KR20240020029A - Antibacterial and anti-inflammatory composition containing asian royal fern extract and composition for enhancing immunity including same - Google Patents

Abstract

The present invention relates to an antibacterial and anti-inflammatory composition containing fern extract and a composition for enhancing immunity containing the same.
The antibacterial and anti-inflammatory composition according to an embodiment of the present invention contains fern extract as an active ingredient, exhibits excellent antibacterial and anti-inflammatory effects, and can be used for a long period of time without problems with side effects such as cytotoxicity. Additionally, various formulations can be prepared and provided as an immune-enhancing composition.

Description

Antibacterial and anti-inflammatory composition containing fern extract and immune-promoting composition containing the same

The present invention relates to an antibacterial and anti-inflammatory composition containing fern extract and a composition for enhancing immunity containing the same. More specifically, the present invention includes fern extract as an active ingredient, exhibits excellent antibacterial and anti-inflammatory effects, and can be used for a long period of time without problems with side effects such as cytotoxicity. In addition, various formulations can be prepared to provide a composition for enhancing immunity.

Food poisoning, one of the diseases that occurs frequently in the summer in Korea, is mainly caused by consuming food contaminated with bacteria such as Staphylococcus aureus, Bacillus cereus, Salmonella, Escherichia coli, and Listeria, which are major food poisoning bacteria. If you are infected with food poisoning bacteria, if it is mild, it may be cured naturally, but if the symptoms are severe, antibiotics may be prescribed.

In general, to treat severe infections caused by Staphylococcus aureus, vancomycin, which is evaluated as one of the most powerful antibiotics developed to date, is sometimes used.

However, these compounds have the disadvantage of developing resistance when used for a long time. For example, in 1996, vancomycin-resistant Staphylococcus Aureus (VRSA), which shows strong resistance to vancomycin, was discovered, and in addition, methicillin-resistant Staphylococcus Aureus (MRSA) and carbapenem-resistant Aureus As a significant number of multidrug-resistant bacteria such as Carbapenem Resistant Acinetobacter Baumanii (CRAB) have been discovered, the demand for new alternatives to synthetic antibiotics to kill these antibiotic-resistant bacteria is increasing.

The problem with the emergence of these superbugs is that the time it takes for bacteria to acquire resistance to antibiotics is incomparably faster than the time it takes to develop new antibiotics. Resistant bacteria spread rapidly throughout animals and plants through a variety of methods, and consumers can potentially harbor resistant bacteria by consuming food containing resistant bacteria.

Therefore, in consideration of the emergence of multi-drug resistant bacteria resistant to these antibiotics and the side effects of antibiotics, interest in antibacterial substances derived from natural plants that are easy to obtain and can be used for a long time due to relatively low side effects such as resistance is increasing. there is.

Regarding antibacterial substances derived from natural plants, Korean Patent Publication No. 2006-119406 discloses an antibacterial composition containing ginkgo leaf extract obtained by extracting ginkgo leaves with an organic solvent as an active ingredient, and Korean Patent Publication No. 2010-20123 discloses an antibacterial composition containing lactobacillus extract as an active ingredient. A composition for preventing and treating food poisoning containing as an active ingredient soybean fermentation product fermented with a complex strain of kimchi lactic acid bacteria such as Bacillus plantarum is disclosed.

However, these compositions did not have a very significant antibacterial effect, so they could not be expected to have an antibacterial effect against antibiotic-resistant superbacteria.

Immune refers to a mechanism that physiologically recognizes and excludes foreign and endogenous foreign substances in the body of humans and animals and maintains homeostasis. Here, foreign substances are called antigens, and numerous types of white blood cells and proteins are involved in the process of removing them. Immunity is largely divided into innate immunity, which is possessed from birth, and acquired immunity, which is acquired through adaptation to daily life.

Innate immunity is also called natural immunity or natural resistance. It reacts non-specifically to antigens and has no special memory effect. The innate immune system includes skin and mucus tissues that block the invasion of antigens, acidic stomach acid, and complement present in the blood. Cells include macrophages and polymorphonuclear leukocytes, which are responsible for phagocytosis, and K cells, which can kill infected cells. Additionally, there are cytokines produced by macrophages. In fact, most infections are protected by the innate immunity. Acquired immunity serves to reinforce innate immunity and is also called acquired immunity and adaptive immunity. When an antigen invades the body, an immune response occurs by lymphocytes, and strong resistance to the antigen is remembered, so that when the antigen invades again, it can react specifically and effectively eliminate the antigen. Here, lymphocytes are one of the white blood cells responsible for immune responses in vertebrates and mammals, accounting for approximately 25% of all white blood cells. They are matured and transformed in the thymus and possess immune antibodies, allowing antigens to enter the body. When invaded, they are cells that primarily respond to the body's immune response to prevent the antigen from causing a harmful reaction within the body.

There are two types of lymphocytes in acquired immunity, which are broadly divided into T cells, which are responsible for cellular immunity, and B cells, which are responsible for humoral immunity. T cells are cells that differentiate into lymphocytes from the epithelial cells of the thymus due to the special internal environment and liquid factors of the thymus. They stimulate antibody production in B cells and directly attack antigens such as tumor cells. B cells are cells that are independent of the thymus and differentiate into lymphocytes in the bone marrow. In response to antigen stimulation and T cell-mediated stimulation, B cells mature into antibody-producing cells and produce and secrete antibodies of IgM, IgG, IgA, and IgE.

The initial recognition of infectious antigens is mainly performed by macrophages. As macrophages phagocytose antigens, cytokines that mediate the immune response are produced.

Cytokines are glycoproteins used as signaling substances that control and stimulate the body's defense system and play important functions in immunity, infectious diseases, hematopoiesis, tissue recovery, and cell development and growth. Cytokines have the function of causing immune cells to gather at the site of infection by causing inflammation in the local area, so they are also called inflammatory cytokines. The most important immune functions are performed by a group of cytokines known as interleukin-1, -6, and -12, interferon gamma (IFN-γ), and tumor necrosis factor (TNF).

Interleukin is a substance that plays an important role in the body's defense function by controlling the immune response by acting on various stages of the immune response. Its types include IL-1, 6, 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 the removal of endogenous antigens by various effector cells, such as macrophages. ), induces the activation of NK cells and T-cells.

NK cells and T-cells activated by stimulation of IL-12 have the characteristics of being major factors mediating the early innate immune response by stimulating the production of IFN-γ. In addition, it is the most important mediator of cellular immunity because it induces the immune system of T-cells to become Th-1-oriented.

Interferon gamma (IFN-γ) is produced by T lymphocytes and macrogranulocytes and is a substance that induces the killing of phagocytosed microorganisms or antigens. In addition, it has various immunomodulatory effects and activates macrophages.

Tumor necrosis factor (TNF) 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, white blood cells in the blood, to the site of infection and induce differentiation and activation of infected cells into effector cells with the ability to kill. The function of TNF is due to its ability to induce the production of cytokines with chemotaxis and to increase the affinity of adhesion molecules between vascular endothelial cells and white blood cells.

In this way, immune function is achieved by the action of a wide variety of cells such as macrophages and lymphocytes (T cells, B cells) and proteins such as cytokines such as interleukin and TNF.

The human body's immune response is an essential element for a healthy life, and modern people are highly interested in various immune function-enhancing supplements and foods effective in enhancing immune function. However, health functional foods that can improve immune function are very poor.

KR 10-1962641 B1 KR 10-2124592 B1

The purpose of the present invention is to provide a composition having anti-inflammatory and antiviral activities using extracts derived from natural products.

The purpose of the present invention is to provide a composition that has the effect of improving immune function.

The purpose of the present invention is to provide a composition using extracts derived from natural products that can exhibit functionality while being used for a long period of time without problems with side effects.

Another object of the present invention is to provide functional preparations such as functional foods and external preparations containing the above composition and manufactured in various dosage forms.

In order to achieve the above object, the antibacterial and anti-inflammatory composition according to an embodiment of the present invention may include a fern extract.

The extract may be extracted using an extraction solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixtures thereof.

The composition includes calamus extract; ginger extract; It may further include any one selected from the group consisting of Dalgae extract and mixtures thereof.

A functional food for antibacterial and anti-inflammatory purposes according to another embodiment of the present invention includes the composition.

An external antibacterial and anti-inflammatory skin preparation according to another embodiment of the present invention includes the composition.

A composition for preventing and improving allergic diseases according to another embodiment of the present invention may include the extract.

A composition for enhancing immunity according to another embodiment of the present invention may include the extract.

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

Inflammation as referred to in the present invention is a type of in vivo response to damage or infection of a specific tissue, and the main mediator is an immune cell. Depending on the damaged or infected area of the tissue, it can be divided into acute inflammation and chronic inflammation. inflammation). When the damaged or infected area of the tissue is small or temporary, it is resolved by acute inflammation, but when the inflamed area is large or chronically infected, it is not resolved by acute inflammation and progresses to chronic inflammation. Chronic inflammation can progress into chronic inflammatory disease.

“Immune disease” as used in the present invention refers to a disease caused by an excessive response of the immune system in the human body, and representative examples include allergic diseases. However, since the inflammatory reaction is ultimately caused by the reaction of the immune system in the body, inflammatory disease and immune disease can be used as terms to refer to similar ranges in a broad sense, and are not used as clearly distinct terms within this specification. , will be used interchangeably.

In addition, the immune disease refers to a disease that causes problems when a specific immune response occurs, and is not limited thereto, but preferably includes autoimmune disease, transplant rejection, and graft-versus-host disease. Autoimmune diseases include Crohn's disease and erythema. Disease, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hyperthyroidism, scleroderma, Behçet's disease, inflammatory bowel disease, multiple sclerosis, severe It may be myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic scleroderma, asthma or ulcerative colitis.

The term “inflammatory disease” used in the present invention refers to a disease caused by an inflammatory reaction derived from external or internal substances, and representative examples include neuroinflammatory disease and arthritis.

The antibacterial and anti-inflammatory composition according to an embodiment of the present invention may include a fern extract.

Gobi (Osmunda japonica) is a perennial herb that grows in plains or mountains. The underground stem is short, thick, tube-shaped, has many leaves in clusters, and is 60 to 100 cm high. The leaves are divided into vegetative leaves and sporophylls, and have dense reddish-brown downy hair when young, but gradually disappear. The vegetative leaves are divided into pinnate shapes twice, and the length of the leaf pieces is 20 to 30 cm, with the lowest one being the largest. The small leaf pieces are lanceolate, wide lanceolate, or long oval lanceolate, 5 to 10 cm long and 1 to 2.5 cm wide, with blunt ends, fine serrated edges, and no stalk.

When the fern extract is used, it can exhibit excellent antibacterial activity and salt-containing effects. Specifically, the extract increases immune activity by increasing cytokine production of TNF-α and IL-12, thereby achieving the effect of preventing and improving allergic diseases.

The composition includes Acinetobacter genus, Alcaligenes genus, Enterobacter genus, Bacillus genus, Escherichia genus, Listeria genus, Proteus genus, Providencia genus, Pseudomonas genus, Salmonella genus, Staphylococcus genus, Candida genus, Saccharomyces genus, Aspergillus genus, for microorganisms selected from the group consisting of Cladosporium, Paecilomyces, Penicillium, Trichothecium, and Klebsiella pneumoniae species. Excellent antibacterial effect.

Cytokines are protein immunomodulators secreted from immune cells and bind to specific receptors during autocrine signaling, paracrine signaling, and endocrine signaling to produce immune response. involved in There are various types of cytokines involved in cell proliferation, differentiation, apoptosis, or wound healing, and many are particularly involved in immunity and inflammation.

Among these, the most important immune functions are performed by a group of cytokines known as interleukin (Interleukin-1, -6, -12), interferon gamma (IFN-γ), and tumor necrosis factor (TNF).

Interleukin (IL) is secreted from white blood cells and is involved in immune system regulation. Currently, more than 30 types are known. Interleukin is a substance that plays an important role in the body's defense function by regulating the immune response by acting on various stages of the immune response. Its types include IL-1, 6, 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 the removal of endogenous antigens by various effector cells, such as macrophages. ), induces the activation of NK cells and T-cells. NK cells and T-cells activated by stimulation of IL-12 have the characteristics of being major factors mediating the early innate immune response by stimulating the production of IFN-γ. In addition, it is the most important mediator of cellular immunity because it induces the immune system of T-cells to become Th-1-oriented.

Interferon (IFN) functions to prevent the proliferation of viruses or control cell proliferation and also plays an important function in the immune system. Interferons come in alpha (α), beta (β), gamma (γ), and omega (ω) types. Interferon-α is mainly secreted by macrophages, and interferon-β is mainly secreted by fibroblasts. Interferon-γ is secreted by Th1 cells and NK cells, and interferon-ω is mainly secreted by trophoblasts or trophoblasts. In particular, interferon-γ (IFN-γ) is produced by T lymphocytes and macrogranulocytes and is a substance that induces the killing of phagocytosed microorganisms or antigens. In addition, it has various immunomodulatory effects and activates macrophages.

Tumor necrosis factor (TNF) is a cell signaling protein related to inflammatory response and is a cytokine that induces 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. .

Therefore, in the case of the immune function improvement effect of the antibacterial, anti-inflammatory, antiviral, and immune function improvement composition of the present invention, natural products without human toxicity and side effects are used, and TNF-α and IL-12, which activate the immune response, are used. It increases the production of cytokines.

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, plant extracts include 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 various 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.

The extract may be extracted using an extraction solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixtures thereof.

The pulverized product of the extract can be eluted with a polar solvent such as an alcohol having 1 to 6 carbon atoms such as water, ethanol, or methanol, or a mixed solvent having a mixing ratio of alcohol and water of 1:0.1 to 1:10. For more details, Alternatively, water can be used as an extraction solvent. At this time, the extraction temperature may be 10°C to 100°C, preferably the extract may be extracted at room temperature.

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

It may be a result obtained by concentrating the filtered extract under reduced pressure with a vacuum rotary concentrator, but is not limited thereto, and includes all extracts, diluted or concentrated extracts, dried products obtained by drying the extracts, and crude or purified products thereof.

The composition includes calamus extract; ginger extract; It may further include any one selected from the group consisting of Dalgae extract and mixtures thereof.

Preferably the composition includes calamus extract; It may include ginger extract and moongave extract. In the case of the above mixed composition, antibacterial and anti-inflammatory activities can be further improved.

More preferably, the composition includes a fern extract, and based on 100 parts by weight of the fern extract, 10 to 30 parts by weight of iris; It may contain 50 to 100 parts by weight of ginger and 1 to 10 parts by weight of moongae.

In the case of the above mixing range, a synergistic effect can be achieved due to the interaction of the active ingredients contained in each extract, and excellent antibacterial and anti-inflammatory activities can be achieved with a smaller amount. Accordingly, the problem of side effects or problems caused by long-term use can be resolved.

More preferably, the composition may further include an extract of the herbaceous plant extract or an extract of the chinensis chinensis extract. When the extract is further included, the effect of additionally increasing overall antibacterial and anti-inflammatory activity can be achieved through different mechanisms of the included active ingredients.

In particular, the composition includes fern extract, based on 100 parts by weight of the fern extract, 10 to 30 parts by weight of iris; 50 to 100 parts by weight of ginger; It may be included in the amount of 1 to 10 parts by weight of Dalgae, 0.1 to 1 part by weight of Dalgae, and 10 to 20 parts by weight of nasturtium.

In the case of the above mixing range, additional synergistic activity is maximized and excellent antibacterial and anti-inflammatory activities can be achieved. Therefore, excellent effects can be achieved over a long period of time with a small amount without problems such as cytotoxicity.

In addition, within the above range, it can be manufactured into various formulations, easily formulated into products for improving immunity, products for preventing and improving allergic diseases, etc., and excellent effects can be achieved with a small amount.

A functional food for antibacterial and anti-inflammatory purposes according to another embodiment of the present invention includes the composition.

The food composition includes health functional foods or general foods.

The health functional food is not particularly limited as long as it can be consumed to improve antibacterial, anti-inflammatory, antiviral and immune activity. When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The active ingredient can be used appropriately depending on its purpose of use (prevention or improvement). Generally, when manufacturing a food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw materials. However, in the case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There are no particular restrictions on the types of health functional foods. The health functional food composition of the present invention can be manufactured into food, especially functional food. The functional food of the present invention includes ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufacturing a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., , xylitol, sorbitol, erythritol, etc.) are preferred. The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may further contain carbonating agents used in beverages.

Taking the general processed food as an example, it is possible to manufacture processed food that contains the extract and can enhance antibacterial, anti-inflammatory, antiviral, and immune function improvement 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 seafood (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 file apple, 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.

An external antibacterial and anti-inflammatory skin preparation according to another embodiment of the present invention includes the composition.

Formulations of the skin external preparation include, for example, solutions, gels, solid or pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspensions, microemulsions, microcapsules, microgranules, or ionic (liposome) and non-ionic types. It may be provided in the form of a vesicular dispersant, cream, skin, lotion, powder, ointment, spray or conceal stick. Additionally, it can be prepared in the form of foam or in the form of an aerosol composition further containing compressed propellant.

In addition, the skin external preparations include cosmetics, such as astringent lotions, softening lotions, nourishing lotions, various creams, essences, packs, foundations, cleansing products, face washes, soaps, treatments, or beauty essences. Specific formulations include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, hand cream, essence, nutritional essence, pack, soap, shampoo, and cleansing foam. , can be manufactured into formulations such as cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, press powder, loose powder, or eye shadow. However, it is not limited to this.

A composition for preventing and improving allergic diseases according to another embodiment of the present invention may include the extract.

A composition for enhancing immunity according to another embodiment of the present invention may include the extract.

One embodiment of the present invention provides a composition having anti-inflammatory and antiviral activities using extracts derived from natural products.

One embodiment of the present invention provides a composition that has the effect of improving immune function.

One embodiment of the present invention provides a composition that can exhibit functionality while being used for a long period of time without the problem of side effects using extracts derived from natural products.

Another embodiment of the present invention provides functional preparations such as functional foods and external preparations containing the above composition and manufactured in various dosage forms.

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: Preparation of extract]

1. Preparation of fern extract

After washing and drying the ferns, they were pulverized. The ground product was subjected to hot water extraction, filtered and concentrated to prepare Gobi extract (AE).

2. Preparation of other extracts

In the same manner as the above-mentioned fern extract, hot water extraction was performed on iris, ginger, scallop, fennel root, and nasturtium herb, resulting in iris extract (BE), ginger extract (CE), fennel extract (DE), chalazion extract (EE), and nasturtium extract. (FE) was prepared.

3. Preparation of mixed formulation

In order to evaluate the synergistic activity through the mixed composition of each extract in addition to the activity of each extract, a mixed formulation was prepared by mixing the AE to FE in the composition shown in [Table 1] below.

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 A.E. 100 100 100 100 100 100 100 100 100 100 100 100 BE 100 - One 10 20 30 40 20 20 20 20 C.E. - 100 25 50 75 100 150 60 60 60 60 60 DE - - 0.1 One 5 10 20 5 5 5 5 5 EE - - - - - - - 0.01 0.1 0.5 One 2 F.E. - - - - - - - One 10 5 20 30

(Unit: parts by weight)

[Experimental example: activity evaluation]

1. Antibacterial activity evaluation

To evaluate the antibacterial activity of each extract and mixed formulation, Staphylococcus aureus KCTC1928 (SA), Escherichia coli (EC), Pseudomonas aeruginosa (PA), and methicillin-resistant Staphylococcus aureus (MRSA) were tested. aureus) was used as a test strain.

The test strain was cultured using a medium (Muller hinton broth) at a temperature of 37°C until the McFarland turbidity reached 0.5, and then used in an antibacterial test.

Evaluation of antibacterial activity was performed using the paper disk diffusion method (Toama et al., 1978). 0.1 ml of culture medium for each of the four strains to be tested was spread on a sterilized agar medium, and the mixed extracts (M1 to M6) prepared in Preparation Example 1 were dissolved in the same solvent as the extraction solvent at a concentration of 10 mg/ml. After preparing the extraction sample, 10 μl or 20 μl was dropped onto a paper disk (Advantac, USA) with a diameter of 0.8 mm and cultured at 37°C. Afterwards, the antibacterial activity was measured based on the presence or absence of growth-inhibiting ring formation and the diameter of the growth-inhibiting ring.

Purified water was used as a control, and the antibacterial activity of each extract was evaluated and shown in [Table 2] below.

For objective comparison, the results of the control group were fixed at an index of 10, and the results of the control group and the results of each extract were compared and evaluated on an index of 1 to 10. As for the index, the lower the number, the better the antibacterial activity.

con A.E. BE C.E. DE EE F.E. SA 10 2 3 2 4 6 5 EC 10 3 3 3 5 7 7 PA 10 3 4 2 6 6 7 MRSA 10 4 4 3 7 6 6

(Unit: index)

Referring to Table 2 above, it can be seen that AE and CE have excellent antibacterial activity. However, when considering the palatability and antibacterial activity of the extract based on its unique scent, when commercializing based on a single extract, using AE may show the best palatability and antibacterial effect compared to the same dose. You can check it.

In addition, in order to determine whether the use of the mixed formulation resulted in an additional synergistic effect on antibacterial activity, M1 to M12 were evaluated in the same manner as above. In addition, for an objective comparative evaluation of the synergistic effect, the result of AE as a control group was fixed at 10, and the antibacterial activity of M1 to M12 was comparatively evaluated with an index of 1 to 10. The lower the number, the better the antibacterial activity. If the index is 10, it means that the antibacterial activity is equal to or lower than that of AE.

The results are shown in [Table 3] below.

A.E. M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 SA 10 10 9 10 6 6 5 8 6 2 2 One 6 EC 10 10 9 9 7 6 5 8 6 2 2 2 6 PA 10 10 9 10 7 7 6 8 7 2 2 2 6 MRSA 10 10 10 10 7 7 6 9 8 3 2 One 7

Referring to [Table 3], it was confirmed that M1 to M2 had an overall low level of antibacterial activity compared to AE, and that a synergistic effect due to mixing was not observed.

However, in the case of the mixing range of M4 to M6, it can be confirmed that the antibacterial effect increases significantly due to the synergistic effect due to the interaction of the active ingredients included in each mixing composition. Therefore, it can be confirmed that excellent antibacterial activity can be achieved with a smaller amount using the above mixed composition. In addition, it can be seen that in the case of M9 to M11, additional synergistic activity appears compared to M5. This means that the above mixed formulations exhibit antibacterial activity through different mechanisms, and it can be assumed that a synergistic effect is achieved through interaction within a certain mixing range. Therefore, when within the above range, even a small amount can exhibit excellent antibacterial activity, making it possible to utilize it in various formulations.

2. Evaluation of anti-inflammatory activity

In order to evaluate the anti-inflammatory activity of each extract and mixed composition, hydrogen peroxide scavenging activity and DPPH free radical scavenging activity were evaluated.

Specifically, in the hydrogen peroxide scavenging ability test, hydrogen peroxide solution (40 mM) was prepared in phosphate buffer (pH 7.4). Black kelp extract was added to hydrogen peroxide solution (0.6mL, 40mM). The absorbance of hydrogen peroxide at 230 nm was measured after 10 minutes against a blank solution containing phosphate buffer without hydrogen peroxide. The percent hydrogen peroxide scavenging capacity of both sample and standard compounds was calculated:

[Equation 1]

Eliminated [H ₂ O ₂ ] = [(AC - AS)/AC] × 100

where AC is the absorbance of the control and AS is the absorbance in the presence of the sample or standard sample. The results are shown in [Table 4] below.

In addition, to evaluate the DPPH free radical scavenging ability, 10 mg DPPH was dissolved in 100 ml methanol (MeOH) to make it 100 ㎍/ml. A stock solution of the extract was prepared by dissolving 25 mg in 50 ml of MeOH. 1 ml DPPH was added to each solution and the solutions were kept dark and allowed to stand for exactly 30 minutes. The UV absorption of each solution was recorded at 516 nm. The reaction was carried out at 37 °C for 30 min and absorbance was monitored with a microplate reader at 516 nm. % radical scavenging activity (% RSA) was determined compared to the DMSO containing control. Scavenging of free radicals by DPPH as radical scavenging activity (%) was calculated as follows:

[Equation 1]

% RSA = (Control Absorbance - Absorbance)

The results are shown in [Table 4] below.

A.E. C.E. M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 H ₂ O ₂ scavenging activity (%) 65 58 71 64 52 48 49 53 63 50 35 32 34 58 DPPH scavenging activity (%) 82 71 88 80 85 60 56 58 76 60 46 45 46 67

Referring to [Table 4], excellent anti-inflammatory activity was observed in AE among single compositions. In particular, it can be seen that it has superior activity compared to CE.

In addition, in the mixed formulation, it can be seen that the anti-inflammatory activity increases due to the interaction of each active ingredient in M4 to M6, and in the case of M9 to M11, it shows a much better effect than the simple combination due to additional synergistic activity compared to M5. You can check the point.

Therefore, in the case of the above mixed formulation, it may be possible to use it in various formulations as a functional product that shows better effects with a smaller amount.

Therefore, the AE or mixed formulation provided in the present invention can exhibit excellent antibacterial and anti-inflammatory activity, and can be provided in various formulations such as food and external preparations. In particular, in the case of the above mixed formulation, excellent effects can be achieved with a small amount, so it can be provided as a functional product that can be used for a long time without problems such as side effects.

3. Evaluation of anti-allergic activity

In order to evaluate the resistance activity of allergic diseases for the AE and each mixed formulation, the production amount of prostaglandin D ₂ (PGD ₂ ) was measured. For measurements, the PGD ₂ EIA kit (Cayman, PGD2-MOX EIA kit, product no. 512011) was used.

Mouse bone marrow-derived mast cells (BMMC) were isolated from the bone marrow of BALB/C mice and cultured in RPMI-1640 medium containing 10% FBS, 100 U/ml penicillin, and 100 ng/ml streptomycin and IL. BMMCs of over 90% homogeneity were obtained by culturing with -3 (a culture medium containing the final 20% supernatant obtained by stimulating rat spleen cells with pokeweed mitogen) for approximately 3 weeks. After 3 ^weeks of culturing the BMMC above, the cells were pre-treated with AE at different concentrations (50 μg/ml, 100 μg/ml, 150 μg/ml, 200 μg/ml, 250 μg/ml) at a cell concentration of 2×10 5 cells for 30 minutes. , PGD ₂ in the supernatant 8 hours after cell stimulation was measured by EIA (Enzyme linked immune assay) using a PGD ₂ analysis kit (Cayman). At this time, in order to inhibit the production of PGD ₂ produced by COX-1 (cyclooxygenase-1), BMMCs were pretreated with 10 ng/ml of aspirin 1 hour before use. Washed four times with washing buffer, treated with 200 μl of substrate solution each, reacted for 5-20 minutes, treated with 50 μl of stop solution, and measured absorbance at 450 nm using ELx800. (BIO-TEK, Instrument, Inc, USA).

The results are shown in [Table 5] below.

50 100 150 200 250 PGD ₂ (pg/ml) 230 115 80 75 60

Referring to [Table 5], it can be seen that when AE is used, the production of PGD ₂ is suppressed in a concentration-dependent manner.

Meanwhile, in order to evaluate the synergistic activity of the mixed formulation, the same anti-allergy evaluation was conducted on M3 to M12, excluding M1 and M2, for which no synergistic effects were observed in antibacterial and anti-inflammatory effects. For objective comparative evaluation, the results of AE were fixed at an index of 10, and the results of M3 to M12 were compared with AE and evaluated with an index of 1 to 10. The lower the index number, the better the anti-allergy effect. Additionally, if the index is 10, it means that the effect is equal to or lower than that of AE and no synergistic effect is observed.

The results are shown in [Table 6] below.

A.E. M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 PGD ₂ (index) 10 10 6 6 6 9 6 4 3 4 7

Referring to [Table 6], it can be seen that in the case of M4 to M6, the anti-allergy effect increases due to additional synergistic activity. In addition, it can be confirmed that in the case of M9 to M11, an additional synergistic effect can be exhibited compared to M5.

Therefore, in the case of the above mixed formulation, a small amount can be used to prevent and improve allergic diseases through synergistic activity. In addition, it can be used continuously without problems such as side effects even after long-term use. In particular, in the case of typical allergic diseases such as asthma, eczema, and rhinitis, it is important to prevent and improve them through continuous use, so the mixed formulation according to the present invention can exhibit significantly excellent prevention and improvement effects.

Therefore, it can be provided in various formulations with preventive and improving effects on immune diseases, including allergic diseases.

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 fern extract
Antibacterial and anti-inflammatory composition. According to clause 1,
The extract is extracted using an extraction solvent selected from the group consisting of water, alcohols with 1 to 6 carbon atoms, and mixtures thereof.
Antibacterial and anti-inflammatory composition. According to clause 2,
calamus extract; ginger extract; Further comprising any one selected from the group consisting of Dalgae extract and mixtures thereof.
Antibacterial and anti-inflammatory composition. Comprising the composition according to claim 1
Functional food for antibacterial and anti-inflammatory purposes. Comprising the composition according to claim 1
Antibacterial and anti-inflammatory topical skin preparation. Containing the extract according to paragraph 1 or 2
Composition for preventing or improving allergic diseases. Containing the extract according to paragraph 1 or 2
Composition for enhancing immunity.