line. It has been described that there is an anti-oxidative effect on the anti-inflammatory effect and an anti-inflammatory effect on inflammation induced for the mouse microglia BV2 cell line.

The present inventors have devised the present invention by focusing on a composition that utilizes ramie leaves and other natural materials, but has a neuroprotective action while being manufactured more economically without going through the process of fermentation.

Republic of Korea Patent Publication No. 10-1584513 (registered on Jan. 6, 2016) Republic of Korea Patent Publication No. 10-1673204 (registered on Nov. 1, 2016)

An object to be solved is to provide a composition that utilizes ramie leaves and other natural materials, but is manufactured more economically without going through the process of fermentation, and has a protective action on brain nerve cells.

A solution is a ramie leaf, dandelion ginseng, turmeric and raw paper mixed with ethanol as an active ingredient, containing a mixed extract extracted with ethanol as an active ingredient, a composition for protecting brain nerve cells.

The mixed extract is characterized in that it is a mixed extract extracted with 30% EtOH after mixing ramie leaf, dandelion ginseng, turmeric and raw paper in a 2:2:1:1 weight ratio.

The mixed extract is characterized in that it contains 50 ~ 200 ug / mL.

The solution is a health functional food having a function of protecting brain nerve cells manufactured using the composition.

The ramie chongmyung tea composition of the present invention has the effect of being economically and safely manufactured without a fermentation process.

In addition, it has a remarkable effect of suppressing excessive inflammatory response of cranial nerve cells through anti-inflammatory activity that suppresses inflammatory mediators that are activated and secreted as LPS in the BV2 cell line, which is a mouse microglia.

In addition, it has a remarkable effect of preventing damage to brain neurons due to oxidative stress through antioxidant activity that suppresses oxidative stress induced by glutamate in the HT22 cell line, a mouse hippocampal cell.

1 to 16 are graphs showing experimental results of an experimental example according to the present invention.

The terms or words used in the present specification and claims should not be construed as being limited to their ordinary or dictionary meanings, and the inventor may properly define the concept of the term in order to best describe his invention. Based on the principle that there is, it should be interpreted as meaning and concept consistent with the technical idea of the present invention.

Therefore, the matters described in the embodiments, reference examples and drawings described in this specification are only the most preferred examples of the present invention and do not represent all of the technical spirit of the present invention, so they can be substituted at the time of the present application It should be understood that various equivalents and modifications may be made.

The following experiments were performed mainly by employing the experimental methods described in Korean Patent Publication Nos. 10-1584513 and 10-1673204 introduced in the prior art literature.

Experimental Example 1. Evaluation of BV2 cytotoxicity by sample of materials

1) Evaluation of toxicity of ramie leaves by solvent in BV2 microglia cells (see Fig. 1)

For ramie leaf extract, DW extract (distilled water extract) 200 μg/mL, 30% (V/V) EtOH extract 200 μg/mL, 50% (V/V) EtOH extract 200 μg/mL, 70% (V/V) ) EtOH extract 200 μg/mL showed cytotoxicity, respectively (*p<0.05).

For additional experiments, 100 μg/mL of DW extract, 30% EtOH extract, 50% EtOH extract, and 70% EtOH extract were all carried out at the highest concentration.

2) Evaluation of toxicity of turmeric by solvent in BV2 microglia cells (see FIG. 2)

For turmeric extract, DW extract is cytotoxic at 200 μg/mL, 30% EtOH extract is 100, 200 μg/mL, 50% EtOH extract is 100, 200 μg/mL, and 70% EtOH extract is 100, 200 μg/mL. appeared (*p<0.05).

For additional experiments, 100 µg/mL of DW extract, 50 µg/mL of 30% EtOH extract, 50 µg/mL of 50% EtOH extract, and 50 µg/mL of 70% EtOH extract were performed at the highest concentration.

3) Evaluation of toxicity by solvent of ginseng in BV2 microglia cells (see Fig. 3)

In the case of ginseng extract, 50% EtOH extract showed cytotoxicity at 200 μg/mL and 70% EtOH extract showed cytotoxicity at 200 μg/mL. *p<0.05).

For additional experiments, 200 µg/mL of DW extract, 200 µg/mL of 30% EtOH extract, 100 µg/mL of 50% EtOH extract, and 100 µg/mL of 70% EtOH extract were performed at the highest concentration.

4) Evaluation of toxicity by solvent of base paper in BV2 microglia cells (see Fig. 4)

In case of raw paper extract, cytotoxicity was observed at 100 µg/mL of DW extract, 100 µg/mL of 30% EtOH extract, 100 µg/mL of 50% EtOH extract, and 100 µg/mL of 70% EtOH extract (*p<0.05).

For additional experiments, 50 μg/mL of DW extract, 30% EtOH extract, 50% EtOH extract, and 70% EtOH extract were all carried out at the highest concentration.

5) Evaluation of toxicity by solvent of Chunma in BV2 microglia cells (see Fig. 5)

In the case of Chunma extract, cytotoxicity was observed at 100 µg/mL of DW extract, 100 µg/mL of 30% EtOH extract, 50 µg/mL of 50% EtOH extract, and 25 and 50 µg/mL of 70% EtOH extract (*p<0.05). .

For additional experiments, 50 µg/mL of DW extract, 50 µg/mL of 30% EtOH extract, 25 µg/mL of 50% EtOH extract, and 12.5 µg/mL of 70% EtOH extract were performed at the highest concentration.

Experimental Example 2. BV2 cell anti-inflammatory evaluation by sample of materials

1) Inhibitory effect of nitrite production by solvent of ramie leaves in BV2 microglia cells (see Fig. 6)

100 μg/mL of ramie leaf DW extract, 30% EtOH extract, 50% EtOH extract, and 70% EtOH extract were all carried out at the highest concentration. As a result, it was confirmed that nitrite production was gradually suppressed at 25, 50, and 100 μg/mL of the ramie leaf DW extract. It was confirmed that the 30% EtOH extract from ramie leaves gradually suppressed nitrite production at 12.5, 25, 50, and 100 μg/mL. It was confirmed that the ramie leaf 50% EtOH extract gradually suppressed nitrite production at 12.5, 25, 50, and 100 μg/mL. It was confirmed that nitrite production was gradually suppressed at 12.5, 25, 50, and 100 μg/mL of ramie leaf 70% EtOH extract (*p<0.05).

2) Inhibition of nitrite production by solvent of turmeric in BV2 microglia cells (see FIG. 7)

100 μg/mL of turmeric DW extract, 50 μg/mL of 30% EtOH extract, 50 μg/mL of 50% EtOH extract, and 50 μg/mL of 70% EtOH extract were carried out at the highest concentrations, respectively.

As a result, the turmeric DW extract did not show a significant effect. It was confirmed that the turmeric 30% EtOH extract gradually inhibited nitrite production at 12.5, 25, and 50 μg/mL. It was confirmed that the turmeric 50% EtOH extract gradually inhibited nitrite production at 25 and 50 μg/mL. It was confirmed that the turmeric 70% EtOH extract gradually inhibited nitrite production at 6.25, 12.5, 25, and 50 μg/mL (*p<0.05).

3) Inhibitory effect of nitrite production by solvent in BV2 microglia cells (see Fig. 8)

200 μg/mL of ginseng DW extract, 200 μg/mL of 30% EtOH extract, 100 μg/mL of 50% EtOH extract, and 100 μg/mL of 70% EtOH extract were used at the highest concentrations, respectively.

As a result, the DW extract of dandelion ginseng had no significant effect. It was confirmed that the 30% EtOH extract of dandelion ginseng inhibited nitrite production at 200 μg/mL. It was confirmed that nitrite production was inhibited in 100 μg/mL of ginseng 50% EtOH extract. It was confirmed that nitrite production was gradually suppressed at 50 and 100 μg/mL in the 70% EtOH extract of dandelion ginseng (*p<0.05).

4) Inhibitory effect of nitrite production by solvent of base paper in BV2 microglia cell (see Fig. 9)

The raw paper DW extract, 30% EtOH extract, 50% EtOH extract, and 70% EtOH extract were all carried out at 50 μg/mL at the highest concentration.

As a result, it was confirmed that nitrite production was gradually suppressed at 25 and 50 μg/mL in the raw paper DW extract. It was confirmed that nitrite production was gradually suppressed at 12.5, 25, and 50 μg/mL of the raw paper 30% EtOH extract. It was confirmed that nitrite production was gradually suppressed at 25 and 50 μg/mL in the 50% EtOH extract of raw paper. It was confirmed that nitrite production was gradually suppressed at 25 and 50 μg/mL in the 70% EtOH extract of raw paper (*p<0.05).

5) Inhibitory effect of nitrite production by solvent of Chunma in BV2 microglia cells (see Fig. 10)

Chunma DW extract 50 μg/mL, 30% EtOH extract 50 μg/mL, 50% EtOH extract 25 μg/mL, and 70% EtOH extract 12.5 μg/mL were each at the highest concentration.

As a result, it was confirmed that nitrite production was gradually suppressed at 25 and 50 μg/mL in the Chunma DW extract. Chunma 30% EtOH extract was confirmed that nitrite production was gradually suppressed at 12.5, 25, 50 μg/mL. Chunma 50% EtOH extract and Chunma 70% EtOH extract had no significant effect (*p<0.05). Chunma showed weak anti-inflammatory action in the whole sample.

Reference Example 1. Comparison of BV2 microglia cell cytotoxicity and nitrite production inhibitory effect according to the extraction solvents of each individual medicinal herb, ramie leaf, turmeric, ginseng, raw paper, and cheonma

1) Concentration that does not show BV2 microglia cell cytotoxicity (μg/mL)

DW 30% EtOH 50% EtOH 70% EtOH ramie leaves 100 100 100 100 curcuma 100 50 50 50 ginseng 200 200 100 100 paper 50 50 50 50 celestial horse 50 50 25 12.5

In summary, it can be seen that the concentration without cytotoxicity was different according to the extraction solvent for each sample (see FIGS. 1 to 5).

2) BV2 microglia cell nitrite inhibitory effect (μg/mL): Statistically (p<0.05) significant concentration summary

DW 30% EtOH 50% EtOH 70% EtOH ramie leaves 25,50,100 12.5,25,50,100 12.5,25,50,100 12.5,25,50,100 curcuma X 12.5,25,50 25,50 6.25,12.5,25,50 ginseng X 200 100 50,100 paper 25,50 12.5,25,50 25,50 25,50 celestial horse 25,50 12.5,25,50 X X

In summary, when examining the nitrite production inhibitory effect at a concentration without cytotoxicity, the 30% EtOH extract had the best effect in all samples (see FIGS. 6 to 10). Therefore, it is judged that it is desirable to extract all samples with 30% EtOH.

Example 1. Moshichongmyung tea composition

According to the cytotoxicity and the inhibitory effect of nitrite production in BV2 microglia cells, the composition ratio of the ramie chongmyung tea composition was set according to the following principle.

1) Nitrite formation inhibitory effect: 30% EtOH is the most effective and is a suitable extraction solvent.

2) Drug composition ratio: When the composition ratio is set based only on the concentration that does not show cytotoxicity of each individual drug in the 30% EtOH extract, it is shown in the table below.

extraction solvent ramie leaves curcuma ginseng paper celestial horse 30% EtOH 2 One 4 One One

3) In the case of the above composition, it was determined that the ratio of ginseng was too high and it was more preferable to lower it to the same ratio (2) as that of ramie leaf. If the ratio of ginseng is 4, the ratio of the total content is 4/9, which is 44.4%. This is because, even at this ratio, ginseng has no toxicity at all. Of course, the composition may be prepared according to the design conditions in the above ratio.

4) It was judged that it would be better to exclude Chunma as it has high toxicity and poor effect.

5) In summary, making all samples with 30% EtOH extract is excellent in effectiveness and economical efficiency, and the most suitable ratio of medicinal herbs is ramie leaf 2: dansam 2: turmeric 1: raw paper 1.

extraction solvent ramie leaves ginseng curcuma paper 30% EtOH 2 2 One One

Experimental Example 3. Evaluation of BV2 cytotoxicity and efficacy of ramie chrysanthemum tea

1) Toxicity evaluation of chamomile tea in BV2 microglia cells (see FIG. 11)

In order to examine the brain inflammation inhibitory activity of ramie chrysanthemum tea (an extract extracted with 30% EtOH after mixing ramie leaf, ginseng, turmeric, and raw paper in a 2:2:1:1 weight ratio), cytotoxicity evaluation was first performed. After processing the extracts of ramie chrysanthemum tea according to the mixing ratio for 24 hours at each concentration, cytotoxicity was evaluated through MTT assay, and it was confirmed that no toxicity was observed up to 200 μg/ml.

2) Inhibitory effect of nitrite production in BV2 microglia cells (see Fig. 12 )

In order to investigate the effect of the R. chrysanthemum tea extract on NO production by LPS induction, BV2 cells were treated with the R. chrysanthemum extract for 3 hours and then LPS (1 μg/ml) was treated for 24 hours. As a result, it was confirmed that NO production was strongly inhibited in a concentration-dependent manner in BV2 cells (*P<0.05, ***P<0.001).

3) Inhibitory effect of PGE2 production in BV2 microglia cells (see Fig. 13)

In order to investigate the effect of the R. chrysanthemum tea extract on the LPS-induced PGE2 production, BV2 cells were treated with the R. chrysanthemum extract for 3 hours and then LPS (1 μg/ml) was treated for 24 hours. As a result, it was confirmed that PGE2 production was inhibited in BV2 cells. (**P<0.01, ***P<0.001)

4) Inhibitory effect of iNOS and COX-2 expression in BV2 microglia cells (see Fig. 14)

In order to investigate the effect of LPS-induced COX-2 and iNOS expression on the LPS-induced LPS-induced LPS, BV2 cells were treated with the R. chrysanthemum tea extract for 3 hours, and then LPS (1 μg/ml) was treated for 24 hours. As a result, it was confirmed that COX-2 and iNOS expression were inhibited in BV2 cells.

Experimental Example 4. Evaluation of HT22 cell toxicity and efficacy

1) Toxicity evaluation of chamomile tea in HT22 cells (see Fig. 15)

In order to examine the brain cell protective activity of ramie chrysanthemum tea, cytotoxicity evaluation was first performed. After treating the extracts according to the mixing ratio of ramie chrysanthemum tea with LPS for 24 hours at each concentration, cytotoxicity was evaluated through MTT assay, and it was confirmed that toxicity did not appear up to 200 μg/ml.

2) Brain cell protective effect of ramie chrysanthemum tea in HT22 cells (see Fig. 16)

In order to examine the brain cell protective activity of R. chrysanthemum tea extract, glutamate (GA, 50 mM) was treated for 12 hours after each concentration was treated for 12 hours, and then the cell viability was confirmed by MTT assay. As a result, it was confirmed that the ramie chrysanthemum tea extract had a protective effect on brain cells. (**P<0.01, ***P<0.001)

Example 2. Moshichongmyung tea composition with brain nerve cell protective function

The composition according to the present invention includes a mixed extract extracted with 30% EtOH after mixing ramie leaf, dandelion ginseng, turmeric, and base paper in a 2:2:1:1 weight ratio as an active ingredient. Depending on the design conditions, ramie leaf, ginseng, turmeric, and raw paper may be mixed in a 2:4:1:1 weight ratio and then extracted.

In addition, based on the results of the experimental example, the mixed extract is to be included in 50 ~ 200 ug / mL.

The present invention can provide a brain nerve cell protection preparation formulated in a pharmaceutical unit dosage form by including the mixed extract as an active ingredient and adding a pharmaceutically acceptable carrier, excipient or diluent. Herein, the carrier, excipient, and diluent include toz, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, undecided. vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical dosage form may be used in the form of a pharmaceutically acceptable salt, and may be used alone or in combination with other pharmaceutically active compounds as well as in an appropriate group. In addition, when formulating the active ingredient, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used. In addition, the pharmaceutical dosage form may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. can

In the solid preparation for oral administration, at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. may be mixed with the extract. It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.

As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The preferred dosage of the mixed extract of the present invention varies depending on the patient's condition and weight, the degree of disease, age, sex, drug form, administration route and period, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the extract of the present invention is preferably administered in an amount of 0.001 to 300 mg/kg, and the administration may be administered once a day or divided into several administrations. The above dosage does not limit the scope of the present invention in any way.

The extract of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous injection.

It is possible to provide a health functional food composition for protecting brain nerve cells by adding a food supplement additive to the active ingredient of the present invention.

Foods to which the active ingredient can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, health functional foods, and the like.

The amount of the active ingredient in the food or beverage may be added 0.01 to 20% by weight of the total food or beverage weight, and the health drink composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml. have.

The health functional beverage composition of the present invention is not particularly limited in other components other than containing the extract, and may contain various flavoring agents or natural carbohydrates as additional components like a conventional beverage. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose; conventional sugars such as disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, erythritol and the like. In addition to the above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used as flavoring agents other than those described above. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, organic acids, protection It may contain a sexual colloid thickener, a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like.

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

Claims (4)

delete delete delete Contains mixed extracts of ramie leaf, ginseng, turmeric and raw paper as active ingredients,
The mixed extract is
- It is an extract extracted with 30% EtOH after mixing ramie leaf, dandelion ginseng, turmeric and raw paper in a 2:2:1:1 weight ratio,
- Characterized in that it contains 50 ~ 200 ug / mL,
A food composition for protecting brain nerve cells.

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