KR101791549B1 - An antibacterial composition comprising the pachyrhizus erosus root bark extract as an effective component - Google Patents

Abstract

The present invention relates to a process for the production of Pyridoxin The present invention relates to an anti-bacterial composition containing an extract of erosus root bark as an active ingredient. More specifically, the present invention relates to an anti-bacterial composition comprising an extract of an extract of a root of a yamvin root (root roots) and a hexane fraction prepared by fractionation thereof, Fractions or mixtures thereof as an active ingredient. The antimicrobial composition containing the Yambin bottom skin extract of the present invention as an active ingredient can be used for the treatment of Gram-positive bacteria and Proteus In particular, the antimicrobial activity of Gram-negative bacteria such as Lactobacillus vulgaris is excellent. In particular, it has excellent thermal stability and does not exhibit a decrease in antibacterial activity even in an acidic condition of pH 2 and plasma, It is a very useful invention to be applied to various applications such as industrial disinfectants, industrial preservatives, antimicrobial household products, antimicrobial foods or pharmaceutical additives as well as pharmaceutical compositions, health functional foods, animal feed additives and the like.

Description

AN ANTIBACTERIAL COMPOSITION COMPRISING THE PACHYRHIZUS EROSUS ROOT BARK EXTRACT AS AN EFFECTIVE COMPONENT As an effective ingredient,

The present invention relates to a process for the production of Pyridoxin The present invention relates to an anti-bacterial composition containing an extract of erosus root bark as an active ingredient. More specifically, the present invention relates to an anti-bacterial composition comprising an extract of an extract of a root of a yamvin root (root roots) and a hexane fraction prepared by fractionation thereof, Fractions or mixtures thereof as an active ingredient.

Humans and microorganisms have coexisted with each other for hundreds of millions of years in a symbiotic or competitive relationship. In particular, bacteria have been used to produce fermented foods such as Chungkukjang, Kimchi, medicines such as antibiotics, raw chemical materials such as ethanol, agricultural raw materials and so on. On the other hand, some germs are known to cause food poisoning or acute or chronic diseases in humans, causing food corruption, corrosion of industrial facilities and personal and social losses. In this regard, mankind has been continuously developing disinfectants, fungicides, antibiotics, etc., and has developed sterilization methods such as high temperature, freezing, and irradiation.

However, in recent years, the efficiency of chemicals and antibiotics capable of selectively disinfecting and sterilizing germs has been greatly reduced, which is due to the emergence of resistant bacteria to various conventional antibiotics and disinfectants. In particular, the emergence of multiple drug resistance bacteria that exhibit resistance to two or more antibiotics with different chemical structures and different mechanisms of action makes existing antibiotics and disinfectants meaningless. Furthermore, antibiotic resistant bacteria such as Methicillin-Resistant Staphylococcus Aureus (MRSA) and Vancomycin-Resistant Staphylococcus Aureus (VRSA) are so called super bacteria that they are practically impossible to control It is known. In 2011, the Korea Centers for Disease Control and Prevention (KIDI) established the Korean Agency for Disease Control and Prevention (CDC) in Korea for the establishment of the Korean Agency for Research on Inflammatory Diseases Intestinal bacteria were included in designated infectious diseases, which are difficult to control. Therefore, it is very urgent to develop antibiotics that exhibit broad antibacterial activity against new new antibiotics, especially various food poisoning bacteria and pathogenic bacteria.

On the other hand, it is necessary to develop an antimicrobial agent for control of harmful microorganisms according to the global warming phenomenon, such as food corruption and an increase in the propagation rate of pathogens, and it is urgent to develop a new antimicrobial agent safely in the food industry as well as in the pharmaceutical industry. In the food industry, various types of processed foods are being developed due to the development of the industry. As food consumption speeds up, safety management of food corruption is very important in processing, distribution and storage of food. In addition, as the proportion of group foodservice and foodservice industry increases, there is an increasing interest in efficient control of foodborne microorganisms that may present a hazard to various foods. Presently, in Korea, preservatives are used to inhibit the growth of harmful food poisoning microorganisms and to prolong the shelf life of food. Thirteen kinds of synthetic preservatives including benzoic acid are approved under the Korean Food Sanitation Act. However, the continuous use of these synthetic preservatives increases the risk of chronic toxicity, carcinogenesis and mutagenesis. Therefore, there is a limit to the use of synthetic preservatives. Also, it is necessary to strengthen the legal regulations on the use of synthetic preservatives and increase the desire for safety and health of consumers. And the avoidance of the use of the synthetic preservative agent is present. Therefore, it is urgently required to develop natural antimicrobial agents to solve these problems and to improve the preservability of foods. Recently, researches on antibacterial active substances and identification of active substances from native and edible plant extracts have been actively conducted.

Pachyrhizus erosus is a species of subtropical legumes derived from Mexico and Central America and is known as Mexican Yam bean, Mexican turnip, Mexican potato or Jicama , Is resistant to insect pests and is easy to cultivate. It is an important edible root plant now widely cultivated not only in Mexico but also in the whole world. Yambin, as its name implies, has edible yam in the ground and beans in the ground. The beans on the ground are mature and contain insecticides called rotenone, which are toxic and can not be used for food (Estrella-Parra EA et al., 2014, Natural Product Res. 28: 1780-1785). The yambin's root mass is wrapped in a thick skin that is easily peeled off by hand like a banana. White peeled husk with its crust removed has crispy texture and apple flavor and pear flavor. It can be used raw, pickled, . In Korea, YAMBIN was cultivated for the first time in Andong, Gyeongbuk Province in 2009 (Kim SK et al., 2009, Korean J. Plant Res. 22, 546-551) Is increasing.

To date, most of the researches related to yambin have been focused on the nutritional analysis of yambin, the toxicity study of yambin seed, the cultivation and storage of yambin (Cantwell M, et al., 1998, American Society for Horticultural Science, Studies on the utilization of starch of yambin and the physiological activity of extract have been reported. Yumbin is known as a low-calorie diet food. Yambin's basal part contains 86-90% of moisture and trace amount of protein, very small amount of lipid component, and contains a large amount of dietary fiber. It shows 37kcal per 100g, Is known to be indicative of the calorie of. In addition, carbohydrates contain inulin, which exhibits diabetic prophylaxis and probiotic effects in addition to starch. It contains 317 mg of calcium per 100 g, 150 mg of potassium, and 93 mg of phosphorus to show a very rich mineral content (Ramos-de-la -Pena AM et al., 2013, Trends in Food Sci% Technol. 29: 44-54). In addition, phytin, lectin, and HCN have been identified as non-insoluble and have been found to be similar in content to potatoes and sweet potatoes (Noman ASM et al., 2007, Food Chemistry, 102: 1112-1118). In addition, it contains vitamin C and mucin in a large amount similar to hemp (mildew), and is known to have excellent skin protection, regeneration and moisturizing effect. The underground skin is also used as a mask pack in a private sector. YAMBIN's starch research has been mainly focused on the characterization of YAMBIN starch itself (Melo EA et al., 2003. Bioresource Technology, 89: 103-106) and the decomposition products of starch (Ramos-de-la-Pena AM et al., 2012, LWT -Food Sci & Technol. 46: 232-238), and studies are being conducted mainly on food characteristics. On the other hand, studies on the seeds of yamvin have been carried out on seed proteins (Morales-Arellano GY. Et al., J. Agric Food Chem. 49. 1512-1516) and seed lipid studies. However, rotenone (Estrella-Parra EA et al., 2014, Natural Product Res. 28: 1780-1785).

Studies on the useful physiological activity of yambin have been limited, and the activities known to date are antioxidant activity and wrinkle improvement effect (Kwon Bong-hee, 2015, wrinkle improvement and antioxidative effect of human fibroblast using yacon and yambin extract, (Phrutivorapongkul et al., 2002, Chem Pharm Bull (Tokyo), 50, 534-7), anti-diabetic activity (Park CJ and Han JS, 2015. Prev Nutr Food Sci. 537), the immune-enhancing activity of the extract of yambin dietary fiber (Kumalasan ID et al., 2013. J. Functional Foods 5: 582-589; Kumalasari ID et al., 2014. Cytotechnology 66, 75-85) and the breast cancer cell growth of yamvin-derived phytoestrogen (Nurrochmad A et al., 2013. Asian Pacific J. Tropical Biomedicine. 3, 847-852). However, no research papers or presentations related to the antibacterial activity of the yam bark have been reported to date.

In addition, about 7 domestic and foreign patents related to yambin are known, and cosmetic compositions based on yambin's antioxidant ability, skin regenerating ability and moisturizing effect are most known. Korean Patent Laid-Open Publication No. 10-2014-0079571 discloses a composition for external application for skin using a yamvin extract, which discloses a composition for external skin application for promoting skin regeneration, promoting collagen biosynthesis, inhibiting wrinkle formation, whitening and moisturizing using yambin extract And Korean Patent No. 10-1458887 (2014-10-31) discloses a skin improving agent which has antioxidant, moisturizing effect and skin irritation mitigation effect containing Hikoma, Cauliflower, Marlow and Icelandic moss extract as active ingredients "Is known. In Japan, "skin external preparation" is disclosed in JP 2004-352658 (2004, 12, 16), "Hair tonic composition" is disclosed in JP-0354523, and "Skin cosmetic" is disclosed in JP- It is known that Yvin has proven its usefulness as a skin cosmetic material. In recent years, Korean Patent Laid-Open Publication No. 10-2015-0006970 (2015-01-20) discloses a composition for preventing or treating constipation containing Allium fucry's and paclitazone erosus as an active ingredient, And JP-0236531 discloses a concentrated seasoning liquid and method for producing the same, which uses yambin starch. However, the patent related to the antimicrobial activity of the chestnut shell has not been known to date.

KR 10-2014-0079571 A KR 10-1458887 B JP 2004-352658 A

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide an antibacterial composition containing an effective ingredient from an extract obtained from the shell of a rootstock of yambin.

In order to solve the above problems, the present invention provides an antibacterial composition containing an extract of Pachyrhizus erosus root bark as an active ingredient.

The extract is preferably an ethanol extract.

Preferably, the extract is a hexane fraction, an ethyl acetate fraction, or a mixture thereof, of the ethanol extract.

The antimicrobial composition is preferably one selected from the group consisting of a pharmaceutical composition, a health functional food, a livestock feed additive, an industrial disinfectant, an industrial preservative, an antimicrobial household product, an antibacterial cosmetic product and an antibacterial food or a pharmaceutical additive.

Wherein containing yambin below-ground bark extract of the present invention as an active ingredient, bacterial composition, as evidenced by the embodiment of the present specification, the gram-positive bacteria and Proteus vulgaris (Proteus vulgaris) and shows a strong antibacterial effect in the same gram-negative bacteria In particular, it is excellent in heat stability and does not exhibit a decrease in antibacterial activity even in an acidic condition of pH 2 and plasma, and is processed into various forms such as extract, powder, ring, and tablet to provide a pharmaceutical composition, a health functional food, And the like, as well as industrial disinfectants, industrial preservatives, antimicrobial household products, antimicrobial foods or pharmaceutical additives, and the like.

FIG. 1A is a graph showing the anti-bacterial activity of the ethyl acetate fraction of the extract of Staphylococcus aureus against Staphylococcus aureus , showing the change in the degree of growth according to the incubation time after addition of the active substance. In the graph,?: No addition,?: Addition of 0.031 mg / ml ethyl acetate fraction,?: Addition of 0.062 mg / ml ethyl acetate fraction, and?: Addition of 0.125 mg / ml ethyl acetate fraction.
Figure 1b is jeneseu L. monocytogenes (Listeria monocytogenes ), showing the change in growth according to the incubation time after addition of the active substance. In the graph,?: No addition,?: Addition of 0.031 mg / ml ethyl acetate fraction,?: Addition of 0.062 mg / ml ethyl acetate fraction, and?: Addition of 0.125 mg / ml ethyl acetate fraction.
FIG. 1C is a graph showing the antimicrobial activity of the ethyl acetate fraction of Bombyx mori extract against Bacillus subtilis , showing the change in the degree of growth according to the incubation time after addition of the active substance. In the graph,?: No addition,?: Addition of 0.031 mg / ml ethyl acetate fraction,?: Addition of 0.062 mg / ml ethyl acetate fraction, and?: Addition of 0.125 mg / ml ethyl acetate fraction.
FIG. 1D is a graph showing the antibacterial activity of the ethyl acetate fraction of the extract of Bombyx mori, against Staphylococcus epidermidis , showing the change in growth according to the time of incubation after addition of the active substance. In the graph,?: No addition,?: Addition of 0.031 mg / ml ethyl acetate fraction,?: Addition of 0.062 mg / ml ethyl acetate fraction, and?: Addition of 0.125 mg / ml ethyl acetate fraction.
Figure 1e is a Proteus vulgaris (Proteus The results are shown in the graphs showing the antibacterial activity of the ethyl acetate fraction of the extract of Bombyx mori. In the graph,?: No addition,?: Addition of 0.031 mg / ml ethyl acetate fraction,?: Addition of 0.062 mg / ml ethyl acetate fraction, and?: Addition of 0.125 mg / ml ethyl acetate fraction.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition containing a yamvin bottom skin extract as an active ingredient, a gram-positive bacterium, and a Proteus wherein due to the effect of inhibiting the growth of gram-negative bacteria of the vulgaris) relates to a bacterial composition.

The inventors of the present invention have found that the extract of the bottom part of yamvin obtained by a certain method shows a strong antibacterial activity and confirmed that the extracts, especially the ethanol extract, exhibit more potent antibacterial activity in the hexane fraction and the ethyl acetate fraction, The components were mixed with Gram positive bacteria and Proteus The present invention relates to an antimicrobial composition capable of inhibiting gram-negative bacteria of the genus Escherichia, vulgaris .

Specifically, the present inventors prepared an ethanol extract of yam bran rootstock skin in order to develop an antimicrobial substance using the yam bran bottom shell, and sequential fractionation with hexene, ethyl acetate and butanol to obtain respective fractions, Negative bacteria, gram-positive bacteria and fungi were evaluated. As a result, it was found that Gam-positive bacteria and Proteus vulgaris ) against gram-negative bacteria.

In addition, the extract of the bottom part of yam bin and the organic solvent fractions thereof were excellent in heat and acid stability, and showed no hemolytic activity against human erythrocytes and did not show acute toxicity, thereby completing the present invention.

Accordingly, the present invention provides an antimicrobial composition comprising an extract of Zambin bark as an active ingredient.

Hereinafter, the extract of the bottom part of the yamvin of the present invention, the method for producing the hexane fraction, the ethyl acetate fraction and the efficacy test will be described in more detail.

The present invention provides a method for preparing an extract, comprising the steps of: preparing an extract from the yam bark bottom; Adjusting the hexane fraction, the ethyl acetate fraction, the butanol fraction, and the water residue after the sequential organic solvent fraction from the yamvin bottom bark extract; Checking the efficacy and stability of the fractions; And examining the minimal inhibitory concentration of the fraction under various conditions.

In the composition of the present invention, " Yambin underground bark extract "is obtained by peeling the bark of Yambin under the skin with a hand or using a tool, drying it by hot air drying to be suitable for extraction, Extraction with a mixed solvent of an organic solvent, filtration of the extract using a filter net of 0.06 mm or less, and concentration under reduced pressure. The solvent used in the present invention is water (cold water or hot water); An organic solvent such as alcohol, alcohol, or anhydrous or hydrous alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol); Or a mixed solvent of water and an organic solvent, and most preferably ethanol that can be used for the production of food ingredients.

In the present invention, Gam-negative bacteria, Gram-positive bacteria and fungal inhibitors against the ethanol extract of the yamvin bottom shell produced according to the preparation method of the present invention and the hexane fraction, ethyl acetate fraction, butanol fraction and water residue sequentially fractionated therefrom Activity was assessed through growth inhibition ring experiments. As a result, it was confirmed that the bark extract and its hexane fraction and ethyl acetate fraction can effectively inhibit the proliferation of Gram-positive bacteria and some Gram-negative bacteria.

The yamvin bottom skin extract of the present invention and its hexane fraction and ethyl acetate fraction can be powdered by a conventional powdering process such as vacuum distillation and freeze drying or spray drying. They maintain their activity even at 100 ° C heat treatment and pH 2 in human body.

Since the extract of the bottom part of the yamvin of the present invention has a strong antibacterial activity, it can be widely used as antimicrobial use in various industrial fields for blocking bacterial infection. For example, the industry includes pharmaceutical compositions for the prevention or treatment of bacterial diseases; Health functional foods for the prevention or improvement of bacterial diseases; Livestock feed additives for the prevention of bacterial diseases; Industrial disinfectants to block bacterial infections; An industrial preservative capable of preventing contamination of objects from bacterial infection and securing a preservation period; Antimicrobial household goods such as office supplies, kitchen utensils and toiletries; And antibacterial food or pharmaceutical additive, but are not limited thereto.

As a preferred embodiment, the Yambin bottom skin extract of the present invention can be used as an active ingredient of a pharmaceutical composition for the prevention or treatment of an infectious disease. It is to be understood that the infectious disease may include bacterial infections and related complications.

The pharmaceutical composition of the present invention may be formulated into various forms such as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, injections of sterilized injection solutions, And can be administered by various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like. Such pharmaceutical compositions may further comprise carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, But are not limited to, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, And the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount of the active ingredient of the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and is generally 1 to 5,000 mg, preferably 100 to 3,000 mg per day, It can be administered every other day or one to three times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like. The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections. In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is either oral or non-oral May be administered orally. The composition of the present invention may also be administered using any device capable of delivering an effective ingredient to a target cell. In the present invention, the term "object" includes, but is not limited to, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig , Preferably a mammal, more preferably a human.

As a preferred embodiment, the active ingredient of the present invention can be used as a health functional food for preventing or improving bacterial infectious diseases.

The health functional food of the present invention can be variously used for foods and beverages effective for prevention or improvement of bacterial infectious diseases. Examples of foods containing the active ingredient of the present invention include various foods, beverages, gums, tea, vitamin complex, health supplement foods and the like, and they can be used in the form of powder, granule, tablet, capsule or beverage . The active extract of the present invention may generally be added in an amount of 0.01 to 15% by weight of the total food, and the health beverage composition may be added in an amount of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml. The health functional food of the present invention may contain, as an essential ingredient, the above-mentioned active ingredient in a prescribed ratio, as well as a food-acceptable food supplementary additive such as natural carbohydrate and various flavoring agents as an additional ingredient. Examples of the natural carbohydrate include sugar sugars such as glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of the flavor agent include tau martin, rebaudioside A, glycyrrhizin, saccharin, and aspartame. The proportion of the above-mentioned flavoring agent is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention may contain various kinds of nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and heavy stabilizers, pectic acid and its salts, alginic acid and its salts, Thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for producing natural fruit juice, fruit juice drink, vegetable drink and the like. These components may be used independently or in combination. The ratio of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active fraction of the present invention.

In a preferred embodiment, the active ingredient of the present invention can be used as a livestock feed additive.

The use of the active ingredient of the present invention as a livestock feed additive can be applied to livestock feed in an appropriate amount depending on the type of livestock, concern of bacterial infection, environmental factors, and preferably 0.1 to 10 wt% May be added.

The livestock may include, but is not limited to, cattle, pigs, chickens, goats, ducks, sheep, turkeys, ostriches, dogs, cats and the like.

In a preferred embodiment, the active ingredient of the present invention can be used as an industrial disinfectant.

The disinfectant is intended to block bacterial infections of a subject, for example, a manufacturing industry, a medical industry, and an animal husbandry industry. For example, 100 parts by weight of the powder of the active ingredient of the present invention may be mixed with 100 to 100,000 parts by weight of distilled water.

In a preferred embodiment, the active ingredient of the present invention may be used as an industrial preservative.

For example, 100 parts by weight of the powder of the active ingredient of the present invention may be mixed with 1,00 to 100,000 parts by weight of distilled water. The preservative may be used in a variety of industrial devices, devices, You can use it.

In a preferred embodiment, the active ingredient of the present invention can be used for the purpose of antimicrobial household products.

The antimicrobial household article may be a commodity such as a kitchen cleaner, a bathroom cleaner, a toilet or the like, but is not limited thereto.

The active ingredient of the present invention may vary depending on the purpose of use, but it may preferably be contained in an amount of 0.1 to 20% by weight in the antimicrobial household article.

As a preferred embodiment, the active ingredient of the present invention may be applied as an antimicrobial food or pharmaceutical additive.

The active ingredient of the present invention may vary depending on the purpose of use, but it may preferably be contained in an amount of 0.1 to 10% by weight of the antimicrobial food or medicinal product.

Hereinafter, the specific method of the present invention will be described in more detail by way of examples. The following examples are only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[Example]

Example  One: Yimbin  Underground Edible portion  And peel extracts

After peeling off the husk from the submerged root of yambin produced in Andong, Gyeongbuk Province in 2014, it was dried in a 60 ° C drier for 2 days and then powdered to be suitable for extraction. 10 times volume of ethanol (95% Daejung Chemical Co., Korea) was added and extracted three times at room temperature for 2 days. On the other hand, the peeled edible part was also prepared in the same manner as above. Each of the extracts prepared was concentrated under reduced pressure to give a powder, which was stored at low temperature until used.

The total flavonoid, total polyphenol, total sugar and reducing sugar content of the extracts of extracts from the bottom part of the prepared yamvin and edible parts were measured. To determine total flavonoid content, each sample was stirred for 18 hours in methanol, 400 ml of 90% diethylene glycol was added to 400 μl of the filtered extract, 40 μl of 1N NaOH was added, and the absorbance at 420 nm was measured at 37 ° C for 1 hour. As a standard reagent, rutin was used. Total polyphenol content was determined by adding 50 μl of Folin-ciocalteau and 100 μl of saturated Na ₂ CO ₃ solution to 400 μl of the extract solution, leaving it at room temperature for 1 hour and measuring the absorbance at 725 nm. Tannic acid was used as a standard reagent. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method. The results are shown in Table 1 below.

As a result, the moisture content and the ethanol extraction efficiency of the submerged part of yamvin were relatively lower than that of the edible part of the yamvin, and the extract of yamvin contained a very high content of polyphenol component of 224.4 mg / g, The content of polyphenol in the extract of the undergrowth was 37 times higher than that of the extract. The content of total flavonoids was 13 times higher than that of the edible part extracts. On the other hand, the bark extract showed lower total sugar and reduced sugar contents than the edible part extract.

Example  2: Yimbin  Underground Edible portion  And peel extract

The antimicrobial activity of the ethanol extract of the edible part and the skin of the yamvin root part of Example 1 was evaluated and the results are shown in Table 2. The antimicrobial activity of the extract was evaluated according to the previously reported method (Kim, Mi Sun et al., 2015. Korean Journal of Life Science, 25: 757-764). Nutrient agar (Difco Co., USA) The medium was evaluated by disc-diffusion method using Sabouraud dextrose (Difco Co. USA) medium for antifungal activity. Escherichia coli KCTC 1682, Proteus was used as an vulgaris KCTC 2433, Salmonella typhimurium KCTC 1926 , Pseudomonas aeruginosa KACC 10186, Gram-positive bacteria are Staphylococcus aureus KCTC 1916, Listeria monocytogenes KACC 10550, Bacillus subtilis KCTC 1924, and Staphylococcus epidermidis ATCC 12228 were used. To evaluate the antifungal activity, Saccharomyces cerevisiae IF0 0233 and Candida albicans KCTC 1940, a causative agent of fungus infections.

In order to evaluate the antibacterial activity, each strain was inoculated into Nutrient broth (Difco Co., USA), and cultured at 37 ° C for 24 hours. Each strain was adjusted to OD6000.1 and cultured on Nutrient agar (Difco Co., (Diameter: 6.5 mm, Whatsman No. 2) was added to each well of a sterilized petri dish (90 × 15 mm, Green Cross, Korea) containing 5 μl of each sample and incubated at 37 ° C. for 24 hours . In the case of fungi, Sabouraud dextrose (Difco Co., USA) was cultured in the same manner at 30 ° C for 24 hours, and the size of the growth inhibition rings was measured to evaluate the antimicrobial activity. As a control, ampicillin, an antimicrobial agent, and miconazole (Sigma Co., USA), an antifungal agent, were used at a concentration of 1 μg / disc, respectively. The size of the growth inhibition ring was the diameter Were measured in mm, and representative results after evaluation of 3 or more times are shown in Table 2 below.

As shown in Table 2, when the ampicillin (1 μg / disc) used as an anti-bacterial activity control showed a growth inhibitory ring of 11.0-28.0 mm, strong antimicrobial activity was confirmed, and myconezol (1 μg / disc) Showed 24 mm growth inhibition against C. albicans .

On the other hand, the edible part extract (500 μg / disc) of ethanol extracts from the bottom of yambin showed no antimicrobial activity against all the experimental bacteria and fungi. However, the bark extracts contained Gram positive bacteria and Proteus Vulgaris Gram negative bacteria. These results suggest that the bark extract of Yambin underground can be used as a new natural antimicrobial production resource.

Example  3: Yimbin  Sequence of the bottom skin extract Organic solvent  Fractions and Fraction  Component analysis

For the preparation of fractions of the extract from the bottom part of the yamvin, the extract was suspended in distilled water and sequenced with hexane, ethyl acetate and butanol, and finally a water residue was obtained. The yields of the extracts of hexane fraction, ethyl acetate fraction, butanol fraction and water residue of yamvin underground bark extract were 18.4%, 18.7%, 25.0% and 37.7%, respectively, with respect to the ethanol extract yield of 5.5%. The total flavonoid, total polyphenol, total sugar, and reducing sugar content of the various fractions of the extract from the bottom part of the prepared yamvin were measured and the results are shown in Table 3.

As shown in Table 3, the total polyphenol content and the total flavonoid content of the organic solvent fractions of the yamvin bottom skin extract were as high as 154.5 mg / g and 36.5 mg / g in the ethyl acetate fraction, respectively, And hexene fractions showed relatively high contents of 69.6 ~ 70.9 mg / g and 1.7 ~ 13.3 mg / g, respectively. The water residue also showed a total polyphenol content of 32.2 mg / ml, indicating that the shell extract contained various lipid soluble and water soluble polyphenol components. In the case of total sugar, water residues> butanol fraction> ethyl acetate fraction> hexene fraction in the order of total residues, and reducing sugar showed a similar pattern. The above results suggest that various fractions of yamvin root bark exhibit high polyphenol and flavonoid contents and thus exhibit various physiological activities.

Example  4: Yimbin  Bottom skin extract and Fraction Anti-bacterial  Activity evaluation

The antimicrobial activity of the ethanol extract of the yamvin bottom skin obtained in Example 3 and its fractions was evaluated in the same manner as in Example 2. The results are shown in Table 4.

As shown in Table 4, mepicillin (1 μg / disc) used as an antibacterial activity control and myconezol (1 μg / disc) as an antifungal agent showed excellent antibacterial and antifungal activity, respectively.

On the other hand, the ethanol extract fraction (500μg / disc) of the yamvin bottom skin showed excellent antimicrobial activity against all Gram positive bacteria and P. vulgaris Gram negative bacteria in the hexane fraction and ethyl acetate fraction. In the butanol fraction, B . subtilis and P. vulgaris . These results suggest that the antimicrobial active substance of the subspecies of yamvin is mainly present in the hexane fraction and the ethyl acetate fraction, and these fractions can be used for various bacterial infections and treatments.

Example  5: Yimbin  Underground skin ethanol extract and its Fraction  Evaluation of hemolytic activity of human erythrocytes

Yubin's root root has been used as a food, and it is an edible plant registered as a food ingredient and secured. Therefore, the safety of the underground skin extract is also secured. The hemolytic activity of human erythrocytes was evaluated to evaluate the possibility of acute toxicity of the ethanol extract of the yamvin bottom skin obtained in Example 3 and its fractions. The results are shown in Table 5. In this case, hemolytic activity was assessed in accordance with the existing report (Jung In-chang, Son Ho Yong, 2014 ㆍ Korean J. Microbiol. Biotechnol. 42: 285-292). In brief, 100 μl of human red blood cells, The reaction mixture was centrifuged at 1,500 rpm for 10 min. After transferring 100 μl of the supernatant to a new microtiter plate, the hemoglobin efflux Was measured at 414 nm. Triton X-100 (1 mg / ml) was used as an experimental control for erythrocyte hemolysis. DMSO (2%) was used as a solvent control of the sample. Hemolytic activity was calculated using the following formula.

( % ) Hemolysis  = [( Abs . S - Abs . C) / ( Abs . T - Abs . C)] x 100

Abs . S: Sample Additive  Absorbance,

Abs . C: DMSO Additive  Absorbance,

Abs . T: Triton X-100 Additive  Absorbance.

First, DMSO and water used as controls did not have hemolytic activity, and triton X-100 showed 100% hemolysis of red blood cells at a concentration of 1 mg / ml. On the other hand, the ethanol extract and its fractions of the yamvin bark showed no red blood cell hemolysis up to a concentration of 0.5 mg / ml, indicating no acute toxicity and erythrocyte hemolytic activity.

Example  6. Yimbin  Bottom skin extract and its bite Hexen Fraction , Plasma, acid and thermal stability evaluation of ethyl acetate fraction

The thermal stability, acid stability and plasma stability of the antibacterial activity of the ethanol extract of the yamvin bottom skin obtained in Example 3 and its hexane fraction and ethyl acetate fraction were confirmed. Each sample was tested for antimicrobial activity against gram-negative and gram-positive bacteria similar to the results in Table 4 above at 2 hours treatment at 100 ° C, 2 hours treatment at pH 2 (0.01 M HCl), 2 hours treatment in plasma The antimicrobial activity of the yamvin root showed high stability against heat, acid and plasma.

Example  7: Yimbin  Bottom skin extract and its bite Hexen Fraction , Determination of the minimum inhibitory concentration of the ethyl acetate fraction

The minimum inhibitory concentration (MIC) for various bacteria was determined on the ethanol extract of the yamvin bottom skin obtained in Example 3 and the fractions thereof. MIC measurements were performed on Nutrient broth (Difco Co., USA) in each well of a 96-well plate in the same manner as the previously reported microbroth dilution method (Lee, Seung-Yoon et al., 2015. J. Food Hygiene & Safety 30: 210-216) to 10 ⁸ colony forming unit / ml was placed ssikeul each strain solution is adjusted to 0.1m, since the yambin below-ground shells of various concentrations of ethanol extract and then adding a fraction thereof and then cultured for 24 hours at 37 ℃, the growth degree using And the concentration at which no growth was observed was determined as MIC. The final concentrations of the bark samples were adjusted to 2.0, 1.5, 1.0, 0.5, 0.25, and 0.125 mg / ml, and the results are shown in Table 6.

As shown in Table 6, the MIC for each bacterium varied depending on the extract and fraction, and the strongest antibacterial activity was confirmed in the ethyl acetate fraction. In the case of ethyl acetate fraction, the growth of S. aureus , S. epidermidis , L. monocytogenes , B. subtilis and P. vulgatis was completely inhibited at the concentration of 0.25-0.5 mg / ml in the above 96 well plate condition.

  The ethyl acetate fraction was measured for MIC under shaking and stirring conditions. The disc-diffusion method in Examples 2 and 4 and the microbroth dilution method in the 96 microplate well described above have an advantage of being able to evaluate the antimicrobial activity of the analytical sample conveniently and rapidly. However, when the diffusivity of the analytical sample is low, There is a problem that it is lower than the actual antimicrobial activity due to the obstruction of diffusion. Therefore, the MIC at the time of shaking stirring is equal to or lower than the MIC at the time of stationary culture. However, a large amount of analytical sample is required for shaking stirring.

In this measurement, 5 ml of each strain prepared by adding 10 ⁸ colony forming units / ml to Nutrient broth (Difco Co., USA) was added to a 25 ml test tube, and then various concentrations of ethyl acetate fraction were added thereto. C for 24 hours at 120 rpm, the change in absorbance of the culture broth was measured at a degree of growth of 600 nm, and a sample concentration at which no change in absorbance was observed was determined as MIC. The final concentration of the ethyl acetate fraction of yamvin bark used was adjusted to be 0.5, 0.25, 0125, 0.062, and 0.31 mg / ml, and the results are shown in Table 7 and FIG.

As shown in Table 7 and FIG. 1, it was confirmed that the ethyl acetate fraction inhibited all of the test bacteria at a concentration of 0.031 to 0.062 mg / ml. A very strong antimicrobial activity and low MIC during shake culture are associated with high hydrophobicity of the ethyl acetate fraction and it is possible to assume that oxidative stress is related to the antibacterial activity of the active substance.

The above results suggest that the extract of Chestnut husk and its hexane fraction and ethyl acetate fraction can be used as antimicrobial agents against various bacteria.

Claims (4)

A composition for inhibiting gram-positive bacteria containing an ethanol extract of Pachyrhizus erosus root bark as an active ingredient. delete The composition for inhibiting Gram-positive bacteria according to claim 1, wherein the ethanol extract is a hexane fraction, an ethyl acetate fraction or a mixture thereof. The composition according to claim 1 or 3, wherein the composition for inhibiting Gram-positive bacteria is a pharmaceutical composition, a health functional food, a livestock feed additive, an industrial disinfectant, an industrial preservative, an antimicrobial household product, Wherein the composition is a composition for inhibiting Gram-positive bacteria.

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