KR20200039267A - Composition for treatment or prevention of neuroinflammatory diseases comprising extract of Carpomitra costata - Google Patents

Abstract

The present invention relates to a composition for preventing and treating neuroinflammation which contains an extract of Carpomitra costata. The composition reduces the production of inflammatory mediators and cytokines to exhibit an anti-neuroinflammatory effect of microglia, and thus can exhibit effects of preventing and treating neuroinflammation having no side effects even when used for treatment. In addition, the present invention is to provide a pharmaceutical composition for preventing and treating neuroinflammation and a functional food composition for preventing and treating neuroinflammation with high preference, wherein the compositions contain the extract of Carpomitra costata.

Description

Composition for treatment or prevention of neuroinflammatory diseases comprising extract of Carpomitra costata}

The present invention is a composition for the prevention and treatment of neuroinflammation containing Joule's end extract, and more specifically, including the Joule's end extract as an active ingredient, neurons through inactivation of NF-κB related to TLR4 pathway and ROS blockade It relates to a composition capable of preventing and treating inflammation.

The central nervous system consists of neurons and glial cells. Glial cells make up about 90% of all brain cells, and by volume they make up about 50% of the entire brain. The glial cells can be further classified into three types: astrocytes, microglia, and oligodendrocytes.

Among them, astrocytes are the cells that occupy the largest number in the nervous system and appropriately remove neurotransmitters secreted by neurons or regulate the ion concentration in the brain and serve to assist neuron activity. Upon stimulation of inflammation, astrocytes proliferate and produce various intercellular mediators such as NO and TNF-α. In addition, microglia are a kind of differentiated macrophage (specialized macrophage), widely distributed in the brain. Microglia act as phagocytes that swallow tissue debris and dead cells, as well as participate in the brain's biodefense.

In addition, microglial cells increase neuroinflammation by secreting various neurotoxins and pro-inflammatory mediators in chronic brain diseases, causing neuronal cell death and demyelination. In response to brain damage or neuroinflammatory stimulation, microglia are nitric oxide (NO), reactive oxygen species (ROS), pro-inflammatory cytokines, prostaglandins and tumor necrosis factor-α (TNF-α) ) To participate in neuroinflammation. Activated microglia migrate to damaged nerve tissue areas, predating and destroying microbes and cell debris.

However, the role of astrocytes and microglia as inflammatory cells is not always beneficial, and the activation of astrocytes and microglia, which are not currently regulated, continuously causes excessive neuroinflammation, causing various central nervous system pathologies including degenerative neurological diseases. It is considered to be.

That is, functionally activated microglia produce and secrete inflammatory mediators, resulting in neuronal cell death, and diseases associated with activation and neuroinflammation of these microglia are brain ischemia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and muscle Various neurological and neurodegenerative planetary diseases such as atrophic lateral sclerosis are known.

More specifically, microglia overexpressed in response to inflammatory signals damage brain neurons and cause the development and progression of various neurodegenerative diseases. In particular, pathogenic toxins bind to Toll-like receptors (TLRs) and induce excessive activation of microglia.

Among them, lipopolysaccharides (LPS) present in the outer membrane of Gram-negative bacteria specifically bind to TLR4. This signal is a nuclear factor-κB through various intracellular signal transduction pathways, including phosphatidyl inositol 3'-kinase (PI3K, phosphatidylinositol 3'-kinase) / Akt and mitogen-activated protein kinase (MAPK). It is a series of inflammatory genes that induce transcriptional activation of (NF-κB) to promote inflammation and neurodegeneration of nerve cells. In addition, LPS-sensitized microglia induce oxidative stress by increasing the production of reactive oxygen species (ROS), which further exacerbates the inflammatory response.

Therefore, preventing excessive activation of microglia is an important tool for delaying the induction and progression of many brain diseases.

Seaweed has long been used as a source of medicine and food by residents of the coast of Asia. By using seaweed, it is necessary to develop a natural extract that has an effect on the prevention and treatment of nerve inflammation through inactivation of NF-κB related to TLR4 pathway and ROS production blockade, and can reduce side effects in the body.

(Patent Document 1) KR 10-2017-0116846 A1

An object of the present invention is to provide a composition for the prevention and treatment of neuroinflammation comprising the extract of Carpomitra costata.

Another object of the present invention is to reduce the production of inflammatory mediators and cytokines, to show the anti-neuro-inflammatory effect of microglia, and to provide a composition for preventing and treating neuro-inflammatory without side effects even when used for the treatment of neuro-inflammatory.

Another object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of neuroinflammation, including the extract of Carpomitra costata.

Another object of the present invention is to provide a functional food composition for the prevention and treatment of nerve inflammation, including the extract of Jouleumwan (Carpomitra costata).

In order to achieve the above object, the composition for preventing and treating neuroinflammation according to an embodiment of the present invention includes an extract of Carpomitra costata, wherein the extract of the line of inflammation is used to produce inflammation mediators and cytokines. When reduced, it may exhibit anti-inflammatory effects of microglia.

The Joule end extract may be extracted with a solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixtures thereof.

The Joule Terminal extract may be a Joule Terminal ethanol extract extracted using ethanol.

The extract of the end of the line may be a fraction obtained by fractionating the extract obtained from the end of the line using an organic solvent.

The Joule's end extract exhibits an antagonistic effect on the binding of LPS to TLR4 in immune cells, thereby inhibiting NF-Κb, PI3K / Akt and MAPKs signal transduction pathways.

The Jouleumwan extract can inhibit the accumulation of Reactive Oxygen Species (ROS) associated with the neuro-inflammatory response.

The pharmaceutical composition for preventing and treating neuroinflammation according to another embodiment of the present invention may include the extract of the end of the line.

Functional food composition for the prevention and treatment of nerve inflammation according to another embodiment of the present invention may include the end of the line extract.

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

In the present invention, "neuritis" refers to an inflammatory reaction occurring in the nervous system, that is, nerve cells, nerve tissues, and the like. The phenomenon of producing and releasing substances such as inflammatory cytokines TNF-α and IL-1β, nitrogen monoxide, prostaglandins, and superoxides, activated by various exogenous and endogenous substances, which are immune cells present in the central nervous system due to various exogenous and endogenous substances It may include. It is known that the production of these substances induces an immune response in the short term, but its excessive production or continuous production induces the death of adjacent nerve cells, which in turn leads to neurodegeneration.

In the present invention, "degenerative cranial nerve disease" refers to a disease that causes various symptoms while degenerative changes appear in neurons of the central nervous system, and specifically, cognitive function, learning or memory is impaired, or the brain is accompanied by a neuroinflammatory reaction. Neurological diseases. Representative degenerative cranial nerve diseases according to the present invention include dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease (Creutzfeld) -Jakob disease, CJD, stroke, multiple sclerosis, cognitive disorder, learning disorder, memory impairment, etc. Among them, Alzheimer's disease (AD) is the most important disease among senile dementia, and it is known that amyloid beta (Aβ) accumulates in the brain and the resulting neurotoxicity is an important cause of development. The Aβ is known to be an amyloid precursor protein (APP) made by the continuous action of the membrane protein hydrolase beta-secretase 1 (BACE1) and gamma-secretase, and thus the BACE1 protein It is obvious that Alzheimer's disease can be prevented or treated by suppressing expression.

The composition for preventing and treating nerve inflammation according to an embodiment of the present invention includes an extract of Carpomitra costata.

Carpomitra costata (Stackhouse) Batters are narrow and flat linear, branched in a car-shaped basin or a tertiary shape, and are brown algae 10-20 cm high and 2-3 mm wide, growing deep in the subtidal zone, Korea The distribution is found in Ulleungdo, Dokdo, Jeju Island, and Munseom Island, and is also distributed in Japan, South Africa, Europe, and Australia.

Recently, attention has been focused on the prevention and treatment of various diseases using marine biological resources. Among them, seaweed has a great potential for development and prevention of nerve inflammation because it is rich in bioactive substances with various pharmacological effects.

However, a number of studies have been conducted showing that seaweed extract has various pharmacological effects so far, but no experiment has been conducted to confirm the anti-inflammatory effect on microglia.

Thus, in the present invention, the anti-inflammatory and anti-oxidative efficacy of ulnar end ethanol extract in BV2 microglia stimulated by LPS was confirmed, and the effect of EECC on activation of the TLR4 signal transduction pathway by LPS was confirmed.

Therefore, in the present invention, by using the extract of the Joule of the Joule, which obtained the extract from the Joule of the Joule, by reducing the production of inflammatory mediators and cytokines, the prevention of neuroinflammation showing anti-inflammatory effects of microglia And it is intended to provide a therapeutic composition.

However, the Joule of the extract in the composition is included in a concentration range of 300 μg / ml or less, and more specifically, in a concentration range of 50 to 300 μg / ml. When the concentration range exceeds 300 μg / ml, due to cytotoxicity, there is a problem in that the prevention or treatment effect of nerve inflammation is insufficient.

The extract of the end of the string can be extracted by the ultrasonic extraction method, and the ultrasonic extraction is after washing and crushing the end of the string, water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, acetone, glycerin, ethyl acetate, butyl After placing in at least one extraction solvent selected from the group consisting of acetate and 1,3-butylene glycol, propylene glycol, dichloromethane, chloroform, ethyl ether, butylene glycol, hexane and mixtures thereof, a temperature condition of 20 to 40 ° C It means to extract for about 1 to 12 hours with an ultrasonic extractor while maintaining pH 5 to 7 at (room temperature). The time of the ultrasonic extraction may vary depending on the amount of the sample to be extracted, and may undergo a process of filtering or purifying the extraction result.

More specifically, the Joule's end extract is extracted with a solvent selected from the group consisting of water, alcohols having 1 to 6 carbons, and mixtures thereof, preferably, the Joule's end ethanol extract extracted with a solvent, but is limited to the above examples. Does not work.

After extraction, the extract can be fractionated by sequentially applying a new fraction solvent. The fractionation solvent used for fractionation is at least one selected from the group consisting of water, hexane, butanol, ethylacetic acid, ethyl acetate, methylene chloride, and mixtures thereof, preferably ethyl acetate or methylene chloride.

After obtaining the extract or fraction, a method such as concentration or lyophilization may be additionally used.

The Joule's end extract exhibits an antagonistic effect on the binding of LPS to TLR4 in immune cells, thereby inhibiting NF-Κb, PI3K / Akt and MAPKs signaling pathways.

In addition, it is possible to inhibit the accumulation of reactive oxygen species (ROS, Reactive Oxygen Species) associated with the neuro-inflammatory response.

Joule's end extract significantly inhibits LPS-induced secretion of pro-inflammatory mediators including nitric oxide (NO) and prostaglandin E2 without significant cytotoxicity.

Consistent with these results, Joule's end extract inhibited LPS-induced expression of inducible NO synthetase and regulatory enzymes such as cyclooxygenase-2. Joule's end extract also down-regulates LPS-induced production and expression of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-1β.

For anti-inflammatory effects, Joule's end extract inhibits nuclear translocation and DNA binding of nuclear factor-κB (NF-κB) by destroying the degradation of the κB-α inhibitor in the cytoplasm.

The lanceolate extract effectively inhibits the expression of Toll-like receptor 4 (TLR4) and bone marrow differentiation factor 88, as well as the binding of LPS and TLR4 in LPS-treated BV2 cells.

The Joule's end extract significantly reduces the production of free radicals (ROS) induced by LPS and exhibits a strong antioxidant effect. In light of these circumstances, the dulcis extract can inhibit LPS-mediated neuro-inflammatory action in BV2 microglia through inactivation of NF-κB signaling by elimination of TLR4 antagonism and / or ROS accumulation.

Preferably, the Joule's end extract is a methylene chloride fraction or an ethyl acetate fraction of the Joule's end ethanol extract.

It was confirmed that the fractions showed better neuroinflammation prevention and treatment effects compared to the ethanol extract of Joule's end.

More preferably, the composition for preventing or treating neuroinflammation of the present invention is a fermentation product of the ulnar end fraction, which is a fermented fraction of the ethanol extract of ulru end.

The fermented product is prepared by using a ethanol extract of Joule's end ethanol by ultrasonic extraction, and after preparing a fraction using ethyl acetate or methylene chloride as a fractional solvent, sugar is mixed with the fraction and inoculated with fermentation bacteria. It was prepared by fermentation at room temperature for 3 to 5 days.

Since the sugar component mixed with the fraction is used as a carbon source in the metabolic process of microorganisms and converted to alcohol, the active ingredient in natural products, especially flavonoids, polyphenols, and catechins, is saccharified and exists as a stable compound, so it is effective through fermentation It is possible to increase the content extraction rate and increase the prevention or treatment of osteoarthritis by the active ingredient active by excluding sugar.

After inoculating microorganisms in a medium at a constant concentration (about 10 ⁵ to 10 ⁷ CFU / ml), culturing while maintaining conditions of pH 5.5 to 6.5, temperature 25 to 35 ° C, and incubation time of 48 to 120 hours, this fermentation product It is possible to obtain a fermentation product of the Joule's end fraction using.

The microorganisms used in the fermentation include Saccharomyces cerevisiae Lactobacillus salivarius, Lactobacillus acidophilus, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus lamvis (Lactobacillus rhamnosus), Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus paracasei, Lactobacillus paracasei Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus gaslue, L. Heritage basil such as (Lactobacillus kefir) L., Lactococcus lactis, Lactococcus plantarum, Lactococcus raffinolactis, Enterococcus faecalis, Enterococcus faecium Bifidobacterium animals, Bifidobacterium animals, lactic acid coxays such as Streptococcus thermophilus, Leuconostoc lactis, Leuconostoc mesenteroides, etc. Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium pseudolongum , Bifidobacterium themophilum, Bifidobacterium adolescentis, etc. It may include Pydobacteria, more preferably Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum Bifidobacterium, Bifidobacterium bre Bifidobacterium breve, and may be one or more selected from the group consisting of Lactobacillus acidophilus.

The composition for the prevention or treatment of nerve inflammation may preferably further include an extract of blood (피).

More preferably, it may include a fermented product of the Joule's end fraction and an H-derived extract.

The above-mentioned bark is extracted using the bark of Cercis chinensis Bunge, which is a deciduous shrub grown to reach 35m in height and has many cortex and white bones. The leaves are repetitive, monocotyledonous, heart-shaped, and have 5 outgrowths at the base. Flowers are magenta and bloom in April. Fruits ripen in 1989 with fruits. Seeds are flat oval, yellow-green.

In order to increase the effect of preventing or treating neuroinflammation, as a result of mixing a small amount of the bark extract in the fermentation product of the ethanol extract of Jouleum, the effect of preventing or treating neuroinflammation is further increased due to the mixing action of each component. .

Thus, the composition for preventing or treating neuroinflammation includes a fermentation product of the Joule's end fraction, and may contain 10 to 20 parts by weight of the bark extract with respect to 100 parts by weight of the fermentation product of the Joule's end fraction.

The bark skin extract may be fermented after preparing the extract using the bark skin. In the case of fermenting the bark extract, it has the advantage of being stably usable at relatively high concentrations due to low cytotoxicity.

Therefore, preferably, the composition for preventing or treating nerve inflammation includes a fermentation product of the Joule's end fraction, and may be included in 10 to 20 parts by weight of the fermented extract of the cortical skin, based on 100 parts by weight of the fermentation product of the Joule's end fraction. have. In the case of the above range, a synergistic effect of a critical significance level is expressed as a synergistic effect due to the interaction of each extract, and when it is outside the above range, the synergistic effect is rapidly reduced or almost absent.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier may be various oral or parenteral formulations. In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used. Solid preparations for oral administration may include tablets, powders, granules, capsules, etc. These solid preparations include at least one excipient in one or more compounds, such as starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, and the like. In addition, lubricants such as magnesium stearate, talc and the like may be used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents, suspension solvents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

In addition, the pharmaceutical composition of the present invention is from the group consisting of tablets, pills, powders, granules, capsules, suspensions, intravenous solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers and suppositories. It can have any one formulation selected.

The extract of Joule of the present invention has been used for edible and medicinal purposes since ancient times, and there is no particular restriction on the dosage, and body absorption, body weight, patient's age, gender, health status, diet, administration time, administration method, It may change depending on the excretion rate, the severity of the disease, etc. In general, Jouleumwan extract can be administered in detail about 10 to 1000 mg per kg of body weight, and more specifically, about 50 to 500 mg per kg of body weight. The pharmaceutical composition comprising the Joule stalk extract of the present invention as an active ingredient is prepared in consideration of an effective amount range, and the unit dosage form formulation formulated in this way is judged by an expert who monitors or observes the administration of a medicament as necessary and the individual. Depending on the requirements of the drug can be administered using a specialized dosing method or several times at regular time intervals.

The present invention also provides a quasi-drug composition for anti-inflammatory activity, which includes Joule's end extract as an active ingredient. The production and configuration of the Jouleumwan extract is as described above.

In the present invention, 'quasi-drug' is a fiber, rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a person or animal, has a weak effect on the human body or does not directly act on the human body, or an apparatus or Non-machinery and similar products, one of the agents used for sterilization, pesticide and similar purposes for the prevention of infection, used for the purpose of diagnosing, treating, alleviating, treating or preventing human or animal diseases This refers to items other than appliances, machinery, or devices that are not intended to be used for the purpose of pharmacologically affecting the structure and function of a person or animal.

When using the Joule's end extract of the present invention as a quasi-drug additive, the Joule's end extract can be added as it is or used with other quasi-drug or quasi-drug components, and can be suitably used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use.

The quasi-drug composition of the present invention is not limited thereto, and may be, in particular, a disinfecting cleaner, shower foam, gagreen, wipes, detergent soap, hand wash, humidifier filler, mask, ointment, or filter filler.

The present invention also provides a health functional food composition for the prevention or treatment of neuroinflammation, which includes Joule's end extract as an active ingredient. The production and configuration of the Jouleumwan extract is as described above. More specifically, the extract of the stem of the present invention can be added to the health functional food composition for the purpose of preventing or treating nerve inflammation.

When using the Joule's end extract of the present invention as a health functional food additive, the Joule's end extract can be added as it is or used with other health functional food or health functional food components, and can be suitably used according to a conventional method. . The mixing amount of the active ingredient can be appropriately determined according to the purpose of use.

There are no particular restrictions on the type of health functional food of the present invention. Examples of health functional foods to which the extract mixture can be added are meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea , Drinks, alcoholic beverages and vitamin complexes, and may include all health functional foods in a general sense, and may include foods used as feed for animals. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin , Alcohol, carbonic acid used in carbonated beverages, and the like. In addition, it may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

However, when using only the extract of the Joule of the Joule as a food composition as described above, or when using a composition that further comprises an extract of the Hyeongpi as a food composition, the taste of the Joule of the Joule extract and the Hyungpi extract is inferior to palatability. The problem arises.

Thus, preferably, it further comprises a suchocho extract, a bibi extract, and a terminator extract, so as to enhance palatability, and to increase the effect of preventing and treating nerve inflammation.

Suchycho (Pachysandra terminalis) grows in the shade of a tree, and the main stem grows sideways, ends up straight, green, and has fine hairs at first, but gradually disappears. It grows to about 30cm in height. The leaves are misaligned, but are gathered in the upper part and appear to stand upside down, with teeth on the upper part. There are fine hairs on the leaf surface veins, and the lower part becomes narrow to become petioles. In addition, the flowers bloom in April or May and are white and hang in water. The female flower runs slightly at the bottom of the ear and the male flower runs a lot at the top. The calyx is divided into 4 and there are no petals. There are 3 to 5 stamens, and the pistil is divided into 2 and folded. The fruit is a core, egg-shaped, and has no hair on the outside. It is distributed in Korea, Japan, Sakhalin Island and China.

Hosta longipes grow well in mountain streams or in humid places and are 30 to 40 cm high. All the leaves sprout from the roots and grow obliquely. Leaves are oval, pointed, 8 or 9 veins. Flowers are light purple, bloom in July or August, slant to one side and run as gunshots, and stalks are 30-40cm long. The fores are thin filmy, purplish white, almost the length of a peduncle. The corolla is split into 6 ends, so the forked piece is slightly retracted, and 6 stamens and 1 pistil are out of the flower. The fruit is a long oval with a nut. The seeds are black with wings on the edges. Edible light planted and planted for ornamental purposes. Wild species are distributed in Korea, Japan, and China. Bibichu is a horticultural variety, and is popular as a garden plant in foreign countries.

Senna tora (L.) Roxb. Is an annual plant that grows up to 2 m in height. The leaf is an even-numbered dominant biplane with 2 to 4 pairs of lobules, and the lobules are obovate, slightly pointed at the tip, and sharp or original. The flowers are yellow with peduncles, and they hang on the leaf axils in June in December. The fruits are curved like a bow.

More preferably, the composition for preventing or treating nerve inflammation includes a fermentation product of the Joule's end fraction, with respect to 100 parts by weight of the fermentation product of the Joule's end fraction, 10 to 20 parts by weight of the fermented coriander extract, suhocho It may include 1 to 5 parts by weight of the extract, 1 to 5 parts by weight of the BB extract and 10 to 15 parts by weight of the extract of the ginseng. In the case of the above range, a synergistic effect of a critical significance level is expressed as a synergistic effect due to the interaction of each extract, and when it is outside the above range, the synergistic effect is rapidly reduced or almost absent. That is, when used as a food composition within the above range, the palatability is increased, and the effect of preventing and treating nerve inflammation is further increased by the interaction of each extract.

The present invention is a composition for preventing and treating neuroinflammation comprising the extract of Carpomitra costata, reducing the production of inflammatory mediators and cytokines, showing anti-inflammatory effects of microglia, and in the treatment of neuroinflammation. Even if used, it can exhibit the effect of preventing and treating nerve inflammation without side effects.

In addition, the present invention is to provide a functional food composition for preventing and treating nerve inflammation and pharmaceutical compositions with high preference for preventing and treating nerve inflammation, including extract of Carpomitra costata.

1 is a result of the effect of EECC and LPS on the cell viability of BV2 microglia.
2 is a result of the effect of EECC and LPS on the cell viability of BV2 microglia.
3 is a result of the NO production by EECC in LPS stimulated BV2 microglia.
Figure 4 is a result of the degree of PGE ₂ production by EECC in LPS-stimulated BV2 microglia.
5 is a result of the inhibition of iNOS and COX-2 expression by EECC in LPS stimulated BV2 microglia cells.
6 is a result of TNF-α measurement to confirm the inhibition of LPS-induced production and proinflammatory cytokine expression by EECC in BV2 microglia.
7 is a result of measuring IL-1β to confirm LPS-induced production by EECC in BV2 microglia and inhibition of proinflammatory cytokine expression.
FIG. 8 shows the results of measuring the expression of TNF-α and IL-1β proteins using the indicated antibody and ECL detection system to confirm the inhibition of LPS-induced production and proinflammatory cytokine expression by EECC in BV2 microglia. to be.
9 is a result of Western blot analysis to confirm inhibition of LPS-induced NF-κB activation by EECC in BV2 microglia cells.
10 is a result of assaying the DNA binding activity of NF-κB by EMSA to confirm inhibition of LPS-induced NF-κB activation by EECC in BV2 microglia cells.
11 is an experimental result for confirming the interaction between LPS and TLR4 by EECC in BV2 microglia, decreased expression of TLR4 and Myd88 by LPS.
12 is an experimental result of measuring the LPS binding on the surface of BV2 cells with a flow cytometer.
13 is an experimental result for confirming the interaction between LPS and TLR4 by EECC in BV2 microglia, decreased expression of TLR4 and Myd88 by LPS.
FIG. 14 is a result of measuring DCF fluorescence by flow cytometry to confirm inhibition of LPS-induced ROS production by EECC in BV2 microglia cells.
15 is a result confirmed using a fluorescent microscope (original magnification, 200 times) after staining with DCF-DA, in order to confirm the inhibition of LPS-induced ROS generation by EECC in BV2 microglia cells.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art to which the present invention pertains can easily practice. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein.

[Production Example 1: Preparation of Jouleumwan extract]

The Joule Horsetail Extract (hereinafter referred to as EECC) used in this study was provided and used by the National Institute of Marine Biological Resources. To prepare for the EECC, C. costata was collected in March 2016 off the coast of Ulleungdo. The collected Joule horse tube was washed with tap water to remove the plate, tip and sand attached to the sample surface and stored at -20 ° C. The frozen tubule samples were lyophilized and homogenized using a grinder prior to extraction. The dried powder was extracted with 70% ethanol (1:10 w / v) for 1 hour (5 times) by ultrasonic extraction, and then the extract (EECC) was rotary evaporator (Rikakikai Co., Ltd., Tokyo, Japan). And dried under vacuum. The extract was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) before use in the experiment.

[Experimental Example 1: Confirmation of the effect of Jouleumwan extract on the prevention and treatment of nerve inflammation]

1. Experimental method

Cell culture and treatment

BV2 microglia are 10% (v / v) fetal bovine serum (FBS, WelGENE Inc.), L-glutamine (2mM), penicillin in humidified conditions containing 5% CO2 and 95% air at 37 ° C. (100 U / ml) and 100 μg / ml streptomycin (100 U / ml, WelGENE Inc.) were cultured in Dulbecco Modified Eagle's Medium (DMEM, Welgene Inc., Korea, Daegu).

To confirm the efficacy of EECC in BV2 cells, medium was exchanged with fresh DMEM and 100ng / ml LPS (Sigma-Aldrich Chemical Co.) was added with or without EECC for the indicated period.

Assessment of cell viability

For cell viability studies, BV2 cells were cultured in 96-well plates at a density of 1x10 ⁴ cells per well. After incubation for 24 hours, cells were treated with various concentrations of EECC for 24 hours, or pretreated with different concentrations of EECC for 1 hour, and LPS (100 ng / ml) was treated after 24 hours. Thereafter, the medium was removed, and 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (3- (4,5-dimethylthiazol-2-yl) was added to each well. -2,5-diphenyltetrazolium bromide, MTT, 0.5 mg / ml, Sigma-Aldrich Chemical Co.) was added and further incubated at 37 ° C for 3 hours. The supernatant was replaced with DMSO to dissolve blue formazan crystals.

After 10 minutes, the optical density was measured at a wavelength of 540 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Dynatech Laboratories, Chantilly, VA, USA).

Measurement of inflammatory mediators and cytokine production

The level of nitrogen monoxide (NO) production was determined indirectly by measuring stable NO carboborate nitrite in the medium by the Griess reaction. In summary, the condition medium was mixed with an equal volume of grease reagent (Sigma-Aldrich Chemical Co.) and incubated at room temperature for 10 minutes. The optical density at 540 nm was measured with an ELISA microplate reader, and the concentration of nitrite was calculated according to a standard curve generated from a known concentration of sodium nitrite. The levels of prostaglandin E2 (PGE2), tumor necrosis factor (TNF) -α and interleukin (IL) -1β in culture medium were measured using a commercial ELISA kit (R & D Systems, Minneapolis, MN, USA).

Protein separation and Western blot analysis

Cytoplasmic and nuclear proteins were isolated using the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's protocol. In the case of Western-blotting, the same amount of protein sample was transferred to a polyvinylidene difluoride membrane (Schleicher and Schuell, Keene, NH, USA) by electrophoresis after separation by sodium-dodecyl sulfate (SDS) gel electrophoresis. The membrane was then blocked for 1 hour with 5% skim milk / Tris buffered saline containing 0.1% Triton X-100 (TBST) and then probed with a specific antibody overnight at 4 ° C. After washing the primary antibody with TBST, the membrane was incubated with an appropriate amount of horseradish-peroxidase (HRP) -conjugated secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for 2 hours at room temperature. Protein bands were detected by an enhanced chemiluminescence (ECL) kit (Amersham Corp., Arlington Heights, IL, USA) according to the manufacturer's instructions.

Electromobility analysis (EMSA)

EMSA was performed using the nuclear extract described in a previous study (Kang et al., 2017). In summary, biotinylation of synthetic complementary NF-κB binding oligonucleotides (NF-κB binding oligonucleotides, Santa Cruz Biotechnology, Inc.) is made using a biotin 3'-terminal DNA labeling kit (Pierce Biotechnology). It was carried out according to the instructions, and annealed at room temperature for 30 minutes. The mixture was separated by electrophoresis on a 4% polyacrylamide gel that was pre-electrophoresis for 60 minutes in 0.5X Tris-borate buffer, and then transferred to a nylon membrane (Hybond ™ N +) for 30 minutes. Biotin-labeled DNA was detected using a LightShift chemiluminescence EMSA kit (Pierce Biotechnology).

Measurement of TLR4 expression on the cell surface

EECC for 1 hour prior to treatment with Alexa Fluor (AF) 488-conjugated LPS (100ng / ml, Invitrogen Life Technologies, Carlsbad, CA, USA) with BV2 cells to investigate the effect of EECC on cell surface TLR4 expression With or without pretreatment with BV2 cells. After incubation for 1 hour, the cells were washed twice with phosphate-buffered saline (PBS), collected with 0.005% ethylenediaminetetraacetic acid (Sigma-Aldrich Chemical Co.), and analyzed by flow cytometry. Alexa 488 was made excited using 488 argon ion laser lines and detected in the FL1 channel using a 530nm emission filter. Fluorescence emission of the samples was recorded by flow cytometry (Becton Dickinson, San Jose, CA, USA) as previously described.

Detection of intracellular ROS levels

The production of intracellular ROS was monitored using a cell permeable fluorescent probe, 5,6-carboxy-2 ', 7'-dichlorofluorescin diacetate (DCF-DA). In summary, cells were treated with EECC for 1 hour or pre-treated with EECC for 1 hour and then incubated for 1 hour with or without LPS. Cells were harvested and stained in dark color with 10 μM DCF-DA (Sigma-Aldrich Chemical Co.) at 37 ° C. for 15 minutes. Cells were then washed twice with PBS and analyzed using a flow cytometer with excitation wavelength 480 nm and emission wavelength 525 nm.

To observe the degree of ROS production by fluorescence microscopy, cells attached to the glass cover slip were stimulated with or without Lng treatment for 100 ng / ml LPS for 1 hour after EECC treatment. Cells were stained with DCF-DA, washed twice with PBS, and then fixed with 4% paraformaldehyde (pH 7.4) for 20 minutes. Fixed cells were analyzed using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Statistical analysis

Data values are expressed as mean ± standard deviation (SD). All statistical analyzes were performed using GraphPad Prism software 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Comparisons between groups were performed through Dunnett's multiple range test. The difference is considered statistically significant at p <0.05.

2. Experimental evaluation

EECC evaluation of cell viability of BV2 microglia

To establish experimental conditions, BV2 cells were treated with extensive EECC for 24 hours and cell viability was examined by MTT analysis. As shown in FIG. 1, the cytotoxic effect was not induced at a concentration up to 200 μg / ml, but the cell viability was significantly reduced in the treatment group at a concentration of 300 μg / ml.

In addition, no significant change was found in the concentration of EECC up to 200 μg / ml even in the presence of 100 ng / ml LPS (FIG. 2). Therefore, in order to study the anti-inflammatory effect of EECC in BV2 cells stimulated with LPS, the maximum concentration of EECC was selected as 200 μg / ml.

Generation of NO and PGE2 by LPS in BV2 microglia of EECC

To determine the inhibitory properties of EECC against the production of NO and PGE2 by LPS, representative inflammatory mediator BV2 cells were pretreated with various concentrations of EECC for 1 hour and then stimulated with or without 100 ng / ml LPS. . The levels of NO and PGE2 in the culture supernatant were determined by Griess reaction analysis and ELISA, respectively. As shown in Figs. 3 and 4, when LPS alone was treated, NO and PGE2 production was significantly increased compared to the group without LPS treatment. . Conversely, EECC significantly inhibited NO and PGE2 secretion by LPS in a concentration-dependent manner in BV2 cells.

LPS-induced NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression levels in EECC's BV2 microglia

It was determined whether the inhibitory effect of EECC on NO and PGE2 production was related to the regulation of the expression of the synthetic enzymes, iNOS and COX-2, respectively. As shown in Figure 5, EECC inhibited the expression of iNOS and COX-2 proteins in LPS-stimulated BV2 cells. This means that EECC inhibits NO and PGE2 production by reducing the expression of their encoding genes.

EECC production and expression of pro-inflammatory cytokines in LPS-stimulated BV2 microglia

The effect of EECC on the production of pro-inflammatory cytokines including TNF-α and IL-6 was investigated in LPS-stimulated BV2 cells. According to the ELISA results, according to FIGS. 6 and 7, the production of cytokines was significantly increased in the culture medium of LPS-stimulated BV2 cells, which decreased in the presence of EECC in a concentration-dependent manner. In addition, inhibition of TNF-α and IL-6 production by EECC is associated with their expression inhibition (FIG. 8).

Inhibition of LPS-induced activation of NF-κB by EECC in BV2 microglia

It was determined whether EECC was able to attenuate the activity of NF-κB induced by LPS in BV2 cells. As a result of immunoblotting using cytoplasmic and nuclear extracts, EECC pretreatment inhibited nuclear accumulation of NF-κB p65 subunits associated with attenuation of κB-α (IκBα) degradation inhibitors in LPS-stimulated BV2 cells ( Figure 4A). Consistent with these results, the DNA binding activity of NF-κB is significantly increased in LPS-treated cells, whereas pretreatment with EECC attenuates transcriptional activation of NF-κB, resulting in LPS-induced NF-κB. DNA binding activity was inhibited (FIG. 10).

EECC inhibits LPS-induced TLT4 and Myd88 expression in BV2 microglia

To determine whether the anti-inflammatory effect of EECC is related to the blocking of the TLR signaling pathway, the effect of LPS and EECC on the expression of TLT4 and myelodifferentiation factor 88 (Myd88) was investigated. According to the immunoblotting results (FIG. 11), the levels of TLT4 and Myd88 proteins were significantly increased in LPS treatment in a time-dependent manner, similar to the results of previous studies (Yoon et al., 2013). However, when EECC was pretreated, the expression of the protein by LPS was suppressed in a concentration-dependent manner (FIG. 12), indicating that EECC can inhibit the LPS activation TLR4 signaling pathway of BV2 cells.

Blocking of LPS-mediated interactions between LPS and TLR4 by EECC in BV2 microglia cells

We further evaluated whether EECC inhibits the interaction between LPS and TLR4 on the surface of BV2 cells treated with LPS. As shown in FIG. 13, the fluorescence intensity of LPS attached to the outside of the cell membrane was significantly increased in cells treated with LPS alone, but was significantly weakened in cells treated with LPS in the presence of EECC. This indicates that the binding of LPS to TLR4 was inhibited in the presence of EECC.

Reduction of LPS-induced ROS production by EECC in BV2 microglia cells

To investigate the antioxidant capacity of EECC, the effect of EECC on LPS-induced ROS production was investigated. As a result of flow cytometry using the fluorescent probe DCF-DA, the level of ROS gradually increased with the treatment of LPS, the peak reached 1 hour, and then decreased. However, in the group treated with EECC alone, ROS generation was not induced, and in the group pretreated with EECC, the ROS level released by LPS was effectively reduced (FIG. 14). The inhibitory effect of EECC on ROS production was similarly observed in experiments using fluorescence microscopy (FIG. 15). In addition, in N-acetyl cysteine (NAC) -pretreated cells used as a positive control, the production of ROS by LPS was completely blocked by EECC due to the strong ROS scavenging effect.

[Production Example 2: Preparation of Joule's end fraction]

The brown algae, C. costata, were collected near Ulleungdo. After collection, C. costata was washed with tap water to remove the plate, linear sand and sand attached to the sample surface, and stored at -20 ° C. The frozen samples were lyophilized and homogenized using a grinder prior to extraction.

The dried powder was obtained for 1 hour (5 times) with 70% EtOH (1:10 w / v) by ultrasonic treatment.

After the suspension of ethanol extract of Joule was suspended in distilled water, the suspension and methylene chloride were added to a separatory funnel at a ratio of 1: 1, the methylene chloride layer and the water layer were fractionated, and the methylene chloride layer was concentrated again under reduced pressure to reduce the Joule end fraction (EECP ).

[Production Example 3: Preparation of fermented product of Joule's end]

5 parts by weight of sugar was mixed with 100 parts by weight of the fraction prepared in Preparation Example 2. Thereafter, Lactobacillus salivarius was inoculated at 10 ⁶ CFU / ml and fermented at 24 hour intervals while incubating for 72 hours while maintaining the conditions of pH 5.5 to 6.5 and temperature 23 to 28 ° C. The microorganism used for fermentation, Saccharomyces cerevisiae KCTC7913, was prepared by liquid culture at 35 ° C. 24 hours before inoculation, and was diluted to be used as an inoculum for fermentation.

70% ethanol is added to the fermentation product at 10 to 20 times the weight of the fermentation product to extract the active ingredient in the fermentation product and remove polymer precipitates and cells using a paper filter of 1㎛ pore size. ).

[Production Example 4: Preparation of a composition for preventing or treating nerve inflammation]

1. Manufacture of bark extract (CCBE)

After washing the bark of the bark tree, and drying it, it was pulverized using a grinder to prepare a powder skin.

The powdery skin powder was obtained by ultrasonic treatment for 1 hour (5 times) with 70% EtOH (1:10 w / v).

2. Preparation of natural extracts

Using the same method as the above-mentioned method for preparing an extract of bark skin, a bibichu extract (HLME), a guardian herb extract (PTSE), and a terminator extract (STRE) were prepared.

3. Preparation of fermented bark extract (CCBB)

The bark skin extract was prepared by using the same method as the fermentation product of Joule's end fermented bark skin.

4. Preparation of a composition for the prevention or treatment of osteoarthritis

A composition for the prevention or treatment of osteoarthritis of the bone was prepared by mixing the Joule's end extract (EECC), an aerated skin extract (CCBE), a BB extract (HLME), a guardian herb extract (PTSE), and a terminator extract (STRE).

The content of the more specific composition is shown in Table 1 below.

SP1 SP2 SP3 SP4 SP5 SP6 SP7 SP8 SP9 SP10 EECC 100 100 100 100 - - - - - - EECP - - - - 100 100 100 100 - - EECB - - - - - - - - 100 100 CCBE 5 10 20 25 5 10 20 25 20 - HLME 0.5 One 5 10 0.5 One 5 10 5 5 PTSE 0.5 One 5 10 0.5 One 5 10 5 5 STRE 5 10 15 20 5 10 15 20 15 15 CCBB - - - - - - - - - 20

(Unit parts by weight) [Experimental Example 2: Anti-inflammatory effect in BV2 microglia]

In Experimental Example 1, LPS-induced NO synthetase (iNOS) and cyclooxygenase-2 (COX-2) expression levels (A), pro-inflammatory cytokine production and expression (B), NF-κB for EECC LPS-induced activation inhibition degree (C), LPS-induced TLT4 and Myd88 expression inhibition degree (D), degree of blockage of LPS-mediated interaction between LPS and TLR4 (E) and degree of decrease in LPS-induced ROS production (F) was measured, and the effect of SP1 to SP10 was compared and tested.

In order to proceed with the relative evaluation with EECC, LPS-induced NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression level (A), pro-inflammatory cytokine production and expression (B), NF-κB LPS-induced activation inhibition of (C), LPS-induced TLT4 and Myd88 expression inhibition (D), LPS-mediated interaction between LPS and TLR4 blocking (E) and LPS-induced ROS production reduction The degree (F) was all set to an index of 5, and the effects on SP1 to SP10 were expressed as an index.

The index is evaluated as 1 to 10, and the higher the score, the better the effect.

EECC SP1 SP2 SP3 SP4 SP5 SP6 SP7 SP8 SP9 SP10 A 5 4 6 7 4 4 7 8 6 8 9 B 5 5 6 7 5 5 7 8 5 8 9 C 5 4 7 7 6 5 7 7 5 8 8 D 5 5 5 8 5 4 8 8 5 9 8 E 5 5 6 7 5 4 7 7 5 8 9

As shown in Table 2, it was confirmed that SP2, SP3, SP6, SP7, SP9, and SP10 exhibit relatively superior effects compared to EECC.

That is, it was confirmed that, when used as a complex extract, compared to the case where only EECC is used alone, the effect of preventing and treating nerve inflammation is excellent.

[Experimental Example 3: Sensory evaluation]

Functionality for flavor and taste according to each combination was evaluated so that the SP1 to SP10 can be provided as a functional food composition.

Therefore, EECC, SP1 to SP10 were used to prepare tea beverages, and sensory evaluation was performed on 20 adult men and women. The sensory evaluation was evaluated by an index of 1 to 10 according to palatability based on aroma and taste, and classified into an average index. The results are shown in Table 3.

On the other hand, the index below is the higher the number, the higher the preference.

EECC SP1 SP2 SP3 SP4 SP5 SP6 SP7 SP8 SP9 SP10 incense 3 3 5 7 4 4 6 7 6 8 9 flavor 2 4 6 7 5 5 7 7 5 8 9 Palatability 3 4 6 6 3 5 7 7 5 8 9

(Unit index) As shown in Table 3 above, when only EECC was used as a tea, it was confirmed that the aroma, taste, and preference were poor. On the other hand, it was confirmed that when prepared as a composite extract, it can be provided as a functional food composition excellent in flavor, taste, and palatability.

The preferred embodiments of the present invention have been described in detail above, but the scope of the present invention is not limited to this, and various modifications and improvements of those skilled in the art using the basic concept of the present invention defined in the following claims are also provided. It belongs to the scope of rights.

Claims (8)

Contains the extract of Carpomitra costata,
The Joule's end extract decreases the production of inflammatory mediators and cytokines, indicating anti-inflammatory effects of microglia.
A composition for preventing and treating nerve inflammation. According to claim 1,
The Joule's end extract is extracted with a solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixtures thereof.
A composition for preventing and treating nerve inflammation. According to claim 1,
The Joule's end extract is a Joule's end ethanol extract extracted using ethanol.
A composition for preventing and treating nerve inflammation. According to claim 1,
The Joule of the end of the extract is a fraction obtained by fractionating the extract obtained from the end of the Joule using an organic solvent
A composition for preventing and treating nerve inflammation. According to claim 1,
The Joule's end extract shows an antagonistic effect on the binding of LPS to TLR4 in immune cells,
NF-Κb, PI3K / Akt and MAPKs which inhibit the signaling pathway
A composition for preventing and treating nerve inflammation. According to claim 1,
The Joule's end extract inhibits the accumulation of reactive oxygen species (ROS) related to the neuro-inflammatory response.
A composition for preventing and treating nerve inflammation. Claim 1 to 6 comprising a composition according to any one of claims
Pharmaceutical composition for preventing and treating nerve inflammation. Claim 1 to 6 comprising a composition according to any one of claims
Functional food composition for preventing and treating nerve inflammation.

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