KR102129461B1 - A composition having anti-bacterial activity comprising Selaginella tamariscina extracts, fractions thereof or compounds isolated therefrom as an active ingredient - Google Patents

Abstract

The present invention relates to an antioxidant or antibacterial cosmetic composition, a food composition, and a pharmaceutical composition comprising an extract of Selaginella tamariscina , a fraction thereof or a compound isolated therefrom as an active ingredient.
The antioxidant or antimicrobial composition comprising the extract of Selaginella tamariscina of the present invention, a fraction thereof or a compound isolated therefrom as an active ingredient has excellent activity of scavenging DPPH free radicals in a concentration-dependent manner, that is, an antioxidant effect Is excellent, and has excellent characteristics of growth inhibition against causative bacteria such as food poisoning, dermatitis, pneumonia, and cellulitis. It can be usefully used in cosmetics, food and medicine for prevention, improvement or treatment.

Description

A composition having anti-bacterial activity comprising Selaginella tamariscina extracts, fractions thereof or compounds isolated therefrom as an active ingredient}

The present invention relates to an antioxidant or antibacterial cosmetic composition, a food composition, and a pharmaceutical composition comprising an extract of Selaginella tamariscina , a fraction thereof or a compound isolated therefrom as an active ingredient.

Oxygen is an essential element in various metabolic reactions to maintain life, and it is necessary for detoxifying poisons in the human body, but oxygen is not only beneficial to the human body, so various pollutants such as enzyme systems, reducing metabolism, chemicals, and photochemical reactions in the body, and physicochemical When it is converted into free radicals, which are highly reactive, such as superoxide radical, hydroxyl radical, hydrogen peroxide, and singlet oxygen, due to environmental factors, etc. It has a double-sided effect that causes fatal oxygen toxicity in the body ( Korea J. Biotechnol. Bioeng. , 2001, 16(6), 592-602). Synthetic antioxidants such as BHT (butylated hydroxy toluene) and BHA (butylated hydroxy anisole), which are synthetic antioxidants for removing these free radicals, have been widely used so far due to their excellent antioxidant effect and economic efficiency. Due to the issue of, the allowed food or consumption is strictly limited. In addition, natural antioxidants such as tocopherol and ascorbic acid have high safety, but have the disadvantages of low chain reaction ability and low cost. For this reason, recently, studies have been actively conducted to separate and use safer, more economical, and more effective antioxidants from natural products. In particular, secondary metabolites of plants as plant-derived substances inhibit the production of free radicals and free radicals. Because it prevents oxidation by removal, many studies have been conducted ( Korean Journal of Plant Res. , 2011, 24(1), 30-39; J. Korean Soc. Food Sci. Nutr. , 2010, 39(9 ), 1249-1256).

On the other hand, in order to maintain the quality of products such as food, cosmetics, textiles, and medicines for a long time, a preservative to prevent spoilage by microorganisms is essential. Especially, in the case of cosmetics, which have a relatively long shelf life and a lot of microbial nutrients, preservatives are more desirable. Is required. The risk factor is always latent compared to the economics of synthetic raw materials, as raised through the recent'oxygen event', and the problem caused when the risk factor is realized is fatal to evaluate profits based on simple arithmetic statistics.

As a conventional preservative, paraben-based preservatives, which are most commonly used in cosmetics, foods, and pharmaceuticals, have been suggested to cause skin allergies and resistant bacteria. To solve these problems, research on natural preservatives that have excellent product safety and economic efficiency Is going on. Natural preservatives (preservatives) are additives used for long-term preservation and extension of shelf life in food and cosmetics, and refer to bio-derived materials used to prevent spoilage by microorganisms, and chemical synthetic preservatives are used and used due to the risk of continuous use. Despite the fact that it is strictly limiting the situation, concerns about various side effects such as chronic and acute toxicity, mutation, and carcinogenesis are constantly raised.

Most of the currently reported natural antibacterial substances have not been commercialized due to color odor, stability deterioration, narrow antibacterial spectrum, and formulation problems, and cypress extract Hinokitiol, magnolia extract Magnolol and grapefruit Very few, such as seed extract DF-100, are commercialized. Among them, DF-100, which is the most advanced in product development, is known to have antibacterial activity due to the organic acid and synthetic preservative benzethonium chloride contained in DF-100, and other natural antibacterial agents have low economic efficiency. There is an urgent need for natural antibacterial agents that can be developed more advanced and commercialized due to problems such as narrow antimicrobial spectrum or limited use range due to physical properties (Korean Patent No. 10-1495272).

Under these backgrounds, the present inventors have been prescribed for various bleeding symptoms such as hemorrhage, fecal bleeding, uterine bleeding, etc., as a result of diligent efforts to find natural products with excellent anti-oxidation or anti-bacterial effects without side effects on the human body, and to relieve pain caused by menstrual pain or bruise DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical scavenging effect, dermatitis, superior in water-saturated butanol fraction obtained from methanol extract of Selaginella tamariscina known to be used The present invention was completed by confirming that the microbial growth inhibitory effect associated with inducing diseases such as food poisoning and pneumonia is excellent.

Korean Registered Patent No. 10-1495272

Korea J. Biotechnol. Bioeng., 2001, 16(6), 592-602 Korean Journal of Plant Res., 2011, 24(1), 30-39 J. Korean Soc. Food Sci. Nutr., 2010, 39(9), 1249-1256

An object of the present invention is to provide a composition for antioxidant or antimicrobial containing a water-saturated butanol fraction of methanol extract of Selaginella tamariscina .

Another object of the present invention is to provide a cosmetic composition comprising the composition for antioxidant or antibacterial.

Another object of the present invention is to provide a food composition comprising the composition for antioxidant or antibacterial.

Another object of the present invention is to provide a pharmaceutical composition comprising the composition for antioxidant or antibacterial.

In order to achieve the above object, the present invention is an antioxidant or antibacterial composition containing the water-saturated butanol fraction of methanol extract of Selaginella tamariscina as an active ingredient, a cosmetic composition, a food composition, and a pharmaceutical composition comprising the antioxidant or antibacterial composition Provided is a composition.

Hereinafter, the present invention will be described in detail.

In one embodiment, the present invention provides a composition for antioxidant or antimicrobial containing a water-saturated butanol fraction of methanol extract of Selaginella tamariscina .

The term "Buddhason ( Selaginella tamariscina )" of the present invention is a fern plant that grows on a sunny rock in the mountains, and is sometimes referred to as Kwonbaek. It is a medicinal plant belonging to the Selaginellaceae family, and has a unique appearance resembling that of the Buddha, and the scientific name is Selaginella tamariscina (P.Beauv.) Spring.

Specifically, the leaves are dense, ovate with 4 rows, and 1.5 to 2 mm long, the ends of which are thread-like protrusions, and have a cupped edge on the edge. The sporophyte is an egg-shaped triangle, and there is a thin and thin side, and the sporangia has large and small. The spore sac (달리子囊穗) runs one by one at the end of the twig, has a square, is 5-15㎜ long and 2㎜ in diameter. The stem reaches 20cm in height, and the branches are split into planes, the surface is dark green, and the back side is whiteish green. When it's wet, the branches spread all over the place, and when it's dry, it rolls inside to become a ball, and when it's wet, it spreads again. The root grows by spreading branches in all directions at the end formed like a stem with many roots and roots intertwined. The root root derived from below is hard and short and has many beard roots.

Almost in Korea, distributed in Japan, Taiwan, China, and Manchuria. It is an evergreen perennial plant that grows mainly on dry, cool rocks or cliff surfaces. It has been used to treat bloody stool, hematuria, and anal hernia in oriental medicine. As blood sugar lowering and the like are known, interest in the effective ingredients of baekbaek is increasing.

The water-saturated butanol fraction of the methanol extract of Selaginella tamariscina is preferably prepared by a manufacturing method including the following steps, but is not limited to this:

1) extracting by adding methanol to Selaginella tamariscina ;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and drying it to prepare a Buddhason methanol extract; And

4) The step of preparing a Buddhason fraction by additionally extracting the methanol extract of Buddhason of step 3) with an organic solvent.

In the above method, the buddha ( Selaginella tamariscina ) of step 1) can be used without limitation, such as those grown or commercially available. In addition, although not limited thereto in step 1), the Buddha hand may be extracted by including all the leaves, branches, or roots.

In the above method, as the extraction method using methanol in step 1), it is preferable to use agitation extraction, Soxhlet extraction or reflux extraction, but is not limited thereto. It is preferable to extract the methanol by adding 1 to 10 times to the portion of the Buddha's hand that has been washed, dried, and powdered. The extraction temperature is preferably 20°C to 100°C, more preferably 20°C to 60°C, and most preferably 50°C, but is not limited thereto. In addition, the extraction time is preferably 4 to 24 hours, more preferably 5 to 12 hours, and most preferably 6 hours, but is not limited thereto. In addition, the number of extractions is preferably 1 to 5 times, more preferably 2 to 4 repetitions, and 3 times is most preferred, but is not limited thereto.

In the above method, it is preferable to use a vacuum decompressor or a vacuum rotary evaporator in step 3), but the present invention is not limited thereto. In addition, the drying is preferably dried under reduced pressure, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

In the above method, it is preferable that the organic solvent of step 4) is normal-hexane ( n- Hexane), methylene chloride, ethyl acetate or water saturated n-butanol. However, it is not limited to this. The fraction was suspended in water with the Buddhason methanol extract, followed by normal-hexane ( n- Hexane), methylene chloride, ethyl acetate, water saturated n-butanol and water ( H ₂ O) is preferably one of a normal-hexane fraction, a methylene chloride fraction, an ethyl acetate fraction, a water saturated n-butanol fraction or a water fraction obtained by sequential fractionation with H, It is more preferably a saturated n-butanol fraction, but is not limited thereto. The fraction can be obtained by repeating the fractionation process 1 to 5 times, preferably 3 times, from the Buddhason methanol extract, and it is preferable to concentrate under reduced pressure after fractionation, but is not limited thereto.

In addition, the water-saturated butanol fraction of the methanol extract of Selaginella tamariscina is preferably prepared by a manufacturing method including the following steps, but is not limited to this:

1) extracting by adding methanol to Selaginella tamariscina ;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and drying it to prepare a Buddhason methanol extract; And

4) The step of preparing the Buddhasonson water-saturated butanol fraction by additionally extracting the Buddhasonson methanol extract of step 3) with water-saturated butanol.

In the above method, the buddha ( Selaginella tamariscina ) of step 1) can be used without limitation, such as those grown or commercially available. In addition, although not limited thereto in step 1), the Buddha hand may be extracted by including all the leaves, branches, or roots.

In the above method, as the extraction method using methanol in step 1), it is preferable to use agitation extraction, Soxhlet extraction or reflux extraction, but is not limited thereto. It is preferable to extract the methanol by adding 1 to 10 times to the portion of the Buddha's hand that has been washed, dried, and powdered. The extraction temperature is preferably 20°C to 100°C, more preferably 20°C to 60°C, and most preferably 50°C, but is not limited thereto. In addition, the extraction time is preferably 4 to 24 hours, more preferably 5 to 12 hours, and most preferably 6 hours, but is not limited thereto. In addition, the number of extractions is preferably 1 to 5 times, more preferably 2 to 4 repetitions, and 3 times is most preferred, but is not limited thereto.

In the above method, it is preferable to use a vacuum decompressor or a vacuum rotary evaporator in step 3), but the present invention is not limited thereto. In addition, the drying is preferably dried under reduced pressure, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

In the above method, it is preferable to fractionate by adding the same amount of water saturated n-butanol to the amount of the methanol extract of Buddhason as the butcherson water-saturated butanol fraction of step 4), but is not limited thereto. The fraction can be obtained by repeating the fractionation process 1 to 5 times, preferably 3 times, from the Buddhason methanol extract, and it is preferable to concentrate under reduced pressure after fractionation, but is not limited thereto.

In addition, although not limited thereto, the water-saturated n-butanol is mixed in an equal amount of butanol and ultrapure water, shaken vigorously, and left to stand to completely separate the layer, then discard the lower layer of the water layer and recover and use the upper layer. Be prepared.

On the other hand, without being limited thereto, the water-saturated butanol fraction includes α-linolenic acid, 3-dioxy-d-mannoic acid, and guaiacol. And it may be to include a butanediol (butanediol) compound.

In addition, but not limited to, the compound is 5 to 10 parts by weight of α-linolenic acid (α-linolenic acid) with respect to 100 parts by weight of the water-saturated butanol fraction, 3-dioxy-di-mananoic acid (3- Deoxy-d-mannoic acid) may be included in 3 to 6 parts by weight, guaiacol in 5 to 10 parts by weight, and butanediol in 0.1 to 3 parts by weight.

The term "antioxidant" of the present invention refers to an action of inhibiting oxidation, and the human body has a balance of an oxidizing accelerator (prooxidant) and an antioxidant (antioxidant), but this balance state is imbalanced by various factors. When it is achieved and tilted toward the promotion of oxidation, oxidative stress is induced, causing potential cell damage and pathological diseases. Reactive oxygen species (ROS), which are the direct cause of these oxidative stresses, are unstable and highly reactive, so they can easily react with various biological materials, attack the polymers in the body, cause irreversible damage to cells and tissues, or mutate, It causes cytotoxicity and carcinogenesis. Reactive nitrogen species (RNS) such as NO, HNO ₂ and ONOO- are produced in large quantities due to the immune response of macrophage neutrophils and other immune cells during an inflammatory reaction, and ROS is also generated. The free radicals as described above oxidize and destroy cells in the body, and thus are exposed to various diseases. Therefore, when the inclusion of the water-saturated butanol fraction of the Buddhason methanol extract of the present invention, in particular, the Buddhasonson methanol extract, can achieve an antioxidant effect, thereby contributing to anti-aging and health promotion.

In a specific embodiment of the present invention, in order to confirm the antioxidant effect of the water-saturated butanol fraction of the Buddhason methanol extract, a result of performing a DPPH free radical scavenging activity measurement and a metal ion catalyst oxidation inhibition test, the water-saturated butanol fraction of the Buddhasonson methanol extract It was confirmed that this concentration-dependent DPPH free radical exhibits a scavenging effect and a protein protective effect, that is, a strong antioxidant effect (FIGS. 5 and 6 ). Therefore, the antioxidant effect of the present invention may be achieved by free radical scavenging activity.

The term "antibacterial" of the present invention refers to the action of inhibiting or controlling the growth and proliferation of microorganisms such as bacteria or fungi, the microorganism of the present invention is not limited thereto, Staphylococcus aureus ( Staphylococcus aureus ), Star may be a pillow nose kusu epidermidis (Staphylococcus epidermidis), or Haemophilus influenzae (Haemophilus influenzae). Staphylococcus aureus , the skin flora, is generally present on the skin and nasal surface of healthy people or livestock, and is known as a bacterium that produces food poisoning by producing heat-resistant exotoxin. Staphylococcus epidermidis is a secondary skin infection after acne infection, and is a bacterium that can generally cause nonpathogenicity, sepsis, urinary tract infection, and endocarditis. Haemophilus influenzae, the causative agent of influenza , is known to cause bacterial meningitis or acute epiglottis.

On the other hand, various types of skin diseases are mainly caused by dermatophytes, and acne and dandruff bacteria are typical. The onset of acne is reported to be the main factor causing the inflammatory response of acne bacteria, and dandruff bacteria proliferate ideally on the skin, such as the back and nape, to cause seborrheic dermatitis. The cause of atopic dermatitis is not yet known, but it is reported as staphylococcus aureus. In order to reduce the incidence of skin diseases caused by these dermatophytes and other bacteria and to protect the skin, the use of preservatives and antibacterial agents is essential, but synthetic materials that are used in the past can cause allergies to the skin, so they use relatively harmless substances. It is important to do.

Therefore, when the Buddhason methanol extract of the present invention, especially the water-saturated butanol fraction of the methanol extract is included in cosmetics, food, or pharmaceuticals, the microorganisms that cause dermatitis, food poisoning, and pneumonia are related By achieving a microbial growth inhibitory effect, it can contribute to the prevention and improvement of diseases such as food poisoning and pneumonia, including skin diseases.

In a specific embodiment of the present invention, in order to confirm the antimicrobial effect of the water-saturated butanol fraction of the Buddhason methanol extract, dermatitis, food poisoning, and pneumonia (Minimum inhibitory concentration assay) Staphylococcus aureus associated with pneumonia induction, Staphylococcus epidermidis (Staphylococcus epidermidis) and Haemophilus influenzae (Haemophilus influenzae) check result of measuring the antimicrobial activity for the three strains in total, which bucheoson saturated butanol fraction the growth inhibitory activity is superior with respect to the three strains (Figs. 10 and 11). Therefore, the antibacterial effect of the present invention may be achieved by growth inhibition activity against food poisoning bacteria and influenza bacteria, including skin microorganisms.

In another aspect, the present invention provides a cosmetic composition comprising an antioxidant or antimicrobial composition containing a water-saturated butanol fraction of methanol extract of Selaginella tamariscina as an active ingredient.

The water-saturated butanol fraction of the Buddhason methanol extract is as described above, and the water-saturated butanol fraction of the Buddhason methanol extract of the present invention has an antioxidant effect and an antibacterial effect, and thus can be added to a cosmetic composition for antioxidant or antibacterial purposes. have.

The cosmetic composition prevents or improves any one skin disease selected from the group comprising acne, atopy, athlete's foot, psoriasis, eczema and dermatitis, and has an effect of preventing or improving aging, wrinkles or loss of elasticity of the skin.

In a specific embodiment of the present invention, the water-saturated butanol fraction of the methanol extract of Buddhasonson has excellent antioxidant effect and Staphylococcus aureus , Staphylococcus epidermidis ( Staphylococcus epidermidis ) and Haemophilus influenzae ( Haemophilus influenzae ) It was confirmed that it shows an antibacterial effect to inhibit the growth of the strain (Fig. 5, Fig. 6, Fig. 10 and Fig. 11).

The present invention provides that the water-saturated butanol fraction of the Buddhason methanol extract has an excellent antimicrobial effect on growth inhibition activity against food poisoning bacteria and influenza bacteria, including skin microorganisms. Antibiotics such as tetracycline have side effects and limitations in use such as the appearance of resistant bacteria, whereas the water-saturated butanol fraction of the Buddhason methanol extract according to the present invention is safe and has no side effects and is suitable as a cosmetic composition.

The cosmetic composition of the present invention can be prepared in the form of general emulsifying and solubilizing formulations. The emulsifying formulations include nutrient makeup, cream, essence, etc., and the solubilizing formulations include softening makeup. Suitable formulations include, but are not limited to, for example, solutions, gels, solid or dough anhydrous products, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) obtained by dispersing an oil phase in an aqueous phase, ionic form It can be in the form of a vesicle dispersant, cream, skin, lotion, powder, ointment, spray or corneal stick. In addition, it may be in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition may additionally include fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, blowing agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metals Commonly used adjuvants such as ion blockers, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredient commonly used in cosmetic compositions. It may contain.

In another aspect, the present invention provides a food composition comprising a composition for antioxidant or antibacterial containing a water-saturated butanol fraction of methanol extract of Selaginella tamariscina as an active ingredient.

The water-saturated butanol fraction of the Buddhason methanol extract is as described above, and when the composition of the present invention is used as a food additive, the water-saturated butanol fraction of the Buddhason methanol extract is added as it is or used with other food or food ingredients. Can be used as appropriate according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment), and may further include a food supplement that is food-acceptable. Since the composition of the present invention uses an extract derived from natural products and a fraction thereof as an active ingredient, there is no problem in terms of stability, and thus there is no great limitation on the mixing amount.

The food composition of the present invention may include all foods in a conventional sense, and can be mixed with terms known in the art, such as functional foods and health functional foods.

The term "functional food" of the present invention refers to food manufactured and processed using ingredients or ingredients having functional properties useful for the human body according to Act No. 6727 of the Health Functional Food Act, and "functional" refers to the structure of the human body. And it means to ingest for the purpose of obtaining useful effects for health purposes such as controlling nutrients for function or physiological action.

In addition, the term "health functional food" of the present invention refers to foods manufactured and processed by methods such as extracting, concentrating, refining, and mixing specific ingredients in food ingredients or using specific ingredients as ingredients for the purpose of supplementary health, A food designed and processed to sufficiently exert bioregulatory functions such as biodefense, biorhythm control, disease prevention and recovery by the above-mentioned components, and the composition for health food is used to prevent disease and prevent disease. It can perform functions related to recovery.

There are no restrictions on the types of foods in which the composition of the present invention can be used. In addition, the composition comprising the water-saturated butanol fraction of the Buddhason methanol extract of the present invention as an active ingredient may be prepared by mixing known additives with other suitable auxiliary ingredients that may be contained in food according to the choice of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gums, ice cream, various soups, beverages, teas, drinks, alcoholic beverages, and There are vitamin complexes, etc., and can be prepared by adding the extract according to the present invention and its fractions as a main component, and adding it to juice, tea, jelly and juice.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., formulas, infants, infant foods, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements , Seasoned foods (e.g. soy sauce, miso, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.) ), beverages (eg, fruit, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavoring agents or natural carbohydrates, etc., as additional components, like a conventional beverage. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin , Alcohol, carbonic acid used in carbonated beverages, and the like. In addition, it may contain flesh for the production of natural fruit juice, fruit juice beverages and vegetable beverages.

In another aspect, the present invention provides a pharmaceutical composition comprising a composition for antioxidant or antimicrobial containing a water-saturated butanol fraction of methanol extract of Selaginella tamariscina as an active ingredient.

The water-saturated butanol fraction of the Buddhason methanol extract is the same as described above, and the water-saturated butanol fraction of the Buddhason methanol extract of the present invention has an antioxidant effect that eliminates free radicals (active oxygen), dermatitis, food poisoning ( Prevents and improves diseases caused by free radicals and microbial infections that can be caused by non-pathogenic or pathogenic microorganisms because it has antibacterial effects on microorganisms related to food poisoning and pneumonia And pharmaceuticals for treatment.

The antioxidant is as described above, and thus may be included in a pharmaceutical composition for the purpose of antioxidant, and may have a prophylactic or therapeutic effect on diseases caused by free radicals. Diseases caused by the free radicals are not limited thereto, but arteriosclerosis, Lou Gehrig's disease, Parkinson's disease, Alzheimer's disease, degenerative neurological diseases including myotrophic sclerosis and Huntington's disease, myocardial infarction, angina pectoris, coronary artery disease, ischemic heart Cardiovascular diseases including diseases, ischemic brain diseases including stroke, diabetes, gastrointestinal diseases including gastritis and gastric cancer, cancer, leukemia, aging, rheumatoid arthritis, hepatitis, atopic dermatitis, and may include various diseases, Preferably, it may be aging caused by free radicals.

In addition, the antimicrobial is as described above, and accordingly, may be included in a pharmaceutical composition for antimicrobial purposes, Staphylococcus aureus , Staphylococcus epidermidis ( Staphylococcus epidermidis ) and Haemophilus influenza ( Haemophilus influenzae ) dermatitis (dermatitis), food poisoning (food poisoning), and may have a prophylactic or therapeutic effect of microbial infections related to pneumonia .

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" of the present invention means that it exhibits non-toxic properties to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art, such as buffers, preservatives, painless agents, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants.

In addition, the pharmaceutical composition of the present invention can be used by formulating in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to a conventional method. have. Furthermore, it can be used in the form of an external preparation for skin in the form of ointments, lotions, sprays, patch, creams, powders, suspensions, gels or gels. Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient, for example, starch, calcium carbonate (calcium) in the water-saturated butanol fraction of the Buddhason methanol extract. carbonate), sucrose or lactose, gelatin. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use may include various excipients, such as wetting agents, humectants, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are simple diluents commonly used for suspending agents, intravenous solutions, emulsions, syrups, etc. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means to introduce a predetermined substance into an individual in an appropriate manner, and the route of administration of the composition can be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, but are not limited thereto.

The term "individual" refers to all animals, including humans, mice, mice, and livestock. Preferably, it can be a mammal, including a human.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's gender, age, and weight Good for factors and other medical fields, including health, condition, type of disease, severity, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of discharge, duration of treatment, combination or drug used concurrently It can be readily determined by a person skilled in the art according to known factors. For the administration, the recommended dosage may be administered once a day, or divided several times.

The water-saturated butanol fraction of the Buddhason methanol extract of the present invention is a natural medicinal plant as a raw material, so even when used as a cosmetic composition, a food composition, or a pharmaceutical composition, it has less side effects compared to a general synthetic compound, so it can be safely contained and useful. .

The antioxidant or antimicrobial composition comprising the extract of Selaginella tamariscina of the present invention, a fraction thereof or a compound isolated therefrom as an active ingredient has excellent activity of scavenging DPPH free radicals in a concentration-dependent manner, that is, an antioxidant effect Is excellent, and has excellent characteristics of growth inhibition against causative bacteria such as food poisoning, dermatitis, pneumonia, and cellulitis. It can be usefully used in cosmetics, food and medicine for prevention, improvement or treatment.

1 is a view schematically showing a process for preparing a methanol extract (STME) and its solvent fraction of Selaginella tamariscina of the present invention according to a sequential solvent fraction.
2 is a diagram showing a resulting, bucheoson of the present invention (Selaginella tamariscina) methanol extract (STME) and their solvents GC / MS (gas chromatograph-mass spectrometer) analysis of the fractions according to the sequential solvent fractionation.
3 is a view schematically showing a process of preparing a methanol extract (STME) and water saturated n-butanol fraction of the present invention ( Selaginella tamariscina ).
4 is a view showing the results of GC/MS (gas chromatograph-mass spectrometer) analysis of the water-saturated butanol fraction of the present invention.
Figure 5 is a graph showing the concentration-dependent DPPH free radical scavenging effect of the Buddhason methanol extract (STME) and water-saturated butanol fractions of the present invention. Here, Asc is ascorbic acid used as a positive control, and Veh is a DMSO (dimethyl sulfoxide) treatment group, which is a solvent used when dissolving the water-saturated butanol fraction of Buddhason.
FIG. 6 is an electrophoresis photograph showing the effect of inhibiting oxidation of a metal ion catalyst (BSA decomposition), that is, a protein protection effect according to the concentration treatment of the Buddhasonson methanol extract and the Buddhasonson supersaturated butanol fraction of the present invention. Here, Asc is ascorbic acid used as a positive control, and Veh is a DMSO (dimethyl sulfoxide) treatment group, which is a solvent used when dissolving the water-saturated butanol fraction of Buddhason.
7 is a graph showing the results of cell activity inhibition according to the treatment by the concentration of the methanol extract of the present invention and the water-saturated butanol fraction of the Buddhason. Here, Ctrl (control) is an untreated group, and Veh is a DMSO (dimethyl sulfoxide) treated group.
8 is a graph showing the results of cytotoxicity test (LDH assay) for mouse splenocytes according to the treatment by the concentration of the Buddhasonson methanol extract and the Buddhasonson saturated butanol fraction of the present invention. Here, as a negative control, hydrogen peroxide (H ₂ O ₂ ), which induces strong cell inhibition, was used, Ctrl (control) is an untreated group, and Veh is a DMSO (dimethyl sulfoxide) treated group.
9 is a view schematically showing the flow of the acute toxicity class test according to oral administration of the methanol extract of Buddhason according to the present invention.
Figure 10 Staphylococcus aureus ( Staphylococcus aureus ) by treatment according to the concentration of water-saturated butanol fraction of the Buddha of the present invention, Staphylococcus epidermidis and Staphylococcus epidermidis and Haemophilus influenza ( Haemophilus influenzae ) is a graph measured by using the minimum inhibitory concentration assay (Minimum inhibitory concentration assay).
FIG. 11 is Staphylococcus aureus by the treatment method according to the concentration of the water-saturated butanol fraction of the Buddha of the present invention by the minimum inhibitory concentration measurement method. Star is a photograph showing an antibacterial effect on the nasal pillow kusu epidermidis (Staphylococcus epidermidis) and Haemophilus influenzae (Haemophilus influenzae).

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Preparation of methanol extract of Buddhasonson and its solvent fraction

In order to secure various extraction components, 100 g of washed, dried, and powdered Buddha ( Selaginella tamariscina ) was immersed in 1 liter of 50% methanol and extracted three times for 6 hours to obtain a Buddhason methanol extract, and the methanol extract was about 10.34. % Yield. The obtained Buddhason methanol extract was filtered and evaporated under reduced pressure to remove methanol, and sequential solvent fractionation was performed in a volume ratio of 1:1 to the Buddhason methanol extract (STME; Selaginella tamariscina metanol extract). Specifically, the sequential solvent fraction was extracted by adding n-hexane (n-hexane) to the methanol extract of Buddhason, and then fractionated by adding methylene chloride to the water layer (aqueous residue) obtained in the process of obtaining the Buddhason hexane fraction. Extracted. Fraction extraction was performed by adding ethyl acetate to the water layer obtained in the process of obtaining the Buddhason methylene chloride fraction, and water fraction n-butanol was added to the water layer obtained in the process of obtaining the Buddhason ethyl acetate fraction to extract the fraction. The water layer obtained in the process of obtaining the n-butanol fraction of the Buddhason was also obtained (FIG. 1).

Example 2: Analysis of the active ingredients of the methanol extract of Buddhason and the solvent fraction thereof

The Buddhason methanol extract obtained in Example 1 and each solvent fraction layer (hexane fraction layer, methylene chloride fraction layer, ethyl acetate fraction layer, water-saturated-n-butanol water) To analyze the active components of the saturated n-butanol (fraction layer) and the remaining water residue (residue), a gas chromatograph-mass spectrometer (GC/MS) analysis was performed.

GC/MS analysis was performed using GC/MS (Agilent Technologies 5975C) equipped with a CTC CombiPAL autosampler system (Palo Alto, CA, USA). At this time, the column used for sample analysis is an HP-5 column (HP-5 column; Agilent Technologies, 250 μm×0.25 μm×30 m), and the analysis conditions are held at 50° C. for 5 minutes and then held at 10° C. per minute. Each was raised, and held at 310° C. for 5 minutes for analysis. All samples (Sample) are methanol and water in a 1:1 mixture of methanol and water for GC/MS analysis, hexane fraction is ethanol, methylene chloride fraction, ethyl acetate fraction, and water-saturated butanol fraction is methanol as solvent. After dissolving at 10 mg/ml, 5 μl was used for splitless injection.

As a result of GC/MS analysis, Buddhasonson methanol extract (STME; Selaginella tamariscina metanol extract) and its fractions (hexane fraction, methylene chloride (MC) fraction, ethyl acetate fraction and water-saturation-n- It was confirmed that oleic acid (9-Octadecenoic acid) and butanediol (BDO) were present in the butanol (water saturated n-butanol fraction), and it was confirmed to be contained in a high content in each of the fractions (FIG. 2).

On the other hand, active ingredients such as oleic acid (9-Octadecenoic acid) and butanediol (BDO) contained in the methanol extract of Buddhasonson and its fractions are known to have antibacterial activity.

Therefore, through the above results, the methanol extract of Buddhason and its solvent fraction (hexane fraction, methylene chloride (MC) fraction, ethyl acetate fraction and water saturated n-butanol) ) It was found that fraction) can be used as a material having antibacterial activity.

Example 3: Preparation and extraction process of water-saturated butanol fraction of methanol extract from Buddhason

Based on the results of Examples 1 and 2, the yield of each extraction process was secured, and an attempt was made to establish an extraction standardization for the fractional extract of Buddhason methanol. Specifically, 100 g of Buddha ( Selaginella tamariscina ) three times for 6 hours at room temperature (room temperature), 35 °C, 50 °C of each temperature condition, and 30%, 50%, 70% of each methanol solvent concentration conditions Extraction to determine the optimal yield extraction method.

As a result, as shown in FIG. 3, the solvent was selected as 70% methanol and the extraction temperature was 50°C to obtain a water-saturated butanol solvent fraction directly from the Buddhason methanol extract through the standardization of extraction for the parent extract, the Buddhason methanol extract. An extraction process that can be done was established. Accordingly, a standard extraction method of 70% methanol for the solvent and 50°C for the extraction temperature was applied to obtain the Buddhason methanol extract in a yield of 12.9%.

Example 4: Identification of major components and analysis of content of water-saturated butanol fraction of Buddhason

GC/MS (gas chromatograph-mass spectrometer; Agilent Technologies 5975C) analysis in order to analyze the main components and contents contained in the water-saturated butanol fraction of the Buddha hand obtained through the extraction process finally established in Example 3 above Was performed.

Specifically, 100 g of Buddha ( Selaginella tamariscina ) was immersed in 1 L of 70% methanol at an extraction temperature condition of 50°C and extracted three times every 6 hours to obtain a Buddhason methanol extract. After methanol extraction, a water-saturated butanol fraction was obtained therefrom, and the yield thereof was 27.4%, showing a yield of about 3.5% compared to 100 g of the Buddha-son powder. GC/MS analysis was performed using the obtained Buddhason water-saturated butanol fraction.

As a result of GC/MS analysis, α-linolenic acid (syn. 9,12,15-Octadecatrienoic acid), 3-dioxy-di-mananoic acid (3-Deoxy-d- mannoic acid), butanediol (butanediol) and was found to contain guaiacol (guaiacol) (Table 1 and Figure 4). Α-linolenic acid (syn. 9,12,15-Octadecatrienoic acid), 3-deoxy-d-mannoic acid contained in the water-saturated butanol fraction , Butanediol (butanediol) and guaiacol (guaiacol) active ingredients are known as compounds having antibacterial and/or anti-inflammatory properties.

Therefore, through the above results, α-linolenic acid (α-linolenic acid; syn. 9,12,15-Octadecatrienoic acid), 3-dioxy-d-mannoic acid, butanediol (butanediol) ) And guiacol (guaiacol), it was found that the Buddhason saturated butanol fraction can be utilized as a material having antibacterial activity and anti-inflammatory efficacy.

No. RT
(retention time, min) Area% Library ID Qual.
(qualitative) One 31.71 11.19 2,4,6-Cycloheptatrien-1-one,3,5-bis-trimethylsilyl- 43 2 24.26 7.78 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- 99 3 13.07 7.37 Guaiacol 93 4 20.06 4.93 3-Deoxy-d-mannoic acid 43 5 23.46 0.37 1,2-Butanediol, 1-(2-furyl)-2,3-dimethyl- 22

Example 5: Analysis of antioxidant effect of methanol extract of Buddhasonson and water-saturated butanol fraction

Through the DPPH radical scavenging efficacy test and the metal ion catalyst oxidation inhibition test, the antioxidant effect of the Buddhason methanol extract and its saturated butanol fraction was analyzed.

Example 5-1: Analysis of antioxidant activity of methanol extract of Buchenson and water-saturated butanol fractions thereof through DPPH radical scavenging assay

To confirm the direct radical scavenging ability of the Bucherson methanol extract (STME; Selaginella tamariscina metanol extract) and its buOH fraction of STME, a DDPH radical scavenging efficacy test was performed. DPPH (1,1-Diphenyl-2-picrylhydrazyl) is a chemically stabilized water-soluble free radical, a purple compound that exhibits characteristic light absorption at 517 nm, and is very stable and emits electrons when it encounters an antioxidant activity. The radical (DPPH) is extinguished and the color changes from purple to yellow in the initial solution, so it is a representative substance that is used in typical antioxidant activity experiments that can easily observe the oxidizing activity with the naked eye.

Specifically, the methanol extract of Buddhasonson and the water-saturated butanol fractions thereof were serially diluted at a 2-fold ratio from 100 µg to 1.56 µg, and reacted with DPPH, and then absorbance at 517 nm was measured to compare radical scavenging activity. At this time, as a positive control, ascorbic acid (50 μM) having excellent antioxidant effect was used.

As a result of the DPPH radical scavenging efficacy test, 12.5 µg in the case of Buddhason methanol extract Significant radical scavenging activity was found to be concentration-dependent at the above test concentrations, and 6.25 ㎍ even in the case of Buddha-saturated butanol fraction. At the above test concentrations, significant radical scavenging activity was confirmed in a concentration-dependent manner. In particular, 50 μg As a result of reacting the water-saturated butanol fraction with DPPH at the above test concentration, it was confirmed that it showed a remarkably excellent antioxidant effect (FIG. 5).

Therefore, through the above results, it was found that the antioxidant activity of the water-saturated butanol fraction of Bucherson, including the methanol extract of Bucherson, was excellent.

Example 5-2: Antioxidant activity analysis of the Buchenson methanol extract and its water-saturated butanol fraction through a metal ion catalyst oxidation protection test

The metal ion catalyst oxidation inhibition test is a method of confirming the effect of an antioxidant that protects a protein or an enzyme from damage caused by reactive oxygen species (ROS) using a metal ion catalyst reaction.

Specifically, BSA (bovine serum albumin, Sigma-Aldrich, MO, USA) was used as a target protein for verifying the oxidation inhibitory effect, and Cu ²⁺ (100 μM) and H ₂ O ₂ were used as the primary reaction. (2.5 mM) was added to generate hydroxyl radicals, and then diluted with BSA and each concentration (6.25 μg, 12.5 μg, 25 μg, 50 μg, 100 μg), butcherson methanol extract or butcherson supersaturated butanol After mixing the fractions, a second reaction was performed. The experimental group for each concentration at which the reaction was terminated was subjected to electrophoresis on 10% SDS polyacrylamide gel to reduce the degree of inhibition of BSA protein degradation by the methanol extract of Buddhason or the water-saturated butanol fraction by each concentration. Confirmed. At this time, ascorbic acid (ascorbic acid, 50 μM) was used as a positive control.

As a result of the metal ion catalyst oxidation inhibition test, a significant protein degradation protection effect was confirmed even in a reaction in which BSA was mixed at a minimum test concentration of 6.25 µg for both the Buddhason methanol extract and the Buddhason saturated butanol fraction. In addition, it was found to have an effect equal to or greater than that of ascorbic acid used as a positive control. Accordingly, it was confirmed that the methanol fraction of Buddhason and the water-saturated butanol fraction of Buddhason had a strong antioxidant effect (FIG. 6 ).

Therefore, through the above results, it was found that the antioxidant activity of the Buddhason methanol extract and the Buddhason saturated butanol fraction was excellent, and in addition, the Buddhasonson methanol extract and the Buddhason saturated butanol fraction had the effect of protecting proteins from radical attack. there was.

Example 6: Toxicity evaluation of methanol extract of Buddhasonson and water-saturated butanol fractions

In the case of extracts derived from natural products, it is often difficult to develop materials due to strong efficacy and accompanying toxicity. Accordingly, the cell viability (CCK) and cytotoxicity (LDH) were checked to evaluate the effect of the Buddhason methanol extract and its saturated butanol fraction on the cells. In addition, through animal experiments, it was confirmed whether acute toxicity following oral administration of the methanol extract of Buddhasonson.

Example 6-1: Cell viability assay according to the treatment of the methanol extract of Buddhasonson and the water-saturated butanol fraction thereof

In the case of extracts derived from natural products, it is often difficult to develop materials due to the strong efficacy and accompanying toxicity, so a cell activity test was conducted for the Buddhason methanol extract and the Buddhason water saturated butanol fraction. In this case, the mouse macrophage cell line Raw 264.7 (mouse macrophage cell line RAW 264.7) was used. The Raw 264.7 cells were adjusted to 37° C., 5% CO ₂ using DMEM complete medium (DMEM (Dulbecco's Modified Eagle's Medium) complete medium; Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (FBS). It was cultured in a CO ₂ incubator.

Cells produce the necessary energy through the mitochondrial electron transport system process. During this process, succinate dehydrogenase (SDH) in the electron transport system degrades the tetrazolium salt to form insoluble formazan. ). Cell viability was measured by measuring the amount of formazan produced using a cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to this principle.

Specifically, the concentrations of the mouse macrophage cell line Raw 264.7 cells by concentration (1.56 µg/ml, 3.12 µg/ml, 6.25 µg/ml, 12.5 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml) After treating each of the Bucherson methanol extract (STME) and the Bucherson saturated butanol fraction separated therefrom, the mitochondrial activity of the cells was analyzed by adding the CCK solution at 1:10 within 24 hours.

As a result, compared with the hydrogen peroxide (H ₂ O ₂ ) treatment group, which induces strong cell inhibition as a negative control group, the methanol activity of the Buddhasonson and the water-saturated butanol fraction of the Buddhasonson were treated even at the highest treatment concentration of 100 μg/ml. It was confirmed that there was no effect on the cells even at a high concentration showing strong antioxidant efficacy because no change was observed (FIG. 7 ).

Example 6-2: Evaluation of cytotoxicity according to the treatment of the methanol extract of Buddhason and the water-saturated butanol fraction thereof ((Lactate dehydrogenase (LDH) release assay)

As an immortalized macrophage, the mouse spleen was extracted and then splenocytes (splenocytes) to determine whether to inhibit the cell viability of the mouse macrophage cell line, Raw 264.7, as well as whether it is directly cytotoxic to primary cells. ) Was isolated to perform a cytotoxicity test. Cytotoxicity test was used for non-radioactive LDH assay kit, CytoTox 96 ® (Promega, Madison, WI, USA) by measuring the amount of LDH released from the damaged cells.

The cytotoxicity test was 1.56 μg/ml, 3.12 μg/ml, 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml as in the cell activity assay of Example 6-1 above. It was measured by treating the spleen cells with the Buddhasonson methanol extract and the Buddhasonson saturated butanol fraction at a concentration of ㎖.

Specifically, after treating the Buddhason methanol extract and the Buddhason saturated butanol fraction by concentration in the splenocytes extracted from the mouse, the LDH solution is added at a 1:1 ratio within 24 hours, and the level of LDH released from the dead cells Was analyzed.

As a result, it was confirmed that even when the Buddhasonson methanol extract and the Buddhason saturated butanol fraction were treated at the highest concentration of 100 μg/ml, it did not cause direct cytotoxicity, and the Buddhasonson methanol extract and the Buddhasonson water saturated butanol fraction had very low cytotoxicity. It was confirmed (Fig. 8).

Example 6-3: Evaluation of acute toxicity following oral administration of Buddhason methanol extract

The single-dose toxicity test was conducted in accordance with the guidelines of the Ministry of Food and Drug Safety, and the methanol extract of Buddhasonson was administered directly to the stomach through an oral administration method and fasted for 4 hours before administration. The concentration of methanol extract of Buddhasonson was administered at a concentration of 300 mg/kg·bw (body weight) for the first time according to the guidelines of the Ministry of Food and Drug Safety, followed by observation of abnormal symptoms for 30 minutes after administration, followed by 4 hours during the first 24 hours. , After that it was observed for 7 days at 8-hour intervals. If there were no signs of abnormality, an additional factor of 2000 mg/kg·bw of Buchenson methanol extract was additionally administered and observed for 7 days, followed by an autopsy to analyze the abnormality factor (FIG. 9) (Cosmetic Toxicity Test Animal Replacement Test Guideline) Ⅱ, Korea Food and Drug Administration, 2008; Guidelines for the Alternatives to Animals in Cosmetic Toxicity Test Ⅶ, Korea Food and Drug Administration, 2015). Animal experiments were conducted with the approval of the Gangneung-Wonju National University Animal Care and Use Committee; Approval no.: GWNU-R2016-29.

No abnormal symptoms were observed for 24 hours immediately after oral administration of the methanol extract of Buddhasonson, and no specificity was observed in the results observed periodically for 14 days thereafter (Table 2). The change in body weight of each individual also showed no difference compared to the control group, and the intake of feed and water also showed no significant difference between each individual, and showed normal activity (Table 3). As a result of observation for 14 days, no abnormal signs appeared, and after euthanasia, it was confirmed whether the internal organs were congested or atrophic. As a result, no change or deformation of organs was found in all the subjects used in the test, and the weight of each organ was also controlled. It was confirmed that there was no damage to the organ because there was no difference from. In addition, there was no significant difference from the control group in all items in the results of performing blood biochemical tests on the Buddhason methanol extract test group to examine whether the toxicity was not externally confirmed (Table 4).

Through the results of Example 6, it was found that both the Buddhasonson methanol extract and the Buddhasonson saturated butanol fraction did not induce cytotoxicity or cytotoxicity, and thus did not show direct cytotoxicity. It was confirmed that the acute toxicity class test according to the administration showed no significant difference from the control group. Accordingly, it was verified that the methanol extract of Buddhason and the water-saturated butanol fraction of Buddhason can be used as a safe material without toxicity through in vitro and in vivo experiments.

Example 7: Analysis of antibacterial effect of water-saturated butanol fraction of Buddhason

Antimicrobial agents such as oleic acid (9-Octadecenoic acid), butanediol (BDO), guaiacol, as active ingredients in the methanol extract of Buddhasonson and the water-saturated butanol fraction of Buddhason through Examples 2 and 4 And/or it was confirmed that a compound having an anti-inflammatory effect was included, and through the examples 5 and 6, the Buddhasonson methanol extract and the Buddhasonson saturated butanol fraction did not have toxicity to cells and experimental animals, and had excellent antioxidant properties. After confirming that it has activity, it is judged that it is possible to develop it as a material for antibacterial and preservatives, the importance of verifying the presence or absence of toxicity based on this, and the star by treatment by fraction concentration of water-saturated butanol fraction of Buddhason obtained from methanol extract of Buddhasonson Staphylococcus aureus , Staphylococcus epi pile confirmed an antimicrobial effect on the display (Staphylococcus epidermidis) and Haemophilus influenzae (Haemophilus influenzae).

Specifically, in order to confirm the direct antibacterial effect of the water-saturated butanol fraction of the Buddha's hand, three known strains known as causative bacteria such as food poisoning, dermatitis, pneumonia, and cellulitis, Staphylococcus ow Russ ( Staphylococcus aureus ), Staphylococcus epi pile was confirmed by using the display (Staphylococcus epidermidis) and Haemophilus influenzae minimum inhibitory concentration assay (Minimum inhibitory concentration assay, MIC assay ) the antimicrobial activity of the (Haemophilus influenzae).

The strains used in the method for measuring the minimum inhibitory concentration were prepared in advance to be 10 ⁸ CFU (colony forming unit)/mL, respectively, according to the Mcfarland standard turbidity (Mcfarland standard), and the maximum concentration in the case of Buddha-saturated saturated butanol fraction was 1,000 μg/mL. It was prepared by serial dilution to 62.5 µg/ml in 2x ratio. The prepared three strains were inoculated at a concentration of 1:100 as a concentration that satisfies the Mcfarland standard turbidity (Mcfarland standard), and then treated and cultured with a fraction of Buddhason saturated butanol at each concentration to observe antibacterial activity.

As a result, Staphylococcus aureus , Staphylococcus aureus , according to the treatment concentration of the water-saturated butanol fraction of Buddhasonson obtained from the methanol extract of Buddhasonson, Staphylococcus epidermidis a significant bacteriostatic effects against (Staphylococcus epidermidis) and Haemophilus influenzae (Haemophilus influenzae) was observed. Specifically, it was confirmed that bacterial growth was significantly inhibited for the above three strains depending on the concentration from the case where the Buddha-Saturated water-saturated butanol fraction was treated at a concentration of 250 to 500 µg/ml or more, particularly at a test concentration of 1,000 µg/ml or more. It was confirmed that it showed an equivalent antibacterial effect compared to ampicillin, a positive control group (FIGS. 10 and 11 ).

Therefore, through the series of results, the Buddhasonson saturated butanol fraction extracted directly from the Buddhason methanol extract without performing sequential solvent fractionation includes Staphylococcus aureus, including strong reactive oxygen species (ROS) scavenging ability and protein protection ability. ( Staphylococcus aureus ), Staphylococcus epidermidis (Staphylococcus epidermidis) and Haemophilus influenzae has been verified that the antibacterial effect of the (Haemophilus influenzae), can be seen that useful as a safe natural materials that can replace synthetic preservatives and antimicrobial The there was.

Preparation Example 1: Preparation of antioxidant or antibacterial cosmetic containing water-saturated butanol fraction of Buddhason methanol extract as an active ingredient

Preparation Example 1-1: Preparation of flexible lotion

The flexible lotion containing the water-saturated butanol fraction of the Buddhason methanol extract as an active ingredient was prepared as shown in Table 5 below.

Raw material Content (parts by weight) Saturated butanol fraction of methanol extract of Buddhason of the present invention 10.00 1,3-butylene glycol 1.00 Disodium LED 0.05 Allantoin 0.10 Dipotassium glyceride 0.05 Citric Acid 0.01 Sodium citrate 0.02 Glyceres-26 1.00 Arbutin 2.00 Hydrogenated castor oil 1.00 ethanol 30.00 Preservative a very small amount coloring agent a very small amount Flavor a very small amount Purified water Balance

Preparation Example 1-2: Preparation of nutrition cream

* The softening nutrient cream containing the water-saturated butanol fraction of the Buddhason methanol extract as an active ingredient was prepared as shown in Table 6 below.

Raw material Content (parts by weight) Saturated butanol fraction of methanol extract of Buddhason of the present invention 10.0 1,3-butylene glycol 7.0 glycerin 1.0 D-panthenol 0.1 Plant extracts 3.2 Magnesium aluminum silicate 0.3 PEG-40 stearate 1.2 Stearic Acid 2.0 Polysorbate 60 1.5 Lipophilic glyceryl stearate 2.0 Sorbitan sesquioleate 1.5 Cetearyl alcohol 3.0 Mineral oil 4.0 Squalane 3.8 Carlylic/Capric Triglyceride 2.8 vegetable oil 1.8 Dimethicone 0.4 Dipotassium glyceride a very small amount Allantoil a very small amount Sodium hyaluronate a very small amount Tocopheryl Acetate Proper Triethanolamine Proper Preservative Proper Flavor Proper Purified water Balance

Preparation Example 2: Preparation of antioxidant or antibacterial food containing water-saturated butanol fraction of Buddhason methanol extract as an active ingredient

Preparation Example 2-1: Preparation of flour food

0.5 to 5.0 parts by weight of the water-saturated butanol fraction of the Buddhason methanol extract of the present invention was added to the flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

Preparation Example 2-2: Preparation of soup and gravy

0.1-5.0 parts by weight of the water-saturated butanol fraction of the Buddhason methanol extract of the present invention was added to soups and gravy to prepare a health-enhancing meat product, noodles soup and gravy.

Preparation Example 2-3: Preparation of ground beef

10 parts by weight of the water-saturated butanol fraction of the Buddhason methanol extract of the present invention was added to the ground beef to prepare a ground beef for health promotion.

Preparation Example 2-4: Preparation of dairy products

5-10 parts by weight of the water-saturated butanol fraction of the Buddhason methanol extract of the present invention was added to the milk, and various dairy products such as butter and ice cream were prepared using the milk.

Production Example 2-5: Preparation of wire

The brown rice, barley, glutinous rice, and yulmu were alpha-polished by a known method to distribute the dried one, and then prepared into a powder having a particle size of 60 mesh with a grinder.

Black soybeans, black sesame seeds, and perilla seeds were also steamed and dried by a known method, and then distributed into a powder having a particle size of 60 mesh with a grinder.

The water-saturated butanol fraction of the Buddhason methanol extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a spray or hot air dryer was pulverized to a particle size of 60 mesh to obtain a dry powder.

The water-saturated butanol fractions of the above-prepared grains, seeds, and the inventive Buddhason methanol extract were prepared in the following proportions:

Grains (30 parts by weight of brown rice, 15 parts by weight of barley, 20 parts by weight of barley), seeds (7 parts by weight of perilla, 8 parts by weight of black soybeans, 7 parts by weight of black sesame seeds), water-saturated butanol fraction of the Buddhason methanol extract of the present invention ( 3 parts by weight), Yeongji (0.5 parts by weight), and sulfur (0.5 parts by weight).

Preparation Example 2-6: Preparation of health drinks

Homogenize 5 g of the water-saturated butanol fraction of the methanol extract of the Buddha's Hand of the present invention with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), and water (75%) After compounding and sterilization at the moment, it was manufactured by packaging it in small packaging containers such as glass bottles and plastic bottles.

Preparation Example 2-7: Preparation of vegetable juice

Vegetable juice was prepared by adding 5 g of water-saturated butanol fraction of the Buddhason methanol extract of the present invention to 1,000 ml of tomato or carrot juice.

Preparation Example 2-8: Preparation of fruit juice

Fruit juice was prepared by adding 1 g of the water-saturated butanol fraction of the Buddhason methanol extract of the present invention to 1,000 ml of apple or grape juice.

Preparation Example 3: Preparation of a pharmaceutical composition for anti-oxidation or antimicrobial containing a water-saturated butanol fraction of the Buddhason methanol extract as an active ingredient

Production Example 3-1: Preparation of powder

1 g of lactose was mixed with 2 g of the water-saturated butanol fraction of the Buddhason methanol extract of the present invention, and filled in an airtight fabric to prepare a powder.

Preparation Example 3-2: Preparation of tablets

The tablets were prepared by mixing the water-saturated butanol fraction of the methanol extract of the Buddhason of the present invention with 100 mg of corn starch, 100 mg of lactose, 100 mg of lactose, and 2 mg of magnesium stearate, followed by tableting according to a conventional tablet preparation method.

Preparation Example 3-3: Preparation of capsules

After mixing the water-saturated butanol fraction 100 mg, corn starch 100 mg, lactose 100 mg, and magnesium stearate 2 mg of the Buddhason methanol extract of the present invention, the capsules are prepared by filling into gelatin capsules according to a conventional capsule preparation method. Did.

Preparation Example 3-4: Preparation of a ring

After mixing the water-saturated butanol fraction 1 g, lactose 1.5 g, glycerin 1 g and xylitol 0.5 g of the Buddhason methanol extract of the present invention, it was prepared to be 4 g per ring according to a conventional method.

Preparation Example 3-5: Preparation of granules

After mixing the water-saturated butanol fraction 150 mg, soybean extract 50 mg, glucose 200 mg, and starch 600 mg of the Buddhason methanol extract of the present invention, 30 mg of 30% ethanol was added and dried at 60°C to form granules. The filling was filled.

Claims (3)

Pharmaceutical composition for the prevention or treatment of pneumonia by Haemophilus influenzae containing the water-saturated butanol fraction of methanol extract of Selaginella tamariscina as an active ingredient.
According to claim 1, wherein the water-saturated butanol fraction of the methanol extract of Buddhasonson is α-linolenic acid, 3-deoxy-d-mannoic acid, and guaiacol ( guaiacol) and butanediol (butanediol) pharmaceutical composition comprising a compound.
According to claim 2, wherein the compound is 5-10 parts by weight of α-linolenic acid (α-linolenic acid) with respect to 100 parts by weight of the water-saturated butanol fraction of the Buddhason methanol extract, 3-dioxy-di-mananoic acid ( 3-Deoxy-d-mannoic acid) is 3 to 6 parts by weight, guaiacol (guaiacol) is 5 to 10 parts by weight, and butanediol (butanediol) is contained in 0.1 to 3 parts by weight. Composition.

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