KR20220018210A - Composition for improving immunity comprising dolgasari extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for improving immunity, which comprises Chondracanthus tenellus extract as an active ingredient so as to exhibit an immune enhancing effect by inducing activation of macrophage phagocytosis without concerns about side effects with the use of natural products. In addition, the present invention relates to a composition for improving immunity, which comprises Chondracanthus tenellus extract as an active ingredient so as to activate an immune response and exhibit an immune enhancing effect by increasing the production of nitric oxide (NO) in macrophages; increasing the production of prostaglandin E2 (PGE2); increasing the expression of immune response-related factors; increasing nuclear factor-kappa B (NF-κB) transcriptional activity; and increasing the expression of toller receptor (TLR4) and myeloid differentiation factor 88 (MyD88).

Description

A composition for improving immunity comprising a stone garfish extract as an active ingredient {COMPOSITION FOR IMPROVING IMMUNITY COMPRISING DOLGASARI EXTRACT AS AN ACTIVE INGREDIENT}

The present invention relates to a composition for improving immunity comprising an extract of Chondracanthus tenellus as an active ingredient. More specifically, it relates to a composition for improving immunity, which has an immune enhancing effect by inducing phagocytosis activation of macrophages by including a stone geranium extract as an active ingredient.

Since 2000, acute new infectious diseases such as acute respiratory syndrome SARS, avian influenza, Ebola virus, and corona virus have appeared around the world, and interest in health is increasing. It is important to improve immunity in order not to catch such infectious diseases or various diseases.

Immunity is a phenomenon in which the internal environment of a living body defends against antigens, which are external factors. It plays a very important role.

In particular, immunity can be divided into innate immunity and acquired immunity depending on the specificity of the antigen that enters the body and produces antibodies. do. On the other hand, acquired immunity is an immune response with specificity that appears only with respect to the antigen that induces the response by acquiring immunity by the antigen when the antigen invades.

As described above, the main cells responsible for immunity in the body are leukocytes in the blood, and among them, macrophages, B cells, T cells, and NK cells that perform phagocytosis are composed of antibodies and immunity to destroy viruses or cancer cells. There are lymphocytes that elicit a response.

The white blood cells account for less than 1% of the total blood volume, and are the only complete cells in the blood having a nucleus and organelles. It is produced from hematopoietic stem cells in the bone marrow and is released into the blood when mature, and consists of five cells that perform immune functions to protect the body against infection or foreign substances.

The five types of cells are neutrophils, eosinophils, basophils, lymphocytes, and monocytes, and account for 40-70% of white blood cells. Eosinophils involved in parasitic infections and allergic reactions, basophils that secrete histamine to dilate blood vessels to inflamed sites in response to allergic reactions and antigens, bind with other foreign invaders such as microorganisms or antigens Helps immune cells to remove them from the body It is made up of monocytes.

The above five cells are used as judgment indicators to monitor bone marrow function or treatment status when a disease causing a problem in leukocyte production is suspected or infection, leukemia is diagnosed, and the like.

Of these, monocytes enter the tissue after circulating in the blood and differentiate morphologically and functionally into macrophages characteristic of the tissue, which act as effector cells that fight against inflammation or initiate an innate immune response. It is the main immune cell.

The macrophages are activated by a number of biological substances as well as microbial components including LPS (LopolySaccharide, LPS), a membrane component of Gram-negative bacteria, and Toll-like resubsides (TLR) recognize this and several signaling pathways , which activates macrophages, which can remove pathogens, foreign substances, and cellular debris through phagocytosis of active macrophages, a cornerstone of the innate immune response.

In addition, activated macrophages neutralize pathogens through the secretion of inflammatory cytokines and mediators, or indirectly participate in immune regulation, and increase immunity-enhancing activity through antigen processing and expression on lymphocytes.

On the other hand, various types of marine organisms have contributed to the prevention and treatment of food sources and diseases for a long time because of their abundant biologically active substances, and many recent studies have demonstrated that extracts or components of edible seaweed have significant immunosuppressive properties.

Seaweed as an immunomodulatory agent can inhibit or enhance host response by interacting with the immune system, various seaweed extracts possess immune stimulants and have been reported to inhibit immunodeficiency virus activity, and certain seaweed components have Studies have also shown that it enhances the immune-modulating effect through the activation of T cells and natural killer cells.

More specifically, polysaccharides isolated from marine algae stimulate macrophage activity while nitric oxide through activation of TLR4/nuclear factor-κB (NF-κB) and/or mitogen-activated protein kinase (MAPKs). By increasing the secretion and production of various cytokines and immune mediators such as (NO) and prostaglandin E2 (PGE2), it can promote macrophage action and enhance macrophage cytotoxicity to tumor cells, so marine algae are immune-modulating It is considered appropriate for use as a treatment or supplement for

Among the marine algae, dolgasari, a red algae, is widely distributed in coastal areas of Japan and China, including Korea, and is rich in minerals such as calcium sulfate polysaccharides and calcium carbonate, and has been used as a food resource for a long time.

Although Piao et al. reported that the protective effect of seaweed extract against ultraviolet B radiation-induced cytotoxicity in human keratinocytes was associated with strong antioxidant activity, currently other pharmacological efficacy studies of seagrass besides antioxidant activity have not It has not been actively done.

For this reason, in the present invention, a composition for improving immunity, which has no side effects and has an effect of improving immunity, has been completed by using a natural product, dolgasari extract, to enhance immune function through macrophage activation.

KR 10-2019-0022017 A

It is an object of the present invention to provide a composition for improving immunity having an immune enhancing effect by inducing phagocytosis activation of macrophages.

Another object of the present invention is that the composition for improving immunity increases the production of nitric oxide (NO), increases the production of prostaglandin E2 (Prostaglandin E2; PGE2), increases the expression of immune response-related factors, nuclear factor-kappa B (Nuclear Factor- Kappa B; NF-κB) transcriptional activity increase, Tolley receptor (TLR4) and myeloid differentiation factor 88 (MyD88) by increasing the expression of the immune response to activate the immune response, and the immune-improving effect of the extract of stonefish (Chondracanthus tenellus) as an active ingredient It is to provide a composition for improving immunity comprising a.

Another object of the present invention is to provide a pharmaceutical composition comprising a composition for improving immunity comprising an extract of Chondracanthus tenellus as an active ingredient.

Another object of the present invention is to provide a food composition comprising a composition for improving immunity comprising an extract of Chondracanthus tenellus as an active ingredient.

In order to achieve the above object, the composition for improving immunity according to an embodiment of the present invention relates to a composition for improving immunity comprising an extract of Chondracanthus tenellus as an active ingredient.

The composition for improving immunity has an immune enhancing effect by inducing phagocytosis activation of macrophages.

The composition for improving immunity is nitric oxide (Nitric Oxide, NO) production increase, prostaglandin E2 (Prostaglandin E2; PGE2) production increase, immune response-related factor expression increase, nuclear factor-kappa B (Nuclear Factor-kappa B; NF-κB) ) to activate the immune response by increasing the transcriptional activity and increasing the expression of Tollieri receptor (TLR4) and myeloid differentiation factor 88 (MyD88).

The immune response-related factors include inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin It is selected from the group consisting of -6 (IL-6) and interleukin-10 (IL-10).

The extract is extracted with a solvent selected from the group consisting of water, C ₁ to C ₆ alcohols and mixtures thereof.

The pharmaceutical composition according to another embodiment of the present invention is prepared by including the composition for improving immunity as an active ingredient.

A food composition according to another embodiment of the present invention is prepared by including the composition for improving immunity as an active ingredient.

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

As used herein, the term 'extract' has the meaning commonly used as a crude extract in the art as described above, but in a broad sense also includes a fraction obtained by additionally fractionating the extract. That is, the plant extract includes not only those obtained using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity), etc. The fraction obtained through the purification method is also included in the plant extract of the present invention.

The extract used in the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying.

The composition for improving immunity according to an embodiment of the present invention includes an extract of Chondracanthus tenellus as an active ingredient.

Stonefish (Chondracanthus tenellus), also called a lagoon, is a member of the order Red algae phylum, and is distributed in Korea (all coasts), Japan, China, the Philippines, and Pacific islands. The thallus is 5~15cm long and 1~2mm wide, and many flat columnar branches alternate or opposite, and the apex is sharp. The body is purple or reddish-purple, cartilaginous, and fluoresces in water. Cystocarps almost globose, small and stalkless. It grows on reefs from the lower intertidal zone to the subtidal zone.

It is also used as a mixed raw material for the manufacture of agar, and is mainly used as a food source because it is rich in minerals such as calcium sulfate polysaccharides and calcium carbonate, and it is reported to have antioxidant activity.

The composition for improving immunity has an immune enhancing effect by inducing phagocytosis activation of macrophages.

Immunity is a process that distinguishes between self and non-self, and recognizes not only microorganisms, bacteria, and viruses that invade from the outside, but also body tissues and unnecessary products as non-self antigens and specifically recognizes them as non-antigens. It is a site that maintains the homeostasis of the living body by producing antibodies in response and removing them, and it is a self-defense system that exists in the body. Immunity, which is such a defense system, is divided into innate immunity, non-specific immunity and acquired immunity, specific immunity depending on the specificity to the antigen.

The innate immunity is also called natural immunity or natural resistance. It responds non-specifically to antigens and has no specific memory function. The innate immune system includes the skin and mucous tissues that block the invasion of antigens, gastric acid of gastric acidity, and complement present in the blood. Cells include macrophages and polymorphonuclear leukocytes responsible for phagocytosis, and natural killer cells (NK cells) that can kill infected cells. There are also cytokines produced by macrophages. In fact, most infections are protected by innate immunity.

In addition, acquired immunity plays a role in reinforcing innate immunity and is also called acquired immunity or adaptive immunity. When an antigen invades the body, an immune response occurs by lymphocytes, and strong resistance to the antigen is remembered.

The initial recognition of antigen infection is mainly performed by macrophages, and cytokines that mediate immune responses are produced in the process of macrophages phagocytosing antigen.

The macrophages quietly exist in a tissue in a state of resident macrophages without a specific stimulus. When activated by an inflammatory response, the macrophages become inflammatory macrophages. Inflammatory macrophages are involved in the inflammatory reaction by making lipid mediators, complement proteins, cytokines, etc.

The cytokine is a glycoprotein used as a signal material to control and stimulate the body's defense system, and has an important function in immunity, infectious disease, hematopoietic function, tissue recovery, and cell development and growth. Cytokines are also called inflammatory cytokines because they have the function of causing immune cells to gather at the site of infection by inducing inflammation in the local area. The most important immune function is caused by a group of cytokines known as interleukin-1,-6,-10,-12,-1β, interferon gamma (IFN-γ), and tumor necrosis factor-α (TNF-α). is carried out

Interleukin (interleukin) is a substance that plays an important role in the body's defense action by regulating the immune response by acting on various stages in which the immune response occurs. Among them, IL-12 is a cytokine composed of p35 and p40 proteins. It has a very important immunological function that connects innate immunity and acquired immunity, and various effector cells involved in the removal of endogenous antigens, i.e., macrophages ), induces activation of NK cells and T-cells.

Tumor necrosis factor (TNF, tumor necrosis factor) is a cell signaling protein related to an inflammatory response, and is a cytokine that induces an acute phase response. Tumor necrosis factor is divided into tumor necrosis factor-α (TNF-α) and tumor necrosis factor-β (TNF-β) called lymphotoxin, but in general, tumor necrosis factor means tumor necrosis factor-α .

The tumor necrosis factor refers to a substance that kills tumors secreted from T cells by lipopolysaccharide, which is an endotoxin that comes out of a bacterial wall, and is the most important inflammatory factor and sometimes causes severe systemic inflammation. However, from an immunological point of view, it is believed that TNF collects neutrophils and monocytes, which are white blood cells, in the blood to the site of infection, and induces differentiation and activation into macrophages with killing ability of infected cells. The function of such tumor necrosis factor depends on the ability to induce the production of cytokines with chemotaxis and to increase the affinity of adhesion molecules between vascular endothelial cells and leukocytes.

As such, in immune function, macrophages secrete and produce proteolytic enzymes, lipid metabolites, cytokines such as interleukins and TNF, and immune mediators such as NO and PGE ₂ , and play an important role in the progression and maintenance of the inflammatory response. , shows an immune response by phagocytosis that removes microorganisms, antigens, dead tissue, and red blood cells.

The composition for improving immunity increases the production of nitric oxide (NO); increase the production of Prostaglandin E ₂ (PGE ₂ ); increase the expression of immune response related factors; increase nuclear factor-kappa B (NF-κB) transcriptional activity; It is to activate the immune response by increasing the expression of Tolleri receptor (TLR4) and myeloid differentiation factor 88 (MyD88).

Nitric oxide (NO) is one of the highly reactive free radicals and is converted into stable nitrogen oxides such as N ₂ O ₃ , NO ₂ , nitrite and nitrate by oxygen or superoxide. Nitric Oxide (NO) is produced from L-arginine in the cell by NO synthase (nitric oxide synthase, NOS). It is classified as inducible NOS (iNOS) produced by In particular, NO produced by iNOS plays an important role in defending the human body from pathogens introduced from the outside, and is known to be closely related to circulatory diseases, inflammatory diseases and cancer.

Like nitric oxide (NO), prostaglandin E2 (Prostaglandin E ₂ ; PGE ₂ ) is synthesized by COX-2 as a mediator of an important inflammatory response mainly involved in the transmission of pain and fever in damaged areas or tissues. When tissue is damaged, not only physical damage but also a large amount _of substances mediating inflammation are produced, causing pain. When produced in excess, it causes an excessive immune response and causes symptoms such as vasodilation, edema, fever and pain, so it is important to produce in an appropriate amount.

In addition, prostaglandins (PGE ₂ ) not only serve as mediators of inflammatory responses, but are also modulators of immune responses that promote Th2 type immune responses and the production of anti-inflammatory cytokines such as IL-10 in macrophages.

Nuclear factor-kappa B (NF-κB) is a major transcription factor of macrophage activation regulating many immunomodulatory mediators, and the inactive NF-κB dimer (p65/50) is an IκB-α in the cytosol interacts with and forms a complex.

The nuclear factor-kappaB inhibitor (IκB-α) is a cellular protein that functions to inhibit the nuclear factor-kappaB (NF-κB) transcription factor, and nuclear factor-kappaB (NF-κB) is inactive in the cytoplasm. isolates and acts as a deterrent.

As the IκB-α is phosphorylated and degraded through the ubiquitin-protein system, the NF-κB dimer is released from the NF-κB/IκB-α complex and converted into the nucleus to initiate the transcriptional activity of the target gene.

TLRs are known to play critical roles in host cell recognition and induction of inflammatory responses by specific patterns of microbial pathogens as well as activation of innate immunity.

Among the TLRs, the Tolleri receptor (TLR4) plays a key role in inflammatory and immune responses and activates TNF receptor-related factor 6 (TRAF6) through a myeloid differentiation factor 88 (MyD88)-dependent pathway, one of the adapter molecules.

Furthermore, TRAF6 functions as an important mediator of signaling pathways regulating immune regulation-related downstream gene expression by promoting activation of NF-κB and MAPK signaling pathways.

The nitric oxide (NO), prostaglandin E2 (PGE2) and increased expression of immune response-related factors, nuclear factor-kappa B (Nuclear Factor-kappa B; NF-κB) transcriptional activity, tollei receptor ( As TLR4) and myeloid differentiation factor 88 (MyD88) expression are increased, immune activity can be enhanced.

The immune response-related factors include inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin It is selected from the group consisting of -6 (IL-6) and interleukin-10 (IL-10).

The extract is extracted with a solvent selected from the group consisting of water, C ₁ to C ₆ alcohols and mixtures thereof.

The stone gas extract can be eluted with a polar solvent such as a C ₁ to C ₆ alcohol such as water, ethanol, methanol, etc., or a mixed solvent having a mixing ratio of 1:0.1 to 1:10 of the alcohol and water. In more detail, ethanol may be used as an extraction solvent. At this time, the extraction temperature may be 10 ℃ to 100 ℃, preferably the extract extracted at room temperature.

The extraction solvent may be used in an amount of 2 to 50 times, preferably 2 to 20 times, based on the weight of the sample. For extraction, the sample may be left for 1 to 48 hours for leaching in the extraction solvent, and in particular, it may be left for 1 to 24 hours.

It may be a result obtained by concentrating the filtered extract under reduced pressure with a vacuum rotary concentrator, but it is not limited thereto, as long as it is a dolgasari extract that can exhibit the immune improvement effect of the present invention, and the extract, the diluted or concentrated solution of the extract, and the extract are dried all of the dried product obtained by

The pharmaceutical composition according to another embodiment of the present invention is prepared by including the composition for improving immunity as an active ingredient.

The pharmaceutical composition may further include one selected from the group consisting of pharmaceutically acceptable carriers, excipients and diluents. Specifically, the pharmaceutical composition is each 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 to be used. can

In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate , calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to simple diluents such as water and liquid paraffin, which are commonly used for suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, the term "pharmaceutically effective amount" of the present invention means to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. It means a sufficient amount, and the effective dose level is the severity of the disease, the drug activity, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the rate of excretion. The duration may be determined according to factors including drugs used in combination or concomitant with the composition of the present invention used and other factors well known in the medical arts.

The pharmaceutical composition of the present invention may be administered alone or in combination with a known immunotherapeutic agent. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects.

A food composition according to another embodiment of the present invention is prepared by including the composition for improving immunity as an active ingredient.

Preferably, in the case of using only the dolgasari extract for use as the food composition, there is a problem in that the palatability of the composition for improving immunity is lowered due to the peculiar smell of dolgasari.

Accordingly, by adding a small amount of red firefly extract and Myeokseochae extract, when preparing a complex composition, the immune improvement effect is equally maintained or improved, and the preference can be increased.

Red firefly (Sanicula rubriflora F. Schmidt ex Maxim) is a perennial plant that grows in a forest valley. The root leaf is 6-20cm in diameter and is deeply divided into 3 parts, and both branches are again divided into 2 parts. Stem leaves are opposite one pair at the top of the stem, and there is no petiole. Flowers bloom in April-June. After 1-5 peduncles appear between stem leaves, several short-stalked flowers hang on each, dark purple. Fruits are stalks, 1-3 each, and there are thorns with curved ends on the outside. It is found north of Gyeongsangbuk-do and Jeollabuk-do in Korea, and distributed in Japan, northeast China, Amur, and Ussuri, Russia. Compared to Arabidopsis firefly, the root leaves are larger, and there are hook-shaped thorns on the upper part of the fruit, so it is distinguished. It is also called red charm body.

Scorzonera austriaca Willd (Scorzonera austriaca Willd) is a perennial herb that grows rarely in sunny grasses along the west coast. Stems are upright, 20-40cm high, and have white hairs like spider webs when young, but they disappear as they grow older. Root leaves are clustered and remain for a long time, linear or lanceolate, 14-18cm long, 10-18mm wide, pointed at the tip, narrowing at the bottom, like a petiole, and flat at the edge. Stem leaves are few or scaly, alternate in phyllotaxis, with a sharp tip, and a broad bottom envelops the stem and is hairless. Flowers bloom from May to June, and 2.5cm diameter hair-shaped flowers hang from the tip of the stem one by one. The head-shaped flower becomes a tongue-shaped bisexual flower, the corolla is yellow, and the tip is deeply divided into 5 parts. Fruits are aqua, linear, and ridged. Umbrella hair is like a feather. It grows wild in South and North Pyongan Provinces, Hwanghae Province, and Gyeonggi Province, and is distributed in China, Mongolia, Russia, and Europe. Compared to Soeschae, the whole is smaller, and the root and stem leaves are different in size and shape, and the stem leaves are small in the shape of scales, and 1 or 2-4 head-shaped flowers hang at the end of the stem. The roots and young leaves are edible.

More preferably, the composition for improving immunity of the present invention may include 5 to 10 parts by weight of a red firefly extract and 5 to 10 parts by weight of an extract of Myeokseochae based on 100 parts by weight of the dolgasari extract. When the extract is further included, it can be provided as a food composition with high palatability by neutralizing the unique seaweed flavor of the dolgasari extract.

As a specific example, it is possible to prepare a processed food capable of enhancing the immune-improving effect by using the food composition. Such processed foods include, for example, sweets, beverages, alcoholic beverages, fermented foods, canned foods, milk products, processed meat foods, noodles, and the like. Confectionery includes biscuits, pies, cakes, breads, candies, jellies, gums, cereals, and the like. The beverage includes drinking water, carbonated beverage, functional ionized beverage, functional ionized beverage, juice (eg, apple, pear, grape, aloe, tangerine, peach, carrot, tomato juice, etc.), sikhye, and the like. Alcoholic beverages include sake, whiskey, shochu, beer, Western liquor, fruit wine, and the like. Fermented foods include soy sauce, soybean paste, red pepper paste, and the like. Canned foods include canned seafood (eg, canned tuna, mackerel, saury, conch, etc.), canned livestock products (canned beef, pork, chicken, turkey, etc.), and canned agricultural products (canned corn, peaches, pineapple, etc.). Processed milk products include cheese, butter, yogurt, and the like. Processed meat products include pork cutlet, beef cutlet, chicken cutlet, and sausage. Includes sweet and sour pork, nuggets, and breadcrumbs. Noodles such as sealed packaged raw noodles are included. In addition to this, the composition may be used in retort foods, soups, and the like.

The composition for improving immunity of the present invention has an immune enhancing effect by inducing phagocytosis activation of macrophages.

More specifically, nitric oxide (NO), prostaglandin E2 (PGE2), immune response-related factors, nuclear factor-kappa B (Nuclear Factor-kappa B; NF-κB) transcriptional activity, tollei receptor ( TLR4) and bone marrow differentiation factor 88 (MyD88) by increasing the expression, relates to a composition for improving immunity, comprising, as an active ingredient, a stone garfish extract capable of exhibiting an immune response activating effect.

1 is a cell viability measurement result according to the treatment of a stone garfish extract according to an embodiment of the present invention.
2 is a result of measuring the phagocytosis activity of RAW 264.7 cells according to the treatment of the stone garfish extract according to an embodiment of the present invention.
3 is an analysis result according to whether the production of NO and PGE ₂ increases according to the treatment of the stone garfish extract according to an embodiment of the present invention.
4 is an ELISA analysis result according to whether cytokine production is increased according to the treatment of stone garfish extract according to an embodiment of the present invention.
5 is a western blot analysis result for confirming whether the expression of iNOS, COX-2 and cytokines according to the treatment of the stone garfish extract according to an embodiment of the present invention is related to NO, PGE ₂ and cytokine production.
6 is a western blot analysis result for the activation degree of TLR4, MyD88, NF-kB and p-IkB-a according to the treatment of the stone garfish extract according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

[Preparation Example 1: Preparation of Stone Garlic Extract]

The rockfish used in this experiment was provided by Jin Jin Hyun (Jeju National University College of Medicine). In order to obtain an ethanol extract (EECT), 80% ethanol was added to 100 g of the dried stone powder, 1.5 L each, and extracted 5 times for 30 minutes in an ultrasonic grinder, and only the supernatant was separated through a filter. The supernatant obtained by repeating this process three times was concentrated under reduced pressure and freeze-dried, and the powder obtained was dissolved in dimethyl sulfate (dimethyl sulfoxide, DMSO, Sigma-Aldrich Co., St. Louis, MO, USA), and a cell culture medium. The final treatment concentration was adjusted before use in the experiment.

[Preparation Example 2: Preparation of complex extract]

1. Preparation of complex extract

Using the same method as the preparation method for the stone garfish ethanol extract (EECT) of Preparation Example 1, red firefly ethanol extract (EERT) and ethanol extract (EEST) were prepared, and immune within the weight range as shown in Table 1 below. A composition for improvement was prepared.

Y1 Y2 Y3 Y4 Y5 EECT 100 100 100 100 100 EERT - One 5 10 15 EEST - One 5 10 15

(Unit: parts by weight)

[Experimental Example 1: Immunomodulatory activity effect experiment on RAW 264.7 cells]

1. Experimental method

cell culture

Rat macrophage-like RAW 264.7 cells were purchased from Korea Cell Line Bank (Korea), 10% (v/v) fetal bovine serum (FBS, WelGene, Inc.), 100 U/mL penicillin, 100 μg /mL Streptomycin (WelGene, Inc.) supplemented with Dulbecco's modified Eagle's medium (Dulbecco's modified Eagle's medium, DMEM, WelGENE, Inc., Daegu, Republic of Korea) using 37 °C and 5% CO ₂ It was cultured under the condition of (v/v).

Culture medium was changed every 2-3 days, and cells were grown to 80-90% confluence in all experiments.

cell viability assay, cell viability assay

Cells were seeded in 96-well plates at a density of 1 x 10 ⁴ cells/ml. After incubation for 16 h, cells were treated with the indicated EECT concentrations (50 μg/ml, 100 μg/ml, 150 μg/ml, 200 μg/ml and 300 μg/ml) and 0.5 ng/ml LPS (Sigma-Aldrich Chemical Co.) .) was treated.

After 24 hours of incubation, 5 mg/ml of 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltrazolium Bromide (MTT; Invitrogen, Carlsbad, CA, USA) solution was added to each well for 3 hours. cultured.

Then, formazan crystals present in each well were dissolved in DMSO, and absorbance at 540 nm was measured using a microplate reader (Molecular Devices Co., Sunnyvale, CA, USA).

Cells treated with LPS were used as a positive control for macrophage activation, and cells cultured in DMEM without other treatment were used as a negative control, and macrophage morphological changes were visualized by phase contrast microscopy (Karl Zeiss, Oberkochen, Germany). .

Phagocytosis analysis and morphology observation

Phagocytosis of RAW 264.7 cells was assessed using a phagocytosis assay kit (Cayman Chemical, Ann Arbor, MI, USA). Cells were plated on 4-well chamber slides (Pocheon, Korea, SPL Life Sciences Co., Co.) and incubated at 37° C. for 16 hours to adhere to the plate, and then treated with EECT or LPS for 24 hours.

Then, latex bead-rabbit IgG-fluorescein isothiocyanate (FITC) complexes were added to the culture medium at a final dilution of 1:200. After incubation at 37° C. for 2 h, cells were gently washed twice with the provided assay buffer and placed under a fluorescence microscope (Carl Zeiss) to observe FITC-labeled latex beads.

In addition, fluorescence intensity was measured by flow cytometry (BD Biosciences, San Jose, CA, USA).

Measurement of NO production

Cells were pre-cultured for 16 hours at a density of 5Х10 ⁴ cell/ml in 24-well culture plates, and then treated with 0.5 ng/ml LPS or EECT at different concentrations. After incubation for 24 hours, the collected culture supernatant was mixed with the same amount of Griess reagent solution (Sigma-Aldrich Chemical Co.) and incubated at room temperature (RT) for 30 minutes. NO concentration was measured with a microplate reader at 540 nm with reference to a standard curve generated with known sodium nitrate (NaNO ₂ ) concentration.

PGE ₂ and measurement of cytokine production

Standard curves prepared from standard samples were used to calculate PGE ₂ and cytokine production.

Culture supernatants were harvested from each group, and PGE ₂ and tumor necrosis factor-α (PGE 2 and tumor necrosis factor-α ( The levels of cytokines including TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-10 (IL-10) were quantified. Standard curves prepared from standard samples were used to calculate PGE ₂ and cytokine production.

Western blot analysis

After treatment, the collected cells were washed with phosphate-buffered saline (PBS) and lysed with a lysis buffer containing a cell protease inhibitor and a phosphate inhibitor (Sigma-Aldrich Chemical Co.).

In a parallel experiment, nuclear and cytoplasmic proteins were isolated using a nuclear extraction kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's procedure, and the protein concentration of the lysate was determined by Bradford assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) was used.

After denaturation, the same amount of protein was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, MA, USA, MA) by electroblotting. .

After incubation with a blocking buffer (containing 5% non-fat milk) for 1 hour at RT, the primary antibody was probed overnight at 4°C with shaking.

Primary antibodies were obtained from Abcam, Inc. (Cambridge, MA, UK, Cambridge, MA, UK), Cell Signaling Technology (Danvers, MA, USA; Danvers, MA, USA) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA; Santa Cruz, CA, USA).

The strips were then incubated with appropriate horseradish peroxidase-conjugated antibodies (Amersham Life Science, Arlington Heights, IL, USA) for 2 hours at room temperature, and an Enhanced Chemiluminescence (ECL) detection system (R&D Systems Inc., Minneapolis, MN, USA). ) and the Fusion FX Image system (Vilber Lourmat, Torcy, France).

statistical analysis

All measurements were performed in triplicate and results are presented as mean ± standard deviation (SD), and to analyze differences between means, statistically significant values were compared using one-way analysis of variance (ANOVA), followed by graph-pad Tukey HSD Post-Hawk Test using Prism (Graph-Pad Prism), version 5 (GraphPad Software, Inc, San Diego, CA, USA) was used. The experimental result was judged to be statistically significant when p < 0.05.

2. Experimental results

Evaluation of Cytotoxicity of Stonefish Extract on RAW 264.7 Macrophages

To establish the experimental conditions, RAW 264.7 cells were treated with various concentrations of EECT (50 - 300 μg/ml) for 24 h, and cytotoxicity was assessed by MTT assay.

As shown in FIG. 1 , there was no cytotoxicity in RAW 264.7 cells regardless of the increased concentration of EECT, and no significant difference was found between the control and EECT-treated cells. Therefore, EECT concentrations of up to 200 μg/ml were chosen for subsequent experiments.

It was confirmed that LPS at a concentration of 0.5 ng/ml used as a positive control for activating macrophages also had no cytotoxicity to RAW 264.7 cells.

Effect of Stonefish extract on the increase of phagocytic activity in RAW 264.7 macrophages

Since the morphological changes of macrophages can be influenced by various extrinsic factors and are sometimes associated with their activation, the effect of EECT on the morphology of RAW 264.7 cells was investigated. As shown in Fig. 2A, RAW 264.7 cells cultured in normal medium were generally round or oval in shape. However, as the EECT treatment concentration increased, the cells became irregular, large, and diamond-shaped, and the surface became rough and coarse. LPS exposure of RAW 264.7 cells as a positive control tended to cause similar morphological changes.

In addition, to investigate whether EECT affects the phagocytic activity of RAW 264.7 cells, latex beads absorbed into the cells were observed under a fluorescence microscope.

As a result, it was shown that the ability of EECT-treated RAW 264.7 cells to entrap fluorescence-labeled latex beads was improved compared to the control group (Fig. 2B and C).

In addition, flow cytometry was applied and it was confirmed that EECT significantly enhanced phagocytosis ( FIGS. 2D and E). The phagocytic activity of RAW 264.7 cells treated with 150 μg/ml EECT was similar to that of the LPS-positive control, confirming that EECT could activate RAW 264.7 cells.

NO and PGE in RAW 264.7 macrophages ₂ Effect of stonefish extract on induction of production

To investigate the effect of EECT on NO and PGE ₂ production, nitrite and PGE ₂ concentrations accumulated in the conditioned medium were quantified using Griess reagent and PGE ₂ ELISA kit, respectively. As shown in Figure 3, as the concentration of EECT treatment increased, the production of NO and PGE2 was significantly increased compared to the control group. It was confirmed that the concentration produced by cells treated with 150 and 200 μg/ml EECT was much higher than that of the LPS positive control.

Effect of Stonefish extract on the enhancement of cytokine production in RAW 264.7 macrophages.

ELISA analysis was performed to evaluate the effect of EECT on the secretion of cytokines such as TNF-α, IL-1β, IL-6 and IL-10 in RAW 264.7 cells.

As shown in Figure 4, EECT significantly enhanced the secretion of all tested cytokines compared to control or LPS-treated cells in a concentration-dependent manner.

Effect of fern extract on the promotion of expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and cytokines in RAW 264.7 macrophages

Western blot analysis was performed to investigate whether the increased production and secretion of NO, PGE ₂ and cytokines by EECT treatment was associated with increased expression of regulatory genes.

As a result, the expression of iNOS and COX-2 involved in NO and PGE ₂ production, respectively, was significantly increased in a concentration-dependent manner in the cells treated with EECT compared to the control group ( FIG. 5A ). These results suggest that EECT can increase the production of NO and PGE ₂ by upregulating the expression of iNOS and COX-2, respectively. Similar to these results, the expression of cytokines was also increased in a concentration-dependent manner by EECT treatment (Fig. 5B).

In addition, the positive LPS control also significantly increased the expression of all these proteins.

Effect of stonefish extract on activation of TLR4, myeloid differentiation factor 88 (MyD88) and NF-κB signaling pathways in RAW 264.7 macrophages

To further analyze whether TLR4, MyD88 and NF-κB signaling pathways were involved in EECT-induced RAW 264.7 macrophage activation, TLR4, MyD88, NF-κB and nuclear factor-kappa B-α inhibitors (IκB- α) was measured by Western blot analysis.

As a result, it was shown that the total protein levels of TLR4 and MyD88 in RAW 264.7 cells treated with EECT for 24 hours increased in a concentration-dependent manner ( FIG. 6A ).

In addition, the expression of both proteins in the cells treated with LPS was significantly higher than that in the non-LPS-treated control group. To investigate the effect of EECT on the NF-κB signaling pathway, cytoplasmic and nuclear proteins were extracted from cells treated with EECT for 1 hour to examine the expression of NF-κB and IκB-α.

Figure 6B shows that expression of NF-κB p65, one important NF-κB subunit for NF-κB activation, is increased in the nucleus and decreased in the cytoplasm of EECT-treated RAW 264.7 cells.

In the cytoplasmic fraction, the total protein expression of IκB-α, a cytoplasmic inhibitor of NF-κB, was gradually down-regulated with increasing concentrations of EECT, whereas the level of phosphorylated IκB-α (p-IκB-α) was decreased in the untreated control group. was upregulated compared with A similar trend was found in RAW 264.7 cells treated with LPS.

[Experimental Example 2: Effect of Complex Extract]

The effects of the composite extracts Y2 to Y5 of Table 1 were compared and shown in the same manner as the experimental method of the ethanol extract (EECT) of the stone garfish shown in Experimental Example 1.

The results are comprehensively shown in Table 2 below, and for an objective comparison, the effect of Y1 (Ethanol extract of stone garfish, EECT) was expressed as an index of 5, and the higher the number, the better the index.

The evaluation indices below were converted to indices of 1 to 10 and shown.

Y1 Y2 Y3 Y4 Y5 immunity boosting power 5 3 6 6 2

(Unit: Index)

As shown in Table 2, the phagocytic activity of macrophages, NO production, PGE ₂ production, nuclear Factor-kappa B (Nuclear Factor-kappa B; NF-κB) increased transcriptional activity, and showed excellent activity of expression of Tollieri receptor (TLR4) and myeloid differentiation factor 88 (MyD88), confirming that the immunostimulating effect was equal or superior. .

In particular, in the case of Y3 and Y4 containing preferred parts by weight of ethanol extract (EERT) and ethanol extract (EEST) of red firefly ethanol extract (EECT) in stone garfish, in the case of including only ethanol extract (EECT) It was confirmed that it exhibited a better immune enhancing effect compared to that.

[Experimental Example 3: Sensory Experiment]

1. Sensory Experiments

After the composite composition of Y1 to Y5 composed of Table 1 was prepared as tea, 40 adult men and women in their 20s to 50s were allowed to taste the prepared tea, and then to evaluate the taste and aroma, the comprehensive preference diagram is shown in Table 2 below indicated.

The evaluation score was requested to be evaluated on a scale of 1 to 10 for each item, and the evaluation result was converted into an average score (rounded off). In the index, the higher the number, the higher the degree of preference.

Y1 Y2 Y3 Y4 Y5 taste 5 3 6 7 3 incense 5 4 7 7 2 Overall preference (average) 5 3 6 7 2

(Unit: Index)

Referring to Table 3 above, it was confirmed that Y3 and Y4 containing ethanol extract of red firefly (EERT) and ethanol extract of Myeoksaechae (EEST) showed a higher overall preference compared to Y1 containing only ethanol extract of stone garfish (EECT). did.

As a result, when it is intended to be used as a food composition including only the ethanol extract (EECT) from stonefish, there is a problem that the preference is lower than that of the immune enhancing effect. It is excellent and can be supplied as a food composition with higher palatability.

Although preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also provided. is within the scope of the

Claims (7)

Containing stone garfish (Chondracanthus tenellus) extract as an active ingredient
A composition for improving immunity. The method of claim 1,
The composition for improving immunity has an immune enhancing effect by inducing phagocytosis activation of macrophages
A composition for improving immunity. The method of claim 1,
The composition for improving immunity includes increased production of nitric oxide (NO), increased production of prostaglandin E2 (PGE2), increased expression of immune response-related factors, nuclear factor-kappa B (Nuclear Factor-kappa B; By increasing NF-κB) transcriptional activity, and increasing expression of Tollieri receptor (TLR4) and myeloid differentiation factor 88 (MyD88)
that activates the immune response
A composition for improving immunity. The immune response-related factors include inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin -6 (IL-6) and interleukin-10 (IL-10) that is selected from the group consisting of
A composition for improving immunity. The method of claim 1,
The extract is extracted with a solvent selected from the group consisting of water, C ₁ to C ₆ alcohols and mixtures thereof.
A composition for improving immunity. Claims 1 to 4 comprising the composition for improving immunity according to any one of claims
pharmaceutical composition. Claims 1 to 4 comprising the composition for improving immunity according to any one of claims
food composition.

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