KR101142598B1 - An Antibacterial composition comprising the essential oil extract of Kjellemaiella crassifolia and the compounds isolated therefrom - Google Patents

Abstract

The present invention relates to an antimicrobial composition containing Kjellemaiella crassifolia extract or a compound isolated therefrom as an active ingredient. Specifically, the extract of the present invention or the compound isolated therefrom is Escherichia coli , Antibacterial activity against Bacillus subtilis , Candida albicans , Streptococcus mutans and Staphylococcus aureus, preventing and preventing bacterial infections It can be used in pharmaceutical compositions and nutraceuticals useful for treatment.

Kelp, compound, bacterial infection

Description

Antibacterial composition comprising the essential oil extract of Kjellemaiella crassifolia and the compounds isolated therefrom}

The present invention relates to an antimicrobial composition containing Kjellemaiella crassifolia extract or a compound isolated therefrom as an active ingredient, and relates to a composition for preventing and treating bacterial infection diseases.

[1] Mi-Kyung Lim, Mi-Ra Kim: Antimicrobial Activity and Component Analysis of Methanol Extract of Portulace oleracea against Food Corruption and Food Toxicity Microorganisms. Korean Journal of Culinary Science 17 (6). p.565, 2001.

[Document 2] Kang, Seong-gu, Seongnak-gye, Yongdu Kim, Soo-cheol Shin, Jae-shin Seo, Kap-seong Choi, Seok-kyu Park: Antimicrobial Activity Screening of Freshly Extracts. Korean Journal of Nutrition, 23 (6): p1008, 1994.

Perry, NB et al., Essential oil from new Zealand Manuka and kanuka Chemotaxonomy of Leptospermum. Phtochemistry , 8 , pp. 1485-1493, 1997.

Document 4 Tom J. MABRY. The systematic Identification of Flavonoids p18--19, 1970.

Kudo, T. and Safa, N., Development of a simple method for antibiotic susceptibility testing in algae using paper disks. Nippon Suisan Gakkaishi , 56 , p.455, 1990.

Document 6 Piddock, LJV; Techniques used for the determiration of antimicrobial resistance and sensitivity in bacteria. J. Appl . Bacteriol, 68: 307, 1990.

Bacterial infectious diseases are caused by pathogenic bacteria, viruses, fungi, parasites, and the like invading the human body, for example, tuberculosis, influenza, typhoid fever, cholera, sore throat, and pneumonia. Bacterial infectious diseases have been able to be treated to some extent through antibiotics and immunization due to the development of medicine. However, the use of antibiotics is increasing due to the emergence and increase of antibiotic resistant bacteria.

Bacterial antibiotic resistance varies from country to country but has already become a serious problem worldwide. The problem of antibiotic resistance encountered in 1992 was serious, and most pathogenic bacteria were often resistant to antibiotics that had been used for primary use. In recent years, the proportion of major resistant bacteria has increased, and in particular, the problem of antibiotic resistance in Korea is very serious.

It is recognized that the emergence and increase of antibiotic resistant bacteria are due to the use of antibiotics. However, antibiotics are the most important treatment for bacterial infections. Therefore, antibiotics cannot be used. Antibiotics should be used because of the risk of worsening the patient's condition by not doing so.

Staphylococcus aureus is an important pathogen commonly isolated from patients. In addition to respiratory infections, Staphylococcus aureus causes infections such as bacteremia, osteomyelitis, and meningitis. The most common infection is skin infection. It has long been rare that S. aureus is susceptible to penicillin, and now, methicillin-resistant S. aureus (MRSA) is almost all in-hospital and its proportion varies greatly from country to country. In Korea, MRSA rates have already exceeded 50% of S. aureus isolates from hospitalized patients in the early 1990s, and a 2002 Korean Nationwide Surveillance of Antimicrobial Resistance (KONSAR) group survey found that S. aureus isolates from most hospitals in Korea. The ratio was 67%. Other non-pathogenic diseases such as conjunctivitis and iris infections, such as Bacillus subtilis , which causes chronic purulent bacteremia , and E. coil , which causes food poisoning, have a steadily increasing resistance rate (Monthly Drug Information (Understanding the latest infectious diseases). ), pp.2-31, 2005), the development of new natural antibiotics without these side effects is urgent.

Escherichia coli is a bacterium that resides in the intestines of humans or mammals. It is a bacterium belonging to the intestinal bacteria and Escherichia . Three coliform groups, including Citrobacter, Klebsiella and Enterobacter . Escherichia coli in a narrow sense is a gram-negative bacillus with a butterfly size of 0.5-0.7 μm and a length of 2-3 μm, but it is main hairy, but some strains do not have flagella. It develops well in ordinary incubators and forms smooth, opaque, white colonies. Escherichia coli breaks down glucose, lactose and mannitol (sometimes it breaks down sugar and salicylic acid), producing acid and gas. As a bacterium Escherichia coli (주로 原 大腸菌; mainly a type of bacterium that causes diarrhea), O-55, O-111, and O-124 have been reported. In general, Escherichia coli is a fungus in the intestinal tract of a mammal, and has a significant value for the host. That is, the above-mentioned degrading power of E. coli is exerted in the intestine, and is also related to the production of vitamins. In addition, E. coli is a standard for fecal contamination, which is a standard for hygiene testing of beverages, foods, and even beaches and public baths. This is because fecal contamination causes the presence of food poisoning bacteria Salmonella and Escherichia coli in addition to typhoid bacteria and typhoid bacteria, or suspicion of such diseases. However, E. coli is not a harmful bacterium and is used as a pollution indicator because it does not proliferate in nature other than the intestinal tract. In addition, Escherichia coli is known to be associated with urinary tract infections, cystitis, kidney infections, urethral infections, gastroenteritis, peritonitis, appendicitis, pneumonia, meningitis, and bone or joint infections.

Bacillus subtilis, also known as Bacillus subtilis , lives mainly in soil and belongs to the Bacillus genus, a Gram positive bacterium. It is aerobic, rod-shaped or cylindrical, of varying size and length, not uniform at the ends, and may have flagella or spores. One to three cells at one end of the cell, or a large number of flagella around the cell can be exercised, there is a bacilli with spores at one end or the center of the cell. The spores of bacteria are the parts where the bacterial cells are at rest and do not live, but are resistant to the influence of the external environment. Therefore, they can survive long-term under bad living conditions, and germinate under proper conditions to form their own bacterial cells. That is, the spores of bacteria are the same as the seeds of bacteria. In addition, Koreans have a deep relationship with fermented microorganisms in Cheonggukjang and are also used as various microbial agents. It is known to be associated with conjunctivitis and iris.

Candida albicans is a common fungus commonly found in the oral cavity and gastrointestinal tract, and is found in 50% in the oral cavity and 90% in feces. C. albicans causes nonspecific clinically insignificant lesions in most cases, but can cause a variety of diseases, depending on the patient's immunity. C. albicans can be found anywhere in the gastrointestinal tract, but most often causes lesions in the throat and esophagus, mostly asymptomatic. Rarely, fungal infections occur in the stomach and duodenum, but most of them are secondary to metastatic proliferation of benign or malignant ulcers.

Streptococcus mutans are bacterial species present in the oral cavity of normal individuals, associated with the early stages of enamel damage and are the main cause of tooth decay. The sugar is broken down to synthesize insoluble Dextran, which aggregates oral bacteria including Mutans, forms plaques, and causes enamel deliming and tooth decay by metabolites (acids) of the plaques.

Staphylococcus aureus is Gram-positive and cocci, and has become a problem as an opportunistic infectious agent in hospitals. Recently, it has been named as a representative superbacteria by MRSA and VRSA. It is a pathogen that causes inflammation and necrosis due to various toxins and various infection sites, but is resistant to penicillin-sensitive Gram-positive bacteria. In 20-30% of adults, it can be found in the nose or skin, but most cases do not cause any problems. However, the infection occurs as the barrier is broken through skin damage or injury. It is related to impetigo, cellulitis, laceration skin syndrome, mastitis, bacteremia, staphylococcal sepsis, staphylococcal pneumonia, endocarditis, and food poisoning coming to the staphylococcus group causes nausea, vomiting, diarrhea and dehydration. do.

With the development of the economy and the improvement of living standards, interest in health is increasing, and preservatives as natural products are attracting attention. Such research has been done before, but no interest or active research has been done. However, as consumers become more aware of the safety issues of chemical synthetic preservatives and their health-oriented desires are growing, the industries are growing rapidly (Mim Kyung-Mim, Kim Mira: Purulane for Food Corruption and Food-Toxic Microorganisms, Methanol) Antimicrobial Activity and Component Analysis of Extracts Korean Journal of Culinary Science 17 (6) .p.565, 2001).

 Natural preservatives currently used in foods include organic acids prepared by ethanol fermentation, spice extracts, pectin degradants, melanoidines, and the like. And natural preservatives containing polymerized phosphate (Gang, Seong-gu, Seongnak-gye, Yongdu Kim, Soo-cheol, Seo Jae-shin, Kap-seong Choi, and Seok-kyu Park: Screening of Antimicrobial Activity of Gat Extract. Korean Journal of Nutrition 23.6 (p1008, 1994). Although there are such natural preservatives, compared to artificial synthetic preservatives, heat, pH, light, etc., and the effect is weak, the practical use in the food industry has become a limit. However, it is significant that the antimicrobial effect is obtained from natural products because consumer demand for natural products is increasing due to the distrust of artificial preservatives. Therefore, many studies on natural preservatives are required, but studies on the antimicrobial effects of harmful microorganisms are insufficient.

Grape seed oil is known to have antioxidant properties as one of the seed oils. BHA is a widely used synthetic antioxidant that has been reported to have antimicrobial properties against S. aureus and E. coli (Perry, NB et al., Essential). oil from new Zealand Manuka and kanuka Chemotaxonomy of Leptospermum. Phtochemistry, 8, pp.1485-1493, 1997).

Laminaria is a genus of brown seaweed kelp, a genus of seaweed, which is distributed along the coast of Hanbok, Korea, and is known to grow in the north east coast, Hamgyongnam-do north of Wonsan, and North Hamgyong in Korea. . It is also distributed in the Pacific coasts of Hokkaido and Tohoku, Japan, Kamchatka Peninsula and Sakhalin Island. Kelp plants grow over 20 species on the Pacific coast. Major species include Laminaria japonica, Laminaria ochotensis, and Laminaria religiosa. Since ancient times, it has been edible in Japan and China.

In addition to various pigments such as carotene, xanthophylls, and chlorophyll, kelp contains a lot of carbohydrates, such as manit and laminarin, which are made by carbon assimilation, and alginic acid, which is a component of cell walls. This contains. The ingredients vary depending on the type, but are generally water 16%, protein 7%, fat 1.5%, carbohydrate 49%, inorganic salts 26.5%, 20% of carbohydrates are fibrin and the rest are polysaccharides such as alginic acid and laminarin. In particular, since there are many inorganic salts such as iodine, potassium, and calcium, it is good to feed kelp a little more often to supply inorganic salts. The amino acid called laminin in kelp is effective in lowering blood pressure.

Accordingly, the present inventors have continuously studied the antimicrobial activity by the extract of kelp (Kjellemaiella crassifolia) or a compound isolated therefrom, seaweed ethanol extract is Escherichia coli, Bacillus subtilis, Candida albicans (Candida albicans), Streptococcus mutans (Streptococcus mutans) and Staphylococcus aureus (Staphylococcus aureus) was confirmed to exhibit excellent antimicrobial activity to complete the present invention.

In order to accomplish the above object, the present invention provides an antimicrobial composition containing an extract of Kelp (Kjellemaiella crassifolia) or a compound isolated therefrom as an active ingredient.

The present invention provides a pharmaceutical composition for the prevention and treatment of bacterial infectious diseases containing Kjellemaiella crassifolia extract or a compound isolated therefrom as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of bacterial infection diseases containing kelp (Kjellemaiella crassifolia) extract or a compound isolated therefrom as an active ingredient.

The antimicrobial composition of the present invention may have the same meaning as an antibiotic which is a generic term for an antimicrobial agent, and may have the same meaning as an antimicrobial agent, a fungicide, a preservative, a preservative or an antimicrobial agent. It means a substance capable of inhibiting or inhibiting function.

The extract as defined herein is water containing purified water, C ₁ to C ₄ lower alcohols, hexane, chloroform, methylene chloride, ethyl acetate, glycerin, butylene glycol or mixed solvents thereof, preferably methanol, water or these It is included in the mixed solvent of, more preferably 50% to 100% methanol, even more preferably 80% to 100% methanol.

Compounds isolated from the extract of Kjellemaiella crassifolia as defined herein are 5,7,8-trihydroxyflavones and 5,7,8-trihydroxyflavone-7-0-glucuro Include needs.

Bacterial infectious diseases defined herein are specifically laryngitis, prostatitis, dermatitis, food poisoning, keratitis, osteomyelitis, meningitis, pneumonia, influenza or tuberculosis due to bacterial infection, preferably food poisoning, dermatitis, pneumonia or influenza, More preferably food poisoning or dermatitis.

The antimicrobial composition defined herein may be an antimicrobial composition having antimicrobial activity against pathogenic microorganisms, specifically Gram-positive bacteria or Gram-negative bacteria, and more specifically, Edwardsiella sp . , Vibrio sp . ), Micrococcus sp., Enterobacter sp., Klebsiella sp., Pseudomonas sp . Salmonella sp., Escherichia At least one selected from the group consisting of Escherichia coli , Bacillus subtilis, Candida albicans, Streptococcus mutans or Staphylococcus aureus the antimicrobial composition having an antimicrobial activity for what, and preferably Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Candida Albi Antibacterial for scan (Candida albicans), Streptococcus mutans (Streptococcus mutans) or Staphylococcus aureus (Staphylococcus aureus), and more preferably Escherichia coli (Escherichia coli) or Bacillus subtilis (Bacillus subtilis) It is an antimicrobial composition having activity.

Hereinafter, the method for obtaining the extract of the present invention will be described in detail.

The extract of the present invention is pulverized dried kelp (Kjellemaiella crassifolia) extract and water containing purified water as extraction solvent, C ₁ to C ₄ lower alcohol, hexane, chloroform, methylene chloride, ethyl acetate, glycerin, butylene glycol Or a mixed solvent thereof, preferably methanol, water or a mixed solvent thereof, more preferably 50% to 100% methanol, about 0.1 to 50 times (v / w), preferably 1 to 25 times the weight of the sample. (v / w), more preferably 2 to 10 times (v / w), and added at 10 ° C to 50 ° C, preferably at 20 ° C to 40 ° C, more preferably at about 30 ° C for about 10 minutes to 2 hours. Extraction methods such as cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, heating extraction, preferably by ultrasonic extraction, and then filtered, concentrated under reduced pressure and dried to obtain the extract of the present invention.

Silica-gel column chromatography, high performance liquid chromatography (Shimadzu LC-20A, Shimadzu, Kyoto, Japan) to purify fractions and purified while confirming their activity to 5,7,8-trihydroxyflavone (norwogonin) or 5,7,8-trihydroxyflavone-7- Compounds of the present invention can be obtained, such as 0-glucuronide compounds and the like.

The present invention provides a pharmaceutical composition for the prevention and treatment of bacterial infectious diseases, containing kelp (Kjellemaiella crassifolia) extract obtained by the above method or a compound isolated therefrom as an active ingredient.

Kelp (Kjellemaiella crassifolia) extract of the present invention or a pharmaceutical composition containing a compound isolated therefrom comprises 0.1 to 50% by weight of the extract or compound based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

Since the extract or compound itself of the present invention has little toxicity and side effects, it is a medicament that can be used safely even for prolonged administration for the purpose of prevention.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents that may be included in the composition comprising the extract or compound 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. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the extract or compound. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract or compound of the present invention is preferably administered at 0.5 g / kg to 5 g / kg, preferably 1 g / kg to 3 g / kg per day. The administration may be carried out once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In another aspect, the present invention provides a health functional food for the prevention and improvement of bacterial infection diseases containing kelp (Kjellemaiella crassifolia) extract obtained by the above method or a compound isolated therefrom as an active ingredient.

The composition containing the extract or the compound of the present invention can be used in a variety of drugs, food and beverages for the prevention and improvement of bacterial infection diseases. Foods to which the extract of the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and can be used in the form of powders, granules, tablets, capsules, or beverages. have.

Extracts or compounds of the present invention may be added to foods or beverages for the purpose of preventing and ameliorating bacterial infectious diseases. At this time, the amount of the extract or compound in the food or beverage is generally added to the health food composition of the present invention 1 to 5% by weight of the total food weight, the health beverage composition is 0.02 to 10 g based on 100 ml, Preferably it can be added in the ratio of 0.3-1 g.

The health beverage composition of the present invention, in addition to containing the extract or compound as an essential ingredient in the indicated ratio, there is no particular limitation on the liquid component, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 mL of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Kjellemaiella crassifolia extract of the present invention or compounds isolated therefrom are Escherichia coli, Bacillus subtilis, Candida albicans, Streptococcus mutans And because it exhibits antimicrobial activity against Staphylococcus aureus (Staphylococcus aureus) , it can be used in pharmaceutical compositions and health functional foods useful for the prevention and treatment of bacterial infectious diseases.

Hereinafter, the present invention will be described in detail by reference examples, examples and experimental examples.

However, the following Reference Examples, Examples, and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Reference Examples, Examples, and Experimental Examples.

Example 1 Preparation of Kelp Methanol Extract

Kelp is a species of wild kelp (Kjellemaiella crassifolia), which is dried and purchased at Jumunjin fish market, and then used to make powder.

100 g of the dried seaweed powder prepared above was placed in a 1000 mL Erlenmeyer flask, 500 mL of 95% methanol was added and mixed, followed by ultrasonic extraction at 30 ° C. for 30 minutes (sonication: Power SONIC 520 HWASHIN). The extracted extract was filtered through filter paper (1 mm-pore-size, Whatman No. 2) to obtain an ethanol extract, concentrated under reduced pressure at 40 ° C. with a rotary depressurizer (Rotavator: R-200, Buchi, switzerland) and freeze-dried ( Freeze-drying with PVIFD30A, Korea Ilshin Lab) 1.7g of the kelp methanol extract (hereinafter referred to as "KC95M") of the present invention was obtained, and stored at -40 ℃ or less, was used as a sample of the following experimental example.

Example 2 Separation of Active Ingredients from Kelp Methanol Extract

2-1. Silica gel column chromatography

Checking by TLC for the purpose of searching and separating the active ingredient from the kelp methanol extract obtained in Example 1 (see FIGS. 1 and 2), silica-gel column chromatography (Merck KGaA 64271 Darmstadt, Germany) Was purified using 94% methanol (MeOH: water = 70:30) as a developing solvent and purified into 10 fractions, and compared with known components by UV detector (Agilent Technologies, Palo Alto, CA, USA). The containing component was confirmed (see FIGS. 8-9).

2-2. High Performance Liquid Chromatography

In order to further purify the active fractions having excellent antibacterial effect among the five fractions obtained in Example 2-1, that is, fractions 2a and 2b (see FIGS. 3 to 4), high performance liquid chromatography (High performance) liquid chromatography; Shimadzu LC-20A, Shimadzu, Kyoto, Japan) were used, and the analytical column was used as a C column, and fractionated into five purified fractions by mixing Methanol and water as organic solvents (70:30). Purifying while confirming their activity, 5,7,8-trihydroxyflavone (norwogonin (hereinafter referred to as "FG1-2a") and 5,7,8-trihydroxyflavone (Trihydroxyflavone) having the following physical properties ) -7-0-glucuronide compound (hereinafter referred to as "FG1-2a") was purely separated (see FIGS. 5 to 7) and confirmed by comparing with physical data described in the literature. (Tom J. MABRY.The systematic Identification of Flavonoids p18--19, 19 70).

UV SPECTRAL DATA (λ nm); (Data of Formula 1) MeOH (281, 364sh); NaOMe (246, 274 (dec.)); AlCl (292 sh, 315, 366 sh); AlCl / HCl (290 sh, 302, 342 sh, 395 sh); NaOAc / HBO (287)

UV SPECTRAL DATA (λ nm); (Data of Formula 2) MeOH (247, 274, 315sh, 342sh); NaOMe (236sh, 281, 357); AlCl (252, 286 sh, 292, 331, 387); AlCl / HCl (248sh, 283sh, 289, 327. 387); NaOAc (264sh, 281, 366); NaOAc / HBO (287)

Reference Example 1. Preparation of Experimental Strains

For antimicrobial screening, Escherichia coli (KCTC7039), Bacillus subtilis (KCTC1021), Candida albicans (KCTC7270), Streptococcus Mutans (Streptococcus mutans (KCTC3065)) and Staphylococcus aureus (KCTC1621) were distributed and used in the following experiments. The bacteria were used by subculture ( Candia albicans : 24 ° C, Bacillus subtilis : 30 ° C, Streptococcus mutans : 37 ° C, Staphylococcus aureus : 37 ° C, Escherichia coli : 37 ° C) .

Experimental Example 1. Determination of antimicrobial activity of methanol extract of kelp

1-1. Determination of antimicrobial activity using paper disc

The test as described below by applying the paper disk method described in the literature in order to search the obtained antibacterial activity of the samples in Examples were conducted ((Piddock, LJV;. Techniques used for the determiration of antimicrobial resistance and sensitivity in bacteria J Appl Bacteriol, 68: 307, 1990).

The strain prepared in Reference Example 1 was inoculated in 5 ml of saline solution by taking 1 platinum for each strain, and 1 ml of this active solution was 4-5 mm thick [ Candida albicans -YM Agar (271210. BD) , Bacillus subtilis -Nutrient Agar (213000 BD), Streptococcus mutans-Brain Heart (241830 BD), Staphylococcus aureus -Tryptic Soy Agar (236950 BD), Escherichia coli -Nutrient Agar (213000 BD) ), Incubator: Eyela, multi Thermo incubator. MTI-202], spread evenly with bent glass rods, place sterilized 6.0 mm filter paper discs (Whatman AA Discs) on the medium, and kelp methanol extract (UP95M), 95% Methanol, and distilled water 1: 5: Samples prepared at 4 (v / v / v) were injected at concentrations of 500 μg / ml, 1000 μg / ml, 1500 μg / ml, 2000 μg / ml and 2500 μg / ml. The solvent of the injected extract was volatilized and incubated at 37 ° C. for 48 hours to measure the diameter of the inhibition zone generated around the paper disc.

1-2. Antimicrobial Activity Determination Using Minimum Growth Inhibition Concentration (MIC)

Kudo et al. (Kudo, T. and Safa, N., Development of a simple method for antibiotic) described in the literature to determine the antimicrobial activity of the kelp methanol extract (KC95M) obtained in the above example and the compounds isolated therefrom susceptibility testing in algae using paper disks.Nippon Suisan Gakkaishi , 56 , p.455, 1990) was used to determine the minimum inhibitory concentration (MIC) as follows.

500 μg / ml, 1000 μg / ml, 1500 μg / ml, 2000 μg / ml of the sample solution prepared with kelp methanol extract (KC95M), 95% Ethanol and distilled water at 1: 5: 4 (v / v / v), respectively , Medium adjusted to a concentration of 2500 μg / ml ( Candida albicans -YM Agar (271210. BD), Bacillus subtilis -Nutrient Agar (213000 BD), Streptococcus mutans-Brain Heart (241830 BD), Staphylo Caucus Aureus -Tryptic Soy Agar (236950 BD), Escherichia coli -Nutrient Agar (213000 BD), Incubator: Eyela, multi Thermo incubator.MTI-202) were aliquoted onto plates and allowed to coagulate. Herein, the strain prepared in Reference Example 1 was inoculated into 5 ml of saline solution by taking 1 platinum for each strain, and 2 ml of this active solution was dispensed into the medium, and then cultured at 37 ° C. for 48 hours ( Candida albicans : 24 ° C.). , Bacillus subtilis : 30 ℃, Streptococcus mutans : 37 ℃, Staphylococcus aureus : 37 ℃, Escherichia coli : 37 ℃) to determine the growth of microorganisms to determine the minimum growth inhibition concentration. .

The experimental results, the growth inhibitory effect with respect to the water layer (water layer), and the fractions were Candida albicans (C. albicans), Escherichia Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) 5,7,8-trihydroxyflavone (Formula 1) and 5,7,8-trihydroxyflavone (-7) isolated from the fractions -Glucuronide compound (Formula 2) (FG1-2a and FG1-2b and the compound has a similar molecular structure, but FG1-2b is a glycoside) Candida albicans ( C. albicans), Escherichia coli exhibited a higher growth inhibitory effect against a (E. coli) and Staphylococcus aureus (S. aureus) (see Table 4).

E.Coli S.aureus C.albicans Sagunjin Dashima Organic ext. - - - Methanol ext. 16mm / 2mg 17mm / 2mg 13mm / 2mg

Hereinafter, a preparation example of a composition including the kelp methanol extract (KC95M) of the present invention will be described, but the present invention is not intended to be limited thereto but only to be described in detail.

Formulation Example 1 Formulation of Powder

Kelp methanol extract (KC95M) 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

Kelp methanol extract (KC95M) 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

Kelp methanol extract (KC95M) 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Kelp methanol extract (KC95M) 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ , 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Kelp methanol extract (KC95M) 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

Kelp methanol extract (KC95M) 1000 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation Example 7 Preparation of Healthy Drink

Kelp methanol extract (KC95M) 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

1 is a diagram showing a typical TLC aspect for silicic acid VLC of the methanol extract of the present invention;

FIG. 2 shows a typical TLC pattern for silicic acid VLC (coating the crude extract with celite and in one test) of the methanol extract of the present invention; FIG.

3 is a diagram showing the Sep-PAK fractionation process of the main antibacterial fractions for VLC;

4 shows a typical TLC pattern for Sep-PAK fractionation of VLC Fraction 7 (VLC fr. No. 7) of the present invention;

5 is a diagram showing the HPLC chromatogram pattern of the antimicrobial active fraction of the methanol extract of the present invention;

6 is a diagram showing the HPLC chromatogram pattern of the antimicrobial active fraction of the methanol extract of the present invention;

7 is a view showing a second HPLC chromatogram of the antimicrobial activity fraction of the methanol extract of the present invention;

8 shows the UV spectral profile of peak 2a of the FG1 fraction separated by HPLC chromatogram;

FIG. 9 shows UV spectral profile for peak 2b of an FG1 fraction isolated by HPLC chromatogram. FIG.

Claims (9)

Escherichia coli, Candida albicans containing 5,7,8-trihydroxyflavone (norwogonin) or 5,7,8-trihydroxyflavone-7-0-glucuronide as an active ingredient Antimicrobial composition against strains. delete delete delete Escherichia coli, Candida albicans containing 5,7,8-trihydroxyflavone (norwogonin) or 5,7,8-trihydroxyflavone-7-0-glucuronide as an active ingredient Pharmaceutical composition for the prevention and treatment of food poisoning or dermatitis by. delete delete Escherichia coli, Candida albicans containing 5,7,8-trihydroxyflavone (norwogonin) or 5,7,8-trihydroxyflavone-7-0-glucuronide as an active ingredient Health functional food for the prevention and improvement of food poisoning or dermatitis by. delete

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