KR102486044B1 - Anti-microbial and anti-viral composition derived from natural product - Google Patents

Abstract

The present invention relates to an anti-microbial composition comprising a lemon extract and a lemon oil. The composition of the present invention not only provides excellent antiviral, antibacterial, and antifungal effects, but also can be used safely without toxicity and side effects because it is derived from natural products so that it can be used for various purposes such as pharmaceuticals, foods, cosmetics, and quasi-drugs.

Description

Antibacterial and antiviral composition derived from natural products {ANTI-MICROBIAL AND ANTI-VIRAL COMPOSITION DERIVED FROM NATURAL PRODUCT}

The present invention relates to an antimicrobial composition comprising lemon extract and lemon oil.

Viruses are representative pathogens that cause many infectious diseases such as smallpox, influenza, and AIDS, but their existence is not properly understood until the 1900s because of their small size. Currently, many vaccines and treatments are being developed because the structure, function, and operation method of viruses are known, but it is difficult to develop effective treatments due to various breeding methods and genetic mutations. For example, hand-foot-and-mouth disease, one of the main causes of which is infection with a picornavirus family of viruses (e.g., Enterovirus 71), is highly contagious and can cause complications such as fever, vomiting, diarrhea, headache and meningitis, mainly in infants and young children. Although it has become a big social problem, effective vaccines and treatments for hand, foot and mouth disease have not been developed to date.

In addition, infections of various microorganisms, such as bacteria, fungi, and yeast, as well as viruses, are also major causes of diseases in mammals, including humans. Numerous antiviral and antibacterial agents have been developed and are in use, but side effects have been reported in some synthetically derived drugs, and resistance to drugs due to the continuous evolution of microorganisms has resulted in a problem of reduced drug effectiveness. Therefore, there is an urgent need to develop an antimicrobial agent derived from natural products that has excellent antimicrobial activity, has no side effects and is safe.

Accordingly, the inventors of the present invention have found that a specific mixed composition derived from a natural product has an excellent effect of inhibiting the activity of various microorganisms, thereby completing the present invention.

One object of the present invention is to provide a natural product-derived antimicrobial composition, and more specifically to provide a pharmaceutical composition, food composition, cosmetic composition and cleaning composition having antimicrobial activity.

In order to achieve the above object, one aspect of the present invention provides an antimicrobial composition comprising a lemon extract and lemon oil as active ingredients.

The term "extract" refers to a product obtained by extraction with an appropriate extraction solvent, and the product includes an extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crude or purified product thereof.

The term "lemon extract" means an extract obtained from a lemon tree, and may be, for example, an extract obtained from flowers, leaves, fruits, fruit peels, seeds, roots and/or stems of a lemon tree. The lemon tree belongs to the evergreen small tree of the dicotyledonous plant class, the rue family, and the scientific name may be Citrus limon . The lemon extract may preferably be an extract obtained from the fruit of a lemon tree.

The term "lemon oil" refers to oil obtained from lemon trees, and may be, for example, oil obtained from flowers, leaves, fruits, fruit peels, seeds, roots and/or stems of lemon trees. The lemon oil may preferably be oil obtained from the fruit of a lemon tree.

The extracts and oils can be prepared using general methods known in the art. For example, methods such as hot water extraction, hot water extraction, chill extraction, reflux cooling extraction, steam distillation extraction, alcohol extraction, alcohol extraction, and/or ultrasonic extraction may be used. The extraction solvent may be selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol, butanol, etc.), organic solvents (hexane, acetone, chloroform, methyl acetate, etc.) and mixed solvents thereof. there is.

As used herein, the term "antimicrobial" means a property that resists microorganisms such as viruses, bacteria, and fungi (eg, yeasts, molds, molds, etc.), and more specifically, inhibits the growth or proliferation of the microorganisms. or stopping or promoting or inducing the death of the microorganisms. "Antimicrobial activity" may be used as an alternative to the above term.

The antimicrobial composition according to the present invention has antiviral, antibacterial and antifungal activity. More specifically, the composition according to the present invention exhibits an activity of inhibiting, stopping or delaying the growth or proliferation of viruses, bacteria and fungi, an activity of promoting or inducing death, an activity of inhibiting infection, and the like.

The term “virus” is used herein as a meaning commonly understood in the art, and includes, but is not limited to, Enterovirus and the like. About 70 types of enteroviruses, such as poliovirus, coxsackievirus A, coxsackievirus B, echovirus, and enterovirus (EV) 71, have been known to date. As disclosed in “Laboratory Diagnosis of Infectious Diseases” published by the Korea Centers for Disease Control and Prevention in 2005, enteroviruses can cause asymptomatic infection, diarrhea, cold, hand-foot-and-mouth disease, herpangina, hemorrhagic conjunctivitis, polio, meningitis, encephalitis, pneumonia, It has been reported to cause various symptoms such as bronchitis, acute paralysis, and myocarditis. In addition to aseptic meningitis, enterovirus 71 often causes hand-foot-and-mouth disease, herpetic stomatitis, acute hemorrhagic conjunctivitis, etc., and causes serious complications in the brain or lungs, leading to death.

The term "bacteria" is used herein as a meaning commonly understood in the art, and includes, but is not limited to, gram-positive bacteria such as Staphylococcus. sp) , Escherichia coli ( E. coli ), Pseudomonas aeruginosa ( Pseudomonas. sp ) and Gram-negative bacteria such as Diseases and symptoms caused by the Staphylococcus sp infection include, for example, food poisoning, suppurative arthritis, suppurative osteomyelitis, folliculitis, otitis media, conjunctivitis, pneumonia, postoperative wound infection, bacteremia, sepsis, endocarditis, cellulitis, etc. reported, but is not limited thereto. Diseases and symptoms caused by E. coli infection have been reported to include, for example, diarrhea, vomiting, fever, abdominal pain, headache, hemorrhagic enteritis, dehydration, hemolytic uremic syndrome (HUS), etc. , but is not limited thereto. Diseases and symptoms caused by the Pseudomonas aeruginosa ( Pseudomonas sp ) infection have been reported to include, for example, pressure sores, pneumonia, bacteremia, sepsis, meningitis, keratitis, otitis media, endocarditis, brain abscess, etc., but are not limited thereto.

The term "fungi" is used herein as a meaning commonly understood in the art, and includes, but is not limited to, yeasts such as Candida, filamentous fungi (also called fungi), and the like. Diseases and symptoms caused by the Candida yeast infection have been reported to include, for example, stomatitis, vaginitis, vulvitis, esophagitis, paronychia, etc., but are not limited thereto. Diseases and symptoms caused by the mold fungus ( Aspergillus. sp ) have been reported to include, for example, athlete's foot, ringworm, jock itch, dermatomycosis, etc., but are not limited thereto.

The antimicrobial composition of the present invention comprises lemon extract and lemon oil. Preferably, the weight ratio of the active ingredient lemon extract to lemon oil included in the composition may be 50:50 or more. More preferably, the weight ratio of the active ingredient lemon extract to lemon oil included in the composition may be 80:20 or more. More preferably, the weight ratio of the active ingredient lemon extract to lemon oil included in the composition may be 90:10 or more. For example, the weight ratio of the active ingredient lemon extract to lemon oil included in the composition may be 99:1.

Another aspect of the present invention provides a pharmaceutical composition comprising the above antimicrobial composition.

In the pharmaceutical composition according to the present invention, the antimicrobial composition is as described above unless otherwise specified.

The pharmaceutical composition may be used to treat or prevent a microbial infection or a disease caused by a microbial infection.

The term "treatment" refers to any activity in which symptoms of a microbial infection or a disease caused by a microbial infection are improved or cured through the administration of the composition according to the present invention. In addition, the term "prevention" refers to any action that suppresses or delays the symptoms of a microbial infection or a disease caused by a microbial infection through the administration of the composition according to the present invention.

The pharmaceutical composition may be preferably used to treat or prevent one or more infections selected from the group consisting of viruses, gram-positive bacteria, gram-negative bacteria, yeasts and filamentous bacteria, or diseases and symptoms caused by these infections.

The content of the antimicrobial composition included in the pharmaceutical composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the progress of the symptoms, the condition of the patient, and the like. For example, the antimicrobial composition of the present invention, based on the total weight of the pharmaceutical composition, is 0.0001 to 99.9% by weight, 0.1 to 90% by weight, 1 to 80% by weight, 1 to 70% by weight, 1 to 60% by weight, or 1 to 60% by weight. It may be contained in an amount of 50% by weight.

The pharmaceutical composition of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. Carriers, excipients and diluents that may be included in the composition include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may be an oral dosage form, and may also be formulated into a suitable dosage form for topical administration using techniques well known to those skilled in the art. In the case of topical formulations, the formulations may include external preparations, effervescent tablets, suppositories, and the like. In one embodiment, the pharmaceutical composition of the present invention may be formulated for external use by mixing the antimicrobial composition with a base that is well known and commonly used in the art. The external preparations may include emulsions, gels, ointments, creams, patches, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, drops, suspensions, and tinctures.

The pharmaceutical composition of the present invention is administered to a subject in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is subject type and severity, age, It may be determined according to factors including sex, activity of drug, sensitivity to drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Another aspect of the present invention provides a cosmetic composition comprising the above antimicrobial composition.

In the cosmetic composition according to the present invention, the antimicrobial composition is as described above unless otherwise specified.

Based on the total weight of the cosmetic composition, the antimicrobial composition of the present invention is 0.0001 to 99.9% by weight, 0.1 to 90% by weight, 1 to 80% by weight, 1 to 70% by weight, 1 to 60% by weight, or 1 to 50% by weight It may be contained in an amount of %.

The cosmetic composition can maximize antiviral, antibacterial, antifungal and cleaning effects by containing the antimicrobial composition of the present invention. This ultimately shows the effect of preventing or improving microbial infections such as viruses, bacteria, fungi, or diseases and symptoms caused by the microbial infections.

The term "improvement" refers to any action that at least reduces a parameter related to the state in which the microbial infection or disease caused by it is being treated, for example, the severity of symptoms.

Of course, the cosmetic composition may be prepared in the form of a general emulsified formulation and a solubilized formulation by using a conventionally known manufacturing method. At this time, the cosmetic composition may be appropriately selected and formulated according to the purpose. For example, it can be prepared in various formulations such as lotions, essences, creams, packs, patches, skin adhesive gels, foundations, and makeup bases, and can be applied to conventional cosmetic preparation methods.

The cosmetic composition according to one aspect may optionally further include various additives in addition to the antimicrobial composition. The above additives include, for example, stabilizers, emulsifiers, thickeners, moisturizers, liquid crystal film strengthening agents, pH adjusting agents, antibacterial agents, water-soluble polymers, coating agents, metal ion sequestering agents, amino acids, organic amines, polymer emulsions, pH adjusting agents, skin nutrients, oxidizing agents, and the like. one or more aqueous additives selected from antioxidants, antioxidants, preservatives, flavoring agents, and the like; and one or more oily additives selected from oils and fats, waxes, hydrocarbon oils, higher fatty acid oils, higher alcohols, synthetic ester oils, and silicone oils.

Another aspect of the present invention provides a food composition comprising the antimicrobial composition.

In the food composition according to the present invention, the antimicrobial composition is as described above unless otherwise specified.

The food composition can improve or prevent microbial infections such as bacteria, viruses, yeasts and fungi, or diseases caused by the microbial infections.

The term "improvement" refers to any action that at least reduces a parameter related to the state in which the microbial infection or disease caused by it is being treated, for example, the severity of symptoms.

The food may be a health functional food. The term "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functionality for the human body. Here, "functionality" means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions.

The food composition according to the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, since the food composition of the present invention contains a natural plant extract as a main active ingredient, there is no side effect or harm to the human body, so it can be consumed as an adjuvant to enhance or improve the treatment effect of microbial infections or diseases caused thereby.

The content of the antimicrobial composition according to the present invention included as an active ingredient in the food composition according to one embodiment may be appropriately determined depending on the purpose of use (prevention, improvement or therapeutic treatment). In general, when preparing food, the antimicrobial composition of the present invention may be included in an amount of 0.001 to 20% by weight, 0.001 to 15% by weight, or 0.001 to 10% by weight of the raw material composition. In the case of health drinks, 0.01 to 2 g, for example, 0.3 to 1 g may be added based on 100 mL. However, in the case of long-term intake for the purpose of health and hygiene or health control, it may be used in an amount smaller than the above range. In the process of preparing the food composition, the content of the antimicrobial composition according to the present invention added to the food composition may be appropriately increased or decreased as necessary.

The food composition may be any one formulation selected from the group consisting of pills, tablets, granules, powders, capsules, and liquid solutions.

Another aspect of the present invention provides a quasi-drug composition comprising the antimicrobial composition.

In the quasi-drug according to the present invention, the antimicrobial composition is as described above unless otherwise specified.

Since the antimicrobial composition of the present invention has very strong inhibitory activity against viruses, bacteria and fungi, it can be used as a quasi-drug composition for antimicrobials. In addition, since the active ingredient is derived from plants, it can be safely used without toxicity or harm to the human body.

The term “quasi-drugs” refers to items excluding items used for pharmaceutical purposes, such as textiles, rubber products, or similar products used for the purpose of treating, alleviating, treating, or preventing diseases of humans or animals; It means items that do not act directly and are not tools or machines and similar, items that fall under one of the agents used for sterilization, insecticidal, and similar purposes to prevent infection, such as disinfectants and disinfectant cleaners , and personal hygiene products.

The quasi-drug composition according to one aspect of the present invention can be used for purposes such as disinfectants and detergents. The disinfectant may be prepared by using the antimicrobial composition of the present invention as it is or by diluting it with a dermatologically and pharmacologically acceptable diluent or solvent to a suitable concentration. The disinfectant may be used on the surface of an organism, preferably on mammalian skin, most preferably on human skin. For example, it may be prepared and used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel. In addition, the disinfectant may be used on surfaces of inanimate objects, such as wood, metal, glass, ceramics, plastics, paper and cloth. The disinfectant may be applied to the surface of the living or inanimate object using a method including dipping, swabbing, spraying and brushing. Preferably, the disinfectant can be used in hospitals and general homes for purposes such as wound surface disinfection, skin disinfection before surgery, disinfection of surgical instruments, disinfection of catheters, and the like. In the case of the disinfectant detergent, it can be easily prepared according to a method known in the art, including one or more excipients and additives generally used in the manufacturing field of detergents in addition to the antimicrobial composition according to the present invention. The detergent may be used for kitchen or food purposes, and may further include anionic surfactants, nonionic surfactants, amphoteric surfactants, and the like in addition to processed citraconic acid anhydride. In the case of a detergent for food, it can be prepared by adding a food-acceptable solvent or diluent to the antimicrobial composition according to the present invention and diluting it to an appropriate concentration. For example, sterilization of fruits, fish and meat, etc. and washing. In addition, the quasi-drug composition may be a personal hygiene product, for example, soap, cosmetics, wet tissue, toilet paper, shampoo, skin cream, face cream, toothpaste, or cleansing gel.

A mixed composition containing lemon extract and lemon oil as active ingredients can effectively inhibit viruses, bacteria, yeast, and mold within a short period of time. Thus, the composition of the present invention provides excellent antiviral, antibacterial, and antifungal effects. In addition, since the composition of the present invention is derived from natural products and can be safely used without toxicity and side effects, it can be used for various purposes such as pharmaceuticals, foods, cosmetics, and quasi-drugs.

1 is a graph showing the enterovirus 71 inhibitory effect in percentage for a single composition of each active ingredient of the present invention and a mixed composition containing each active ingredient in various ratios.

Hereinafter, the present invention will be described in more detail by examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of Lemon Oil

After adding 30 g of dried lemon to a steam distillation extractor set containing 570 g of water, steam distillation extraction treatment was performed for 1 hour at 100 ° C in an extractor equipped with a cooling condenser. The steam distillation extract obtained by cooling was subjected to stationary cooling at 5° C. for 24 hours to obtain an upper separated oil. The lemon oil thus obtained was referred to as "Sample 1" and was used in the following test examples.

Example 2. Preparation of Lemon Extract

After immersing 30 g of dried lemon in 570 g of water, it was treated with hot water (reflux) for 1 hour at 60 ° C. in an extractor equipped with a cooling condenser. After completion of the hot water (reflux) treatment, the extract was separated by centrifugation for 20 minutes at 8,000 rpm and 15° C. using a centrifuge (Supra 22K, Hanil, Korea). The separated extract was filtered through 0.8 μm and 0.45 μm filters to obtain a filtrate, and the filtrate was concentrated for 2 hours at 60° C. and 30 hPa using a concentrator (Rotary Evaporator NE-series, Eyela, Japan). did This concentrate was pretreated by freezing at -21 ° C and then freeze-dried at -80 ° C for 48 hours using a freeze dryer (Bondiro, Ilshin, Korea) to obtain an extract. The lemon extract thus obtained was referred to as "Sample 6" and was used in the following test examples.

Example 3. Preparation of Lemon Extract and Lemon Oil Mixtures

Using a homogenizer (IKA T25, IKA, JPN), the lemon oil and lemon extract obtained in Examples 1 and 2 were mixed in various ratios, such as 50:50, 80:20, 90:10, and 99:1 by weight. and vigorously homogeneously mixed. The mixed compositions thus obtained are referred to as "Sample 2", "Sample 3", "Sample 4", and "Sample 5" respectively in the order of weight ratios of 50:50, 80:20, 90:10, 99:1, It was used in the following test examples.

Test Example 1. Inhibitory effect of enterovirus 71 infection

1-1. Measurement of antiviral activity rate using SRB assay

In order to confirm the effect of inhibiting enterovirus 71 infection of the composition according to the present invention, SRB (sulforhodamine B) assay (Choi et al, Antiviral activity of raoulic acid from Raoulia australis against Picornaviruses, Phytomedicine, 2009) was applied and tested.

Vero cells were dispensed in a well plate at a concentration of 3×10 ⁴ cell/well, and cultured for 24 hours in DMEM medium containing 10% FBS. After removing the medium and washing with PBS, enterovirus 71 suspension and FBS medium were added to the wells containing each sample prepared in the above example at a concentration of 100 μg/mL, and then in a CO ₂ incubator (Sanyo, JPN). It was cultured for 2 days under 5% CO ₂ conditions. Then, it was washed with PBS, and 100 μl of 70% acetone was added to each well, left at room temperature for 30 minutes, and then removed. After drying using a dry oven (Hanbaek, KOR) for 10 minutes, 100 μl of 0.4% SRB staining reagent containing 1% acetic acid was added and allowed to stand at room temperature for 30 minutes. After removing SRB and washing the plate several times with a co-solvent containing 1% acetic acid, it was dried again in a dry oven and observed using a phase contrast microscope (Nikkon, JPN). After dissolving each well in a Tris-base solution and leaving it to stand, the absorbance value (OD) at 560 nm was measured using an ELISA reader (EPOCH2, Biotek, USA).

As a result, the measured absorbance value (OD) was incorporated into Equation 1 below, and the antiviral activity of Samples 1 to 6 was calculated as a percentage. Here, the antiviral activity rate represents the activity of the sample to inhibit the growth and proliferation of Enterovirus 71, which can also be interpreted as meaning the cell protection rate from Enterovirus 71 infection.

[Equation 1]

The antiviral activity rate (%) of each composition calculated according to Equation 1 is shown graphically in FIG. As can be seen in FIG. 1, it was confirmed that samples 1 and 6 had almost no antiviral activity against enterovirus 71 infection when compared to the virus-treated control group. On the other hand, in the case of the mixed compositions (samples 2, 3, 4 and 5), it was confirmed that the cell protective effect against enterovirus 71 infection was significant regardless of the mixing ratio. In particular, samples 4 and 5 in which lemon extract and lemon oil were mixed at a ratio of 90:1 to 99:1 showed the best antiviral activity.

1-2. Confirmation of antiviral activity through western blot analysis of VP protein

To re-verify the antiviral efficacy of the composition according to the present invention, Western blot was performed on the VP protein of Enterovirus 71.

After heating the supernatant containing the VP protein of Enterovirus 71 at 100° C. for 10 minutes, electrophoresis was performed on a 12% acrylamide gel at 100 V for 1 hour. Then, the proteins in the acrylamide gel were transferred to an iBlotTransfer Stack, PVDF, regularsize (IB401001, invitrogen) for 7 minutes at 20 V. The membrane was reacted in 5% skim milk (232100, Difco) for 1 hour at room temperature, and then washed three times with phosphate buffered saline (PBS) Tween-20.

Thereafter, the membrane was reacted with a mouse anti-enterovirus 71 monoclonal antibody (MAB 979, Millipore) diluted 1000-fold in 5% skim milk on the membrane for 2 hours at room temperature, and washed several times with PBS Tween-20. Control α-tubulin was performed in the same manner using a mouse IgG1 monoclonal antibody (SC-32293, Santa Cruz) as a primary antibody.

As a secondary antibody to identify the VP protein of enterovirus 71 and the control α-tubulin, polyclonal goat anti-mouse IgG (H+L) HRP (SA001-500, GenDEPOT) was used. After diluting polyclonal goat anti-mouse IgG (H+L) HRP in 5% skim milk at a ratio of 5000, the membrane was reacted at room temperature for 1 hour, washed with PBS Tween-20, and Western blotting was performed.

As a result, α-tubulin was confirmed in all of the virus-untreated control group, the virus-treated control group, and the virus+sample-treated experimental group. The VP protein of enterovirus 71 was absent in the virus-free control group, whereas it was very prominent in the virus-treated control group. However, the VP protein of Enterovirus 71 was hardly observed in the experimental group in which samples 2 to 5 belonging to the present invention were treated with the virus, respectively.

Thus, it was confirmed that a significant level of antiviral activity is exhibited when the composition according to the invention is treated against viruses.

Test Example 2. Antibacterial and antifungal effects

In order to test the effect of the antibacterial and antifungal activity of the samples obtained in the above example, solid culture dilution method (Agar Serial Dilution Method) and disk diffusion method (Paper disc method) experiments were performed.

Staphylococcus aureus (KCTC 6910) as Gram-positive bacteria, Pseudomonas aeruginosa (KCTC 1637) and Escherichia coli (E. Coli, KCTC 1039) as Gram-negative bacteria, Candida albicans as yeast albicans, KCTC 7965), and Aspergillus niger (KCTC 6910) as filamentous fungi, a total of 5 strains were used, all of which were distributed from the Korea Research Institute of Bioscience and Biotechnology. Specific experimental methods and results are as follows.

2-1. Experiments according to the solid culture dilution method

The antibacterial activity of the composition was confirmed by measuring the minimum inhibitory concentration by the solid culture dilution method.

For the antibacterial test, bacteria were inoculated on tryptic soy agar medium and pre-cultured at 37 ° C for 24 hours. For yeast, bacteria were inoculated on potato dextrose agar medium and pre-cultured at 25 ° C for 2 days. In the case of , the bacteria were inoculated on a potato dextrose agar medium and pre-cultured in an incubator at 25 ° C for 7 days, and then the spores of the filamentous fungus formed on the surface of the medium were recovered using a smear rod and diluted in sterilized saline.

2 mL of each composition diluted with 5% dimethylsulfoxide (DMSO) physiological saline solution was added to a sterilized petri dish. As a control, 5% DMSO physiological saline solution was used. 18 mL each of tryptic soy agar medium and potato dextrose agar medium that had been sterilized and cooled to 48° C. was added to each petri dish, stirred, and allowed to stand to solidify.

Thereafter, each of the pre-cultured bacteria was plated on a Petri dish (smear cell concentration: about 1 × 10 ^{6 CFU/mL of bacteria, about 1 × 10 5 CFU} /mL of yeast, about 1 × 10 ⁴ CFU/mL of mold). . After culturing bacteria at 37°C for 24 hours, yeast at 25°C for 3 days, and filamentous fungi for 7 days in an incubator at 25°C, the formation of colonies in each compartment was observed.

The minimum sample concentration (i.e., minimum inhibitory concentration (MIC)) of the non-growth plate was confirmed, and the results are shown in Table 1 below. At this time, the lower the value of the minimum inhibitory concentration, the higher the antibacterial effect.

Minimum inhibitory concentration (MIC, μg/mL) * Mixing ratio: lemon extract: lemon oil strain Sample 1
(0:100) sample 2
(50:50) sample 3
(80:20) sample 4
(90:10) sample 5
(99:1) sample 6
(100:0) S. aureus 2,000 1,000 500 200 100 1,000 P. aeruginosa 1,500 1,000 500 200 150 2,500 E. Coli >5,000 2,500 500 200 200 >5,000 C. albicans >5,000 3,000 300 200 200 >5,000 A. niger >5,000 3,000 300 200 200 >5,000

As can be seen in Table 1, the mixed compositions (Samples 2, 3, 4, and 5) of the present invention are all strong antibacterial against various bacteria, yeast, and filamentous fungi when compared to single substances (Samples 1 and 6). showed effect.

2-2. Experiment according to the disk diffusion method

In order to test the antibacterial effect of the composition, a disk diffusion method experiment was performed.

1 Platinum amount of each strain cultured on the plate medium was cultured in 10 mL of liquid medium for 24 hours and activated. After inoculating 0.1 mL of the cultured bacterial solution in 10 mL of liquid medium and incubating for 6 hours, the bacterial solution per plate medium was inoculated to about 10 ⁷ cfu / mL and spread evenly. Thereafter, a sterilized paper disk (6 mm, Satorius, Germany) was placed on a solid plate medium, and the sample dissolved in the solvent was absorbed to a concentration of 0.05 ml/disk and cultured. Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were cultured at 37°C, and fungi Candida albicans and Aspergillus niger were cultured at 27°C for 24 hours and 120 hours, respectively. The diameter of the transparent zone around the paper disk was measured.

The measurement results are shown in Table 2 below. At this time, the transparent zone around the paper disk corresponds to an index of the degree to which the proliferation of the strain is inhibited. Therefore, the larger the diameter (mm) of the transparent zone, the higher the antibacterial effect of the sample against the corresponding strain.

Clear zone diameter (mm) * Mixing ratio: lemon extract: lemon oil strain Sample 1
(0:100) sample 2
(50:50) sample 3
(80:20) sample 4
(90:10) sample 5
(99:1) sample 6
(100:0) S. aureus 7 8 13 20 25 8 P. aeruginosa 8 8 13 22 24 7 E. Coli - 7 15 22 21 - C. albicans - 7 17 20 20 - A. niger - 7 16 17 17 -

As shown in Table 2, samples 1 and 6, which are single substances, had little antibacterial effect. On the other hand, in the case of the mixed composition, all compositions showed significantly better antibacterial effects against various bacteria, yeast, and fungi than single extracts.

Hereinafter, lotion and lotion are exemplified as formulation examples of the present invention, but the formulation of the composition according to the present invention should not be construed as being limited thereto, and ordinary changes by those skilled in the art are possible within the scope of the present invention.

Formulation Example 1. Lotion ingredient content(%) One Purified water balance 2 Butylene Glycol 5.00 3 composition of the present invention 2.00 4 glycerin 4.00 5 Disodium EDTA 0.02 6 carbomer 0.10 7 betaine 0.50 8 hyaluronic acid 0.01 9 ethanol 4.00 10 PEG-60 Hydrogenated Castor Oil 0.30 11 Methylparaben 0.10 12 Phenyl Trimethicone 0.05 13 flavoring agent a very small amount 14 coloring agent a very small amount

Formulation Example 2. Lotion ingredient content(%) One Purified water balance 2 Methylparaben 0.20 3 composition of the present invention 5.00 4 glycerin 5.00 5 Butylene Glycol 7.00 6 carbomer 0.10 7 xanthan gum 0.05 8 Cetearyl Alcohol 3.00 9 Glyceryl Stearate 0.05 10 Propylparaben 0.10 11 mineral oil 3.00 12 vegetable squalane 3.00 13 Cetylethylhexanoate 3.00 14 arginine 0.10 15 flavoring agent a very small amount

Formulation Example 3. Cream ingredient content(%) One Purified water balance 2 Methylparaben 0.20 3 composition of the present invention 5.00 4 glycerin 5.00 5 Butylene Glycol 7.00 6 carbomer 0.20 7 xanthan gum 0.08 8 Cetearyl Alcohol 3.00 9 Glyceryl Stearate 0.50 10 behenyl alcohol 2.00 11 hydrogenated lecithin 0.20 12 Propylparaben 0.10 13 mineral oil 3.00 14 vegetable squalane 3.00 15 Cetylethylhexanoate 3.00 16 arginine 0.20 17 flavoring agent a very small amount

Formulation Example 4. Essence ingredient content(%) One Purified water balance 2 Methylparaben 0.20 3 composition of the present invention 5.00 4 hyaluronic acid 0.05 5 Butylene Glycol 2.00 6 carbomer 0.08 7 xanthan gum 0.04 8 Cetearyl Alcohol 0.50 9 Glyceryl Stearate 0.50 10 Propylparaben 0.10 11 mineral oil 1.00 12 vegetable squalane 2.00 13 Cetylethylhexanoate 1.00 14 arginine 0.08 15 ethanol 4.00 16 PEG-60 Hydrogenated Castor Oil 0.30 17 flavoring agent a very small amount

Formulation Example 5. Pack ingredient content(%) One Purified water balance 2 glycerin 10.00 3 composition of the present invention 5.00 4 Butylene Glycol 5.00 5 kaoline 4.00 6 Caprylic/Capric Triglyceride 4.00 7 cyclomethicone 1.00 8 Magnesium aluminum silicate 1.00 9 PEG-100 Stearate, Glyceryl Stearate 2.00 10 xanthan gum 0.10 11 ethanol 3.00 12 Methylparaben 0.20 13 clophenesin 0.10 14 titanium dioxide 0.50 15 flavoring agent a very small amount

Claims (5)

a) a lemon extract obtained by filtering and concentrating a hot water extract of lemon at 60 ° C and freeze-drying; and b) lemon oil separated from the upper portion after cooling the steam distilled extract of lemon at a weight ratio of 90 to 99: 1 to 10,
An antiviral composition characterized in that it inhibits the activity and infection of Enterovirus 71. A pharmaceutical composition comprising the antiviral composition according to claim 1,
A pharmaceutical composition for treating or preventing an enterovirus 71 infection or a disease caused by an enterovirus 71 infection. A cosmetic composition comprising the antiviral composition according to claim 1,
A cosmetic composition for preventing enterovirus 71 infection or symptoms caused by enterovirus 71 infection. A food composition comprising the antiviral composition according to claim 1,
A food composition for preventing or improving enterovirus 71 infection or symptoms caused by enterovirus 71 infection. A quasi-drug composition comprising the antiviral composition according to claim 1,
A quasi-drug composition for preventing or improving enterovirus 71 infection or symptoms caused by enterovirus 71 infection.

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