KR102224078B1 - A composition for treating, improving and preventing of papilloma virus infection comprising Ecklonia kurome Okamura extract or Ecklonia kurome Okamura fraction - Google Patents

Abstract

The present invention relates to a composition for treating or preventing human papillomavirus (HPV) infection, and by containing an extract or fraction of Ecklonia kurome Okamura as an active ingredient, it is possible to treat or prevent human papillomavirus (HPV) infection. It can be used as an antiviral composition, and because it is not toxic, it can be consumed in the form of food.

Description

{A composition for treating, improving and preventing of papilloma virus infection comprising Ecklonia kurome Okamura extract or Ecklonia kurome Okamura fraction}

The present invention relates to a composition for the treatment, improvement or prevention of human papillomavirus (HPV) infection, which contains the extract or fraction of Ecklonia cava as an active ingredient.

In general, papillomavirus (human papillomavirus, HPV) has been known to infect epithelial cells of various tissues of animals and cause benign tumors commonly called warts. In particular, among human papillomaviruses (HPV), types 16 and 18 are associated with malignant tumors in the genital organs, oral cavity, and skin of men and women, and have been found to be major factors causing cervical cancer, which accounts for more than 90% of female uterine cancers.

Specifically, human papillomavirus (HPV) is a small DNA virus that causes benign malignancies with approximately 8,000 base sequences, and up to now, more than 100 human papillomavirus (HPV) subtypes have been identified according to genomic differences. , About 90 HPVs were completely genotyped. Among these types, the high-risk human papillomavirus (HPV) type (eg, HPV-16, 18, 31, 33, 35, 45, 51, 52, 56) is associated with almost 90% of cervical cancer.

More than 50% of cervical cancers infected with human papillomavirus (HPV) are related to HPV-16 type, followed by HPV-18 (12%), HPV-45 (8%), and HPV-31 (5%). ) Is related to the type. Cervical cancer is a cancer that can be prevented through early examinations in obstetrics and gynecology, but it still ranks second in the mortality rate after breast cancer among cancers in women around the world. Currently, there is no cure for papillomavirus (HPV), and vaccines are mainly used for prophylaxis, but its side effects have also been reported. There are two preventive vaccines for papillomavirus (HPV) developed to date: Cervarix (Cervarix: GSK) and papillomavirus (HPV) 16 and 18 types, including papillomavirus (HPV) 16 and 18 types. And papillomavirus (HPV) type 6 and 11, a tetravalent vaccine Gardasil (Merck). therefore. There is a need to develop an alternative treatment that will solve the side effects of preventive vaccines and the absence of therapeutic drugs.

Korean Registered Patent No. 1309538

Therefore, the object of the present invention was conceived to solve the above problems, a composition capable of treating or preventing human papillomavirus (HPV) infection by containing an extract or fraction of Ecklonia kurome Okamura as an active ingredient. It is in providing.

In addition, another object of the present invention is to provide a food composition capable of improving or preventing infection of human papillomavirus (HPV) by containing an extract or fraction of Ecklonia kurome Okamura as an active ingredient.

According to an embodiment of the present invention in order to achieve the above object, the composition for treating or preventing human papillomavirus (HPV) infection may contain an extract or fraction of Ecklonia kurome Okamura as an active ingredient.

The black snail extract may be an extract obtained by immersing the dried black snails in an extraction solvent.

The black-and-white composition may be extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

The mixed solvent may be an aqueous solution of 20 to 98% by volume of methanol, ethanol, butanol or propanol.

The black snail fraction may be obtained by fractionating the black snail extract with dichloromethane, ethyl acetate, n-butanol, or water.

The composition may be a pharmaceutical composition for the treatment or prevention of human papillomavirus infection.

In addition, according to another embodiment of the present invention, the composition for cleaning the human body may contain an extract or fraction of Ecklonia kurome Okamura as an active ingredient.

In addition, according to another embodiment of the present invention, the female cleanser composition may contain an extract or fraction of Ecklonia kurome Okamura as an active ingredient.

The pharmaceutical composition for the treatment or prevention of human papillomavirus infection may be a liquid preparation, an external preparation for skin, or an oral preparation.

In addition, according to another embodiment of the present invention, the food composition for improving or preventing human papillomavirus infection may contain an extract or fraction of Ecklonia kurome Okamura as an active ingredient.

According to the composition for the treatment, improvement or prevention of human papillomavirus (HPV) infection containing the extract or fraction of the present invention as described above, it suppresses the infection caused by human papillomavirus (HPV) without side effects. Or, it is possible to prevent cervical cancer and laryngeal cancer caused by the viral infection with an antiviral active substance that prevents or can be usefully used as a pharmaceutical composition and a food composition.

The effects according to the present invention in the above are not limited to the above, and other effects of the present invention are clearly described by those of ordinary skill in the field to which the present invention belongs through the matters described in the examples and claims to be described later. It will be understandable.

Figure 1 shows the extraction and fractionation scheme of the black Ecklonia cava of the present invention.
Figure 2 shows the human papillomavirus inhibitory activity according to the concentration of the ethanol extract of the present invention.
Figure 3 shows the human papillomavirus inhibitory activity according to the concentration of the solvent fraction of black Ecklonia cava of the present invention.
Figure 4 shows a comparison of protective efficacy against attack inoculation using HPV16 pseudovirion of the ethanol extract and chloromethane fraction of the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a composition for the treatment, improvement or prevention of human papillomavirus (HPV) infection containing an extract or fraction of Ecklonia kurome Okamura as an active ingredient.

The composition capable of treating or preventing human papillomavirus (HPV) infection of the present invention contains an extract of Ecklonia kurome Okamura or a fraction thereof as an active ingredient.

According to the present invention, the extract or fraction of Ecklonia kurome Okamura of the present invention is used as an antiviral agent to inhibit or prevent infection of HPV, or cervical cancer caused by infection of human papillomavirus (HPV). And it can be usefully used in pharmaceutical compositions and health functional foods for the prevention of laryngeal cancer.

The extract and some fractions may be used as an antiviral agent that inhibits or prevents HPV infection, or may be usefully used in pharmaceutical compositions and health functional foods for the prevention of cervical cancer and laryngeal cancer caused by the viral infection.

The Ecklonia kurome Okamura is a large perennial brown algae belonging to Phaeophyta, Phaeophyceae, Laminariales, and Laminariales, distributed in Korea and Japan. It has an attachment device and is 5-15cm in diameter. The stem is cylindrical, 8-100cm long, 10-15mm in diameter, and the strength of secreting sticky liquid is irregular ring shape and scattered in the cortex. The upper part of the leaf is flat, hard like leather, tough, slightly wrinkled, dark brown, 2-3mm thick, deeply divided into pinnates 1-2 times, the lobe on both sides is long tongue-shaped, the bottom part is wedge-shaped, and usually at the edge of the leaf. There are thick teeth. The sporangia is formed on the leaf surface and spores are produced in September-November. In Korea, it inhabits the east coast, the south coast, and the rocky area near the low Joseon coast of Jeju Island.

In an embodiment of the present invention, the extract of Ecklonia kurome Okamura is an extract extracted for 10 minutes to 72 hours, preferably 10 minutes to 36 hours after mixing the dried Ecklonia kurome Okamura with an extraction solvent at a ratio of 1:2 to 20 to be. Preferably, the Gumdung Ecklonia cava extract is obtained by performing an extraction method such as cold needle extraction, hot needle extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, and more preferably cold needle extraction and hot needle extraction at room temperature.

For example, the extract of Gumdung Ecklonia cava is cold-soaked in an extraction solvent of 20 to 26°C for 10 minutes to 5 hours, then immersed in an extraction solvent of 50 to 80°C, warmed for 10 minutes to 5 hours, and then concentrated. Can be obtained. At this time, it is preferable to use the same extraction solvent as the extraction solvent used for the cold needle and the extraction solvent used for the warm needle.

The extracting solvent for extracting the black snail extract may include water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, which can preferably act in the treatment or prevention of human papillomavirus infection. The mixed solvent is not particularly limited, but is preferably an extract extracted with 20 to 98% by volume of methanol, ethanol, butanol or propanol aqueous solution, and more preferably 60 to 95% by volume of an aqueous ethanol solution.

As another example, the composition capable of treating or preventing human papillomavirus infection of the present invention contains the Ecklonia kurome Okamura fraction as an active ingredient.

The Ecklonia kurome Okamura fraction is mixed with water of 5 to 30 times (w/w), preferably 8 to 20 times (w/w) in the concentrated Ecklonia kurome Okamura extract , A polar solvent fraction and a non-polar solvent fraction are obtained, respectively, by performing a nucleation process using a polar solvent or a non-polar solvent. Examples of the polar solvent include n-butanol and water, and examples of the non-polar solvent include dichloromethane and ethyl acetate.

According to an embodiment of the present invention, a composition containing the extract or fraction of Ecklonia kurome Okamura as an active ingredient may be a pharmaceutical composition.

In addition, according to an embodiment of the present invention, it is possible to provide a food composition or health functional food containing the extract or fraction of Ecklonia kurome Okamura as an active ingredient.

In the present invention, the extract'or'fraction' used while referring to the extract or fraction of Ecklonia kurome Okamura in the present invention includes not only the extract or fraction obtained by treating the extraction solvent, but also the processed product of the Ecklonia kurome Okamura extract or fraction. do. For example, the extract or fraction of Ecklonia kurome Okamura may be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In addition, the extract or fraction of Ecklonia kurome Okamura of the present invention is a processed product of Ecklonia kurome Okamura formulated to administer Ecklonia kurome Okamura to animals in a broad sense, such as Ecklonia kurome Okamura. Ecklonia kurome Okamura (Ecklonia kurome Okamura) is meant to include powder. Although the experiment was conducted with extracts or fractions of Ecklonia kurome Okamura in the present invention, it will be expected by those skilled in the art that the desired effect can be achieved even in the same form as the processed product of Ecklonia kurome Okamura. .

On the other hand, in the present specification, the term'containing as an active ingredient' means to contain an amount sufficient to achieve the efficacy or activity of the extract or fraction of Ecklonia kurome Okamura . As an example, the seaweed Ecklonia kurome (Ecklonia kurome Okamura ) extracts or fractions are used at a concentration of 10 to 1500 μg/ml, preferably 100 to 1000 μg/ml. Since the extract or fraction of Ecklonia kurome Okamura is a natural product and does not have side effects on the human body even if it is administered in excess, the quantitative upper limit of the extract or fraction of Ecklonia kurome Okamura included in the composition of the present invention is within an appropriate range for those skilled in the art. You can choose to do it.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, lubricants. Agents or flavoring agents may be used.

For administration, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients, and may be preferably formulated into a pharmaceutical composition.

The formulation form of the pharmaceutical composition may be granules, powders, tablets, external preparations for skin, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules (oral preparations), the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, trackcacanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

In addition, as another embodiment of the present invention, a composition for cleaning the human body is provided.

Specifically, the “human body cleaning composition” refers to a composition used for cleaning the skin such as face and body, for example, a material such as soap and foam cleansing, and the type is not limited.

The composition for cleaning the human body according to the present invention contains an extract or fraction of Ecklonia kurome Okamura as an active ingredient.

For example, soap, which is one of the compositions for cleaning the human body, may be prepared including a skin moisturizer, an emulsifier, a water softener, and the like as an additive. As the base of the soap, vegetable oils such as palm oil, palm oil, soybean oil, perm oil, olive oil, and palm kernels, or animal oils such as tallow, pork fat, yangji, fish oil, etc. can be used, and as the skin moisturizer, glycerin, erythritol, polyethylene Glycol, propylene glycol, butylene glycol, pentylene glycol, hexyl glycol, isopropyl myristate, silicone derivatives, aloe vera, sorbitol, etc. can be used, and as the emulsifier, natural oils, wax fatty alcohols, hydrocarbons, natural plant extracts And the like may be used, and as the water softening agent, tetrasodium IDTA and the like may be used.

The soap composition corresponding to one example of the composition for cleaning the human body of the present invention also contains an antimicrobial agent, a dispersant, a foam inhibitor, a solvent, a scale inhibitor, a corrosion inhibitor, a fragrance, a colorant, a metal ion sequestering agent, an antioxidant, a preservative, etc. as an additive. May contain additionally.

In the soap composition corresponding to one example of the composition for cleaning the human body of the present invention, the soap base or additive may be included in an amount generally used in the art, and the soap base is generally based on the total weight of the soap composition. When doing so, it may be added in an amount of 99.999% to 50% by weight, and an additive may be added in an amount of 1% to 20% by weight.

The female cleanser composition according to the present invention may contain an extract or fraction of Ecklonia kurome Ok amura as an active ingredient.

The “feminine cleansing composition” is generally used for the purpose of preventing secretions, itchiness, odor, etc., and reducing or eliminating discomfort, which are symptoms that appear as a result of infection by bacteria invading the vagina of a woman from the outside, and the present invention Since the feminine cleansing composition has an antiviral effect on HPV, it may include a preventive and therapeutic effect on diseases that may be caused by the virus, such as warts, vaginitis, and cervical cancer.

The feminine cleansing composition minimizes chemical preparations and replaces chemical preparations with naturally derived ingredients to realize excellent anti-inflammatory, antiviral, and antibacterial effects and hypoallergenic to the skin.

In the case of the feminine cleansing composition, it may be formulated by a general method in the art. Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA for the formulation of external preparations for skin may be referred to.

Specifically, the feminine cleansing composition may be prepared in a general emulsified formulation and a solubilized formulation. For example, a lotion such as a flexible lotion or a nutritional lotion; Emulsions such as facial lotion and body lotion; Creams such as nourishing cream, etc.; essence; Cosmetic ointment; spray; Gel; pack; Foundations such as liquid type, solid type or spray type; powder; It may be formulated with a cleansing cream, a cleansing lotion, or a detergent such as a cleansing foam, soap, or body wash, but is not limited thereto. In addition, the feminine cleansing composition may be formulated as an ointment, patch, gel, cream, or spray, but is not limited thereto.

The feminine cleansing composition may be appropriately blended with other ingredients within a range that does not impair the object according to the present invention, depending on the type or purpose of use, in addition to the essential ingredients in each formulation.

The feminine cleansing composition may include a generally acceptable carrier, for example, oil, water, surfactant, moisturizing agent, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately blended, but is limited thereto. It is not.

The acceptable carrier may vary depending on the formulation. For example, when formulated as an ointment, paste, cream or gel, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or these Mixtures can be used.

When the feminine cleansing composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component, and chlorofluoro It may further include a propellant such as hydrocarbon, propane, butane or dimethyl ether.

When the feminine cleansing composition is formulated as a solution or emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol , 1,3-butylglycol oil may be used, and in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used. .

When the feminine cleansing composition is formulated as a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

The feminine cleansing composition is a fatty substance, organic solvent, solubilizer, thickener, gelling agent, emollient, antioxidant, suspending agent, stabilizer, foaming agent, which are commonly used in the industry depending on the quality or function of the final product. Fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, commonly used in cosmetics It may additionally contain adjuvants commonly used in the field of cosmetology or dermatology, such as any other ingredients that are used.

However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect the desirable properties of the female cleanser composition according to the present invention.

As acceptable pharmaceutical carriers in the composition formulated as a liquid solution, as sterilization and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injection formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Further, it can be preferably formulated according to each disease or ingredient using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, vaginal administration, etc., preferably oral It is administration.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, and is usually As such, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001 to 10 g/kg.

The pharmaceutical composition of the present invention may be prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient, or may be prepared by incorporating it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

In addition, the present invention provides a food composition for improving or preventing human papillomavirus (HPV) infection containing an extract or fraction of Ecklonia kurome Okamura as an active ingredient.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gums, ice creams, vitamin complexes, health supplements. Etc.

The food composition of the present invention may include an extract or fraction of Ecklonia kurome Okamura as an active ingredient, as well as ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, It includes seasoning and flavoring agents. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavoring agents [taumatin, stevia extract (for example, rebaudioside A, Glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made of drinks and beverages, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts, in addition to the extract or fraction of the present invention, Ecklonia kurome Okamura And the like may additionally be included.

The present invention provides a health functional food comprising a food composition for the treatment or prevention of human papillomavirus (HPV) infection comprising the seaweed blackhead (Ecklonia kurome Okamura) extract or fraction as an active ingredient. Health functional foods are foods prepared by adding extracts or fractions of Ecklonia kurome Okamura to food ingredients such as beverages, teas, spices, gums, confectionery, or made into capsules, powders, suspensions, etc. It means bringing a specific effect on health, but unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long time by using food as raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of the extract of Ecklonia kurome Okamura in such a health functional food is the type of the health functional food that is the target. Although it may not be uniformly defined depending on the food, it may be added within a range that does not impair the original taste of the food, and is usually 0.01 to 50% by weight, preferably 0.1 to 20% by weight, based on the target food. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

In addition, the present invention provides a use of an extract or fraction of Ecklonia kurome Okamura for the manufacture of medicine or food for the treatment or prevention of human papillomavirus (HPV) infection. As described above , the extract or fraction of Ecklonia kurome Okamura may be used for the treatment or prevention of human papillomavirus (HPV) infection.

In addition, the present invention provides a method for treating or preventing human papillomavirus (HPV) infection comprising administering an effective amount of Ecklonia kurome Okamura extract or fraction to a mammal.

The term "mammal" as used herein refers to a mammal that is the subject of treatment, observation or experimentation, and preferably refers to a human.

The term "effective amount" as used herein refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, doctor or other clinician, which Includes an amount that induces relief of symptoms of the disease or disorder. The effective amount and frequency of administration for the active ingredient of the present invention may be changed according to the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, and general health of the patient. It can be adjusted according to various factors including condition, sex and diet, time of administration, route of administration and rate of secretion of the composition, duration of treatment, and drugs used concurrently. In the prevention, treatment, or improvement method of the present invention, in the case of adults, when the extract of Ecklonia kurome Okamura is administered once to several times a day, it is administered at a dose of 0.001 g/kg to 10 g/kg. desirable.

In the treatment method of the present invention, a composition comprising an extract or fraction of Ecklonia kurome Okamura as an active ingredient is oral, rectal, vaginal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, It can be administered in a conventional manner via the intraocular or intradermal route.

In the embodiment of the present invention, ethanol extract and four organic solvent fractions of Ecklonia kurome Okamura were obtained, and for them, using HPV-16 pseudovirus, bioluminescence (SEAP) in 293TT cells was used. The antiviral activity of the extracts and fractions of Black Ecklonia cava was confirmed. Among the four fractions fractionated by the extract and solvent extraction method, strong antiviral activity was confirmed in the ethanol extract, and the strongest activity was observed in the dichloromethane fraction. Ethyl acetate and butanol fractions also showed significant anti-HPV activity.

Hereinafter, a preferred embodiment is presented to aid in the understanding of the present invention, but it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the present invention and the scope of the technical idea, but the following examples are only illustrative of the present invention, It is natural that such modifications and modifications fall within the appended claims.

Example 1. Preparation of extracts and fractions of Black Ecklonia cava

1-1. Extraction of Black Ecklonia

Freeze-dried black pepper (386g) was chopped, cooled twice at room temperature with ethanol (3.9L), warmed once in a water bath at 60°C, and concentrated to obtain an ethanol extract.

1-2. Organic Solvent Fraction of Black Egret Extract

58.0 g of the ethanol extract obtained in 1-1 above was suspended in 580 mL of distilled water, and fractionated with dichloromethane, ethyl acetate, and n-butanol three times according to the conventional method, and dichloromethane, ethyl acetate, n-butanol and water fractions were obtained. Got it.

1-3. Results

386 g of freeze-dried black pepper was extracted three times with ethanol, filtered, and concentrated under reduced pressure to obtain 62.07 g of ethanol extract. Some of these were stored and the remainder was suspended in distilled water and partitioned with an organic solvent to obtain CH2Cl2 (10.63g), EtOAc (6.50g), n-BuOH (4.71g) and H2O (36.56g) fractions (Fig. 1) .

Example 2. In vitro antiviral activity of extracts and fractions of E.

2-1.Experiment preparation

2-1-1. Cell preparation

293TT used for HPV pseudovirus production and in vitro assay (a cell line that expressed SV40 large T antigen in 293T cells made by manipulating human embryonic kidney cells to transform into adenovirus E1a, provided by Schiller Lab), and the cells were Dulbecco's Cultured using a medium containing modified Eagles medium (DMEM (SH30243, Hyclone, UT, USA)) with heat inactivated 10% FBS (26140079, Hyclone, UT, USA) and maintained at 37℃ with 5% CO2 Became.

2-1-2. Production of HPV-16 pseudovirus

2-1-2-1. Plasmid: HPV-SEAP pseudovirus was produced for in vitro antiviral assay, and HPV-Luc PV was produced for in vivo challenge test. For the production of HPV-SEAP PV, p-SEAP and p16L1L2 plasmid, and pc-Luc and p16L1L2 plasmid were used to produce HPV-Luc PV. Each plasmid was provided by Schiller Lab (Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, USA).

2-1-2-2. Infection: 293TT cells were seeded in a 75T flask with 5 X 106 cells and incubated for 16 hours at 37°C and 5% CO2, followed by 19 μg of p16L1/L2 and 19 μg of pSEAP or pc-Luc plasmid with Lipofectin Reagent. (18292-011, Invitrogen, CA, USA) was used for co-transfection. After 6 hours of transfection, it was exchanged with complete media and incubated at 37°C for 48 hours, and the cells were harvested by trypsinization. The harvested cells were washed with Dulbecco's Phosphate-Buffered Saline (DPBS, 14190-250, Invitrogen, CA, USA).

2-1-2-3. Cell harvesting and virion maturation: After resuspending in 1 ml of DPBS, 5% Triton X-100 (9002-93-1, Sigma, MO, USA), 25 mM ammonium sulphate (pH 9) (A4418 , Sigma-Aldrich, MO, USA) 0.2% of Benzonase (9025-65-4, Benzonas, Sigma, UK) was added and added, and the virus was maturated by incubation at 37°C for 24 hours.

2-1-2-4. Salt extraction: After cooling the matured virion on ice for 5 minutes, add 0.17 volume of 5N NaCl and incubate on ice for another 20 minutes. Thereafter, the virion solution was collected and transferred to an e-tube, centrifuged at 4°C and 12,000 rpm for 10 minutes, and then only the supernatant was collected and stored at -80°C or opti-prep ultracentrifugation.

2-1-2-5. Purification: A 46% optiprep gradient solution is prepared by mixing 23 ml DPBS/0.8M NaCl in 77 ml of SIGMA density gradient medium, and 37%, 33%, 39% gradient solutions are made into DPBS/0.8M NaCl solution using this ( 27%: 9.3ml DPBS/0.8M NaCl+13.2ml 46% Optiprep, 33%: 6.4ml DPBS/0.8M NaCl+16.1ml 46%, 39%: 3.4ml DPBS/0.8M NaCl+19ml 46%). Carefully add 1 ml each of 39%, 33%, and 27% gradient solution to 5 ml of Beckman ultracentrifuge tube (361625, Beckman Coulter, USA) to prevent the layer from breaking, and load 1 ml of virion supernatant thereon. do. Using an Ultracentrifuge (Optima L 90K, Beckman Coulter Ultracentrifuge, USA), centrifugation was performed at 47,800 rpm, 16° C. for 4 hours. The virion fraction was collected and stored at -80 °C.

2-1-2-6. Titration of HPV PVs: 293TT cells were seeded in 96-well plates at 5 × 103 increments and incubated for 16 hours at 37°C and 5% CO2. After 5-fold serial dilution of HPV PVs, cells of each well were infected and incubated for 72 hours. The titer measurement of HPV-SEAP PV was performed by taking the cell culture supernatant and confirming the activity of secreted alkaline phosphatase (SEAP) using the Great EscAPE™ SEAP Chemiluminescence Kit (631738, Clontech, CA, USA). In addition, for titer measurement of HPV-Luc PV, the cell culture supernatant was taken and luciferase activity was confirmed using the BioLux® Gaussia Luciferase Assay Kit (0301008, New England biolabs, MA, USA). For chemiluminescent detection, relative light units (RLU) values were obtained using a luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA), and each HPV PVs titer was calculated.

2-2. In vitro antiviral efficacy search

Screening assays and HPV inhibition assays for samples were performed by Shaneyfelt's method (Shaneyfelt et al., 2006) and Schiller Lab's method (Roden et al., 1996, Unckell et al., 1997, Touze et al., 1998, Selinka et al., 1998). al., 2003, and Klasse et al., 2002) were applied. Before the start of the in vitro antiviral assay, 293TT cells were seeded in a 96-well plate of 5 × 103 and incubated at 37°C and 5% CO2. After 16 hours, the culture solution was removed, and the extract and each fraction were diluted to a concentration of 400, 200, 100, 50, 25, and 12.5 μg/ml, and each 100 μl was dispensed into the cells. After the extract and each fraction were treated for 16 hours, the culture medium was removed and the cells were washed twice with PBS. 100 μl of HPV PVs of 106 RLU/ml were infected into the cells, followed by incubation at 37°C and 5% CO2 for 48 hours. Antiviral activity was measured by taking the cell culture supernatant and using the Great EscAPE™ SEAP Chemiluminescence Kit (Clontech, CA, USA) to confirm the activity of secreted alkaline phosphatase (SEAP). For SEAP activity, relative light units (RLU) values were obtained using a luminescence counter (Micro beta triLux 1450, PerkinElmer, CT, USA), and the RLU values in the cells not treated with the extract and each fraction were compared with the treated cells. Therefore, the decrease in SEAP activity was regarded as the inhibition effect on HPV PVs.

2-3. In vitro antiviral efficacy search results

As a result of the anti-HPV in vitro experiment, as shown in Figs. 2 to 3, it was possible to confirm the viral inhibitory ability in the ethanol extract and 4 kinds of solvent fraction samples. Ethanol extract of Black Ecklonia cava showed a 94% virus inhibitory effect at a sample concentration of 200 μg/ml, and an IC50 value of 12.5 μg/ml or less showed strong activity. Among the solvent fractions, the dichloromethane fraction showed the strongest antiviral activity, and its value showed a virus inhibitory effect close to 100% at a sample concentration of 200 μg/ml or more. Ethyl acetate and butanol fractions also showed a significant inhibitory effect on HPV16 PVs.

Example 3. HPV16 Pseudovirus Inhibitory Efficacy Test in Mouse Vivo of Black Ecklonia C.

3-1. Experiment method

In order to confirm the inhibitory activity of the sample obtained in the above example against in mice treated with luciferase-containing pseudovirion, the following experiment was performed by applying the method disclosed in the literature (Roberts, JN, et al., (2007). Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan. Nat Med 13; 857-861. ).

3-1-1. Sample treatment

A 6-week-old female Balb/C mouse (Orientbio Co., Korea) was used, and an ethanol extract or dichloromethane fraction was administered externally as follows.

Ethanol extract or dichloromethane fraction was used as an external agent for genital tract, mixed with 0.5% methyl cellulose, and administered at 100 mg/kg once a day for 3 days. 4 hours after the last administration, HPV16 PVs were injected into the genital tract. As a negative control, mice to which extracts and fractions were not administered were used.

Depot medroxyprogesterone acetate (Depo-Provera™, Pharmacia, Belgium) at 6 weeks old female Balb/C mice (Orientbio Co., Korea) at 3 mg per head 4 days before the luciferase-containing pseudovirion challenge. Was injected subcutaneously on the back.

3-1-2. Challenge test in the vagina

The mice to which the sample was administered were treated with 20 μl of 4% nonoxynol-9 (Sigma Aldrich, MO, USA) in the vagina 6 hours before HPV16-Luc PVs challenge, and then 20 μl of 3% carboxymethylcellulose (Sigma Aldrich, MO, USA) and HPV16-Luc PVs were injected into the vagina at ^{5 × 10 6 RLU.}

Three days after the challenge inoculation, all mice were anesthetized and 30 μl of luciferin (caliper, MA, USA) was injected at a concentration of 7 mg/ml. In HPV16-Luc PVs, the plasmid that carries the luciferase gene, pLucf, is encapsulated, allowing for pseudoinfection (Jeff Roberts, 2007). After leaving for 10 minutes, each mouse was detected luciferase expression using an IVIS 200 bioluminescence imaging system (Xenogen, NJ, USA), and the images were compared and analyzed. In addition, for the image, the expression level of luciferase was quantified using Living Image 2.20 software (Xenogen, NJ, USA), and the HPV defense efficacy of the sample was compared and analyzed.

3-2. In vivo test results

As a negative control, a 6-week-old female Balb/C mouse to which no sample was administered was used, and the attack vaccination method was the same as the above method.

In mice challenged through the vaginal tract, the expression of luciferase was localized in the genital region, and it was confirmed that luciferase activity was detected by HPV16 PVs infection. In addition, since the luminescence in the negative control group was confirmed, the protective efficacy in the experimental group could be compared and confirmed.

Mice to which the ethanol extract was administered externally showed weak luminescence, and it was found that they strongly defended against HPV16 PVs.

The luminescence was not observed in the mice subjected to the challenge test for HPV by mixing the dichloromethane fraction with 0.5% methyl cellulose and applying it to the vaginal tract at 100 mg/kg once a day for 3 days, so HPV16 PVs were almost perfect. It was confirmed to have defended it.

As a result of quantifying the amount of luminescence by Luciferase, the amount of luminescence was 12% and 4% or less in the mice to which the ethanol extract and dichloromethane fraction were administered externally compared to the mice without administration (FIG. 4). Therefore, when the ethanol extract and the dichloromethane fraction were administered externally, the HPV virus could be killed or the infection could be suppressed, thereby confirming the complete protective effect against the attack vaccination.

Hereinafter, examples of the formulation of the composition containing the powder of the present invention will be described, but the present invention is not intended to limit it, but is intended to be described in detail.

Formulation Example 1 Preparation of powder

500 mg of extract powder obtained in Example 1

100 mg lactose

10 mg of talc

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

Preparation Example 2 Preparation of tablets

300 mg of extract powder obtained in Example 1

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

Formulation Example 3 Preparation of Capsule

200 mg of extract powder obtained in Example 1

3 mg of crystalline cellulose

148 mg lactose

Magnesium stearate 02 mg

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

Formulation Example 4 Preparation of Injection

600 mg of extract powder obtained in Example 1

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na2HPO4,12H2O 26 mg

It is prepared in the amount of the above ingredients per ampoule according to a conventional injection preparation method.

Formulation Example 5 Preparation of liquid formulation

4 g of extract powder obtained in Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of liquid formulations, add and dissolve each component in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water and add purified water to adjust the total to 100 g, then fill in a brown bottle for sterilization. To prepare a liquid formulation.

Formulation Example 6 Preparation of granules

1,000 mg of extract powder obtained in Example 1

The right amount of vitamin mixture

70 ug vitamin A acetate

1.0 mg of vitamin E

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

Vitamin B12 0.2 ug

10 mg of vitamin C

Biotin 10 ug

1.7 mg of nicotinic acid amide

50 ug folic acid

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dicalcium phosphate 55 mg

90 mg of potassium citrate

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for granules, but it is possible to arbitrarily modify the mixing ratio. After mixing the above ingredients according to a conventional granule preparation method, the granules And can be used to prepare a health functional food composition according to a conventional method.

Formulation Example 7 Preparation of Functional Beverage

1,000 mg of extract powder obtained in Example 1

1,000 mg citric acid

100 g oligosaccharides

2 g of plum concentrate

1 g taurine

900 mL total by adding purified water

After mixing the above ingredients according to the normal health drink manufacturing method, stirring and heating the mixture at 85°C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and then stored in a refrigerator. It is used to prepare the functional beverage composition of the present invention.

The composition ratio is a mixture of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, the country of demand, and the purpose of use.

Claims (10)

Ecklonia kurome Okamura (Ecklonia kurome Okamura) human papillomavirus (human papillomavirus, HPV) infection treatment or prevention composition containing as an active ingredient one or more fractions selected from the group consisting of ethanol extract or its dichloromethane, n-butanol and water. delete delete delete delete delete A composition for cleaning the human body comprising the composition of claim 1. A feminine cleansing composition comprising the composition of claim 1. delete Ecklonia kurome Okamura (Ecklonia kurome Okamura) A food composition for improving or preventing human papillomavirus infection, containing as an active ingredient, one or more fractions selected from the group consisting of ethanol extract or its dichloromethane, n-butanol, and water.

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