KR20200084215A - Anti-inflammatory composition comprising extract of Polyopes affinis - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing a Polyopes affinis extract exhibiting an anti-inflammation effect and anti-oxidant activity as an active component. The present invention can be provided as a food, cosmetic, or pharmaceutical composition for use in alleviating inflammatory diseases, relieving inflammatory skin irritation, etc.

Description

A composition for anti-inflammatory comprising tuna black meat extract as an active ingredient. {Anti-inflammatory composition comprising extract of Polyopes affinis}

The present invention relates to an anti-inflammatory composition comprising an extract of kamjamaksal as an active ingredient, more specifically, to inhibit the expression and antioxidant activity of inflammatory cytokines, nitric oxide (NO) and COX-2, which are substances that mediate inflammation. The present invention relates to an anti-inflammatory composition comprising the extract of kamjamaksal.

Inflammation is a local biological defense response that appears in response to physical trauma, harmful chemicals, infections with bacteria, fungi, viruses, or pathological conditions caused by irritating substances in metabolites in vivo. Inflammation is triggered by a variety of inflammatory mediators produced from damaged tissue and moving cells. During an inflammatory reaction, plasma accumulates in the inflammatory site, diluting the toxicity secreted by bacteria, increasing blood flow, and accompanying symptoms such as erythema, pain, edema, and fever. In the normal case, the living body neutralizes or eliminates the onset factors through the inflammatory reaction and regenerates the upper tissue to restore normal structure and function, but in other cases, it progresses to a disease state such as chronic inflammation.

Recently, due to the development of molecular biology, many studies have been conducted on the inflammatory response at the molecular level, and various biochemical phenomena are involved in the inflammatory reaction. In particular, macrophage (Macrophage) is caused by chemical stimulation, etc. It is known that it produces an inflammatory cytokine and plays an important role in the inflammatory reaction (Ito T., etal, Curr Drug Traget Inflamm Allergy, 2(3);257-265, 2003), nitric oxide is a nitric oxide synthase (nitricoxide It is synthesized from L-arginine by the action of synthase (NOS). NOS has several isoforms. B/NOS (brain NOS) present in the brain, nNOS (neuronal NOS) present in the nervous system, and endothelial NOS (eNOS) present in the vascular endothelial system are always expressed at a certain level in the body, and small amounts are generated by them Nitrogen monoxide (NO) plays an important role in maintaining homeostasis by performing various physiological reactions related to blood pressure regulation, neurotransmission, learning, and memory. On the other hand, iNOS (induced NOS), whose expression is induced by a certain stimulus, generates excessive NO, and nitric oxide (NO), which is excessively generated by iNOS, reacts with superoxide to cause peroxynitrite. To form. It acts as a powerful oxidizing agent and damages cells, thereby engaging in various pathological processes including inflammation and cancer (Gupta SC et al., Exp Biol Med., 236;658-671, 2011; Riehemann etal, FEBS Lett, 442;89-94, 1999; Stamler et al., Science, 358;1898-1902, 1992).

Meanwhile, cyclooxygenase (COX) has the function of COX and hydroperoxidase (HOX) activity to synthesize intermediates PGG₂ and PGG₂ from arachidonic acid, and these compounds include PGE₂, PGF₂, PGD₂, Pro Stacyclin and thromboxane A2 (TxA2) are produced, and there are two isoforms in COX. While COX-1 is always expressed in most tissues to synthesize prostaglandins (PGs) necessary for cell protection, COX-2 is rapidly inducing its expression during an inflammatory reaction, and is important for causing an inflammatory reaction by generating PGE₂. Plays a role (Weisz A., Biochem. J., 316:209-215, 1996; (Miller MJ et al., Mediators of inflammation, 4:387-396, 1995: Appleton L. et al., Adv. Pharmacol., 35:27-28, 1996).

PGE₂ is best known as an agent that mediates inflammation, and is involved in tissue damage by inducing the production of matrix metalloproteinases (MMPs) in inflammatory diseases such as periodontitis. In macrophages obtained from patients with rheumatoid arthritis, a large amount of PGE₂ is produced and generated PGE₂ plays an important role in the mechanism of inflammatory response and tissue destruction in rheumatoid arthritis.

PGE₂ not only acts as a mediator of the well-known inflammatory response, but also promotes the Th2 type immune response and suppresses the Th1 type immune response. In macrophages, TNF-α, IL-1β, IL-8, IL-12, etc. The role of a modulator of the immune response in inhibiting the production of inflammatory cytokines and promoting the production of anti-inflammatory cytokines such as IL-10 has been found in many studies in recent years.

Expression of iNOS and COX-2, which induce overproduction of NO and PGs, which serve as a mediator of this inflammatory response, is regulated by the nuclear transcription factor NF-κB, which is a Rel gene family. As a nuclear protein of, in the cytoplasm, it is bound to I-κB and exists in an inactive form, but when I-κB kinase is activated by some factor, I-κB is released through phosphorylation to activate it. The activated NF-κB moves to the nucleus and induces gene expression of iNOS and COX-2 that induce an inflammatory response (Oh, G. T. et al., Atherosclerosis, 159(1):17-26, 2001).

Accordingly, the present inventors activate the transcription factor NF-κB, and by using bacterial endotoxin LPS (lipopolysaccharide), which can induce the expression of iNOS and COX-2, NO, inflammatory cytokines, PGE₂, and other inflammation control substances The study was conducted by secreting.

The results of these studies are related to inhibiting NO production or suppressing the production of inflammatory cytokines such as TNF-α and IL-6. As a drug that suppresses the expression of iNOS or COX-2, it has the potential to be an effective anti-inflammatory agent. .

It is known that non-steroidal anti-inflammatory drugs (NSAIDS), which are currently widely used as anti-inflammatory agents, cause serious side effects such as gastrointestinal disorders, liver disorders and renal impairment.

Therefore, it can be said that there is still a need for the development of new drugs with anti-inflammatory activity and low side effects and lasting effects.

On the other hand, seaweed has historically been widely used by humans for food or industrial purposes for a long time. In Asia, food ingredients have only been used for a long time, while in the West, it has been used to manufacture valuable chemical materials, and research has been conducted. Aquatic biological resources are rich in seaweeds that contain slightly different physiologically active substances from terrestrial organisms, so it can be said to be a report of bioactive substances. It is known that seaweed contains various active ingredients such as polyphenol and polysaccharide components, functional peptides, various antioxidants and essential minerals.

However, the antioxidant activity of seaweeds to date has been limited to edible seaweeds, such as seaweed, greens, capsicum, kelp, and seaweed, and studies on the antioxidant activity of various seaweeds have not been deepened. Among them, red algae, such as the true purple radish (Symphyocladia latiuscula), Chindurus ocellatus, Carpopeltis affinis, and Polyopes affinis, contain a large amount of various amino acids, minerals, phenolic compounds, and flavonoids. It has antioxidant activity and nitrite scavenging ability.

Active oxygen species (superoxide anion, hydrogen peroxide (H2O2), hydroxyl radical (OH), etc.) generated by oxidative stress in the human body are produced during normal metabolic processes of mitochondria, phagocytes, or cytoplasm. It plays an important role (Aruoma and Cuppette 1997; Cavas and Yurdakoc 2005). However, overexpressed free radicals damage cell membranes, proteins, DNA and enzymes in vivo, and cause harmful reactions to cells and tissues, causing diseases such as cancer, stroke, diabetes and arteriosclerosis. In addition, reactive oxygen species are known to be closely related to aging.

Therefore, in the functional food and pharmaceutical industry, synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and propyl gallate (PG), which are inexpensive and have high antioxidant activity, have been mainly used to remove free radicals. These synthetic antioxidants show intracellular toxicity or cause side effects such as cancer in animal models, and thus it is necessary to develop safer and more effective antioxidants than synthetic antioxidants.

(Patent Document 1) KR 10-2018-0064635 A1

It is an object of the present invention to provide an anti-inflammatory composition comprising an extract of poly black pepper (Polyopes affinis) as an active ingredient.

Another object of the present invention is to provide an anti-inflammatory composition comprising an extract of polyopes affinis, which can exhibit excellent anti-inflammatory and antioxidant activity, by inhibiting the expression of a substance that mediates inflammation.

Another object of the present invention is to provide a food composition comprising an extract of tuna black pepper showing an anti-inflammatory effect and antioxidant activity as an active ingredient.

Another object of the present invention is to provide a cosmetic composition comprising an extract of tuna black pepper showing anti-inflammatory effect and antioxidant activity as an active ingredient.

Still another object of the present invention is to provide a pharmaceutical composition comprising an extract of black tuna with anti-inflammatory effect and antioxidant activity as an active ingredient.

In order to achieve the above object, the anti-inflammatory composition according to an embodiment of the present invention includes a black pepper (Polyopes affinis) extract as an active ingredient.

The anti-inflammatory composition comprises inhibiting the expression of inflammatory cytokines (Cytokine), nitric oxide (NO) and COX-2, which are substances that mediate inflammation.

The anti-inflammatory composition includes those having antioxidant activity through free radical scavenging activity.

The polychaeps (Polyopes affinis) extract is extracted using an extraction solvent selected from the group consisting of water, C₁ to C ₅ alcohol, C₁ to C ₅ alcohol aqueous solution and mixtures thereof.

Food composition according to another embodiment of the present invention includes the composition.

The cosmetic composition according to another embodiment of the present invention includes the composition.

The pharmaceutical composition according to another embodiment of the present invention includes the composition.

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

The anti-inflammatory composition according to an embodiment of the present invention includes an extract of Polyopes affinis as an active ingredient.

As used herein, "active ingredient" refers to a component that exhibits the desired activity alone or can itself exhibit activity with an inactive carrier.

In addition, "anti-inflammatory" in the present specification is meant to include the improvement (reduction of symptoms) of inflammatory diseases as defined below, and the inhibition or delay of the development of such diseases.

In addition, in the present specification, the "inflammatory disease" refers to an inflammatory reaction specified by a local or systemic bio-defense response to an external physiological stimulus or an infection or autoimmunity of an external infectious agent such as bacteria, fungi, viruses, and various allergens. It can be defined as the pathological symptom it causes. These inflammatory reactions include various inflammatory mediators and activation of enzymes related to immune cells (iNOS, COX-2), secretion of inflammatory mediators (secretion of NO, TNF-α, IL-6, etc.), infiltrating fluids, cell migration, tissue It involves a series of complex physiological reactions, such as destruction, and is externally manifested by symptoms such as erythema, pain, swelling, fever, and a decrease or loss of certain functions in the body.

The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so as long as any disease is included in the definition of such inflammatory disease, it is acute, chronic, ulcerative, allergic, or Necrotic or not.

The polychaes affinis (Polyopes affinis) is distributed in the west coast of the North Pacific, and is a seaweed belonging to the genus family of the red algae plant. And wider, with a slightly swollen tip. In addition, the stem has a thickened form as the cortex thickens, and the cortex is made of small oral-shaped cells lined up vertically on the surface of the plant, and is made of cells that are slightly serpentine, slightly growing in the intertidal zone. It is known.

In addition, the red algae plant (Polyopes affinis) is a substance that has various physiological activities such as anti-cancer, anti-inflammatory, immune-enhancing, and anti-viral, and is constantly being researched to develop new pharmaceuticals, cosmetics, and functional foods. As a type of seaweed, it contains a large amount of polysaccharides, vitamins, functional peptides, various antioxidants, and essential minerals.

The anti-inflammatory composition inhibits the expression of inflammatory cytokines (Cytokine), nitric oxide (NO) and COX-2, which are substances that mediate inflammation.

IL-6, TNF-α), PGE₂, COX-2 및 iNOS의 생성을 억제하여, 항염증 효과를 나타낼 수 있다.More specifically, polyopes affinis extract contains nitrogen oxide (NO), inflammatory cytokines (IL-1)

IL-6, TNF-α), PGE₂, COX-2 and iNOS can be suppressed to produce anti-inflammatory effects.

In general, in the inflammatory response, LPS (lipopolysaccharide) is an intracellular toxin derived from the cell wall of Gram-negative bacteria and activates transcription factors NF-κB by binding with toll-like receptor 4 (TLR4), iNOS and COX-2 Induces the expression of NO, inflammatory cytokines and PGE₂ secretion of various inflammation control substances.

, IL-6, TNF-α), PGE₂, COX-2 및 iNOS의 생성을 억제하는 것으로 항염증제의 용도로 사용될 수 있다.During this process, polyopes affinis extract is concentration-dependently NO, inflammatory (IL-1

, IL-6, TNF-α), PGE₂, COX-2 and iNOS to inhibit the production of anti-inflammatory agents.

The PGE₂ is one of the inflammatory mediators, which is synthesized by enzymatic action of phospholipase A2. It begins with the production of arachidonic acid from membrane phospholipid.

The arachidonic acid becomes prostaglandin G2 by enzymatic action and again becomes prostaglandin H2, an unstable metabolite.

These two processes are promoted by cyclooxygenase (COX), and there are two or more isoenzymes of COX, of which COX-1 is continuously expressed to platelet aggregation, gastric mucosa protection, and renal function regulation. It is responsible for the physiological functions of the back and COX-2 is expressed by irritation such as inflammation.

PGH₂ produced by COX is an unstable intermediate metabolite and is metabolized to PGE₂, PGD₂, PGI₂, PGF₂, thromboxane A2 (TXA₂) by various prostanoid synthases depending on cell types or stimuli.

In addition, PGE synthase (PGES), which is involved in the metabolism of PGH₂ to PGE₂, has three known isoforms: cytosolic PGES (cPGES), microsomal PGES-1 (mPGES-1), and mPGES-2. Among mPGES-1, expression is increased by inflammatory stimuli such as lipopolysaccharid (LPS), inflammatory cytokines such as IL-1β and TNF-α, and nitric oxide (NO), functionally close to COX-2 And is involved in the production of PGE₂.

That is, the production of PGE₂ is functionally closely related to COX-2 and plays an important role in the inflammatory reaction.

Accordingly, according to the present invention, the use of polyopes affinis extract as an active ingredient suppresses the production of COX-2 and iNOS, and the expression of inflammatory cytokines (Cytokine), nitric oxide (NO) and COX-2 By inhibiting, it may exhibit excellent anti-inflammatory effects.

The anti-inflammatory composition includes those having antioxidant activity through free radical scavenging activity.

More specifically, the black pepper (Polyopes affinis) extract contains a large amount of polyphenols exhibiting antioxidant activity, and thus exhibits ABTS radical scavenging activity and DPPH radical scavenging activity and thus can be used as natural antioxidants with high antioxidant activity.

The ABTS radical scavenging ability is a method of measuring the antioxidant activity of the extract by expressing the characteristic blue-green color of the ABTS radical in the plasma by the antioxidant component, which is expressed as an absorbance value. Antioxidant activity of natural products donates electrons to active radicals and inhibits fat oxidation in food, and plays a role in inhibiting aging by free radicals in the human body, and radical scavenging action prevents human diseases and aging Plays a very important role.

Accordingly, in the present invention, after the formation of radicals by oxidation of potassium persulfate and ABTS in a method based on the principle that the absorbance of cationic radicals of ABTS is reduced by antioxidant active substances, the radical scavenging activity of each sample is measured. The antioxidant capacity of the sample was measured.

The DPPH radical is reduced by antioxidant components such as vitamin C and tocopherol, and is used to measure the electron donating ability of antioxidants by discoloring dark purple. The electron donating ability is a measure of inhibiting oxidation by donating oxidizing active free radical electrons involved in the chain reaction of lipid peroxidation.

The polychaeps (Polyopes affinis) extract is extracted using an extraction solvent selected from the group consisting of water, C₁ to C ₅ alcohol, C₁ to C ₅ alcohol aqueous solution and mixtures thereof.

The extraction solvent is water, C₁ to C ₅ alcohols (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF) , Dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixture obtained by leaching using a solvent, extract obtained using a supercritical extraction solvent such as carbon dioxide, pentane, or fractions of the extract It means the obtained fraction, and the extraction method may be any method such as cold immersion, reflux, warming, ultrasonic radiation, and supercritical extraction in consideration of the polarity, extraction degree, and storage degree of the active material.

In the case of fractionated extracts, the fractions obtained by suspending the extract in a specific solvent and mixing and policing with a solvent having a different polarity, adsorb the crude extract on a column filled with silica gel, etc., and then add a hydrophobic solvent, a hydrophilic solvent or a mixed solvent thereof. It means that it contains the fraction obtained as a mobile phase.

In addition, the meaning of the extract includes a concentrated liquid extract or a solid extract in which the extraction solvent is removed by lyophilization, vacuum drying, hot air drying, spray drying, or the like. Preferably, it means an extract obtained using water, ethanol or a mixed solvent thereof as an extraction solvent, and more preferably an extract obtained using ethanol as an extraction solvent.

Although it was confirmed that it exhibits excellent anti-inflammatory and antioxidant effects even when using the tuna extract alone, in the present invention, as it is used as a composite extract mixed with a natural extract in the tuna extract, more excellent anti-inflammatory and antioxidant properties I want to show the effect.

The natural extract may further include an extract selected from the group consisting of Soksagae extract, Eumjigye extract, Sambaekcho extract and mixtures thereof.

The Equisetum hyemale L. is an evergreen perennial plant that grows in the valley wetlands 30-60 cm in height, looks like a stem, has no branches, and has 10-18 ridges between nodes. The degenerated scale-like leaves are attached to each other to completely surround the node, forming a leaf sheath, serrated at the end and alternately running with each ridge. The sporangium runs at the end of the main stem, is conical, and has a pointed tip.

The Eumjigye is a perennial herb that grows in sunny forests or grasses with sceptridium ternatum (Thunb.) Lyon. The thick fleshy roots spread all over the place. The leaf is divided into 2, and it is composed of a nutritive lobe and a true lobe, and the lobe has long petiole, split into 3, and has serrate on the edge. The spore lobe is longer than the broad lobe and the upper part is split long, and the spore sac hangs on the branch.

The three hundred seconds (Saururus chinensis (Lour.) Baill.) is a perennial plant that grows in wetlands and is 50 to 100 cm high and has a white root. The leaves are coarse, pointed at the end, the bottom is heart-shaped, and the edges are flat. Flowers are benign, bloom white in June-August, and inflorescences cope with leaves. Fruits are round and consist of 3 to 5 threads, with 1 seed in each thread.

Preferably, the anti-inflammatory composition includes a black yam extract, soksaek extract, Eumjigye extract and Sambaekcho extract. Compared to the case of using kamaksal extract alone, when it is composed of a natural complex extract, it suppresses the production of COX-2 and iNOS, and the inflammatory cytokines (Cytokine), nitric oxide (NO) and COX-2 By inhibiting expression, it is possible to exhibit a better anti-inflammatory effect.

More preferably, the anti-inflammatory composition may include 40 to 80 parts by weight of Soksaek extract, 40 to 80 parts by weight of Eumjigye extract, and 40 to 80 parts by weight of Sambaekcho extract, based on 100 parts by weight of the tuna extract. When used within the above content range, it can exhibit a synergistic effect of the anti-inflammatory effect according to the mixed use between each component. Food composition according to another embodiment of the present invention includes the composition.

The food composition may be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, ionic beverages, processed oils such as milk, yogurt, gums, rice cakes, Chinese medicine, breads, confectionery, cotton, and the like. Tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, etc. can be prepared as health functional food preparations.

In addition, any classification of products may be taken as long as it meets the applicable laws and regulations at the time of manufacture and distribution. For example, it is a health functional food in accordance with the Korea “Act on Health Functional Food”, or a food fair (Notification of the Ministry of Food and Drug Safety, Standards and Standards of Food) in the Korean “Food Sanitation Act”, depending on each food type, confectionery, soybean, tea, beverage, It may be a special purpose food.

The food composition may include food additives in addition to the active ingredient of polyopes affinis extract.

Food additives can be generally understood as substances added to foods to be mixed or infiltrated in manufacturing, processing, or preserving foods, and their safety must be ensured because they are consumed daily and for a long time with foods. Food additives in accordance with national laws governing the manufacture and distribution of food ("Food Sanitation Act" in Korea) are limited in terms of ingredients or functions in which food additives with guaranteed safety are limited. In the “Ministry of Food and Drugs Standards and Standards”), the food additives are classified in terms of chemical chemical products, natural additives, and mixed preparations. These food additives are sweeteners, flavors, preservatives, and emulsifiers in terms of function. , Acidulant, thickener, etc.

Sweeteners are used to impart moderate sweetness to food, and both natural and synthetic can be used in the composition of the present invention. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar syrup such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to enhance taste or aroma, and both natural and synthetic ones can be used. Preferably, it is the case of using a natural thing. In addition to flavor, when using natural ones, the purpose of enhancing nutrition can also be combined. As a natural flavoring agent, it may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, or the like, or may be obtained from green tea leaves, perilla, large leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo can be used. Natural flavors may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

As a preservative, sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin and the like, and as the acidulant, arithmetic, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid and the like can be used. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms in addition to the purpose of enhancing taste.

As a thickener, a suspending agent, sedimentation agent, gel forming agent, swelling agent, etc. may be used.

The cosmetic composition according to another embodiment of the present invention includes the composition.

When the anti-inflammatory composition is identified as a cosmetic composition, its use can be understood as relief of inflammatory skin irritation, and is a stable component that is commonly used in cosmetic compositions other than the active ingredient of polyopes affinis extract. It may include conventional adjuvants and carriers such as agents, solubilizers, surfactants, vitamins, pigments and flavors.

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, and solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, It can be formulated as an emulsion foundation, wax foundation and spray.

The pharmaceutical composition according to another embodiment of the present invention includes the composition.

The pharmaceutical composition may be prepared as an oral dosage form or a parenteral dosage form according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier, in addition to the active ingredient of the black pepper (Polyopes affinis) extract. . Here, "pharmaceutically acceptable" means that the target of application (prescription) has no toxicity beyond adaptability without inhibiting the activity of the active ingredient.

In addition, when the pharmaceutical composition is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc., according to methods known in the art with suitable carriers It can be prepared as a formulation. At this time, examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, starch such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethyl cellulose, Celluloses such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable And the like. In the case of formulation, if necessary, it can be formulated by including diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like.

When prepared in parenteral formulations, they can be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories according to methods known in the art with suitable carriers. Suitable carriers for formulation with injectables include sterile water, ethanol, polyols such as glycerol or propylene glycol or mixtures thereof, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine or sterilized for injection Isotonic solutions such as water and 5% dextrose can be used. When formulated as a transdermal dosage form, it may be formulated in the form of ointments, creams, lotions, gels, external solutions, pasta agents, linen agents, aerosols, and the like. In the case of nasal inhalants, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide can be formulated in the form of an aerosol spray using a suitable propellant, and when formulated as a suppository, Witthesol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters and the like can be used.

When the natural extract of the present invention is intended to be provided as a food composition, a cosmetic composition, and a pharmaceutical composition, the food composition and the pharmaceutical composition should consider taste and aroma, and even in the case of a cosmetic composition, the aroma has an important effect on preference. will be.

Thus, as described above, the anti-inflammatory composition of the present invention includes a black tuna extract, sesame seed extract, Eumji cigarette extract and Sambaekcho extract, but when the natural extract is composed in the form of a complex extract as described above, it is unique. There is a problem that the preference is lowered due to the aroma and taste of the.

In order to prevent such a problem, a sweetener, a flavoring agent, etc. may be used as described above, but when using such a chemical component, there is a problem that side effects may occur in some cases.

In order to prevent such a problem, the second natural extract is intended to be mixed to alleviate the intrinsic flavor and taste of the natural extract to enable the provision of the food composition, pharmaceutical composition, and cosmetic composition with high preference.

The second natural extract, in order to prevent such a problem, more preferably in the present invention, in order to improve palatability, may further include a widower flower (Chloranthus japonicus Siebold) extract and a Nahariaceae (Siraitia grosvenorii) extract.

Preferably, the composition is based on 100 parts by weight of the black seaweed extract, 40 to 80 parts by weight of the Soksaek extract, 40 to 80 parts by weight of the extract from Eumji, 40 to 80 parts by weight of the extract of Sambaekcho, 10 to 30 parts by weight of the wisteria flower extract, and Nahanwa extract 10 to 30 parts by weight may be included. The wisteria flower extract and Nahanwa extract are included in a relatively small amount, thereby alleviating the taste and aroma of the natural extract, thereby providing a highly palatable composition, as well as enhancing the anti-inflammatory and antioxidant effects.

As it further includes the wisteria flower extract and the Nahanwa extract, compared to the case of using only the kamjaksal extract, soksaek extract, umji cigarette extract, and sambaekcho extract, the anti-inflammatory effect is high and the functional food composition excellent in palatability, drug It makes possible to provide to a pharmaceutical composition and a cosmetic composition.

The anti-inflammatory composition of the present invention uses an extract of Polyopes affinis, which is a natural material, and exhibits antioxidant activity while exhibiting excellent anti-inflammatory effects.

In addition, using the extract of the black pepper (Polyopes affinis) of the present invention may be provided as a food composition, a cosmetic composition and a pharmaceutical composition for the purpose of improving inflammatory diseases, alleviating inflammatory skin irritation, and the like.

1 is a result of the cytotoxicity of the ethanol extract of black pepper (Polyopes affinis) according to an embodiment of the present invention.
Figure 2 is the experimental results for the inhibition of nitric oxide (NO) production of the ethanol extract of black pepper (Polyopes affinis) according to an embodiment of the present invention.
Figure 3 is the experimental results for the inhibition of PGE production of ethanol extract of kamakmaksal according to an embodiment of the present invention.
Figure 4 is the experimental results for the inhibition of TNF-α production of ethanol extract of kamakmaksal according to an embodiment of the present invention.
Figure 5 is a graph of the results for IL-1β production of ethanol extract of kamakmaksal according to an embodiment of the present invention.
6 is a result of Western blot analysis on the reduction of expression of cytokines according to the treatment of ethanol extract of blackcurrant 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 to which the present invention pertains can easily practice. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein.

1. Preparation of tuna black meat extract

Polyopes affinis samples were collected from Jeju Island, and evidence samples were purchased from Jeju Bio-Resource Extract Bank.

In order to remove salt and other impurities from the Polyopes affinis sample, it was washed with water and freeze-dried.

The freeze-dried blackcurrant was added to 70% ethanol, extracted at room temperature for 24 hours, and concentrated under pressure with a rotary concentrator to obtain an ethanol-soluble component.

2. Preparation of natural extracts

Using the same method as the preparation of the kamjamaksal extract, Soksaek extract, Eumjigyeok extract, Sambaekcho extract, wisteria flower extract and Nahanwa extract were prepared.

3. Preparation of complex extracts

The yam extract (YA), Soksaek extract (AR), Eumjigye extract (HJ), Sangbaekcho extract (HH), wisteria flower extract (JC) and Nahanwa extract (HB) in the composition ratio shown in Table 1 below. Mixed to prepare a complex extract.

WP1 WP2 WP3 WP4 WP5 WP6 YA 100 100 100 100 100 100 AR - 30 40 60 80 100 HJ - 30 40 60 80 100 HH - 30 40 60 80 100 JC - 5 10 20 30 40 HB - 5 10 20 30 40

(Unit parts by weight) [Experimental Example 1: Anti-inflammatory activity test]

Cell and reagent

The mouse macrophage cell line RAW 264.7 cells were pre-sold from American type culture collections (ATCC), and using Dulbecco's Modified Eagle's Medium (DMEM, WELGENE), medium containing 10% Fetal bovine serum (FBS, WELGENE) at 37°C, 5 % CO ₂ conditions.

DMEM medium and FBS were purchased from Invitrogen-Gibco (Grand Island, NY), and ELISA kits for quantifying TNF-α, IL-1β, IL-6 and PGE2 were obtained from R&D system, Inc.(St. Louis, MO) and BD biosciences (San Diego, CA).

Cytotoxicity measurement

MTT analysis was performed to determine the concentration to be used in the experiment. Cell culture 6 well plate mouse macrophage cell line RAW 264.7 was dispensed and cultured for 24 hours, and then YA was treated by concentration, and the group treated with LPS (100ng/ml) treated with YA and after 1 hour, LPS (100ng/ml ) To induce an inflammatory reaction for 24 hours.

After 24 hours, all of the medium was removed, and 0.5 mg/ml MTT solution was added, followed by incubation at 37°C for 5 hours under 5% CO2 conditions to remove all of the solution. The absorbance was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader by placing DMSO (AMRESCO) in formazan (formazan) and dissolving it in 200 μl in a 96-well plate.

The survival rate for each cell was compared and analyzed by expressing the difference in absorbance to the control as a percentage.

1 is a result of confirming the cytotoxicity of YA in the mouse macrophage cell line RAW 264.7, did not show cytotoxicity to a concentration of 500 μg/ml, and similar effects were confirmed in the group treated with LPS simultaneously.

NO production inhibitory activity experiment

To confirm the effect of YA on the production of NO, the mouse macrophage cell line RAW 264.7 was dispensed into a 6 well plate and cultured for 24 hours at 37° C., 5% CO ₂ , and then treated with YA by concentration and LPS (100 ng/ml) for 24 hours.

형태로 측정하기위해 그리스(Griess) 시약 A는 distilled water (DW) 35 ml, 100% acetic acid 15 ml, sulfanilamide 0.5 g을 혼합시켜 만들었으며, 그리스(Griess) 시약 B는 DW 20 ml, 100% acetic acid 30 ml과 N-(1-Naphthyl) ethylenediamine 0.05 g을 넣어 준비하였다.The amount of NO generated is present in the cell culture medium using a grease reagent.

To measure the morphology, Greases Reagent A was made by mixing 35 ml of distilled water (DW), 15 ml of 100% acetic acid, and 0.5 g of sulfanilamide, and Greases Reagent B was DW 20 ml, 100% acetic It was prepared by adding 30 ml of acid and 0.05 g of N-(1-Naphthyl) ethylenediamine.

형태이며, 생성된 NO의 양은 Sodium nitrite (NaNO₂)를 기준으로 비교하였다. 대조군은 LPS만의 처리군으로 하였다.100 μl of cell culture supernatant and 100 μl of Grease reagent were reacted for 10 minutes at room temperature, and absorbance was measured at 540 nm, which is present in the cell culture solution.

Form, and the amount of NO generated was compared based on Sodium nitrite (NaNO ₂ ). The control group was treated with LPS only.

As can be seen in Figure 2, the results confirmed that YA has a high NO production inhibitory activity.

Inhibitory activity experiment of inflammatory cytokines and PGE2 production

After verifying the effect of YA on the production of inflammatory cytokines such as Prostaglandin E ₂ (PGE ₂ ), Interleukin (IL)-1β, Tumor Necrosis Factor-α (TNF-α), after culturing the cells under the above conditions, The supernatant was collected and the contents of PGE ₂ , IL-1β, and TNF-α (Enzo LifeScience) were quantified using an ELISA kit.

As shown in Figures 2, 3, 4, and 5, the results confirmed that YA significantly inhibited NO, PGE2, TNF-α and IL-1β.

Checking the expression change of iNOS, COX-2, TNF-α, IL-1β through Western blot analysis To observe the expression change of the transcription factors related to immune regulation at the protein level, the cells were subjected to the above conditions. After cultivation, Western blot analysis was performed.

Prepared cells are protein using lysis buffer containing 250 mM NaCl, 25 mM Tris-HCL (pH 7.5), 10 mM ethylenediaminetetraacetic acid, 1% Nonidet P-40, 0.1 mM phenyl-methysulfonylfuoride and protease inhibitor. (protein) was dissolved. The concentration of the isolated protein was measured using a protein quantification reagent (Bio-Rad, Herculs, CA, USA). Equal amounts of proteins were separated using 10% sodium dodecy sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher% Schuell, Keene, NH, USA). Each membrane was treated with 5% skim milk for 1 hour to block non-specific proteins.

The corresponding antibodies and the enhanced chemiluminescence (ECL, Amersham Corp. Arlington Heights, IL, USA) solution were treated, followed by photosensitization with an X-ray film to confirm protein expression. The primary antibody used in this experiment was Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Cell signaling (Beverly, MA, USA). Secondary antibodies were purchased from Amersham Life Science Co. and used.

As can be seen in Figure 3, it was confirmed that the production of NO is reduced in the mouse macrophage cell line RAW 264.7 by YA treatment. It was confirmed that this decrease was due to a decrease in expression of imducible NOS (iNOS).

, TNF-α의 생성 감소로 인해 항염증 효과를 나타낸다고 할 것이다. 복합 추출물의 항염증 활성 시험Therefore, in mouse macrophage cell line RAW 264.7, YA is NO, PGE2, IL-1

, It will be said to have anti-inflammatory effect due to reduced production of TNF-α. Anti-inflammatory activity test of complex extract

As above, the anti-inflammatory activity test results for WP1 were confirmed. Accordingly, an anti-inflammatory activity test was conducted on the complex extract, and for relative evaluation, the anti-inflammatory level of WP1 was set to an index of 5, and the degree of anti-inflammatory effect on other complex extracts was described as an index.

The anti-inflammatory activity test results for the complex extracts are shown in Table 2 below.

WP1 WP2 WP3 WP4 WP5 WP6 NO production suppression 5 4 6 9 7 5 Inhibition of inflammatory cytokine production 5 4 6 8 6 4 Inhibition of PGE2 production 5 3 5 8 7 5

As shown in Table 2 above, in relation to NO production inhibition, inflammatory cytokine production inhibition, and PGE2 production inhibition to confirm the anti-inflammatory effect, relative comparison results to WP1, excellent anti-inflammatory effect equal to or higher in WP3 to 5 It was confirmed that indicates.

[Experimental Example 2: Analysis of free radical scavenging activity]

To confirm the free radical scavenging activity of the extract according to the present invention prepared through Preparation Example 1, DPPH radical scavenging activity and ABTS radical scavenging activity were measured.

DPPH radical scavenging activity measurement

100 μl of each sample and 100 μl of 0.1 mM DPPH were dispensed into a 96 well plate, reacted for 30 minutes at room temperature, and absorbance was measured at 212 nm of a microplate reader (EL-800, BioTek Instruments, Winooski, VT, USA).

As a control, ethanol was added instead of the sample, and a synthetic antioxidant BHA was used for comparison.

DPPH radical scavenging capacity (%) = [(Ac As)/Ac] × 100

(Ac: control absorbance, As: sample group absorbance)

As a result of the measurement, the DPPH radical scavenging ability increased as the concentration of YA increased, and showed a low free radical scavenging ability of 20% or less at a concentration of 1.0 mg/ml.

ABTS radical scavenging activity measurement

After 7 mM ABTS and 2.45 mM potassium persulfate were added and allowed to stand in the dark for 16 hours, dilution with distilled water at 734 nm led to a light intensity of 0.7-0.8.

Absorbance of 1.5 ml of diluted ABTS was measured at 734 nm.

As a control, ethanol was added instead of the sample, and a synthetic antioxidant BHA was used for comparison.

ABTS radical scavenging capacity (%) = [(Ac As)/Ac] × 100

(Ac: control absorbance, As: sample group absorbance)

As a result of the measurement, YA showed ABTS radical scavenging activity at a concentration of 1.0 mg/ml at 13.6%.

Antioxidant activity test of complex extract

As described above, the antioxidant activity test results for WP1 were confirmed. Accordingly, an antioxidant activity test was conducted for the composite extract, and for relative evaluation, the degree of antioxidant of WP1 was set to an index of 5, and the degree of antioxidant effect on other composite extracts was described as an index.

The anti-inflammatory activity test results for the complex extracts are shown in Table 2 below.

WP1 WP2 WP3 WP4 WP5 WP6 DPPH radical scavenging ability 5 4 6 9 7 5 ABTS radical scavenging ability 5 4 7 8 7 5

As shown in Table 3, for the DPPH radical scavenging ability and ABTS radical scavenging ability to confirm the antioxidant effect, relative comparison to WP1, it was confirmed that WP3 to 5 exhibited superior antioxidant effects equal to or higher.

[Experimental Example 3: Sensory evaluation]

Functional evaluation was performed on the WP1 to WP6. After preparing WP1 to WP6 as tea, after providing them to 20 adult men and women, evaluation of taste and aroma was requested.

The evaluation score was requested from 1 to 10 points for each item, and the evaluation results were expressed in terms of the average score.

WP1 WP2 WP3 WP4 WP5 WP6 flavor 2 2 5 7 6 4 incense 2 3 6 8 6 4

Table 4 shows the results of the sensory evaluation.

Referring to Table 4, it was confirmed that the anti-inflammatory and anti-oxidative effects were excellent in WP1, but the preference was lowered due to the unique taste and aroma of tuna. Similarly, in the case of WP2 and WP6, it was confirmed that the palatability decreased by using other natural extracts. On the other hand, in the case of WP3 to 5, in addition to excellent anti-inflammatory and antioxidant effects, it was confirmed that it exhibits a relatively excellent taste and aroma, confirming that it is possible to provide a functional food composition with high preference.

Claims (7)

Contains Polyopes affinis extract as an active ingredient
Anti-inflammatory composition. According to claim 1,
The composition inhibits the expression of inflammatory cytokines (Cytokine), nitric oxide (NO) and COX-2, which are substances that mediate inflammation.
Anti-inflammatory composition. The method of claim 1
The composition has an antioxidant activity through free radical scavenging activity
Anti-inflammatory composition According to claim 1,
The extract is extracted using an extraction solvent selected from the group consisting of water, C 알코올 to C ₅ alcohol, C₁ to C ₅ alcohol aqueous solution and mixtures thereof.
Anti-inflammatory composition Claim 1 to 4 comprising a composition according to any one of
Food composition Claim 1 to 4 comprising a composition according to any one of
Cosmetic composition Claim 1 to 4 comprising a composition according to any one of
Pharmaceutical composition

Images