KR102671693B1 - Composition for antioxidant and anti-inflammatory comprising extract of chondracanthus tenellus hommersand as an active ingredient - Google Patents

Abstract

The present invention relates to an anti-inflammatory and antioxidant composition containing stone agar extract as an active ingredient, which not only suppresses reactive oxygen species (ROS) caused by oxidative stress, but also inhibits inflammation. To provide an anti-inflammatory and antioxidant composition that can suppress inflammation by suppressing the production of NO (nitric oxide), which causes , and suppressing the production of TNF-α, IL-6, and IL-10.

Description

Anti-inflammatory and antioxidant composition containing stone fish extract as an active ingredient {COMPOSITION FOR ANTIOXIDANT AND ANTI-INFLAMMATORY COMPRISING EXTRACT OF CHONDRACANTHUS TENELLUS HOMMERSAND AS AN ACTIVE INGREDIENT}

The present invention relates to an anti-inflammatory and antioxidant composition containing stone agar extract as an active ingredient. More specifically, it relates to a composition containing stone agar extract as an active ingredient and having excellent anti-inflammatory and antioxidant effects.

Oxygen is the most important molecule essential for maintaining life. For example, brain cells begin to break down if they are deprived of oxygen for just 30 seconds. ‘Mitochondria’ is an organ that acts as a core factory that creates energy within cells.

Oxygen transported to the mitochondria produces energy by breaking down glucose made when a person consumes nutrients (carbohydrates, proteins, fats). During the energy production process, water, carbon dioxide, and oxygen are generated, and this oxygen is called reactive oxygen species (ROS). Reactive oxygen species are generated during normal intracellular activity and are involved in various biological processes, including cell differentiation, gene expression, and response to cytokines. Oxidative stress is a phenomenon in which ROS within cells increases due to an imbalance between the production of ROS and the antioxidant reaction that removes it, reacting with and damaging DNA, proteins, and lipids. This is known to be a key cause of aging and heart-related diseases. .

Most reactive oxygen species are produced as by-products during mitochondrial electron transfer. Additionally, ROS forms intermediates necessary for metal-catalyzed oxidation reactions. The oxygen atom has two auxiliary electrons in different orbits of its outer electron shell. This electronic structure makes oxygen susceptible to radical formation. Sequential degeneration of oxygen by the addition of electrons leads to the formation of ROS such as superoxide, hydrogen peroxide, hydroxyl radical, hydroxyl ion, and nitric oxide.

Detoxification of produced oxygen species is of utmost importance for the survival of all aerobic life forms. Many of the commonly mentioned defense mechanisms provide a balance between the removal and production of ROS and have evolved to meet this need. The imbalance between ROS production and removal is referred to as oxidative stress. Cells possess a variety of defense mechanisms to ameliorate the harmful effects of ROS. Superoxide dismutase (SOD) catalyzes the conversion of two superoxide anions inserted into hydrogen peroxide (H ₂ O ₂ ) and oxygen (O ₂ ) molecules. In the peroxisomes of eukaryotic cells, the catalase enzyme converts H ₂ O ₂ into water and oxygen and completes the detoxification of reactive oxygen species starting from SOD.

There are many non-enzymatic small molecule antioxidants that play an important role in this detoxification. Glutathione plays the most important role in intracellular defense against the harmful effects of reactive oxygen species. Reaction with ROS molecules oxidizes Glutathione, but the reduced form is regenerated in redox by NADPH-dependent reductase. The ratio of the oxidized form of glutathione (GSSG) to the reduced form, reduced glutathione (GSH), is a dynamic indicator of oxidative stress in organisms.

In order to maintain normal immune function related to inflammation, it is very important to always maintain a healthy state. Immune responses involve various immune-related cells and immunomodulatory factors, but sufficient nutrient intake is necessary for the immune system to function normally, and some immunomodulatory factors do not allow environmental factors to significantly affect the expression of immune function. It is known that

The purpose of this inflammation is to suppress initial cell damage, remove necrotic cells and injured tissue from the wound area, and simultaneously regenerate tissue. Macrophages are known to be major cells involved in inflammatory reactions. They are activated by exposure to stimuli or cytokines secreted by immune cells, and play an important role in biological defense by producing cytokines in the early stages of infection. When macrophages are treated with lipopolysaccharide (LPS), known as a component of the cell wall of Gram-negative bacteria, the mitogen-activated protein kinase (MAPK) and NF-kB signaling pathways are activated by toll like receptor (TLR)-4. It is induced.

Meanwhile, in recent years, research focusing on the evaluation of natural seaweed products has received much attention in the food, cosmetics, and pharmaceutical industries. A variety of bioactive secondary metabolites have been identified in seaweed, including polyphenols, terpenoids, sulfated polysaccharides, alkaloids, polyunsaturated fatty acids, mycosporin-like amino acids, peptides, and halogenated compounds. These molecules exhibit activity against various diseases. Unlike most natural products isolated from terrestrial organisms, these molecules have unique structural features, so extensive research has been conducted, ranging from characterization to synthesis of structural analogs.

In particular, a lot of research is being conducted on pharmaceutical and food materials with antioxidant and anti-inflammatory properties from seaweed, and research efforts are being made to find natural products with fewer side effects.

KR 10-2019-0070259 B1

The purpose of the present invention is to suppress reactive oxygen species (ROS) caused by oxidative stress by including stone agar extract as an active ingredient, as well as suppress the production of NO (nitric oxide), which causes inflammation, and inhibit TNF-α. , to provide an anti-inflammatory and antioxidant composition that can suppress inflammation by suppressing the production of IL-1β and IL-6.

In order to achieve the above object, the anti-inflammatory and antioxidant composition according to an embodiment of the present invention includes Chondracanthus tenellus (Harvey) Hommersand) extract as an active ingredient.

The composition inhibits cyclooxygenase-2 (COX-2) activity, nitric oxide (NO), PGE2, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and It exhibits an anti-inflammatory effect by inhibiting the expression of interleukin-10 (IL-10).

The composition exhibits an antioxidant effect through inhibition of reactive oxygen species (ROS) activity.

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

A pharmaceutical composition according to another embodiment of the present invention is prepared containing the anti-inflammatory and antioxidant composition as an active ingredient.

A food composition according to another embodiment of the present invention is prepared by including the anti-inflammatory and antioxidant composition as an active ingredient.

A cosmetic composition according to another embodiment of the present invention is prepared by including the anti-inflammatory and antioxidant composition as an active ingredient.

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

The term 'extract' used in this specification has the meaning commonly used in the art as a crude extract, as described above, but in a broad sense also includes fractions obtained by additional fractionation of the extract. In other words, the Dolgasi extract includes not only those obtained using the above-described extraction solvent, but also those obtained by additionally applying a purification process. For example, fractions obtained by passing the extract through an ultrafiltration membrane with a certain molecular weight cut-off value, separation by various chromatographs (designed for separation according to size, charge, hydrophobicity, or affinity), etc. Fractions obtained through purification methods are also included in the plant extract of the present invention.

The extract used in the present invention can be prepared in powder form by additional processes such as reduced pressure distillation and freeze drying or spray drying.

In the present invention, “anti-inflammation” refers to suppressing inflammation, which is one of the defense responses of living tissue to certain stimuli and causes complex lesions that occur together with three factors: tissue deterioration, circulatory failure and exudation, and tissue proliferation. says More specifically, inflammation is part of innate immunity, and like other animals, human innate immunity recognizes cell surface patterns that are specific to pathogens. Phagocytes recognize cells with such a surface as non-self and attack the pathogen. If pathogens break through the body's physical barriers, an inflammatory response occurs. The inflammatory response is a non-specific defense function that creates a hostile environment for microorganisms invading the wound area. In an inflammatory response, when a wound occurs or an external infectious agent enters the body, white blood cells responsible for the initial immune response flock in and express cytokines. Therefore, the expression level of intracellular cytokines becomes an indicator of inflammatory response activation.

The term “antioxidant” in the present invention refers to the action of inhibiting oxidation. The human body has a balance of prooxidants and antioxidants, but when this balance becomes unbalanced due to various factors and leans toward pro-oxidation, oxidative stress occurs. It can cause potential cell damage and pathological diseases. Reactive oxygen species (ROS), which are the direct cause of this oxidative stress, are unstable and highly reactive, easily reacting with various biological substances, and attacking macromolecules in the body, causing irreversible damage to cells and tissues, mutations, and mutations. It causes cytotoxicity and carcinogenesis.

The anti-inflammatory and antioxidant composition according to an embodiment of the present invention includes Chondracanthus tenellus (Harvey) Hommersand) extract as an active ingredient.

Chondracanthus tenellus (Harvey) Hommersand, also known as Lagoon, is a member of the order Asteridae in the phylum True Red Algae, and is distributed in Korea (all coastal areas), Japan, China, the Philippines, and the Pacific Islands. The thallus is 5 to 15 cm long and 1 to 2 mm wide, and has many flat, columnar branches that are alternate or opposite, and the apex is sharp. The body is purple or red-purple, cartilaginous, and fluoresces in water. The cystic fruit is almost spherical, small, and has no stalk. It grows on reefs from the lower intertidal zone to the subtidal zone. Stone agar is also used as a mixed raw material in the manufacture of agar. It is rich in minerals such as calcium sulfate polysaccharide and calcium carbonate, so it is mainly used as a food resource and is reported to have antioxidant activity.

The composition inhibits cyclooxygenase-2 (COX-2) activity, nitric oxide (NO), PGE2, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and It exhibits an anti-inflammatory effect by inhibiting the expression of interleukin-10 (IL-10).

The inflammatory response is a defense response to tissue damage, external physical and chemical stimuli, and various infectious agents, and is a mechanism to repair and regenerate damaged tissues. During an inflammatory reaction, plasma accumulates at the site of inflammation, diluting the toxins secreted by bacteria, blood flow increases, and symptoms such as erythema, pain, swelling, and fever occur. In normal cases, the body neutralizes or removes disease-causing factors through an inflammatory response and regenerates damaged tissues to restore normal structure and function. However, a chronic inflammatory response that occurs continuously or excessively causes tissue damage. This inflammatory response leads to the progression of stomach, colon, bladder, and prostate cancer, and is the cause of various diseases such as rheumatoid arthritis and chronic infections.

When an inflammatory response occurs, immune cells such as macrophages and monocytes produce nitric oxide (NO), prostagladin E ₂ (PGE ₂ ), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, Secretes inflammatory mediators such as IL-8 and IL-12.

A representative example of an inflammatory response is the activation of the toll like receptor (TLR) signaling system of macrophages by pathogen associated molecular patterns (PAMPs). Lipopolysaccharide (LPS), a type of endotoxin present in the outer membrane of Gram-negative bacteria, belongs to PAMPs, which are inflammatory mediators present in external microorganisms. When bacteria infiltrate into the human body, TLR4 on the surface of macrophages recognizes LPS with the help of MD-2 or CD14, induces activation of downstream signaling systems, and ultimately causes the transcription factor nuclear factor-κB (NF- Activates κB).

NF-κB, which has moved to the nucleus, is activated by inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8, IL-12, etc.), inducible nitiric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). It induces gene expression, and nitric oxide (NO) generated by iNOS intensifies inflammation. Nitric oxide overproduced by iNOS in macrophages reacts with superoxide to form peroxynitrite, which acts as a strong oxidant and damages cells, causing various pathological diseases including inflammation and cancer. It is known to be involved in the process.

However, synthetic drugs such as ibuprofen, antihistamines, steroids, cortisone, immunosuppressants, and immunostimulants are used to treat inflammation, but the therapeutic effect is temporary or has many side effects such as simple symptom relief, hypersensitivity reactions, and worsening of the immune system. Therefore, it is difficult to fundamentally treat inflammation.

The present invention includes stone fish extract as an active ingredient, nitric oxide (NO), PGE ₂ , tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL) Through the effect of inhibiting the expression of -10), it can exhibit anti-inflammatory activity, and the problem of side effects can be reduced by using natural products.

The tumor necrosis factor (TNF) is a cell signaling protein related to 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), also called lymphotoxin, but tumor necrosis factor generally refers to tumor necrosis factor-α. .

The tumor necrosis factor refers to a substance that kills tumors secreted by T cells by lipopolysaccharide, an endotoxin released from the bacterial wall. It is the most important inflammatory factor and sometimes causes severe systemic inflammation. However, from an immunological perspective, TNF is believed to aggregate neutrophils and monocytes, which are white blood cells in the blood, to the site of infection and induce differentiation and activation into macrophages with the ability to kill infected cells. The function of tumor necrosis factor is its ability to induce the production of cytokines with chemotaxis and the ability to increase the affinity of adhesion molecules between vascular endothelial cells and white blood cells.

Interleukin is a substance that plays an important role in the body's defense by regulating the immune response by acting on various stages of the immune response. Its types include IL-1, 6, 10, and 12. Among these, IL-12 is a cytokine composed of p35 and p40 proteins, and has a very important immunological function in linking innate and acquired immunity. It is also involved in various effector cells, such as macrophages, involved in the removal of endogenous antigens. ), induces the activation of NK cells and T-cells.

In this way, in terms of anti-inflammatory activity, macrophages play an important role in the progression and maintenance of the inflammatory response by secreting and producing proteolytic enzymes, lipid metabolites, cytokines such as interleukins and TNF, and immune mediators such as NO and PGE _2. It produces an immune response through phagocytosis, which removes microorganisms, antigens, dead tissues, red blood cells, etc.

The composition exhibits an antioxidant effect through inhibition of reactive oxygen species (ROS) activity.

Reactive oxygen species (ROS) is a superoxide anion (reactive oxygen species), which is a singlet oxygen generated during the oxidation and reduction process of triplet oxygen, the most stable form of oxygen. Superoxide anion, hydroxyl radical, free radical, and hydrogen peroxide (H2O2) are unstable and have an increased oxidizing power, so they easily react with biological substances, causing oxidative stress if they are not removed from the human body. It causes it. Since this oxidative stress is linked to the inflammatory response, it induces lipid peroxidation in various metabolic processes and damages proteins, cell membranes, and DNA, thereby inducing cellular aging and deformation, leading to stroke, cancer, arteriosclerosis, Alzheimer's disease, and Parkinson's disease. , which causes various diseases such as arteriosclerosis.

The stone agar extract of the present invention protects against oxidative substances generated due to oxidative stress in the body by removing reactive oxygen species (ROS), and can be provided as an antioxidant and anti-inflammatory composition.

The extract is extracted using an extraction solvent selected from the group consisting of water, C ₁ to C ₆ lower alcohols, and mixtures thereof.

Specifically, to prepare the extract, there are steps of washing the natural product; Drying after washing; Grinding the natural product after drying; leaching the pulverized material using an organic solvent; Leaching and drying the sample; Leaching using water; And including the step of leaching, a pine cluster extract can be obtained.

The natural extract extracted using the organic solvent may further include the step of fractionation using the organic solvent.

The method of preparing the extract may be a conventional extraction method in the art, such as ultrasonic extraction, leaching, and reflux extraction. Specifically, it may be an extract obtained by extracting a natural product from which foreign substances have been removed by washing and drying with water, alcohol having 1 to 6 carbon atoms, or a mixed solvent thereof, and may be an extract obtained by sequentially applying the solvents to the sample.

The reflux extraction method is based on 100 mL of water and alcohol having 1 to 6 carbon atoms, 10 to 30 g of pulverized natural product, reflux time of 1 to 3 hours, and alcohol or water having 1 to 6 carbon atoms.

More specifically, based on 100 mL of alcohol with 1 to 6 carbon atoms or 100 mL of water, 10 to 20 g of pulverized natural product, reflux time of 1 to 2 hours, and alcohol or water with 1 to 4 carbon atoms.

The leaching method is carried out at 15 to 30°C for 24 to 72 hours, and water or an alcohol with 1 to 6 carbon atoms is used as an extraction solvent. More specifically, the extraction is performed at 20 to 25°C for 30 to 54 hours, and the extraction solvent is water or an alcohol having 1 to 6 carbon atoms.

The ultrasonic extraction method proceeds at 30 to 50°C for 0.5 to 2.5 hours, and the extraction solvent is water or an alcohol with 1 to 6 carbon atoms. Specifically, extraction is performed at 40 to 50°C for 1 to 2.5 hours, and the extraction solvent is water or an alcohol with 1 to 6 carbon atoms.

The extraction solvent can be used in an amount of 2 to 50 times, more specifically, 2 to 20 times the weight of the sample. For extraction, the sample may be left for 1 to 72 hours, more specifically, 24 to 48 hours for leaching in the extraction solvent.

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

After obtaining the extract or fraction, additional methods such as concentration or freeze-drying can be used.

Preferably, the anti-inflammatory and antioxidant composition of the present invention further includes Carex nubigena var. albata extract, Primula jesoana var. pubescens extract, and Artemisia sylvatica Maxim. extract.

When the extract is further included, the unique seaweed flavor of the stone agar extract can be neutralized to provide a highly palatable food composition, and the excellent anti-inflammatory and antioxidant effects of the stone agar extract can be increased.

The above-mentioned sedge (Carex nubigena var. albata) is a perennial herb of the monocot family Cyperaceae and grows in mountains and fields. The rhizome is short, and the stem is thin, long, triangular, clustered, and stands upright. The height is about 30cm. The leaves are clustered, line-shaped, and have sharp ends. The leaves are shorter or longer than the stem and are about 3mm wide. Flowers bloom from April to June. The flower spike stands up straight at the end of the stem, has a cylindrical shape, and is about 25-40 mm long. There are short, rope-shaped bracts at the base, and the spikelets are bisexual and about 5 mm long. The female flower inflorescence is egg-shaped, has a pointed tip, and is dark brown. The fruit sac is egg-shaped, 4-5 mm long, longer than the inflorescence, and has two styles. It is distributed in Jeollabuk-do, Gyeonggi-do, South Pyongan Province, North Pyongan Province, South Hamgyong Province, and North Hamgyong Province.

The hairy primrose (Primula jesoana var. pubescens) is a perennial of the primrose family of the primrose order of the dicotyledonous plant group. Compared to the primrose, the petiole and flower stem have more long hairs, and the rhizome extends sideways without the main stem. The flowers are whole, reddish purple flowers with a diameter of 1.5 to 2.5 cm that bloom in July to August. The fruit produces long oval capsules measuring 7 to 12 mm in length. Distributed in Korea, Japan, China, etc.

Artemisia sylvatica Maxim. is a perennial plant of the dicotyledonous plant Campanula Asteraceae and grows in mountainous forests. The leaves growing from the roots remain until the flowers bloom, spread out in a rose shape, are egg-shaped or long oval, and have pointed ends. It is 11 to 20 cm long and 7 to 9.5 cm wide. It has slightly curly hairs on the surface, spider web-like hairs on the back, and sharp saw teeth on the edges. The leaves growing from the stem are similar to the leaves growing from the roots, but their size is different.

More preferably, the anti-inflammatory and antioxidant composition of the present invention comprises 5 to 10 parts by weight of sedge extract, 5 to 10 parts by weight of primrose extract, and 5 to 10 parts by weight of shade extract, based on 100 parts by weight of stone agar extract. It can be included.

If the above range is used, the anti-inflammatory and antioxidant synergistic effects of the stone agar extract can be maximized, and the palatability is excellent, so it can be manufactured into a highly palatable food composition.

A pharmaceutical composition according to another embodiment of the present invention is prepared containing the antioxidant and anti-inflammatory composition 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 may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. You 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, gum acacia, alginate, gelatin, and calcium phosphate. , calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract and its fractions with at least one excipient such as starch, calcium carbonate, It is prepared by mixing sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups, and may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to the commonly used simple diluents such as water and liquid paraffin. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogenatin, etc. can be used.

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

The pharmaceutical composition of the present invention can be administered alone or in combination with a known immunotherapeutic agent. It is important to consider all of the above factors and administer the amount that will achieve the maximum effect with the minimum amount without side effects.

A food composition according to another embodiment of the present invention is prepared by including the antioxidant and anti-inflammatory composition as an active ingredient.

As a specific example, the above food composition can be used to produce processed food that can enhance antioxidant and anti-inflammatory effects. These processed foods include, for example, snacks, beverages, alcoholic beverages, fermented foods, canned foods, processed milk foods, processed meat foods, noodles, etc. Confectionery includes biscuits, pies, cakes, bread, candy, jellies, gum, cereals, etc. Beverages include drinking water, carbonated beverages, functional ionic beverages, functional ionic beverages, juices (e.g., apple, pear, grape, aloe, tangerine, peach, carrot, tomato juice, etc.), sikhye, etc. Alcoholic beverages include sake, whiskey, soju, beer, liquor, and fruit wine. Fermented foods include soy sauce, soybean paste, and red pepper paste. Canned food includes canned marine products (e.g., canned tuna, mackerel, saury, canned conch, etc.), canned livestock products (canned beef, pork, chicken, turkey, etc.), and canned agricultural products (canned corn, peaches, canned pineapple, etc.). Milk processed foods include cheese, butter, yogurt, etc. Processed meat products include pork cutlet, beef cutlet, chicken cutlet, and sausage. Includes sweet and sour pork, nuggets, neobiani, etc. Includes noodles such as raw noodles in sealed packages. In addition, the composition can be used in retort foods, soups, etc.

A cosmetic composition according to another embodiment of the present invention is prepared by including the antioxidant and anti-inflammatory composition as an active ingredient.

The cosmetic composition of the present invention can be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing agents. , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it can be manufactured in the form of nutritional cream, astringent lotion, softening lotion, lotion, essence, nutritional gel, or massage cream.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, gum tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier ingredients. It can be.

When the formulation of the present invention is a powder or spray, suction, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, fatty esters of glycerol, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar or tracant gum can be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

The cosmetic composition of the present invention further contains fatty substances, organic solvents, solubilizers, thickening and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, film formers, water, Ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any commonly used in cosmetics. It may contain adjuvants commonly used in the fields of cosmetology or dermatology, such as other ingredients. Additionally, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

The anti-inflammatory and antioxidant composition of the present invention contains stone agar extract as an active ingredient, which not only suppresses reactive oxygen species (ROS) caused by oxidative stress, but also suppresses the production of NO (nitric oxide), which causes inflammation. The purpose is to provide an anti-inflammatory and antioxidant composition that has the effect of suppressing inflammation by suppressing the production of TNF-α, IL-6, and IL-10.

Figure 1 shows the results of cell viability measurement according to treatment with stone fish extract (EECT) according to an embodiment of the present invention.
Figure 2 shows the results of measuring phagocytic activity of RAW 264.7 cells according to treatment with stone agar extract (EECT) according to an embodiment of the present invention.
Figure 3 shows the results of analysis according to whether NO and PGE ₂ production increases due to treatment of stone fish extract (EECT) according to an embodiment of the present invention.
Figure 4 shows the results of an ELISA analysis according to whether cytokine production increases due to treatment with stone agar extract (EECT) according to an embodiment of the present invention.
Figure 5 shows the results of Western blot analysis of the degree of activation of NF-kB and IkB-a according to treatment with stone agar extract (EECT) according to an embodiment of the present invention.
Figure 6 shows the results of DCF-DA analysis of ROS content according to treatment of stone agar extract (EECT) according to an embodiment of the present invention.

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

[Preparation Example 1: Preparation of natural extract]

1. Preparation of stone fish extract

C. tenellus collected from the coast of Jeju Island, South Korea, was washed and stored at -20°C. Afterwards, it was pulverized using a grinder and dried. The dried pulverized material was sonicated at 1 hour intervals and extracted 5 times with 70% ethanol (EtoH) 1:10 W/V. The extracted stone agar extract was evaporated under vacuum and then dissolved in DMSO (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) to prepare stone agar extract (EECT).

2. Preparation of other extracts

Using the same method as the method for producing the above-mentioned stone agar extract (EECT), eucalyptus sedge extract (EECN), hairy primrose extract (EERJ), and eucalyptus extract (EEAS) were prepared.

3. Preparation of complex extract

A complex extract was prepared by mixing the prepared above-prepared sedge extract (EECT), sedge extract (EECN), hairy primrose extract (EERJ), and shade extract (EEAS) in the content range as shown in Table 1 below.

AN1 AN2 AN3 AN4 AN5 EECT 100 100 100 100 100 EECN - One 5 10 15 EERJ - One 5 10 15 EEAS - One 5 10 15

(Unit: parts by weight)

[Experimental Example 1: Evaluation of cytotoxicity of stone fish extract (EECT)]

In order to confirm the cytotoxicity of the prepared stone fish extract (EECT), the cell viability of EECT on RAW 264.7 macrophages was evaluated through MTT analysis.

cell culture

RAW 264.7 macrophages were purchased from the Korea Cell Line Bank (Korea) and cultured in DMEM (WelGENE, Inc., Daegu, Republic) containing 100 U/mL penicillin, streptomycin, and 10% fetal bovine serum. of Korea) were used to culture RAW 264.7 macrophages under conditions of 37°C and 5% CO ₂ .

Cell viability assay

Cell viability of RAW 264.7 cells added with EECT at concentrations of 0 μg/ml, 100 μg/ml, 200 μg/ml, 300 μg/ml, 400 μg/ml, and 500 μg/ml and 100 ng/ml. After treatment with LPS (Sigma-Aldrich Chemical Co.), stonefish extract (EECT) at concentrations of 0 μg/ml, 50 μg/ml, 100 μg/ml, 150 μg/ml, 200 μg/ml, and 300 μg/ml was used. Treated cell viability was evaluated.

After the above processing. The medium was removed, and MTT (5 mg/ml, Sigma-Aldrich Chemical Co.) solution was dispensed into each well and reacted at 37°C for 3 hours.

The supernatant after the reaction was removed, formazan crystals were dissolved in DMSO, and the absorbance at 540 nm was measured using ELISA (Dynatech Laboratories, Chantilly, VA, USA). Changes in macrophage morphology were captured in cell images through phase contrast microscopy (Carl Zeiss, Oberkochen, Germany).

The results are shown in Figure 1 below, and the cytotoxicity evaluation of stone fish extract (EECT) on RAW 264.7 macrophages showed no cytotoxicity at a concentration of less than 300 μg / ml, as shown in Figure 1 (A). However, it was confirmed that significant cytotoxicity was observed in cells treated with 400 μg/ml or more.

As shown in B) of Figure 1 below, EECT of stone agaric extract (EECT) on RAW 264.7 macrophages treated with 100ng/ml LPS did not show any cytotoxicity and did not have any effect on cell viability even at a concentration of less than 300 μg/ml. It was confirmed that there were no side effects.

[Experimental Example 2: Anti-inflammatory effect of stone fish extract (EECT)]

Phagocytosis assay

Phagocytosis can deactivate macrophages, suppress inflammatory responses, promote TGF-β expression and prostaglandin E ₂ production, and inhibit T cell proliferation. Under these conditions, it produces NO and IL-10, regulates chronic inflammation and immune responses, and promotes tissue regeneration. Conversely, phagocytosis of necrotic cells causes a proinflammatory response.

In order to analyze the phagocytic activity of the prepared stone agar extract (EECT), and to confirm the cytotoxicity of the prepared stone agar extract (EECT), the phagocytic activity of RAW 264.7 cells according to addition was tested using a phagocytosis assay kit (Cayman Chemical, Ann Arbor, MI, USA).

RAW 264.7 cells were pretreated for 1 hour with EECT at the concentration shown in Figure 2, and then cultured for 24 hours with or without 100ng/ml LPS treatment.

After 24 hours of treatment, latex bead-rabbit IgG-fluorescein isothiocyanate (FITC) complexes were added to each well at a dilution of 1:200. Then, it was cultured at 37°C for 2 hours.

Cells were washed twice using the assay buffer provided in the kit, washed additionally twice with phosphate-buffered saline (PBS), and measured for absorbance at 620 nm using flow cytometry (BD Biosciences, San Jose, CA, USA). was measured.

The results are shown in Figure 2 below, and the degree of phagocytosis of RAW 264.7 macrophages in response to stone agar extract (EECT) treatment is that LPS treatment stimulates phagocytosis in RAW 264.7 cells, whereas the stone agar extract of the present invention stimulates phagocytosis in RAW 264.7 cells. It was confirmed that phagocytosis was inhibited in a concentration-dependent manner by treatment with (EECT).

NO and PGE ₂ measurement of production

RAW 264.7 cells were pretreated for 1 hour with EECT at the concentration shown in Figure 3, and then cultured for 24 hours with or without 100ng/ml LPS treatment. After 24 hours of incubation, the collected culture supernatant was mixed with an equal amount of Griess reagent solution (Sigma-Aldrich Chemical Co.) and incubated at room temperature (RT) for 10 minutes.

NO concentration was measured with an ELISA reader at 540 nm, referring to a standard curve generated with known sodium nitrate (NaNO ₂ ) concentration. Culture supernatants were collected from each group, and the corresponding commercial enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical, Ann Arbor, MI, USA) was used according to the vendor's instructions, and NO, PGE ₂ and iNOS, COX The levels of cytokines including -2 and Actin were quantified.

The results are shown in Figure 3 below, and as shown in Figure 3 A) and B), LPS treatment significantly increased the expression of NO and PGE ₂ compared to the untreated control group, but the stone agar extract ( It was confirmed that following EECT) treatment, the expression of NO and PGE ₂ was suppressed.

The results are shown in Figure 3 below, and as shown in Figure 3C), it was confirmed that EECT extract inhibits the expression of iNOS and COX-2 by LPS.

Measurement of inflammatory cytokine production

To confirm the effect of EECT extract on the production and expression of inflammatory site kines, including TNF-α, IL-6, and IL-10, protein isolation and Western analysis were performed.

First, the cultured RAW 264.7 cells were washed with phosphate-buffered saline (PBS) and lysed with lysis buffer. Nuclear and cytoplasmic proteins were isolated using a nuclear extraction kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's procedures, and protein concentrations in lysates were determined using Bradford assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). It was measured using .

The same amount of protein was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, MA, USA). The PVDF membrane to which the protein was delivered was blocked with blocking buffer (containing 5% non-fat milk) for 1 hour at room temperature (RT), and then measured at 4°C overnight.

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

The results are shown in Figure 4 below, and as shown in Figure 4, EECT extract increases TNF-α (Figure 4A) and IL-6 (Figure 4B) in LPS-treated RAW 264.7 cells. and IL-10 (Figure 4 C).

Ability to inhibit NF-κB signaling pathway activity

RAW 264.7 cells were inoculated into a 4-well culture plate and stabilized for 24 hours. Afterwards, RAW 264.7 cells were treated with EECT at a concentration of 300 μg/ml, then treated with 100 ng/ml LPS for 1 hour or untreated, fixed with methanol for 10 minutes, and washed with PBS.

Afterwards, cells were blocked for 1 hour using 5% fetal bovine serum albumin (BSA Sigma-Aldrich Chemical Co.) with PBS-T (PBS containing 0.1% Triton Afterwards, cells were washed with PBS-T and incubated with secondary antibody (Alexa Fluor 594, Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour.

Afterwards, cells were washed with PBS, counterstained with 4', 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich Chemical Co.) for 20 minutes, and cell fluorescence was observed under a fluorescence microscope (Carl Zeiss). .

The results are shown in Figure 5 below. As shown in Figure 5 below, when RAW 264.7 cells were stimulated with LPS, the expression of NF-κB was confirmed to be significantly increased compared to the control group. In contrast, in the cytoplasm, It was confirmed that NF-κB was activated.

However, when treated with stone agaric extract (EECT), it reduced the nuclear accumulation of NF-κB p65 and the degradation of IκB-α induced by LPS, and reduced the level of NF-κB p65 observed in the nucleus of LPS-treated cells. It was confirmed that it also decreased significantly.

[Experimental Example 3: Antioxidant effect of stone fish extract (EECT)]

Reactive oxygen species (ROS) measurement

Reactive oxygen species (ROS) were measured using 5,6-carboxy-2', 7'-dichlorofluorescein diacetate (DCF-DA, Sigma-Aldrich Chemical Co.), and RAW 264.7 cells were incubated with various concentrations of stonefish extract ( EECT) for 1 hour, then treated with or without 100 ng/ml LPS for 1 hour and stained with 10 μM DCF-DA for 15 minutes at 37°C in the dark.

Afterwards, cells were washed with PBS and analyzed using flow cytometry (BD Biosciences, San Jose, CA, USA). To compare the level of ROS production, cells were stained with DCF-DA for 15 minutes at 37°C. did. After staining, the cells were fixed with paraformaldehyde solution (4%, pH 7.4) for 20 minutes, washed with PBS, and analyzed for ROS fluorescence intensity using a fluorescence microscope.

The results are shown in Figure 6 below, and it was confirmed that EECT extract inhibits oxidative stress in LPS-treated RAW 264.7 cells.

As shown in Figures 6 A) and B), the ROS content increased upon LPS stimulation, but it was confirmed that the ROS content decreased in a concentration-dependent manner upon treatment with EECT.

In addition, as shown in Figure 6C) below, it was confirmed that DCF-DA fluorescence intensity was weakened upon treatment with EECT.

[Experimental Example 4: Effect of complex extract]

The effects of the complex extracts AN2 to AN5 in Table 1 were compared and shown in the same manner as the experimental method for the EECT extract shown in Experimental Examples 1 to 3 above.

Similar to the cytotoxicity of EECT) conducted in Experimental Example 1, cell viability was tested at concentrations of EECN, EERJ, and EEAS extract of less than 300 μg/ml. It was confirmed that no side effects were observed, and the results of testing the anti-inflammatory effect in Experimental Example 2 and the antioxidant effect in Experimental Example 3 are comprehensively shown in Table 2 below.

For objective comparison, the effect of AN1 (stonefish extract, EECT) was expressed as an index of 5, and the higher the index number, the better.

The evaluation indices below are converted to indices of 1 to 10.

AN1 AN2 AN3 AN4 AN5 anti-inflammatory effect 5 6 8 9 7 Antioxidative effect 5 5 8 8 7

(Unit: index)

As shown in Table 2, excellent macrophages in the case of AN2 to AN5, which further include sedge extract (EECT), sedge extract (EECN), hairy primrose extract (EERJ), and shade extract (EEAS). Excellent expression inhibitory activity of phagocytic activity, nitric oxide (NO), PGE ₂ , tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) It was confirmed that the anti-inflammatory effect was excellent.

In addition, it was confirmed that the activity of inhibiting the activity of reactive oxygen species (ROS) was excellent, and that not only the anti-inflammatory effect but also the antioxidant effect was excellent.

In particular, in the case of AN3 and AN4, which contain preferred parts by weight of eucalyptus sedge extract (EECN), hairy primrose extract (EERJ) and shade extract (EEAS) in the ethanol extract of eucalyptus (EECT), It was confirmed that it exhibits better anti-inflammatory and antioxidant effects compared to the case containing only ).

[Experimental Example 5: Evaluation of palatability]

After AN1 to AM5 prepared in Preparation Example 1 were manufactured into tea, 40 adult men and women in their 20s to 50s were asked to taste the prepared tea. The overall preference was shown in Table 2 below to evaluate the taste and aroma after tasting.

The evaluation score was requested to be 1 to 10 points for each item, and the evaluation results were converted to an average score (rounded). The higher the number, the higher the preference for the index.

AN1 AN2 AN3 AN4 AN5 taste 3 4 6 7 5 incense 3 5 7 7 5 Comprehensive preference (average) 3 4 6 7 5

(Unit: index)

Referring to Table 3, AN2 to AN5, which further contain Echinacea sedge extract (EECN), Primrose extract (EERJ), and Elephant sedge extract (EEAS), are superior to AN1, which contains only EECT. It was confirmed that it shows overall preference, which means that the unique seaweed flavor of EECT can be neutralized and provided as a food composition with high overall flavor and taste preference while maintaining excellent anti-inflammatory and antioxidant effects.

Although the 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 made by those skilled in the art using the basic concept of the present invention defined in the following claims are also possible. falls within the scope of rights.

Claims (7)

Contains Chondracanthus tenellus (Harvey) Hommersand) extract, sedge sedge extract, hairy primrose extract, and shade extract as active ingredients,
Inhibition of cyclooxygenase-2 (COX-2) activity, nitric oxide (NO), PGE2, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) or interleukin-10 It exhibits anti-inflammatory effects by inhibiting the expression of (IL-10).
Anti-inflammatory composition. delete delete According to clause 1,
The extract is extracted with a solvent selected from the group consisting of water, C ₁ to C ₆ alcohols, and mixtures thereof.
Anti-inflammatory composition. delete Containing the anti-inflammatory composition according to paragraph 1
Anti-inflammatory food composition. Containing the anti-inflammatory composition according to paragraph 1
Anti-inflammatory cosmetic composition.