KR20200055624A - Antibiotic composition containing extract of Syzygii Flos and extract of Magnoliae Cortex - Google Patents

Abstract

The present invention relates to a composition comprising a Syzygium aromaticum extract and a Machilus thunbergii extract as active ingredients. Since the composition comprising a Syzygium aromaticum extract and a Machilus thunbergii extract as active ingredients has a synergistic antibacterial activity against various microorganisms, the composition can be usefully used for antibacterial purposes to inhibit the proliferation or growth of various bacteria and fungi.

Description

Antimicrobial composition containing extract of Syzygii Flos and extract of Magnoliae Cortex

The present invention relates to an antibacterial composition comprising a clove extract and a peppermint extract as an active ingredient, and more particularly, to a composition comprising a clove extract and a peppermint extract having synergistic antibacterial activity.

Direct or indirect damage caused by pathogenic microorganisms causes many problems in the economy, environment, and medicine. For example, the entire society, such as the loss of decay during food distribution in the food industry, the harmfulness and environmental pollution of excessive use of chemical pesticides for agricultural products in the agricultural industry, and the emergence of antibiotic-resistant strains due to misuse of antibiotics. Causes damage by pathogenic microorganisms.

An antibacterial agent means a substance that has an antibacterial effect against microorganisms, specifically, a substance that has an antimicrobial effect by inhibiting a system in which a microorganism synthesizes a cell wall or a protein, or is produced from such a substance. Antibacterial agents are widely used to treat diseases caused by bacterial infections, and have been mainly produced by a method of chemically synthesizing or separating from natural products including fungi. Most of the antibiotics currently being used are manufactured through chemical synthesis, and have a high cost and have many limitations such as causing side effects.

In recent years, in order to solve various problems caused by the use of chemical antibacterial agents, the main task is to develop an antimicrobial composition using natural extracts, but in most natural extracts, microorganisms having antibacterial effects It is limited, and even if it has an antibacterial effect, its function is exhibited at a very high concentration, and thus it has to be used in combination with an additional antibacterial agent. Therefore, it is necessary to develop an antibacterial substance derived from nature that reduces side effects, which is the biggest disadvantage of antibacterial agents, and simultaneously has antibacterial activity against various bacteria.

Accordingly, the present inventors have the antimicrobial effect on various microorganisms at the same time without any side effects, and have completed the present invention on the antimicrobial composition by confirming that the composition of the present invention has synergistically antibacterial activity compared to the individual cloves extract or thick pepper extract.

Republic of Korea Patent Publication No. 10-2003-0003671

An object of the present invention is to provide a composition for antibacterial comprising clove extract and peppermint extract as an active ingredient.

One aspect of the present invention provides an antibacterial composition comprising a clove extract and a peppermint extract as an active ingredient.

In the present invention, "Clove (丁香, Syzygii Flos)" is a bud of a syrup tree ( Syzygium aromaticum (L.) Merrill et. Perry) of tropical evergreen glue, and uses powder or clove oil for food and medicinal purposes. Cloves are used for vomiting, stomach cancer, abdominal indigestion and increased sexual function, gum inflammation and gum pain. As a pharmacological action, gastric secretion promotion, analgesic, anticonvulsant, anti-inflammatory, antioxidant, antithrombotic, anthelmintic and hypotensive effects have been reported.

Clove used in the present invention can be purchased in nature or commercially available, but is not limited thereto.

In the present invention, "hubak (厚朴, Magnoliae Cortex)" refers to the stem or root bark of the afternoon tree ( Magnolia officinalis Rehd. Et Wils.), Alpha, beta-eudesmol (α, β) It is known to contain -eudesmol, magnolol, honokiol, obovatol, etc. (Biopharmaceutical Research Society, Modern Pharmacy, Hakchangsa, 567-571, 2003). Fubak, bitter taste, warm properties, has been used in the treatment of indigestion, sputum, asthma, etc. in oriental medicine. Recently, various effects have been reported on anticancer, neurological diseases (anxiety, depression, Alzheimer's disease, stroke, etc.), inflammatory diseases, cardiovascular diseases, gastrointestinal diseases, asthma and liver diseases.

The aftertaste used in the present invention can be purchased from nature or commercially available, but is not limited thereto.

In the present invention, "antibacterial" means the ability to resist microorganisms and the ability to inhibit the growth or proliferation of microorganisms, and refers to all mechanisms that are made to defend against the action of microorganisms such as bacteria, fungi, yeast, and the like.

In the present invention, "bacteria" is a creature belonging to bacteria, the most prosperous species among living organisms, most of which are pathogenic bacteria and have the characteristics of prokaryotes, but do not have a nuclear membrane, mitochondrial, chloroplast-like structure. The size varies from 0.5㎛ to 0.5mm.

In the present invention, "fungus" refers to a group of microorganisms composed of more than 72,000 species including fungi, yeast, mushrooms, belongs to eukaryotic organisms with a nuclear membrane, and cell organelles such as mitochondria, vesicles, etc., are developed, and chitin , Glucan and the like. Most of them are characterized by forming a mycelium that is cellular, renal, developing, and sexual and asexual reproduction. It forms a spore as a propagation body, but some fungi have unicellular proliferation. As a by-product, it is mainly involved in organic decomposition in nature, but some parasitic or symbiotic with plants and animals.

The composition comprising the clove extract and the peppermint extract according to one embodiment of the present invention has superior synergistic antibacterial activity compared to the individual clove extract or the peppermint extract, and has a wide range of antimicrobial activity against microorganisms including bacteria and fungi. .

In the present invention, "synergistic (synergistic) antibacterial activity" or "synergistic (synergistic) antimicrobial effect" is a combination of antibacterial effects of a single extract in which the effect of the composition with a mixture of clove extract and peppermint extract is reasonably expected It means that it shows better antibacterial activity than the additive effect. Therefore, the antimicrobial composition according to an embodiment of the present invention has a synergistic antibacterial activity by mixing a clove extract and a thick pepper extract, and can effectively inhibit the growth or growth of microorganisms even at low concentrations without additional antibacterial agents.

According to one embodiment of the present invention, the clove extract and the aftertaste extract may be prepared according to conventional methods known in the art. For example, after crushing the dried cloves and thick peppers, a solvent commonly used for extraction may be added, respectively, and extracted at an appropriate temperature and pressure to produce cloves extracts and thick pepper extracts.

According to one embodiment of the invention, the clove extract is water, alcohols having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane and these It may be obtained from a solvent extraction method using an extraction solvent selected from the group consisting of a mixture or a supercritical extraction method.

According to one embodiment of the present invention, the aftertaste extract is water, alcohol having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane and these It may be obtained from a solvent extraction method using an extraction solvent selected from the group consisting of a mixture or a supercritical extraction method.

According to an embodiment of the present invention, the alcohol having 1 to 4 carbon atoms may be ethanol or methanol.

In the present invention, the "extract" is an extract obtained by the extraction treatment of herbal medicines, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a crude product or a purified product of the extract, such as a mixture thereof, and the extract itself It contains extracts of all formulations that can be formed.

According to one embodiment of the present invention, the clove extract and the aftertaste extract may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

According to an embodiment of the present invention, the clove extract and the aftertaste extract include not only extracts by the above-described extraction method, but also extracts that have been subjected to a conventional purification process. For example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods may also be included in the extract of the present invention.

In the present invention, the "supercritical extraction method" refers to an extraction system in which the principles of the conventional distillation and extraction techniques are combined, and extracts a substance using the dissolving power of a supercritical fluid. A "supercritical fluid" is a substance that does not change its state when heat and pressure are applied to a substance, and when heat and pressure are applied beyond a specific temperature (critical temperature) and pressure (critical pressure), the supercritical fluid is a liquid. It is a state in which the state of and gas cannot be distinguished, and has unique characteristics different from those of a general liquid or gas. The supercritical fluid is a compressive fluid that has a medium property between gas and liquid, so it can diffuse well like a gas and dissolve other substances like a liquid, and can also adjust the physical properties to a desired state by changing pressure and temperature.

According to one embodiment of the present invention, the supercritical fluid may be carbon dioxide, water, methane, ethane, propane, ethylene, propylene, methanol, ethanol, or acetone, but is not limited thereto. The conditions of the supercritical fluid should be chemically safe, no corrosion in the device, low critical temperature and pressure, and good solubility. In this regard, carbon dioxide is mainly used in the process. Supercritical carbon dioxide is relatively inexpensive, is harmless to the human body, and is non-flammable and chemically stable. In addition, it is easy to change the properties with a low critical point (73.8 bar, 31.1 ° C) and can be recycled.

The supercritical extraction method according to an embodiment of the present invention can extract a temperature-sensitive material without denaturation and decomposition, and enables rapid extraction and phase separation.

According to one embodiment of the present invention, the antimicrobial composition may include a clove extract and a peppermint extract respectively extracted by the above extraction method, or may include an extract obtained by extracting a mixture of clove and peppermint herbs by the above extraction method.

According to an embodiment of the present invention, the clove extract and the aftertaste extract have a weight ratio of 100: 1 to 1: 1000, a weight ratio of 100: 1 to 1: 500, a weight ratio of 100: 1 to 1: 100, and a 50: 1 to Weight ratio of 1:50, weight ratio of 30: 1 to 1:30, weight ratio of 20: 1 to 1:20, weight ratio of 20: 1 to 1:15, weight ratio of 20: 1 to 1:10, 10: 1 to Weight ratio of 1:10, weight ratio of 2: 1 to 1:10, weight ratio of 1: 1 to 1:10, weight ratio of 1: 1 to 1: 100, weight ratio of 1: 1 to 1:30, or 1: 1 It may be included in a weight ratio of 1 to 15.

According to one embodiment of the present invention, the clove extract and the aftertaste extract may be included in an amount of 0.001% by weight to 99.99% by weight, preferably 0.1% by weight to 99% by weight, based on the total weight of the composition, which is used for an antibacterial composition Depending on the method and purpose of use, it may be appropriately selected by those skilled in the art to which the present invention pertains.

According to one embodiment of the present invention, the antimicrobial composition is Escherichia sp., Pseudomonas sp., Staphylococcus sp., Candida sp., Malassezia sp., Salmonella sp., Acinetobacter sp., Corynebacterium sp., Mycobacterium sp., And it may be to inhibit the growth of microorganisms selected from the group consisting of Micrococcus sp.

According to one embodiment of the present invention, the microorganism is Escherichia coli , Pseudomonas aeruginosa , Candida albicans , Candida tropicalis , Malesacea Pachidermatis ( Malassezia pachydermatis), Salmonella typhimurium (Salmonella typhimurium), Acinetobacter John Sony (Acinetobacter johnsonii), Corynebacterium Ako Lawrence (Corynebacterium accolens), Corynebacterium Army cola Tomb (Corynebacterium amycolatum), Corynebacterium Corynebacterium xerosis , Corynebacterium jeikeium , Staphylococcus aureus , Staphylococcus haemolyticus , Staphylococcus haemolyticus , Staphylococcus war Staphylococcus warneri , Staphylococcus saprophyticus , Staphylococcus cohnii , Staphylococcus hominis , Staphylococcus hominis , Staphylococcus xylos Staphylococcus xylosus , Staphylococcus simulans , Mycobacterium fortuitum , and Micrococcus luteus .

In the present invention, " Escherichia sp. ( Escherichia sp.) Strain" is a Gram-negative strain, which is a type of intestinal bacteria and is a causative strain such as food poisoning. For the purposes of the present invention, Escherichia sp. Strains include, without limitation, microorganisms that can be used as targets of the antimicrobial composition provided by the present invention. In the present invention, the strain of Escherichia genus may be, for example, E. coli, but is not limited thereto. Escherichia coli constitutes a normal bacterial layer in the intestine, and is usually non-pathogenic, but may be a causative agent of biliary tract, cystitis, pyelonephritis, and urinary tract infection.

By using the antimicrobial composition according to one embodiment of the present invention can suppress the growth or growth of the strain of Escherichia genus, it is possible to prevent or improve the disease caused by the strain of Escherichia genus.

In the present invention, " Pseudomonas sp. ( Pseudomonas sp.) Strain" is a gram-negative strain, which may be a pathogen or a rot, but is also used for the production of amino acids by amino acid fermentation. For the purposes of the present invention, Pseudomonas strains include, without limitation, microorganisms that can be used as targets of the antimicrobial composition provided by the present invention. The strain of the genus Pseudomonas of the present invention may be, for example, Pseudomonas aeruginosa, but is not limited thereto. Pseudomonas eruginosa is a Gram-negative aerobic bacillus, which has weak pathogenicity to humans, but is widely distributed in the natural environment, causing mixed infections and secondary infections to exacerbate the disease state. In particular, when bio-resistance, such as surgery and burns, is lowered, septicemia may occur due to infection, and diarrhea may be caused by invading the mucous membrane of the digestive system.

By using the antimicrobial composition according to one embodiment of the present invention can suppress the growth or growth of the strain Pseudomonas genus, it is possible to prevent or improve the disease caused by the strain Pseudomonas genus.

In the present invention, " Staphylococcus sp. ( Staphylococcus sp.) Strain" belongs to the Micrococcaceae family (Micrococcaceae) and is present in the intestines of humans and animals, as well as in the skin and mucous membranes. S. aureus , S. epidermidis, S. saprophyticus , S. haemolyticus , S. hominis, or S. warneri are isolated from human disease. For the purposes of the present invention, strains of the genus Staphylococcus include, without limitation, microorganisms that can be used as targets of the antimicrobial composition provided by the present invention. The strains of the genus Staphylococcus of the present invention include, for example, Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus warnery, Staphylococcus saprophyticus, Staphylococcus Couscous, staphylococcus hominis, staphylococcus xylos or staphylococcus simulations, but is not limited thereto. Staphylococcus aureus is a gram-positive, anaerobic bacterium, which normally exists on the skin and nasal surfaces of healthy people or livestock, produces heat-resistant exotoxins, causes food poisoning, purulent skin, otitis media, cystitis, etc. It is known to cause disease.

By using the antimicrobial composition according to an embodiment of the present invention it is possible to inhibit the growth or growth of the strains of the genus Staphylococcus, thereby preventing or improving the disease caused by the strains of the genus Staphylococcus.

In the present invention, " Candida sp. ( Candida sp.) Strain" is a genus of incomplete bacteriophage, which is a single cell spherical or oval, and is present in the mouth, skin, etc. of the human body or animal, and is harmless to the human body under normal conditions, but has long-term antibiotics. When used or when the body's resistance to immunity is weakened, it multiplies abnormally in the body and causes candidiasis. In areas of high infection, the mucous membranes of the mouth and genitals, etc., cause itching and pain of the mucous membranes.

For the purposes of the present invention, Candida genus strains include, without limitation, microorganisms that can be used as targets of the antimicrobial composition provided by the present invention. The Candida genus strain of the present invention may be, for example, Candida albicans or Candida tropicalis, but is not limited thereto. Candida albicans is a Gram-positive fungus, a kind of skin fungus that is also present in the oral, vaginal, intestinal, and digestive tracts of healthy people. However, representatively, the female vagina is inflamed, causing candidal vaginitis. It has been reported that about 75% of women suffer from candidal vaginitis more than once in the vagina and vulva, and 45% of the female population is a common disease that occurs more than once a year. Candida tropicalis is the leading causative agent of sepsis and spread candidiasis in people with lymphoma, leukemia and diabetes, and is usually found on the gastrointestinal tract and skin surface.

By using the antimicrobial composition according to an embodiment of the present invention can inhibit the growth or growth of the genus Candida strains, it is possible to prevent or improve the disease caused by the genus Candida strains.

In the present invention, the “ Malassezia sp.” Strain is mainly a lipid-affecting bacterium and belongs to the normal microflora of the skin, and naturally exists on the skin surface. Survives mainly by forming colonies. Colony formation by Malassezia is known to occur most often from late adolescence to early adulthood, when the sebaceous glands are active from birth. Malassezia grows by eating fats and proteins from the skin, and thus has high lipase activity and increases the production and secretion of inflammatory cytokines, causing pityrosporum folliculitis, pityriasis versicolor, seborrheic dermatitis, and It can cause dandruff.

For the purposes of the present invention, strains of the genus Malassezia include, without limitation, microorganisms that can be used as targets of the antimicrobial composition provided by the present invention. The strain of the genus Malassezia of the present invention may be, for example, Malesacea pakidermatis, but is not limited thereto.

By using the antimicrobial composition according to an embodiment of the present invention can inhibit the growth or growth of the strain of Malassezia, it is possible to prevent or improve the disease caused by the strain of Malassezia.

In the present invention, " Salmonella sp. ( Salmonella sp.) Strain" is a Gram-negative strain, which is not only cold and frozen but also resistant to drying and is a causative strain such as typhoidosis, acute gastritis, or food poisoning.

For the purposes of the present invention, the strain of Salmonella genus includes, without limitation, microorganisms that can be used as targets of the antimicrobial composition provided by the present invention. The strain of Salmonella genus of the present invention may be, for example, Salmonella typhimurium, but is not limited thereto. Salmonella typhimurium is mainly found in the intestine of the small intestine, and its toxicity is caused by LPS present in the outer membrane of the bacteria and causes acute gastroenteritis, food poisoning, and the like.

By using the antimicrobial composition according to an embodiment of the present invention can suppress the growth or growth of the Salmonella genus strain, it is possible to prevent or improve the disease caused by the Salmonella genus strain.

In the present invention, " Corynebacterium sp." Is a genus of Actinomycetales, parasitic on the mucous membrane with a rod-like Gram-positive strain, well detected from healthy bodies, and usually combined with pathogenicity and toxin production. do.

For the purposes of the present invention, strains of the genus Corynebacterium include, without limitation, microorganisms that can be used as targets of the antimicrobial composition provided by the present invention. The strain of the genus Corynebacterium of the present invention may be, but is not limited to, Corynebacterium acorens, Corynebacterium amicolatum, Corynebacterium xerosis, Corynebacterium J. caeum, for example.

When using the antimicrobial composition according to an embodiment of the present invention can inhibit the growth or growth of the strain of genus Corynebacterium, the disease caused by the strain of Salmonella can be prevented or improved.

The antimicrobial composition according to one embodiment of the present invention has a wide range of antimicrobial activity against bacteria and fungi, and includes an active ingredient extracted from natural substances, causing toxicity or skin allergies, skin irritation, and environmental hormones compared to chemical synthetic products. There are few side effects, such as potential and causing resistant bacteria.

According to one embodiment of the present invention, the antimicrobial composition disclosed herein may be a composition for skin, scalp, oral, ocular or vaginal. According to one embodiment of the invention, the antimicrobial composition is shampoo, body cleanser, soap, foam cleanser, skin, lotion, cream, ointment, spray, toothpaste, wipes, animal wipes, kitchen cleaners, bathroom cleaners, pets It can be used as a cleaning agent, an antibacterial agent for textiles, a lens cleaner, and a female cleaner.

According to one embodiment of the present invention, the antimicrobial composition may be used as a preservative, preservative, detergent, or cleaner.

In the present invention, preservatives inhibit or sterilize the proliferation of one or more harmful microorganisms or spoilage microorganisms selected from the crowd consisting of bacteria, fungi, and yeast to prevent deterioration or spoilage, chemical changes in food, feed, cosmetics, and medicines, thereby improving nutritional value and freshness. It means the additive used to maintain the. In a narrow sense, it means a substance that can stop or sterilize the growth or growth of decaying microorganisms such as bacteria, fungi, and yeast.

As an ideal condition as a preservative, the composition should be non-toxic and should be effective even in trace amounts. Since the antimicrobial composition according to one embodiment of the present invention is a natural ingredient and is not harmful to the human body, it can be safely applied to food, medicine, cosmetics or animal feed.

In one embodiment of the present invention, the preservative may further include additional additional additives appropriately selected according to the purpose and purpose of use, and other active ingredients such as additives commonly used, pigments, surfactants, preservatives, etc. It can be used in admixture with additives. The additive may be prepared by powdering, granulating, tableting or liquefying depending on the purpose and use of the additive, and a conventional method may be used for packaging for productization.

In one embodiment of the present invention, the amount of the antimicrobial composition used in the preservative may be appropriately used according to the purpose of use and application form, application site, and object to be applied, and includes, for example, 0.01 to 50 parts by weight based on the total composition. It can, but is not limited to. Since the antimicrobial composition according to one embodiment of the present invention is a natural component extracted from herbal medicines and is not harmful to the human body, it can be used in various amounts if it is possible to achieve the application purpose due to the characteristics of the product.

Since the antimicrobial composition according to one embodiment of the present invention has excellent antimicrobial activity against various microorganisms, it can be used as a preservative added to food, quasi-drug or cosmetic compositions by replacing synthetic preservatives.

The antimicrobial composition according to one embodiment of the present invention may be used as a cleaning agent, and the cleaning agent may be a detergent for clothes, a dishwashing agent, a food cleaning agent, and a household appliance cleaning agent. Since the antimicrobial composition according to one embodiment of the present invention contains natural ingredients extracted from herbal medicines and is not harmful to the human body, it can be used for the purpose of washing or imparting antimicrobial properties to various household products, including kitchenware or food washing. have.

In one embodiment of the present invention, the cleaning agent may further include an appropriately selected additive according to the purpose and purpose of use, and mixed with additives such as other active ingredients, pigments, surfactants, and preservatives, such as commonly used cleaning agents. Can be used.

According to one embodiment of the present invention, the cleaning agent may be manufactured by pulverizing, granulating, tableting or liquefying depending on the purpose and use of the cleaning agent, and a conventional method for packaging for productization may be used.

In one embodiment of the present invention, the amount of the antimicrobial composition used in the cleaning agent may be appropriately used according to the purpose of use and application form, application site, object to be applied, etc., for example, 0.01 to 50 parts by weight based on the total composition However, the content of the antimicrobial composition is not limited thereto. Since the antimicrobial composition according to one embodiment 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 it can achieve the application purpose due to the characteristics of the product.

According to one embodiment of the present invention, the present invention can be applied as an antimicrobial composition as a cleaning agent. The cleaning agent according to one embodiment of the present invention can be used for a hand, mouth or female cleaning agent in a simple manner to perform a systemic infection of a pathogenic microorganism, which is often susceptible to infection in everyday life. Since the antimicrobial composition according to one embodiment of the present invention is a natural ingredient and is not harmful to the human body, it can be safely applied even when used as a hand, mouth or female cleanser.

The antimicrobial composition according to one embodiment of the present invention causes inflammation in the female genitals, causing vaginal infections, itching, and odors, which can cause female genitourinary system diseases, such as Candida albicans and Staphylococci It has excellent antibacterial activity against Staphylococcus aureus and does not cause skin irritation, so it can be used as a cleanser for women.

The composition comprising the clove extract and the peppermint extract as an active ingredient has a synergistic antimicrobial activity against various microorganisms, and thus can be usefully used as an antibacterial application to inhibit the growth or growth of bacteria and fungi.

Figure 1 shows the results of evaluating the growth inhibitory effect on C. albicans according to the concentration of clove hot water extract.
Figure 2 shows the results of evaluating the growth inhibitory effect on C. albicans according to the concentration of the supercritical superthin extract.
Figure 3 shows the results of evaluating the growth inhibitory effect on C. albicans according to the concentration of clove methanol extract.
Figure 4 shows the results of evaluating the growth inhibitory effect on C. albicans according to the concentration of thick methanol extract.
5 and 6 show a synergistic antimicrobial effect on C. albicans when mixed with clove hot water extract and thick pepper supercritical extract. Specifically, FIG. 5 shows the C. albicans cell density measured when each of the cloves hydrothermal extract and the thick thin supercritical extract are added or mixed, and FIG. 6 shows the growth rate of the C. albicans strain when each extract is added to the control group.
Figure 7 shows a synergistic antimicrobial effect on C. albicans when mixed with clove methanol extract and thick methanol extract, specifically, shows the growth rate of C. albicans strain when each extract is added to the control group.
8 and 9 show a synergistic antimicrobial effect against S. aureus when mixing the clove hot water extract and thick supercritical extract. Specifically, FIG. 8 shows S. aureus cell density measured when each of the clove hot water extract and the super thin supercritical extract is added by mixing, and FIG. 9 shows the growth rate of the S. aureus strain when each extract is added to the control group.
Figure 10 shows the growth rate of C. albicans according to the mixing ratio of clove hot water extract and super thin supercritical extract.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of herbal extracts

1-1. Preparation of clove hot water extract

After crushing the cloves (Jiundang Oriental Pharmacy, Gyeongdong Market, Seoul, Korea) to a size of 5 cm or less, add 100 ml of distilled water to 10 g of the crushed cloves and extract for 4 hours and 30 minutes at 100 ° C temperature condition using a reflux device. Did. After extraction, the filter paper (Whatman ™ No.1 qualitative filter paper; GE Healthcare Life Sciences) was filtered using a pressure reducing device to remove solids. The hot water extract from which solids were removed was dried using a freeze dryer. The amount of clove hot water extract obtained from 10 g of cloves was 1.78 g, and the yield was 17.81%.

0.08 g of clove hot water extract obtained by lyophilization was dissolved in 2 ml of RPMI1640 medium to prepare a stock of 40 g / L concentration, and then immersed in water at 50 ° C. for 15 minutes to allow the extract to dissolve in the medium as much as possible. The quenched extract was sufficiently dissolved by vortexing, and then centrifuged at 13,000 rpm for 10 minutes, and the supernatant except the insoluble material was taken and used for further experiments.

1-2. Preparation of thick supercritical extract

Supercritical extraction was performed by commissioning Hubak (Jiyundang Herbal Medicine, Gyeongdong Market, Seoul, Korea) to EnSNature. 3200 g of thick foil was introduced into a 10 L supercritical extractor, and extracted for 5 hours using supercritical carbon dioxide under conditions of 350 bar and 50 ° C. After extraction, impurities were removed using a filter paper (whatman ™ grade 4 qualitative filter paper; GE Healthcare Life Sciences). The amount of supercritical extract obtained from 3,200 g of thick foil was 55.0 g, and the yield was 1.72%. 0.1012 g of the supercritical extract was dissolved in 1 ml of 50% tween20 diluted in RPMI 1640 medium to prepare a stock at a concentration of 100 g / L, and then vortexed to uniformly dissolve the extract and used in later experiments.

1-3. Preparation of cloves and thick pepper extracts using methanol solvent

After crushing the cloves and thick foil to a size of 5 cm or less, 30 g of the crushed cloves or 30 g of the thin foil were placed in a flask, and extracted by immersing in 300 ml of 99.5% methanol as a solvent in a constant temperature water bath at 50 ° C. for 3 hours. The flask was shaken 4 to 6 times at 30 minute intervals to mix the solvent and the herbal medicine. After extraction, the solids were filtered off using a pressure reducing device using a filter paper (Whatman ™ No.1 qualitative filter paper; GE Healthcare Life Sciences). The filtered extract was concentrated using a rotary concentrator and left to stand in a dryer at 60 ° C. for up to 2 weeks to remove methanol and obtain a solid extract. The solid content of 5.48 g of clove extract and the solid content of 4.45 g of peppermint extract were obtained from 30 g of cloves and 30 g of thick peppers, respectively.

50 mg, 25 mg, 13 mg, 6 mg of solid content of clove methanol extract and 100 mg, 50 mg, 25 mg, 13 mg, 6 mg, 3 mg, and 2 mg of solid content of thick methanol extract were dissolved in 1 ml of methanol solvent, respectively. After dissolving, the extract was dissolved in a solvent as much as possible through a water bath at 50 ° C for 5 minutes. After vortexing to sufficiently dissolve, after centrifugation at 13,000 rpm for 10 minutes, the supernatant excluding the insoluble material was taken and used for further experiments.

Example 2. Measurement of antibacterial activity according to the concentration of clove extract or thick pepper extract

2-1. Preparation of strain and culture medium

Candida albicans to measure the antimicrobial effect of the herbal extract (less than Candida albicans, C. albicans) KCTC 7965 and Staphylococcus aureus (Staphylococcus aureus, less than S. aureus) Biological Resource Center of KCTC 3881 (Korean Collection for Type Cultures, Korea).

The composition of the medium used in the experiment is shown in Table 1 below.

The SDA medium was used to make the C. albicans bacteria stored at -80 ° C active growth state, and the RPMI 1640 medium was used to cultivate the C. albicans bacteria which became active growth state through the SDA medium. The finished medium was stored at 4 ° C.

YPD medium made of solid medium including agar is a medium that makes C. albicans bacteria stored at -80 ° C active growth state, and YPD liquid medium prepared without agar is C. albicans which has become active growth state. It was used when culturing the bacteria.

The including agar solid medium made of a TSB culture medium medium to create a S. aureus strain that is kept at -80 ℃ in active growth state, and TSB liquid medium prepared without containing the agar is a S. aureus growth conditions as the active It was used to cultivate the bacteria .

2-2. Antibacterial effect according to the concentration of clove hot water extract

To measure the antibacterial effect on C. albicans according to the concentration of the clove hot water extract, the CLSI M27-A2 method was used.

C. albicans KCTC 7965 strain stock was streaked on SDA agar plates and cultured for 2 days at 35 ° C. A single colony was then taken, suspended in 0.145 mol / L saline and vortexed for 15 seconds. Measure the absorbance of the suspension at 530 nm using an Optizen 2120uv spectrophotometer (Mecasys Co., Korea) to check if the cell density is 0.5 McFarland standard and, if necessary, 0.5 McFarland turbidity. Saline was added as much as possible. Saline was added to a suspension having a McFarland turbidity of 0.5, diluted 100-fold, and further diluted 10-fold using RPMI 1640 medium to dilute to a concentration of 1x10 ³ to 5x10 ³ CFU / ml immediately before being processed on the microplate. One C. albicans fungus suspension was dispensed in 100 µl each on a microplate.

The clove hot water extract stock (40 g / L) prepared in Example 1-1 was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was taken and diluted 640 times with RPMI1640 medium so that the concentration was 0.0625 g / L. Clove hydrothermal extracts at a concentration of 0.0625 g / L were serially diluted twice in RPMI1640 medium to 31.25 mg / L, 15.63 mg / L, 7.81 mg / L, 3.91 mg / L, 1.95 mg / L, and 0.98 mg / L concentration The clove hot water extract of C. albicans fungus suspension was dispensed 100 μl each on a microplate and 100 μl of RPMI1640 medium containing no clove hot water extract was dispensed as a control.

As described above, the microplate containing C. albicans bacteria and clove hot water extract was placed for 48 hours at 35 ° C., and then cultivated by pipetting the suspended bacteria and absorbed at 540 nm with a microplate reader (Dynex Opsys MR ™ Microplate Reader). It was measured and expressed as the cell density of C. albicans . This cell density value reflects the proliferation or growth results of C. albicans .

Figure 1 shows the results of evaluating the growth inhibitory effect on C. albicans according to the concentration of clove hot water extract. In Fig. As shown in Figure 1, at the lower concentration than 7.81㎎ / L did not show the growth inhibitory effects of C. albicans 15.63㎎ / L concentrations since exhibited an excellent bactericidal effect against C. albicans.

2-3. Antibacterial effect according to the concentration of thick supercritical extract

In the same manner as in Example 2-2, 100 µl each of the suspensions of C. albicans bacteria diluted to a concentration of ¹ × 10 ³ to 5 × 10 ³ CFU / ml immediately before treatment on the microplate was dispensed into the microplate.

The super thin supercritical extract stock prepared in Example 1-2 (100 g / L) was diluted 50-fold with RPMI1640 medium to a concentration of 2 g / L. 2 g / L super thin supercritical extract at a concentration of 1 g / L, 0.5 g / L, 0.25 g / L, 0.125 g / L, 62.5 mg / L, 31.25 mg / L concentration by serially diluting 2 times with 1% tween20 100 μl of the supercritical extract of C. albicans was dispensed into each microplate containing the suspension of C. albicans , and 100 μl of RPMI1640 medium containing 1% tween 20 without the supercritical extract as a control was dispensed.

As described above, the microplate containing C. albicans and super thin supercritical extract was placed at 35 ° C. for 48 hours to suspend the cultured and submerged bacteria, and absorbed at 540 nm with a microplate reader (Dynex Opsys MR ™ Microplate Reader). It was measured and expressed as the cell density of C. albicans .

Figure 2 shows the results of evaluating the growth inhibitory effect on C. albicans according to the concentration of the supercritical superthin extract. As shown in FIG. 2, it can be confirmed that the growth inhibitory effect of C. albicans increases as the concentration of the supercritical superthin extract increases, and at the maximum concentration of 1 g / L, growth inhibition of C. albicans by about 22% compared to the control group It showed an effect.

2-4. Antibacterial effect of clove methanol extract or thick methanol extract

C. albicans KCTC 7965 strain stock was streaked on YPD agar plates and cultured at 26 ° C for 2 days. Thereafter, a single colony was taken and inoculated into 5 ml of YPD medium, followed by pre-incubation for 18 hours to 24 hours under conditions of 250 rpm and 26 ° C in a test tube. Using the Optizen 2120uv spectrophotometer (Mecasys Co., Korea), the absorbance was measured at 600 nm to obtain the pre-cultured C. albicans cell density, and using this, the C. albicans strain was inoculated into each test tube so that the OD ₆₀₀ was 0.05. do.

The concentration of the clove methanol extract in each test tube was 0.5 g / L, 0.25 g / L, 0.13 g / L, or 0.06 g / L, respectively, and the concentration of the thick methanol extract was 0.99 g / L, 0.5 g / L, Clove methanol extract or thick methanol extract prepared in Example 1-3 was added to be 0.25 g / L, 0.13 g / L, or 0.06 g / L, 0.03 g / L, and 0.02 g / L. Methanol was added to 5 ml of YPD medium to set YPD medium containing 1% methanol as a control.

The strain was inoculated into the test tube as described above, and the extract was added to incubate at 26 ° C. for 18 hours. The culture was measured by absorbance at 600 nm using an Optizen 2120uv spectrophotometer (Mecasys Co., Korea), which was expressed as the cell density of C. albicans (left graph in FIGS. 3 and 4), compared with a 1% methanol treatment control group. The growth rate of C. albicans according to the concentration of the clove methanol extract or the thick methanol extract was shown (right graph in FIGS. 3 and 4).

Figure 3 shows the results of evaluating the growth inhibitory effect on C. albicans according to the concentration of clove methanol extract. When the concentration when growth of C. albicans is inhibited by 20% is drawn by drawing a linear trend line on the graph of FIG. 3, the trend of the clove extract is y = -152.01x + 98.194, from which the growth of C. albicans compared to the control group It can be seen that the concentration at 20% inhibition is 0.12 g / L. Clove methanol extract of this concentration was used in later experiments.

Figure 4 shows the results of evaluating the growth inhibitory effect on C. albicans according to the concentration of thick methanol extract. As shown in FIG. 4, since the growth of C. albicans at the concentration of 0.06 g / L was suppressed by 22.44%, the thick-shell methanol extract at a concentration of 0.06 g / L was used in a subsequent experiment.

Example 3. According to the mixture of clove extract and thick pepper extract C. albicans Synergistic antibacterial effect

3-1. Synergistic Effect of Mixing Clove Hot Water Extract and Thick Squash Supercritical Extract

Based on the results of Examples 2-2 and 2-3, even if the concentration of the extract is doubled, the effect of growth inhibition is not more than 50%, and the concentration does not show growth inhibition even at the concentration, and cloves hydrothermal extract 0.005 g / L, superthin supercritical extract 0.25g / L was used to examine whether there is a synergistic antibacterial effect on C. albicans according to the mixing of cloves hot water extract and superthin supercritical extract. The concentration of each extract used in the experiment is listed in Table 2.

Each extract was treated at the concentration shown in Table 2, and absorbance was measured at 540 nm in the same manner as in Example 2-2, and this was expressed as the cell density of C. albicans . When RPMI1640 medium was added as a control (indicated as control in FIG. 5), the growth rate of C. albicans was calculated as a percentage when the clove hot water extract was added, and 0.25 g / L of super thick supercritical extract of 0.25 g / L contained 0.25% tween 20. RPMI1640 medium containing 0.25% tween 20 as a control (represented as 0.25% tween 20 in FIG. 5) was added as a control for the growth rate of C. albicans as a percentage, and 0.5 g / L super thin supercritical Since the extract contains 0.5% tween 20, when 0.5 g / L super thin supercritical extract is added, RPMI1640 medium containing 0.5% tween 20 is used as a control (represented as 0.5% tween 20 in FIG. 5) to increase the growth rate of C. albicans . It was calculated as a percentage.

Since the mixture of the clove extract and the peppermint extract contains 0.25% tween 20, when the mixture is added, RPMI1640 medium containing 0.25% tween 20 is used as a control (indicated as 0.25% tween 20 in FIG. 5) and the growth rate of C. albicans is expressed as a percentage. It was calculated. The relative strain growth rate (Relative cell density,%) was calculated by Equation 1 below and the results are shown in Table 2.

When the growth rate (%) of microorganisms when the clove extract was treated was A, and when the thick extract was treated, the growth rate (%) was B, and the expected growth rate (%) was calculated by Equation 2 below.

When the growth rate (%) when the composition containing the thick extract and the cloves extract is treated is A + B, if the value of A + B is lower than the expected growth rate calculated by Equation 2, the growth of microorganisms Or, since the growth was suppressed than expected, it is judged that there is a synergistic antibacterial effect by mixing the extract.

5 and 6 show a synergistic antimicrobial effect on C. albicans according to mixing the clove hot water extract and super thin supercritical extract. Specifically, FIG. 5 shows the C. albicans cell density measured when each of the cloves hydrothermal extract and the super thin supercritical extract are added by mixing, and FIG. 6 shows the C. albicans growth rate (%) of each extract compared to the control group.

5 and 6, the clove hot water extract and the thick supercritical extract do not show a C. albicans growth inhibitory effect compared to the control group. On the other hand, when treating the composition of a mixture of supercritical superthin extract and clove hot water extract, it showed a growth inhibitory effect of C. albicans of about 27% compared to the control group. The A + B value at this time is 73%, which is lower than the expected growth rate of 105% due to the mixing of the clove hot water extract and the super thin supercritical extract, so that it is superior to C. albicans by mixing the cloves hot water extract and the super thin supercritical extract. It was confirmed that it exhibits a synergistic antibacterial effect.

Moreover, the clove hot water extract and super thin supercritical extract do not show a growth inhibitory effect on C. albicans , but antibacterial activity may be exhibited by increasing the concentration of the extract according to the mixing of the cloves hot water extract and super thin supercritical extract, Table 2 , As shown in Figures 5 and 6, when the clove hot water extract was treated at a double concentration (0.01 g / L) and when the thick supercritical extract was treated at a double concentration (0.5 g / L), the clove hot water extract was And a thick supercritical extract mixed composition exhibit excellent C. albicans growth inhibitory effect. Therefore, it can be seen from this that the composition of the present invention has an excellent synergistic antibacterial effect, and effectively inhibits the growth or proliferation of C. albicans even at low concentrations.

3-2. Synergistic Effect of Mixing Clove Methanol Extract and Thick Methanol Extract

Based on the results of Example 2-4, even if the concentration of the extract is doubled, the effect of growth inhibition is not more than 50%, and the concentration does not show growth inhibition even at the concentration, Clove methanol extract 0.12g / L, thick methanol The antimicrobial effect of C. albicans according to the mixture of clove methanol extract and thick methanol extract was examined using 0.06 g / L extract. The concentration of each extract used in the experiment is listed in Table 3.

As in the method described in Example 2-4, each extract and C. albicans fungus suspension at the concentrations listed in Table 3 were dispensed, and absorbance was measured at 600 nm after cultivation, and this was expressed as the cell density of C. albicans . The growth rate (%) of C. albicans was calculated and described in Table 3 as described in Example 3-1.

Figure 7 shows a synergistic antibacterial effect on C. albicans when mixing the clove methanol extract and thick methanol extract, specifically, shows the growth rate of C. albicans when each extract is added to the control group.

As shown in FIG. 7, when the clove methanol extract and the aftertaste methanol extract were treated individually, the growth of C. albicans was inhibited by 8% of the clove extract and 16% of the aftertaste extract compared to the control group. When the expected growth rate was calculated by the method described in Example 3-1, it was 77.28%. On the other hand, when mixed with clove methanol extract and thick methanol extract, it showed about 58% of C. albicans growth inhibition compared to the control group. From the above results, it can be seen that the mixture of clove methanol extract and aftertaste methanol extract has a growth inhibitory effect on C. albicans of 7 times and 3.5 times, respectively, than when the clove extract and aftertaste extract were individually treated. In this case, the A + B value at this time was 42%, which was significantly lower than the expected growth rate value (77.28%), so it was confirmed that it exhibited excellent synergistic antibacterial effects of the clove methanol extract and the thick methanol extract.

Moreover, antibacterial activity may be exhibited by increasing the concentration of the extract according to the mixing of the clove methanol extract and the thick methanol extract, but when treated by increasing the concentration of the clove methanol extract twice (0.24 g / L), it grows 31% compared to the control group. Inhibition effect was observed, C. albicans growth inhibitory effect was increased by approximately 1.9 times when the concentration of single clove extract was treated twice (0.24 g / L) when mixed with clove methanol extract and thick methanol extract. Was confirmed.

Therefore, it can be seen from this that the composition of the present invention has an excellent synergistic antibacterial effect, and effectively inhibits the growth or proliferation of C. albicans even at low concentrations.

Example 4. According to the mixture of clove extract and thick pepper extract S. aureus Synergistic antibacterial effect

To examine the antimicrobial effect on S. aureus , 0.5 g / L of clove hot water extract and 0.5 g / L of superthin supercritical extract, cells of S. aureus upon extract treatment according to the modified CLSI M27-A2 method Density was examined.

S. aureus KCTC 3881 strain stock was streaked on TSB agar plates and cultured for 1 day at 37 ° C. Thereafter, a single colony was taken and inoculated into TSB medium, followed by pre-incubation for 18 to 24 hours under conditions of 250 rpm and 37 ° C in a test tube. Thereafter, the pre-cultured bacteria were measured at 600 nm with a spectrophotometer, diluted with TSB medium so that the absorbance (ABS) value was 0.1, and then diluted with 100 μl of S. aureus bacteria suspension on a microplate.

A composition containing 50 µl of clove hot water extract or 50 µl of supercritical superthin extract and 50 µl of TSB medium, and 50 µl of clove hot water extract and 50 µl of superthin supercritical extract (micro total 100 μl), respectively, dispensed with S. aureus fungus suspension as to the method described in example 2-2 by frequency division to the plate, by measuring the absorbance at 595nm, looking at a cell density of S. aureus, by the method described in examples 3-1 of this by calculating the growth rate of S. aureus The antibacterial effect of the composition of the invention on S. aureus was examined. Table 4 shows the concentration of each extract used in the experiment and the growth rate of S. aureus .

8 and 9 show a synergistic antimicrobial effect against S. aureus when mixing the clove hot water extract and thick supercritical extract. Specifically, FIG. 8 shows the S. aureus cell density measured when each of the clove hot water extract and the super thin supercritical extract are mixed or added, and FIG. 9 shows the growth rate of the S. aureus strain of each extract compared to the control group.

At this time, the relative strain growth rate and expected growth rate were calculated by the method described in Example 3-1. The growth rate of S. aureus when treated with clove hot water extract was 105.6%, and that of S. aureus when treated with super thin supercritical extract. The growth rate is 75.4%. Therefore, the expected growth rate when mixing these two extracts is 79.6%. Results cloves and growth rate is 52.7%, so, because the expected growth rate compared to the S. aureus growth of approximately 33.8% inhibition clove second hot-water extract and Magnolia high rise also to S. aureus by a mixture of extracts of the threshold when mixed with magnolia extract It was confirmed that it exhibits a synergistic antibacterial effect.

Example 5. Synergistic antibacterial effect according to the mixing ratio of clove extract and thick pepper extract

Cloves of the hot-water extract and Magnolia seconds to investigate the antimicrobial activity of the C. albicans when different mixture ratio by using a threshold extract, as described in Example 3-1 by changing the mixing ratios of the extract of C. albicans Growth rate was calculated. Table 5 shows the concentrations of specific extracts and the growth rate of C. albicans .

Figure 10 shows the growth rate of C. albicans according to the mixing ratio of clove hot water extract and super thin supercritical extract. As shown in Table 5 and Figure 10, the antibacterial effect on the maximum C. albicans was shown in the group treated with a composition comprising 0.5 g / L of peppermint extract and 0.01 g / L of clove extract.

Claims (7)

Antibacterial composition comprising clove extract and peppermint extract as an active ingredient.
The method according to claim 1, The clove extract is water, alcohol having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane and mixtures thereof Antibacterial composition obtained from a solvent extraction method or a supercritical extraction method using an extraction solvent selected from the group consisting of.
The method according to claim 1, The thick extract is water, alcohol having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane and mixtures thereof Antibacterial composition obtained from a solvent extraction method or a supercritical extraction method using an extraction solvent selected from the group consisting of.
The method according to claim 1, The clove extract and the thick extract is an antimicrobial composition that is included in a weight ratio of 1: 1 to 1: 100.
The method according to claim 1, wherein the antimicrobial composition is Escherichia sp., Pseudomonas sp., Staphylococcus sp., Candida sp., Malassezia Genus ( Malassezia sp.), Salmonella sp., Acinetobacter sp., Corynebacterium sp., Mycobacterium sp., And Micrococcus genus ( Micrococcus sp.) Antimicrobial composition that inhibits the growth of microorganisms selected from the group consisting of.
The method according to claim 5, Escherichia coli , Pseudomonas aeruginosa , Candida albicans , Candida tropicalis , Malassezia pachydermatis , Salmonella typhimu Salmonella typhimurium , Acinetobacter johnsonii , Corynebacterium accolens , Corynebacterium amycolatum , Corynebacterium xerosis , Corynebacterium jeikeium , Staphylococcus aureus , Staphylococcus haemolyticus , Staphylococcus warneri , Staphylococcus warneri , Star Staphylococcus saprophyticus , Staphylococcus cohnii , Staphylococcus hominis , Staphylococcus xylosus , Staphylococcus xylosus , Staphylococcus xylosus Antimicrobial composition that is at least one selected from the group consisting of Staphylococcus simulans , Mycobacterium fortuitum , and Micrococcus luteus .
The composition according to any one of claims 1 to 6, wherein the antimicrobial composition is used as a preservative, preservative, detergent, or cleaner.

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