KR102287391B1 - Antimicrobial composition comprising the extract of moringa root - Google Patents

Abstract

The present invention relates to an antibacterial composition containing an extract of Moringa oleifera as an active ingredient, and more particularly, to a broad-spectrum antimicrobial composition containing a hexene fraction or an ethyl acetate fraction of an extract of Moringa oleifera as an active ingredient. will be. Moringa underground extract as an active ingredient of the broad-spectrum antibacterial composition of the present invention, as demonstrated through the examples of the present specification, is for antibacterial pharmaceutical compositions, health functional foods, cosmetic compositions, animal feed compositions, food, cosmetics or animal feed It has an excellent effect that it can be used as an additive, various detergents, and various cleaning agents. In addition, the hexene fraction and ethyl acetate fraction of the Moringa underground extract of the present invention have excellent thermal stability, and no loss of activity even under acidic conditions of pH 2 and plasma, liquid, cream, powder, pill, tablet, etc. Since it can be easily processed into various forms, it can be very usefully used in the pharmaceutical industry, food industry, livestock industry, health industry, and cosmetics industry.

Description

Antibacterial composition comprising Moringa underground extract

The present invention relates to an antibacterial composition containing an extract of Moringa oleifera as an active ingredient, and more particularly, to a broad-spectrum antimicrobial composition containing a hexene fraction or an ethyl acetate fraction of an extract of Moringa oleifera as an active ingredient. will be.

Humans have used various antibacterial agents and preservatives to defend against the invasion of pathogenic microorganisms and to control food spoilage microorganisms. However, in the case of chemical antibacterial agents and antiseptics, they show excellent antibacterial activity against various bacteria and fungi, but when used for a long time, many side effects such as redness due to allergy, angioedema, bone marrow suppression, anemia, gastritis, and degenerative brain disease appear. do. In fact, in the United States, the use of antibiotics with severe side effects, such as chloramphenicol, has been banned. Therefore, various studies on the development of natural antibacterial agents using natural products that have been used for a long time while ensuring safety are in progress.

According to the U.S. Center for Disease Control and Prevention, nosocomial bloodstream infections are one of the leading causes of death in the United States. In addition, infection of pathogenic bacteria and fungi through the skin and soft tissues is recognized as a universal medical disease, and it is understood that it is mainly caused by trauma or surgery. Many pathogenic bacterial and fungal infections have different treatment methods depending on the severity of the infection, but successful treatment is possible with oral antimicrobial agents and topical cleaning. However, more severe or complex infections that commonly occur in patients with underlying risk factors (eg, vascular blood flow abnormalities, diabetes) and/or infections caused by difficult-to-treat or multi-resistant bacteria and fungi include intravenous Measures such as antimicrobial treatment and invasive surgical removal may be necessary. For example, the glycopeptide antibiotic vancomycin has been used successfully against severe nosocomial infections caused by multi-drug-resistant Gram-positive bacteria, particularly MRSA, coagulase-negative Staphylococcus and Enterococcus. However, it has been recently reported that the isolation of strains resistant to vancomycin is increasing, and it is true that current antibiotics are very limited in treating severe infections caused by some of the pathogens. Therefore, the need for the development of new more effective antibiotics against increasingly resistant bacteria is increasing.

On the other hand, Moringa oleifera is the most well-known member among 13 species of trees belonging to the Moringacease family. The flowers are white and fragrant, and the fruits with long pods are 30-40 cm tall, and the whole fruits, leaves, flowers, buds, pods, stems and roots are used as food. In particular, in Southeast Asia, leaves with 40% protein content, seeds and pods rich in vitamins and essential oils are routinely eaten, and the roots are chopped and used as seasonings like horseradish.

Although the origin of Moringa, a tropical plant, is India, it is currently cultivated everywhere in the world. In the case of seeds, they contain proteins with strong aggregation ability, so they are often used to purify contaminated water. On the other hand, the underground part of Moringa is being discarded without any specific use, and it is only used as a spice in some areas.

Moringa is called drumstick tree or horse radish tree due to its shape, and known ingredients include flavonoid, isothiocyanates, glucosinolates, thiocarbamates, etc. In fact, Moringa leaves are known for their antioxidant, anti-inflammatory, anti-cancer activity, anti-hypertensive and hair loss prevention effects.

Academic research on Moringa is mainly focused on leaves, fruits, and seeds, and research on the constituents and useful activity of the underground part (root) is still at a rudimentary level. First, the antioxidant, physicochemical and sensory properties of seolgitteok with moringa leaf hot water extract (Eun-Joo Choi et al., 2015, Journal of the Korean Society for Food and Culinary Science 31: 335-343), hair protection of moringa leaf extract treatment during cosmetic hair permanent wave treatment The effect (Soon-deok Lee, 2015, Wonkwang University doctoral thesis) is known, and in the case of moringa fruit, anti-inflammatory activity (Hyo-jin Lee, 2012, Daegu Catholic University master’s thesis), research on anti-aging cosmetics of the above-ground part of moringa (Min-sook Shin, 2015) , Daegu Haany University master's thesis), etc. are known. However, regarding the physiological activity of the Moringa subterranean (root) extract, the renal fibrosis inhibitory effect of the Moringa root extract (Park Suhyeon et al., 2012. J. Life Sci. 22: 1371-1377), PMA-induced in breast cancer cell lines Inhibitory effect on cancer cell infiltration (Hyun Jo et al., 2014. J. Life Sci. 24: 8-13), Inhibitory effect of Moringa root extract on TNF-α-induced inflammation in HaCaT keratinocytes (Hyojin Lee) et al., 2012. J. Life Sci. 22: 1254-1260) are only known.

As for antibacterial studies related to Moringa, most of the studies have been on Moringa seeds and Moringa leaves. Report showing activity (Miller GJ et al., 2017. J Microbiol Biol Educ. 2017 18(3). pii: 18.3.56; doi: 10.1128/jmbe.v18i3.1306), thiocarbamate of Moringa seeds is dagene There is a report (Albuquerque Costa R et al., 2017, Biomed Res Int. 7963747. doi: 10.1155/2017/7963747) showing antibacterial activity against St. Vibrio. In addition, antibacterial activity in Moringa leaves (Gothai S. et al., 2017, Pharmacogn Mag. 13(Suppl 3):S462-S469) is known, and Gram-positive bacteria of Staphylococcus aureus and Streptococcus mutans of Moringa subterranean extract are recently known. It has been reported that antibacterial activity against (Elgamily H et al., 2016, J. Medical Sciences 4: 585-590). However, antifungal activity has not been reported yet, and the antibacterial activity of the underground part of Moringa was found to be very weak compared to the leaves and seeds of the above-ground part. In particular, it is not known that a specific fat-soluble component (Essential oil of Moringa subterranean) of the Moringa subterranean extract exhibits strong broad-spectrum antibacterial activity to date.

Most of the patents related to Moringa are related to functional cosmetics using the powerful antioxidant, whitening, and hair loss prevention activities of Moringa, and the Republic of Korea Patent No. 10-1572155 states "Nanoemulsion natural cosmetic composition containing Moringa extract" manufacturing method", "Cleaning composition containing peat and moringa" in Patent No. 10-1306841, "Cosmetic mask pack" using Moringa powder in Registration No. 10-1558967, Registration No. 10- No. 1540333 discloses "a method for producing a natural extract having an enhanced anti-hair loss effect and a shampoo for preventing hair loss containing a natural extract prepared by the method".

In addition, as patents related to health food and pharmaceutical compositions, "Tofu containing Moringa leaf and its manufacturing method" in Registration No. 10-1193120 and "Vitamins and Moringa leaf extract" in Registration No. 10-1408837 Beverage composition and its manufacturing method comprising", Registration No. 10-1386879, "Method for manufacturing fermented food by complex lactic acid bacteria fermentation mainly with brown rice and moringa and complex lactic acid bacteria fermentation manufactured by the manufacturing method Food" is disclosed, and Publication No. 10-2015-0089241 discloses "Ethanol extract from Moringa leaves that promotes the development, complexity and synaptic generation of neuronal cell construction and a method for preparing the same", Publication No. 10-2015-0091460 "Composition for the treatment or prevention of cancer comprising a water-soluble extract of Moringa leaf as an active ingredient, and a method for producing an extract of Moringa leaf" in No. 10-2015-0144963, "Radiation combination containing Moringa leaf extract" Composition for treatment" and No. 10-2015-0127442 discloses "a pharmaceutical composition for preventing and improving obesity comprising an active ingredient of Moringa oleifera leaves".

In particular, as an antibacterial patent related to Moringa, Korean Patent Registration No. 10-1452326 discloses "An antibacterial agent in which Moringa oleifera extract is collected in porous zinc oxide and a method for manufacturing the same", and the antibacterial activity of Moringa leaves is Patent Publication No. 10-2018-0111106 discloses "Antibacterial composition comprising yeast fermentation broth of Moringa extract and soap comprising the same" It is not known that it exhibits antibacterial activity.

US 10-1452326 B US 10-2018-0111106 A

Elgamily H. et al., Microbiological Assessment of Moringa Oleifera Extracts and Its Incorporation in Novel Dental Remedies against Some Oral Pathogens, 2016, J. Medical Sciences 4: 585-590.

The present invention has been devised to solve the problems of the prior art as described above, and an object to be solved in the present invention is to provide a broad-spectrum antibacterial composition containing a Moringa underground extract as an active ingredient.

In order to solve the above problems, the present invention provides an antibacterial composition comprising an extract of Moringa oleifera underground.

The Moringa subterranean extract is preferably a hexene fraction or an ethyl acetate fraction obtained from the Moringa subterranean extract.

Preferably, the antibacterial activity is antifungal.

The antimicrobial composition is preferably selected from the group consisting of pharmaceutical compositions, health functional foods, cosmetic compositions, animal feed compositions, food additives, cosmetic additives, animal feed additives, detergents and cleaners.

Moringa underground extract as an active ingredient of the broad-spectrum antibacterial composition of the present invention, as demonstrated through the examples of the present specification, is for antibacterial pharmaceutical compositions, health functional foods, cosmetic compositions, animal feed compositions, food, cosmetics or animal feed It has an excellent effect that it can be used as an additive, various cleaning agents, and various cleaning agents. In addition, the hexene fraction and ethyl acetate fraction of the Moringa subterranean extract of the present invention have excellent thermal stability, and no loss of activity even under acidic conditions of pH 2 and plasma, liquid, cream, powder, pill, tablet, etc. Since it can be easily processed into various forms, it can be very usefully used in the pharmaceutical industry, food industry, livestock industry, health industry and cosmetics industry.

1 is a photograph of the underground part of Moringa;
2 is a photograph of Moringa above-ground leaves;
Figure 3 is a photograph of the above-ground stem of Moringa.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepared a Moringa underground extract by a certain method in order to test the antibacterial and antifungal efficacy of the Moringa underground part, and the hexene fraction and ethyl acetate fraction thereof were recovered as antibacterial active ingredients, and the hexene fraction and ethyl acetate fraction showed no hemolytic activity against human red blood cells, but had excellent thermal stability and acid stability.

In particular, the active fractions exhibit strong antibacterial activity against gram-positive and gram-negative bacteria as well as fungi, and thus can be developed as broad-spectrum antibacterial agents for various bacterial and fungal infectious diseases, thereby completing the present invention.

Specifically, the present inventors prepared a hot water extract and an ethanol extract for the above-ground part of Moringa and the underground part of Moringa in order to develop an antibacterial composition using Moringa, which is known to have anti-inflammatory, hepatoprotective, and anti-hypertensive effects. After sequential organic solvent fractions were prepared for the extracts, their antibacterial activity against various bacteria and fungi was evaluated. As a result, strong antibacterial and antifungal activity was shown in the hexene fraction and ethyl acetate fraction of the Moringa subterranean extract. It was confirmed that it can be used as an antibacterial agent.

Accordingly, the present invention provides an antibacterial composition comprising an extract of Moringa oleifera underground.

The Moringa subterranean extract is preferably a hexene fraction or an ethyl acetate fraction obtained from the Moringa subterranean extract.

Preferably, the antibacterial activity is antifungal.

The antimicrobial composition is preferably selected from the group consisting of pharmaceutical compositions, health functional foods, cosmetic compositions, animal feed compositions, food additives, cosmetic additives, animal feed additives, detergents and cleaners.

Hereinafter, the preparation method and efficacy experiment of the Moringa underground extract of the present invention will be described in more detail.

The inventors of the present invention in order to achieve the object of the present invention, the steps of preparing an extract from Moringa above ground and underground; Preparation of sequential organic solvent fractions of hexene, ethyl acetate, and butanol from the extracts of Moringa aboveground and underground parts and preparing the water residue obtained thereafter; Antimicrobial activity evaluation step of the extract and fractions; and a step of investigating the stability of the hexene fraction and the ethyl acetate fraction of the Moringa subterranean ethanol extract.

"Moringa underground extract" included in the composition of the present invention can be obtained by extracting the underground part of Moringa with an organic solvent and filtering the extract using a filtration network of 0.06 mm or less, and concentrating it under reduced pressure. .

The solvent used in the present invention is water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, etc. available, hot water or 95% ethanol extraction is most preferred.

In a preferred embodiment, the Moringa subterranean extract of the present invention may be extracted with hot water or ethanol. In addition, the hot water or ethanol extract may be fractionated sequentially or separately with an organic solvent of hexene, ethyl acetate and butanol to additionally obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction and a water residue.

The Moringa subterranean extract and its hexene fraction and ethyl acetate fraction of the present invention may be prepared into a powder through a conventional pulverization process such as drying under reduced pressure, freeze-drying, or spray-drying.

The hexene fraction and the ethyl acetate fraction of the Moringa subterranean extract are not decomposed by various degrading enzymes in plasma, and maintain antibacterial activity even at 100°C heat treatment and at a pH of 2 in the human stomach.

The hexene fraction and ethyl acetate fraction of the Moringa subterranean extract of the present invention exhibit strong antibacterial activity, and thus are related to the prevention or treatment of pathogenic bacteria and fungi infection, pharmaceutical composition, health functional food, cosmetic composition, animal feed composition, food, cosmetics , can be used as an additive for animal feed and as a material for various cleaning agents.

In a preferred embodiment, the antimicrobial composition of the present invention can be applied as a pharmaceutical composition.

The pharmaceutical composition is formulated in various forms, such as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and injections of sterile injection solutions, according to conventional methods for each purpose of use. It can be used, and it can be administered through various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like. The pharmaceutical composition may further include a carrier, excipient, or diluent, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil and the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, 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 is determined by the type, severity, activity of the drug, and the type of disease in the patient; Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The effective amount of the component having antibacterial activity in the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, and generally 1 to 5,000 mg per body weight, preferably 100 to 3,000 mg per body weight, is administered daily or every other day Or it can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, disease severity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way. The pharmaceutical composition of the present invention may be administered to a subject through various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intra-cerebroventricular injection. 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 oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to a target cell. In the present invention, "subject" is not particularly limited, but includes, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. and preferably a mammal, more preferably a human.

In a preferred embodiment, the antimicrobial composition of the present invention can be applied as a dietary supplement.

The health functional food of the present invention can be used in various ways, such as effective foods and beverages that can lower the possibility of infection from a subject with a high risk of infection by pathogenic microorganisms. Foods containing the active ingredient having excellent antibacterial activity of the present invention include, for example, various foods, beverages, gum, tea, vitamin complexes, health supplements, and the like, in powder, granule, tablet, capsule or beverage form. can be used as In general, the active ingredient of the present invention may be added in 0.01 to 15% by weight of the total food weight, and the health beverage composition may be added in a proportion of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml. In addition to containing the above compound as an essential ingredient in the proportion indicated, the health functional food of the present invention may contain food pharmaceutically acceptable food supplement additives, such as natural carbohydrates and various flavoring agents, as additional ingredients. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and conventional sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, thaumatin, rebaudioside A, glycyrrhizin, saccharin, aspartame, and the like may be used. The ratio of the 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 includes various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloids It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain natural fruit juice, fruit juice for the production of fruit juice drinks, vegetable drinks, and the like. These components may be used independently or in combination. The proportion of these 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 antimicrobial composition of the present invention can be applied as a cosmetic composition.

The cosmetic composition of the present invention can be prepared in the form of, for example, essence, nourishing cream, body lotion, conditioner, shampoo, body cleanser, lotion, skin, mask pack, etc. It is not limited here. Preparation in the above form can be easily carried out according to various manufacturing processes known to those skilled in the art.

In a preferred embodiment, the antimicrobial composition of the present invention can be applied as an animal feed composition.

The animal is a group of organisms corresponding to plants, mainly ingesting organic matter as nutrients, and has differentiated digestive, embryonic and respiratory organs, specifically echinoderms, crustaceans, molluscs, fish, amphibians, reptiles, birds, may be mammals, preferably echinoderms such as sea urchins or sea cucumbers, arthropods including crustaceans such as crabs, shrimps and lobsters, molluscs such as cephalopods, gastropods or bivalves, red sea bream, sea bream, cod, flounder , fish such as halibut, birds including poultry such as pheasants or chickens, or mammals such as pigs, cattle, and goats.

The composition for animal feed may further include grains, vegetable protein feed, animal protein feed, sugar or dairy products to the antimicrobial composition comprising the antimicrobial component of the present invention. The grain may be specifically pulverized or crushed wheat, oats, barley, corn and rice, and the vegetable protein feed may specifically include rapeseed, soybean and sunflower as main components, and the animal protein feed is specifically may be blood meal, meat meal, bone meal, and fish meal, and the sugar or dairy product may be a dry component consisting of various types of powdered milk and whey powder.

The composition for animal feed may additionally contain ingredients such as nutritional supplements, digestion and absorption enhancers, vocal tract promoters or disease prevention agents.

The antimicrobial composition included in the composition for animal feed of the present invention may vary depending on the purpose and conditions of use of the feed, for example, 0.1 g to 100 g of the antimicrobial composition may be included based on 1 kg of the finally produced feed.

In addition, the feed composition may be prepared as a hard viscous granulated or granular material depending on the degree of pulverization of the ingredients, and the composition may be supplied as a mesh or formed into a desired separated shape for further processing and packaging, and storage For ease of storage, it may be subjected to pelletization, expansion or extrusion process, and preferably excess water may be removed by drying.

In a preferred embodiment, the antimicrobial composition of the present invention can be applied as an additive for food, cosmetics or animal feed.

The additive is added to impart preservation to the food, cosmetic or animal feed used, and the antibacterial composition is a natural ingredient and is not harmful to the human body, so it can be safely applied to food, cosmetics or animal feed.

The additive may further include additional other additives appropriately selected according to the purpose and purpose of use, and may be used by mixing with other active ingredients such as commonly used additives or additives such as pigments, surfactants, and preservatives. . The additive may be manufactured by powdering, granulating, tableting or liquefying according to the purpose and use thereof, and a conventional method for packaging may be used for productization.

In addition, the amount of the antimicrobial composition used among the additives may be appropriately used depending on the purpose of use, application form, application site, application target article, etc., for example, it may include 0.01 parts by weight to 50 parts by weight based on the total composition, but The content of the composition is not limited thereto. Since the antibacterial composition of the present invention is a natural component and is not harmful to the human body, it can be used in various amounts as long as the application purpose can be achieved due to the characteristics of the product.

In a preferred embodiment, the present invention can be applied as an antibacterial composition for use as a cleaning agent.

The cleaning agent may be preferably a detergent for clothes, a dishwashing agent, a food cleaner, and a household appliance cleaner. Since the antibacterial composition is a natural component, it is not harmful to the human body, and more preferably, it can be used for the purpose of washing various household items including kitchenware or food washing or for imparting antibacterial properties.

The detergent may further include an additive selected appropriately according to the purpose and purpose of its use, and may be used by mixing with other active ingredients such as commonly used detergents or additives such as pigments, surfactants, and preservatives. The detergent may be prepared by powdering, granulating, tableting or liquefying according to the purpose and use thereof, and a conventional method for packaging may be used for productization.

In addition, the amount of the antimicrobial composition used in the cleaning agent may be appropriately used depending on the purpose of use, application form, application site, application target article, etc., and may include, for example, 0.01 parts by weight to 50 parts by weight based on the total composition, but The content of the composition is not limited thereto. Since the antimicrobial composition of the present invention is a natural component and is not harmful to the human body, it can be used in various amounts if the application purpose can be achieved due to the characteristics of the product.

In a preferred embodiment, the present invention can be applied as an antibacterial composition for use as a cleaning agent.

The cleanser may be used as a hand, oral or feminine cleanser in a simple way in order to prevent infection of pathogenic microorganisms that are easily infected in daily life in a simple way. Since the antibacterial composition is a natural component and is not harmful to the human body, it can be safely applied even when used as a hand, oral, and feminine cleanser.

The cleaning agent may further include additional additives appropriately selected according to the purpose and purpose of use, and may be used by mixing with other active ingredients such as commonly used additives or additives such as pigments, surfactants, and preservatives. . The cleaning agent may be manufactured by powdering, granulating, tableting, or liquefying depending on the purpose and use thereof, but in the case of a liquid type, convenient use will be possible.

In addition, the amount of the antibacterial composition used in the cleaning agent may be appropriately used depending on the purpose of use, application form, application site, etc., for example, it may include 0.01 parts by weight to 50 parts by weight based on the total composition, but the content of the antibacterial composition is The present invention is not limited thereto. Since the antibacterial composition of the present invention is a natural component and is not harmful to the human body, it can be used in various amounts as long as the application purpose can be achieved due to the characteristics of the product.

Hereinafter, the present invention will be described in more detail through examples. The following examples are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[Example]

Example 1: Preparation of hot water and ethanol extracts of the underground and above-ground parts of Moringa and analysis of useful components thereof

After removing foreign substances from the underground and above-ground parts of Moringa purchased in Cambodia in 2015, they were used to prepare ethanol extracts and hot water extracts. When preparing the ethanol extract, 10 times the amount of ethanol was added to the Moringa used part, extracted twice at room temperature, the extract was collected, filtered, and concentrated under reduced pressure to prepare a powder. In the case of hot water extract, 10 times of distilled water is added to the Moringa used part in the same manner, extracted by heating at 100 ° C. for 1 hour, left to cool, repeat the above process once again, collect the extract, filter, and concentrate under reduced pressure. It was prepared as a powder. The results of each extraction efficiency and component analysis of the extract are shown in Table 1.

Component analysis determined total polyphenols, total flavonoids, total sugars and reducing sugar contents. For the total polyphenol content, 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution were added to 400 μl of the extraction sample, left at room temperature for 1 hour, and absorbance was measured at 725 nm. As a standard reagent, tannic acid was used. For the total flavonoid content, each sample was extracted with methanol for 18 hours, and 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample, 40 μl of 1 N NaOH was added, and the absorbance was measured at 420 nm after reaction at 37° C. for 1 hour. As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 1] Extraction efficiency and useful component analysis of ethanol extract and hot water extract for each part of Moringa

As shown in Table 1, the ethanol extraction efficiency of Moringa above and below the ground was 7.2~7.5%, whereas the hot water extraction efficiency was 12.5~13.2% at the above and below. As a result of measuring the total polyphenol content of the Moringa extract, the hot water and ethanol extracts showed a content of 35.8 to 54.7 mg/g in the above-ground part, whereas the hot water and ethanol extract in the underground part showed a content of 7.4 to 13.0 mg/g. Compared to the above-ground part, the content was 20~23% lower. The total flavonoid content also showed a similar pattern, and the underground extract showed a very low content of 6-8% compared to the above-ground part. On the other hand, the total sugar content was higher in the underground part than in the above-ground part, and the reducing sugar content was similar in the above-ground part and the underground part in the ethanol extract, but in the case of the hot water extract, it was 1.7 times higher in the underground part than the above-ground part. These results mean that the above-ground and underground extracts of Moringa have many differences in their composition.

Example 2: Evaluation of antibacterial activity of Moringa underground and above-ground extracts

The antibacterial activity of the extracts of Moringa underground and above-ground parts prepared in Example 1 was evaluated, and the results are shown in Table 2. As strains used for the evaluation of antibacterial activity, Escherichia coli KCTC 1682, Proteus vulgaris KCTC 2433, Pseudomonas aeruginosa KACC 10186, Salmonella typhimurium KCTC 1926 were used as Gram-negative bacteria, and Staphylococcus aureus KCTC 1916, Staphylococcus epidermidis ATCC 12228, Staphylococcus epidermidis were used as Gram-positive bacteria. Listeria monocytogenes KACC 10550 and Bacillus subtilis KCTC 1924 were used. Meanwhile, for the evaluation of antifungal activity, Saccharomyces cerevisiae IF0 0233 and Candida albicans KCTC 1940, a causative agent of Candida fungal infection, were used.

First, in the case of antibacterial activity evaluation, each bacteria was inoculated in Nutrient broth (Difco Co., USA) and cultured at 37° C. for 24 hours, and then each strain _{was adjusted to OD 600} 0.1 to Nutrient agar (Difco Co. , USA) 100 μl of each sample is smeared on a sterile petri dish (90 mm, Green Cross, Korea) containing the medium, and 5 μl of each sample is added to sterile disc-paper (diameter 6.5 mm, Whatsman No. 2), at 37° C. for 24 hours. In the case of fungi, the antibacterial activity was evaluated by measuring the size of the growth inhibition ring after incubation for 24 hours at 30°C in the same manner using Sabouraud dextrose (Difco Co. USA). As a control, the antibacterial ampicillin and the antifungal myconazole (miconazole, Sigma Co., USA) were each used at a concentration of 1 μg/disc. It was measured in mm, and after evaluation three or more times, it is shown as a representative result.

[Table 2] Antibacterial activity of Moringa underground and above-ground extracts

As a result, as shown in Table 2, ampicillin, which is used as an antibacterial agent in clinical practice, showed a wide range of antibacterial activity even at 1 μg/disc, but showed relatively weak growth inhibitory rings (9-11 mm) against Pseudomonas aeruginosa and Salmonella typhimurium. It was. miconazole, used as an antifungal agent in clinical practice, strongly inhibited the growth of Saccharomyces cerevisiae and Candida albicans even at 1 μg/disc. On the other hand, ethanol and hot water extracts from above and below Moringa did not show growth inhibition even at a concentration of 500 μg/disc. For reference, it is necessary to note that in the evaluation of the antibacterial activity of the previously known extracts of Moringa leaves, seeds, and underground parts, 20 μl of 400 mg/ml of the extract per disc was added, and a very high concentration was used than the experiment in this example. (Elgamily H. et al., 2016, J. Medical Sciences 4: 585-590).

Example 3: Preparation of sequential organic solvent fractions of hot water and ethanol extracts from the underground and above-ground parts of Moringa and analysis of their useful components

A hexene fraction, ethyl acetate fraction, butanol fraction and water residue after organic solvent fraction for each extract were prepared through sequential organic solvent fractionation of the Moringa underground and above-ground extracts prepared in Example 1. The yield of the prepared fraction and the analysis result of its useful components are shown in Table 3.

[Table 3] Fractionation efficiency and useful component analysis of fractions obtained from extracts of each part of Moringa

As shown in Table 3, in the case of above Moringa, the highest fractionation efficiency was found in the water residue (44.1% in the case of ethanol extract and 63.7% in the case of hot water extract), and the highest fractionation efficiency in the underground part of Moringa as well. Efficiency was shown for water residues (71.5% for ethanol extracts and 97.0% for hot water extracts). On the other hand, the hexene fraction of the fat-soluble component showed 0.6% (hot water extract) and 8.2% (ethanol extract), respectively, in the above-ground part of Moringa, while <0.1% (hot-water extract) and 4.8% (ethanol extract) in the underground part of Moringa. indicated. Therefore, it was found that the composition of the above-ground part and the underground part of Moringa were significantly different.

As a result of measuring the total polyphenol content of the fractions obtained from the above extract of Moringa, the ethyl acetate fraction showed the highest content in both the hot water and ethanol extracts, and the butanol fraction > water residue > hexene fraction was shown in the order. However, in the case of the underground part, the ethyl acetate fraction > the butanol fraction > the hexene fraction > the water residue was shown in the order. The ethyl acetate fraction of the above-ground part of Moringa showed a total polyphenol content of about 3.7 times and a total flavonoid content of 34.6 times that of the ethyl acetate fraction of the underground part, whereas, in the case of the hexene fraction, the hexene fraction of the underground part was 1.3 times higher than the hexene fraction of the above-ground part. It showed ~12.0 times higher total polyphenol content. These results mean that there are many differences in composition in the above-ground and underground extracts of Moringa, as well as in its fractions.

Example 4: Evaluation of antibacterial activity of fractions obtained from Moringa underground and above-ground extracts

The antibacterial activity of the fractions of the extracts of Moringa underground and above-ground parts prepared in Example 3 were evaluated as antibacterial and antifungal activities in the same manner as in Example 2. The results are shown in Table 4.

[Table 4] Antibacterial activity of fractions obtained from Moringa underground and above-ground extracts

As a result, as shown in Table 4, the hexene fraction and ethyl acetate fraction of the ethanol extract and hot water extract of Moringa underground showed a wide range of antibacterial activity. It showed strong antibacterial activity against both negative bacteria and fungi. In addition, the hexene fraction of the underground ethanol extract showed strong antibacterial activity against gram-positive bacteria, fungi, and gram-negative bacteria, Proteus vulgaris, the causative bacteria of allergic food poisoning. On the other hand, in the case of the above-ground extract fraction, excellent antibacterial activity against Gram-positive bacteria was confirmed in the hexene and ethyl acetate fractions of the ethanol extract, but no antibacterial activity against Gram-negative bacteria and fungi, which was found in Moringa leaves, seeds, and roots. The extract had antibacterial activity against Gram-positive bacteria at a very high concentration (800 μg/disc), but antifungal activity against fungi such as Candida albicans was confirmed with similar results to the previous report showing that it did not appear even at 800 μg/disc (Elgamily H. et al., 2016, J. Medical Sciences 4: 585-590).

Therefore, the above results suggest that when preparing the hexene fraction and ethyl acetate fraction of ethanol and hot water extract using the underground part of Moringa, which does not have a separate use, it can be effectively used to control food poisoning-causing bacteria and pathogenic bacteria.

Example 5: Evaluation of erythrocyte hemolytic activity of Moringa extracts for each part and fractions thereof

Moringa is a safe edible plant that has been used as a food ingredient for a long time. To investigate the acute toxicity potential of hot water extracts and fractions thereof, ethanol extracts and fractions of Moringa above ground and underground, human erythrocyte hemolytic activity was evaluated, and the results are shown in Table 5.

At this time, the hemolytic activity was evaluated according to an existing report (Hoyong Son et al., 2014 ㆍKorean J. Microbiol. Biotechnol. 42: 285-292), and simply 100 μl of human red blood cells washed three times with PBS was placed in a 96-well microplate. 100 μl of sample solutions of various concentrations were added, followed by reaction at 37° C. for 30 minutes. Then, the reaction solution was centrifuged (1,500 rpm) for 10 minutes, and 100 μl of the supernatant was transferred to a new microtiter plate. Measured at 414 nm. DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1 mg/ml) was used as an experimental control for hemolysis of red blood cells. Hemolytic activity was calculated using the following formula.

[Table 5] Human erythrocyte hemolytic activity of Moringa above ground, underground ethanol extract and hot water extract, and sequential fractions thereof

As shown in Table 5, DMSO and water used as controls had no hemolytic activity, and it was confirmed that triton X-100 hemolyzed red blood cells 100% at a concentration of 1 mg/ml. In the case of empotericin B, which is used as an anticancer agent, more than 80% of red blood cells were hemolyzed at a concentration of 0.025 mg/ml. On the other hand, the Moringa extract and its fractions did not show any red blood cell hemolysis at a concentration of 0.5 mg/ml in all samples except for the hexene fraction of the above-ground part, confirming that there was no acute toxicity and no hemolytic activity. Therefore, it was confirmed that the hexene fraction and ethyl acetate fraction of the underground part of Moringa, which have excellent stability, can actually be used as a broad-spectrum antibacterial agent.

Example 6: Evaluation of Plasma, Acid and Thermal Stability of Hexene Fraction and Ethyl Acetate Fraction of Moringa Underground Extract

Plasma stability, thermal stability, and acid stability with respect to antibacterial activity were confirmed for the hexene fraction and ethyl acetate fraction of the ethanol extract of Moringa underground obtained in Example 3 above. As a result, the active fraction showed no significant loss of antibacterial activity even after 1 hour heat treatment at 100° C., 1 hour treatment at pH 2 (0.01 M HCl), and 1 hour treatment in plasma. Therefore, the hexene fraction and ethyl acetate fraction of the Moringa subterranean extract are expected to maintain excellent antibacterial activity even under the processing process of pharmaceuticals, foods, and various antibacterial products.

Claims (4)

Moringa ( Moringa oleifera ) Antifungal composition comprising a hexene fraction or ethyl acetate fraction of the underground hot water extract or ethanol extract. delete delete The antifungal composition according to claim 1, wherein the antifungal composition is selected from the group consisting of a pharmaceutical composition, a health functional food, a cosmetic composition, an animal feed composition, a food additive, a cosmetic additive, an additive for animal feed, a detergent and a cleaning agent. composition.

Images