KR102622016B1 - Composition preventing or treating inflammation or cancer for vagina and cervix using steam extract of punarnava herb - Google Patents

Abstract

The present invention discloses the prevention or treatment of vaginal inflammation and cervical cancer using the steamed and dried product of Punarnava herb . The steamed and dried product is prepared using Punarnava herb , wild yam ( Dioscorea ) and Echinacea , which are extracted with alcohol and used for vaginal cleansing and to inhibit cell proliferation against vaginal inflammation and cervical cancer. Provides preventive or therapeutic effects through.

Description

Composition for preventing or treating inflammation or carcinoma of the vagina or cervix using the steamed dried product of Punarnava {COMPOSITION PREVENTING OR TREATING INFLAMMATION OR CANCER FOR VAGINA AND CERVIX USING STEAM EXTRACT OF PUNARNAVA HERB}

The present invention relates to a composition that uses the steamed and dried product of Punarnava to exhibit preventive or therapeutic properties and vaginal cleansing properties through inhibition of cell proliferation for inflammation or carcinoma of the vagina or cervix.

A healthy woman's vagina has an acidic pH of about 3.5 to 4, and has a self-cleaning effect that prevents pathogens from entering and multiplying in the vagina. However, if there is an abnormality in vaginal acidity, pathogens, etc. invade the vagina, causing inflammation, and this causes vaginal discharge, which has a foul odor and increases in vaginal secretion, causing cold discharge. And in cases where this type of coldness is severe, the amount of coldness may increase to the point where you need to change your underwear frequently, and a foul odor may be so strong that even people around you can smell it.

In addition, cervical cancer refers to a malignant tumor that occurs in the cervix, which is connected to the vagina, among the corpus and cervix that make up the uterus. Cervical cancer is the second most common cancer among women worldwide, and its number is so vast that it ranks fourth among the causes of cancer death in women. In addition, the incidence of cervical cancer is gradually increasing due to changes in lifestyle habits and environmental factors. In the past, it occurred more often in middle-aged women, but now the incidence is increasing in younger age groups in their 20s and 30s.

The above-mentioned vaginitis and cervical cancer are all cell proliferative diseases, so cell proliferation inhibition is required first.

Punarnava herb is a plant species (Boerhavia diffusa) belonging to the genus Boerhavia. It is widespread throughout India, the Pacific, and the southern United States. It has small flowers and its leaves are a green vegetable consumed in many parts of India. It is known to have an analgesic effect.

Wild yam ( Dioscorea ) is the root of the yam (Dioscorea japonica Thunb) of the Yam family. Yam is native to Korea and is distributed in mountainous areas throughout the country except mountainous areas where the climate is very cold. It is a vine-like perennial herb plant. The leaves are opposite, but sometimes alternate, and the petioles are long, 5 - 10 cm long, 2 - 2.5 cm wide, long oval or narrow triangular, and have sharp ends. The flowers are dioecious and bloom from June to July. The stems are wrapped around the body and have many branched branches, and the roots have a long, cylindrical shape. In oriental medicine, yam has been used since ancient times to treat frostbite, polyuria, urinary incontinence, and skin eczema. Recently, it has also been used to treat diabetes, and is a medicinal herb that has been used as nourishment, tonic, and tonic. It is known that the active ingredients contained in yam include diosgenin, vitamin C, polyphenoloxidase, glycoprotein, and saponin. However, nothing has been disclosed or disclosed anywhere in the literature regarding the muscle function enhancing performance of wild yam.

Echinacea is a perennial flower of the Asteraceae family with thick, elongated stems, and flowers of various colors bloom at the top of the stem. The name Echinacea comes from the stamen part in the center of the flower. At first glance, it looks like a sea urchin with sticky thorns, so it was named echinacea, which comes from the Greek word enhinos, meaning sea urchin. Thanks to its sharp, cone-shaped stamens, it is also called cornflower in English. It is native to North America and is often used by Native Americans, giving it the name 'Indian herb'. However, nothing has yet been disclosed or disclosed in the above literature regarding the ability of wild yam or echinacea to prevent or treat vaginal inflammation, cervical cancer, and vaginal cleansing ability.

Accordingly, while conducting research on Punarnava, the present inventors completed the present invention by confirming that Punarnava can simultaneously exhibit preventive or therapeutic properties and vaginal cleansing properties in terms of inhibiting cell proliferation for inflammation or carcinoma of the vagina or cervix. did.

Korean Patent Publication No. 2005-0059286

The purpose of the present invention is to provide a composition that simultaneously exhibits the ability to prevent or treat inflammation or carcinoma of the vagina or cervix by inhibiting cell proliferation using the steamed dried product of Punarnava herb and to simultaneously exhibit vaginal cleansing ability.

The above and other objects of the present invention can all be achieved by the present invention described below.

In order to achieve the above object, the present invention

Prevention of inflammation or carcinoma of the vagina or cervix by inhibiting cell proliferation containing powder or extracts of steamed dried products using Punarnava herb, wild yam ( Dioscorea ) and Echinacea as active ingredients. or provides a therapeutic composition.

The Punarnava herb, wild yam ( Dioscorea ), and echinacea ( Echinacea ) may satisfy a weight ratio of 1: 0.5 to 1.5: 0.5 to 1.5.

The steamed dried product is first steamed at 90 to 110°C for 1 to 24 hours, the first steamed dried product is secondarily steamed at 90 to 110°C for 1 to 24 hours, and the secondarily steamed dried product is steamed at 90 to 110°C for 1 hour. The steamed product that has been steamed three times for 24 hours may be dried at 60 to 92°C for 1 to 8 hours and then dried at -30°C or lower to have a moisture content of 10% or less.

The steamed dried product may have been subjected to alcohol extraction.

The alcohol post-extraction may be extraction at 60 to 90° C. for 2 to 5 hours under reflux.

The composition may have a vaginal cleansing effect, an antibacterial effect against vaginitis-causing bacteria, or a cell proliferation inhibitory effect on vaginal epithelial cell lines or cervical cancer cells.

According to the present invention, the powder or extract of the steamed dried product of Punarnava, which simultaneously exhibits the ability to prevent or treat inflammation or carcinoma of the vagina or cervix by inhibiting cell proliferation and the ability to cleanse the vagina, is used as an active ingredient to treat vaginal inflammation and inflammation. It may provide prevention or treatment for cervical cancer.

Figure 1 is a graph comparing ABTS and DPPH radical scavenging activities by concentration for the sample obtained in Example 1 and the four samples obtained in Comparative Examples 1 to 3.
Figure 2 is a graph comparing the results of vaginal epithelial cell line cytotoxicity evaluation for the sample obtained in Example 1 and the four samples obtained in Comparative Examples 1 to 3.
Figure 3 is a graph comparing the results of LPS inflammation-induced vaginal epithelial cell line cytokine evaluation for the sample obtained in Example 1 and the four samples obtained in Comparative Examples 1 to 3.
Figure 4 is a graph comparing the results of evaluation of cervical cancer cell proliferation inhibition for the sample obtained in Example 1 and the four samples obtained in Comparative Examples 1 to 3.

Hereinafter, the present invention will be described in more detail to facilitate understanding of the present invention.

Terms and words used in this specification and claims should not be construed as limited to their common or dictionary meanings, and the inventor may appropriately define the concept of terms in order to explain the invention in the best way. Therefore, it should be interpreted with meaning and concept consistent with the technical idea of the present invention.

In the present disclosure, inflammation of the vagina or cervix includes viral and bacterial diseases.

As confirmed in the Examples and Experimental Examples below, the present invention relates to the manufacture of powders or extracts of steamed dried products using Punarnava herb , wild yam ( Dioscorea ), and Echinacea . The extract increases the ability to inhibit and kill bacteria in the vagina or cervix and simultaneously expresses the ability to inhibit cell proliferation, thereby providing a composition for cleaning the vagina and preventing or treating inflammation or carcinoma of the vagina or cervix by inhibiting cell proliferation. can be provided.

The hot water extract of the steamed dried product of Punarnava increased the inhibition and killing ability against bacteria in the vagina or cervix and did not show cell proliferation inhibition ability at the same time. Also, although not shown in the experimental example below, Punarnava herb , if the content ratio of wild yam ( Dioscorea ) and Echinacea ( Echinacea ) does not satisfy the weight ratio of 1: 0.5 to 1.5 : 0.5 to 1.5, the inhibition and killing ability against bacteria in the vagina or cervix increases and the cell proliferation inhibition ability simultaneously increases. did not indicate

The composition of the present invention that increases the inhibition and killing ability of bacteria in the vagina or cervix and simultaneously implements the cell proliferation inhibition ability is provided based on the results of these experiments, and is used to prevent inflammation or carcinoma of the vagina or cervix of the present invention. And the therapeutic composition is a powder of the steamed dried product obtained by mixing Punarnava herb , wild yam ( Dioscorea ) and Echinacea in a weight ratio of 1: 0.5 to 1.5: 0.5 to 1.5. It is characterized by containing extract as an active ingredient.

In this description, unless otherwise specified, an active ingredient refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

In this description, unless otherwise specified, the steamed and dried product is first steamed at 90 to 110°C for 1 to 24 hours, the first steamed and dried product is secondarily steamed at 90 to 110°C for 1 to 24 hours, and the second steamed. The dried product may be steamed for the third time at 90 to 110°C for 1 to 24 hours, dried at 60 to 92°C for 1 to 8 hours, and then dried at -30°C or lower to a moisture content of 10% or less. In this case, it is desirable because it can provide an increased effect of the active ingredient.

At this time, a normal steaming device can be used for the steaming, a normal drying device such as a hot air dryer can be used for drying, and a normal drying device such as a freeze dryer can be used for drying to control the moisture content. .

The steamed dried product of the present invention is preferably obtained by using the steamed dried product whose moisture content has been adjusted to 10% or less through drying pretreatment.

In addition, more preferably, in the first steam drying, the steaming time is 6 to 15 hours, in the second steam drying, the steaming time is 8 to 15 hours, and in the third steam drying, the steaming time is 8 to 12 hours. It is preferable to obtain it by doing so.

The steamed dried product can be pulverized to 10 to 500 mesh, for example, 50 to 150 mesh, and then used for alcohol extraction.

The grinding can be performed in a conventional grinding device.

The steamed dried product may have been subjected to alcohol extraction.

The powder or extract can be obtained by subjecting the steamed product to alcohol extraction.

The alcohol extraction is preferably performed using 3 to 20 times the weight of ethanol as the steamed dried product.

The alcohol is not specific, but considering reaction efficiency, it is preferable to use an alcohol with 1 to 4 carbon atoms at a concentration of 60 to 90%.

In the present disclosure, the alcohol post-extraction is performed under reflux at 60 to 90° C. for 2 to 5 hours. In this case, the extraction efficiency of active ingredients from the steamed and dried product can be maximized.

In this description, unless otherwise specified, the powder of the steamed dried product or its extract refers to the solid material itself or the powder to be extracted with lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, acetone, hexane, Supercritical extraction solvents such as extracts obtained by leaching using ether, chloroform, ethyl acetate, butylacetate, dimethylformamide, dimethyl sulfoxide, 1,3-butylene glycol, propylene glycol, or mixed solvents thereof, carbon dioxide, pentane, etc. It refers to an extract obtained using or a fraction obtained by fractionating the extract, and the extraction method is applied by any method such as cold immersion, reflux, heating, ultrasonic radiation, or supercritical extraction, considering the polarity, degree of extraction, and degree of preservation of the active substance. can do.

In the case of fractionated extracts, the extract is suspended in a specific solvent, mixed with a solvent of different polarity, and the obtained fractions and crude extract are adsorbed on a column filled with silica gel, etc., and then a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof is used as the mobile phase. It is meant to include fractions obtained by.

The meaning of the extract includes concentrated liquid extract or solid extract from which the extraction solvent has been removed by methods such as freeze-drying, vacuum drying, hot air drying, spray drying, etc. Preferably, it refers to an extract obtained using water, a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), or a mixed solvent thereof as an extraction solvent.

In the present disclosure, the shape of the solid material is not particularly limited and may be in the form of pills, granules, powder, etc.

The composition of the present invention can be used for various purposes requiring a vaginal cleansing effect, an antibacterial effect against vaginitis-causing bacteria, or a cell proliferation inhibitory effect against vaginal epithelial cell lines or cervical cancer cells.

For example, it may be used as a preventive or therapeutic agent for that purpose.

The prophylaxis or treatment herein may be treated or modified or treated and modified to enhance the immunosuppressive or immunoprivileged properties of the cells for administration to human subjects; And optionally, cells can be identified using cells that have been treated or modified to enhance amplification of the virus in the cells. Alternatively, this can be done using cells that are permissive for oncolytic virus amplification, accumulate in the tumor, and/or are not recognized by the subject's immune system for a sufficient time to deliver the virus to the tumor in the subject.

Commercial allogeneic cell lines that can be used at this time include: mesenchymal stem cells, such as, for example, APCETH-201, APCETH-301 (APCETH), Cx601 (TIGENIX), TEMCELL, MSC-100-IV, Prochymal ( MESOBLAST); Induced pluripotent stem cells (iPSCs) such as, eg, ToleraCyte (Fate Therapeutics); Fibroblast cells such as CCD-16Lu, WI-38; Tumor-associated fibroblasts, such as Malme-3M, COLO 829, HT-144, Hs 895.T, hTERT PF179T CAF, etc.; Endothelial cells, e.g. HUVEC, HUVEC/TERT 2, TIME; Embryonic epithelial cells, such as HEK-293, HEK-293 STF, 293T/17, 293T/17 SF, HEK-293.2sus; Embryonic stem cells, such as hESC BG01V; and epithelial cells, such as NuLi-1, ARPE-19, VK2/E6E7, Ect1/E6E7, RWPE-2, WPE-stem, End1/E6E7, WPMY-1, NL20, NL20-TA, WT 9-7 , WPE1-NB26, WPE-int, RWPE2-W99, BEAS-2B.

As a specific example, the cell viability expressed by Equation 1 below in a vaginal epithelial cell line may be 90 to 100%, or 92 to 100%.

[Equation 1]

Cell viability (%) = [(Exp. - Blank)/Control] Х 100

(In Equation 1 above, Exp: Absorbance of extract containing cells, Blank: Absorbance of extract without cells, Control: Absorbance of distilled water containing cells.)

As another specific example, the cell survival rate expressed by Equation 1 in cervical cancer cells may be 45 to 60%, or 50 to 55%.

As used herein, the composition may be a pharmaceutical composition.

The pharmaceutical composition may include a pharmaceutically acceptable carrier or additive. As used herein, the meaning of “pharmaceutically acceptable” means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond what the subject of application (prescription) can adapt to. The “carrier” is defined as a compound that facilitates the addition of a compound into cells or tissues.

The pharmaceutical composition of the present specification can be administered alone or in mixture with any convenient carrier, etc., and the dosage form may be a single dose or repeated dose form. The pharmaceutical composition containing the pharmaceutical composition may be a solid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, etc. Solid preparations may include, but are not limited to, carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, etc. Liquid preparations include, but are not limited to, solutions such as water and propylene glycol solutions, suspensions, and emulsions, and can be prepared by adding appropriate colorants, flavoring agents, stabilizers, viscosifiers, etc. For example, powders can be prepared by simply mixing the pharmaceutical composition, which is the active ingredient of the present specification, with a suitable pharmaceutically acceptable carrier such as lactose, starch, or microcrystalline cellulose. Granules include the pharmaceutical composition of the present specification; A suitable pharmaceutically acceptable carrier; and a pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropyl cellulose, and then mixed with a wet granulation method using a solvent such as water, ethanol, or isopropanol, or a dry granulation method using compression force. You can. Additionally, tablets can be manufactured by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate and then compressing the mixture into tablets using a tablet press.

The pharmaceutical composition of the present specification may be an oral agent, an injectable agent (e.g., intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), inhalation agent, or intranasal administration depending on the disease to be treated and the condition of the individual. , may be administered vaginally, rectally, sublingually, transdermally, or topically, but is not limited thereto. Depending on the route of administration, it may be formulated in an appropriate dosage unit form containing commonly used and non-toxic pharmaceutically acceptable carriers, excipients, and vehicles.

In particular, the pharmaceutical composition may be administered topically or intravaginally. When compositions of the invention are prepared for intravaginal administration, they are generally combined with pharmaceutically acceptable carriers, diluents or excipients to form a pharmaceutical formulation or unit dosage form. For vaginal administration, the compositions may be presented as solutions, suspensions, emulsions, powders, granular formulations, or natural or synthetic polymers or resins. The active composition may also be presented as a bolus or paste. The intravaginally administered therapeutic compositions of the invention may also be formulated for sustained release, for example, the compositions may be coated, microencapsulated, or placed within a sustained release delivery device. The total active ingredients of the formulation may comprise 0.1 to 99.9% by weight of the formulation.

The pharmaceutical composition of the present specification may be administered in an amount of about 0.0001 mg/kg to about 10 g/kg daily, and a daily dosage of about 0.001 mg/kg to about 1 g/kg is preferred. However, the dosage may vary depending on the degree of purification of the mixture, the patient's condition (age, gender, weight, etc.), and the severity of the condition being treated. If necessary, for convenience, the total daily dose may be divided and administered several times during the day.

When the composition of the present specification is used as a pharmaceutical composition, the content of the pharmaceutical composition in the composition can be appropriately adjusted to an effective amount capable of exhibiting anti-inflammatory activity depending on the symptoms of the disease, the degree of progression of the symptoms, the patient's condition, etc., for example, the pharmaceutical composition The amount of the composition may be 0.0001% by weight or more, specifically 0.001% by weight or more, and 80% by weight or less, specifically 50% by weight or less, based on the total weight of the entire composition, but is not limited thereto.

In this specification, the composition may be a food composition.

When the composition of the present specification is used as a food composition, it may contain acceptable food auxiliary additives and may further include appropriate carriers, excipients, and diluents commonly used in the production of food.

In this specification, food refers to a natural product or processed product containing one or more nutrients, and specifically means that it has gone through a certain degree of processing to become ready to eat. In the usual sense, various foods, It is used to include functional foods, beverages, food additives, and beverage additives. Examples of the above foods include various foods, beverages, gum, tea, vitamin complexes, functional foods, etc. Additionally, foods in this specification include special nutritional foods (e.g., milk formula, infant and toddler food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), health supplements, and seasoned foods ( (e.g., soy sauce, soybean paste, red pepper paste, mixed paste, etc.), sauces, confectionery (e.g., snacks), dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.), beverages (e.g. Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, ice cream, etc.), natural seasonings (e.g., ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages, and other health supplements. The functional foods, beverages, food additives or beverage additives can be manufactured by conventional manufacturing methods.

The above-mentioned “functional food” refers to a food group or food composition that has added value to a food group or food composition by using physical, biochemical, biotechnological methods, etc. to function and express the function of the food for a specific purpose, such as regulation of biological defense rhythm, disease prevention and recovery. It refers to food that has been designed and processed to fully express the body's regulatory functions related to the body, etc., and may specifically be a health functional food. The functional food may include food auxiliary additives that are foodologically acceptable, and may further include appropriate carriers, excipients, and diluents commonly used in the production of functional foods.

In addition, in the food composition, the amount of the active ingredient of the pharmaceutical composition may be 0.00001% by weight or more, specifically 0.1% by weight or more, and 800% by weight or less, specifically 50% by weight or less, more specifically 40% by weight or less, of the total weight of the food composition. It may be included in weight percent or less, and if the food is a beverage, it should be 0.001 g or more, specifically 0.01 g or more, 50 g or less, specifically 10 g or less, and more specifically 2 g or less based on 100 ml of the total volume of the food. It may be included as a ratio, but is not limited thereto.

The food composition of the present specification may include sweeteners, flavoring agents, bioactive ingredients, minerals, etc. in addition to the active ingredients. Sweeteners can be used in amounts to give foods an appropriate sweet taste and can be natural or synthetic. Specifically, when using a natural sweetener, natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose. Flavoring agents can be used to improve taste or aroma, and both natural and synthetic ones can be used. Specifically, this is the case when using natural products. When using natural products, they can serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, coriander leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. You can also use things obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo nuts. Natural flavoring agents may be liquid concentrates or solid pharmaceutical compositions. In some cases, synthetic flavoring agents may be used, and the synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, etc. As physiologically active substances, catechins such as catechin, epicatechin, gallocatechin, and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, and riboflavin can be used. As minerals, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc, etc. can be used.

In addition, the food composition of the present specification may contain preservatives, emulsifiers, acidulants, thickeners, etc., as necessary, in addition to the sweeteners.

These preservatives, emulsifiers, etc. are preferably added in very small amounts as long as they can achieve the purpose for which they are added. When expressed numerically, trace amounts mean in the range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition. Preservatives that can be used include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, Examples include sodium benzoate, potassium benzoate, and EDTA (ethylenediaminetetraacetic acid). Emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin, etc. Acidulants that can be used include malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. These acidulants may be added to ensure that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms in addition to improving taste. Thickening agents that can be used include suspending agents, settling agents, gel forming agents, bulking agents, etc.

The composition of the present invention can be usefully used for the prevention or treatment of vaginitis by inhibiting cell proliferation. In particular, it has the purpose of strengthening vaginal epithelial cell tissue and can be used for the prevention and treatment of vaginitis as an alternative to existing hormone therapy. You can.

Furthermore, in addition to the above-mentioned aspects, it can be used without limitation for any purpose that requires simultaneous expression of preventive or therapeutic ability through inhibition of cell proliferation for inflammation or carcinoma of the vagina or cervix and vaginal cleansing ability.

The pharmaceutical composition and functional food composition of the present invention can be prepared according to the method for producing the composition commonly performed in the art, except that it contains active ingredients known in the art.

In explaining the prevention or treatment of vaginal inflammation and cervical cancer of the present disclosure, it is stated that other conditions or equipment not explicitly described can be appropriately selected within the range commonly practiced in the art and are not particularly limited. .

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily practice it. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[Example]

<Example 1> Preparation of multi-stage steamed ethanol Punarnava extract

(1) Punarnava herb , wild yam ( Dioscorea ), and Echinacea were thoroughly washed and dried at 60°C.

(2) Dry raw materials were mixed at a constant weight (100 g: 100 g: 100 g).

(3) The mixed raw materials were steamed at 100°C for 12 hours.

(4) The above steaming was repeated two more times.

(5) The steamed raw material was dried with hot air at 70°C for 6 hours, then freeze-dried at -80°C and the steamed product was ground to 100 mesh.

(6) 70% ethanol 10 times the weight of the steam-dried raw material was added, a reflux tube was connected for 6 hours at 70°C, and reflux extraction was repeated three times. Afterwards, the obtained extract was filtered through a 0.45 μm membrane filter, and then ethanol was removed using a reduced pressure concentrator to prepare a multi-stage steamed alcoholic Punarnava extract (sample PDE-4).

<Comparative Example 1> Preparation of steamed hot water Punarnava extract 1

(1) Punarnava herb , wild yam ( Dioscorea ), and Echinacea were thoroughly washed and dried at 60°C.

(2) Dry raw materials were mixed at a constant weight (100 g: 100 g: 100 g).

(3) The mixed raw materials were steamed at 100°C for 12 hours.

(5) The steamed raw material was dried with hot air at 70°C for 6 hours, then freeze-dried at -80°C and the steamed product was ground to 100 mesh.

(6') Distilled water 10 times the weight of the steam-dried raw material was added, a reflux tube was connected at 70° C. for 6 hours, and reflux extraction was repeated three times. Afterwards, the obtained extract was filtered through a 0.45 μm membrane filter and concentrated under reduced pressure to prepare steamed hot water Punarnava extract (sample PDE-1).

<Comparative Example 2> Preparation of multi-stage steamed hot water Punarnava extract 2

(1) Punarnava herb , wild yam ( Dioscorea ), and Echinacea were thoroughly washed and dried at 60°C.

(2) Dry raw materials were mixed at a constant weight (100 g: 100 g: 100 g).

(3) The mixed raw materials were steamed at 100°C for 12 hours.

(4) The above steaming was repeated two more times.

(5) The steamed raw material was dried with hot air at 70°C for 6 hours, then freeze-dried at -80°C and the steamed product was ground to 100 mesh.

(6') Distilled water 10 times the weight of the steam-dried raw material was added, a reflux tube was connected at 70° C. for 6 hours, and reflux extraction was repeated three times. Afterwards, the obtained extract was filtered through a 0.45 μm membrane filter and concentrated under reduced pressure to prepare a multi-stage steamed hot water Punarnava extract (sample PDE-2).

<Comparative Example 3> Preparation of single-steamed ethanol Punarnava extract

(1) Punarnava herb , wild yam ( Dioscorea ), and Echinacea were thoroughly washed and dried at 60°C.

(2) Dry raw materials were mixed at a constant weight (100 g: 100 g: 100 g).

(3) The mixed raw materials were steamed at 100°C for 12 hours.

(5) The steamed raw material was dried with hot air at 70°C for 6 hours, then freeze-dried at -80°C and the steamed product was ground to 100 mesh.

(6) 70% ethanol 10 times the weight of the steam-dried raw material was added, a reflux tube was connected for 6 hours at 70°C, and reflux extraction was repeated three times. Afterwards, the obtained extract was filtered through a 0.45 μm membrane filter, and then ethanol was removed using a reduced pressure concentrator to prepare a single-steamed ethanol Punarnava extract (sample PDE-3).

<Experimental example>

<Experimental Example 1> Antioxidant test

ABTS radical scavenging activity and DPPH method radical scavenging activity tests were performed to evaluate the radical scavenging ability of the samples as an antioxidant test for the samples obtained in Example 1 and Comparative Examples 1 to 3.

Specifically, the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3 were diluted in water to prepare test solutions with concentrations of 100 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL. did. 7mM ABTS and 2.45mM potassium persulfate were mixed and reacted at room temperature for 12 hours in the dark to form ABTS cations. Afterwards, ethanol was added to adjust the absorbance value to 0.70 ± 0.02 at 734 nm. 100 μL of the sample solution and 100 μL of the prepared ABTS solution were added to a 96 well plate, reacted at room temperature for 7 minutes, and measured at 734 nm.

Compared to the blank test solution, the ABTS radical scavenging rate was calculated as a percentage (%) according to Equation 2 below, and the results are shown in Figure 1 below.

[Equation 2]

ABTS radical scavenging ability (%) = [Control - (Sample - Blank)]/Control Х 100

(In the above formula, Control: Absorbance of ABTS reagent, Sample: Absorbance of Sample + ABTS reagent, Blank: Absorbance of Sample + Blank)

In addition, the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3 were diluted in water to prepare test solutions with concentrations of 100 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL. . 100 μL of the test solution and 100 μL of 0.2 mM DPPH were added to a 96 well plate, and after 30 minutes, the absorbance was measured at 517 nm using a microplate reader.

Compared to the blank test solution, the DPPH radical scavenging rate was calculated as a percentage (%) according to Equation 3 below, and the results are shown in Figure 1 below.

[Equation 3]

DPPH radical scavenging ability (%) = [Control - (Sample - Blank)]/Control Х 100

(In the above formula, Control: Absorbance of DPPH reagent, Sample: Absorbance of Sample + DPPH reagent, Blank: Absorbance of Sample + Blank)

As shown in Figure 1 below, as a result of DPPH radical analysis, the antioxidant activity of ascorbic acid used as a control was the best, and was confirmed to be 99.5% at 1000 μg/mL.

In addition, among the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3, the experimental group with the highest antioxidant ability was PDE-4 of Example 1, which was confirmed to be 91.2% at 1000 μg/mL.

Next, PDE-3 in Comparative Example 3 was confirmed to be 82.8%, and was analyzed in the order of PDE-2 in Comparative Example 2 and PDE-1 in Comparative Example 1, respectively, at 75.4% and 71.3% at 1000 μg/mL. It has been done.

The ABTS radical analysis results showed the same trend as the above-described DPPH radical analysis results, in the following order: PDE-4 in Example 1, PDE-3 in Comparative Example 3, PDE-2 in Comparative Example 2, and PDE-1 in Comparative Example 1. It was analyzed as 91.2%, 89.1%, 74.5%, and 70.9% at 1000 μg/mL. For reference, ascorbic acid, the control group, was analyzed at 99.6% at 1000 μg/mL.

<Experimental Example 2> Antibacterial activity test

The antibacterial activity against vaginitis-causing bacteria and vaginal useful bacteria for the samples obtained in Example 1 and Comparative Examples 1 to 3 was measured.

Vaginitis-causing bacteria and vaginal bacteria cell culture

The vaginitis-causing bacteria used in the experiment were Candida albicans (ATCC 28367) , Chryseobacterium gleum ( KCTC 2904) , Sphingomonas paucimobilis (KCTC 2510) , and Protus mummy. There were 4 types of bilis (Proteus mirabilis , KCTC 2834), the vaginal bacteria were Lactobacillus coleohominis ( KCTC 21007), and all strains were purchased from the Biological Resources Center (KCTC).

Lactobacillus coleohominis (KCTC 21007), a vaginal bacterium, was cultured at 35°C in Lactobacilli MRS broth medium, and Chryseobacterium gleum ( KCTC 2904), a vaginitis-causing bacterium, and Protus mirabili. Proteus mirabilis ( KCTC 2834) was cultured in nutrient broth medium at 28°C and 35°C, respectively. Sphingomonas paucimobilis ( KCTC 2510) was cultured at 28°C in R2A broth, and Candida albicans (ATCC 28367) was cultured at 28°C in YM broth medium and used in the experiment. did.

Specifically, the antibacterial test for vaginitis-causing bacteria and vaginal bacteria is cultured vaginitis-causing bacteria Candida albicans (ATCC 28367) , Chryseobacterium gleum (KCTC 2904) , and Sphingomonas pouch. Four types of Sphingomonas paucimobilis (KCTC 2510) and Proteus mirabilis ( KCTC 2834) and the vaginal bacteria Lactobacillus coleohominis ( KCTC 21007) are used at 1 Х 10 ⁶ CFU/mL . did. 100 μL of each strain was smeared on solid medium (agar 20 g/L) using a sterile cotton swab. The samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3 were diluted in water to prepare a concentration of 500 μg/mL, and 20 μL per disc was placed on a paper disc (diameter 6mm, Roshi Kaisha., Tokyo, Japan). After being absorbed slowly, the solvent was volatilized through a drying process. The paper discs on which the samples of each Example 1 and Comparative Examples 1 to 3 had been absorbed were placed in close contact with the plate medium on which the strains had been smeared, then cultured, and the antibacterial activity was compared by measuring the clear zone (mm) created around the disc. The results are shown in Table 1 below.

As shown in Table 1 above, as a result of the test, the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3 were confirmed to have an antibacterial effect on all four types of vaginitis-causing bacteria, especially in PDE-4 of Example 1. The clear zone diameters of Candida albicans, Chryseobacterium gleum, Sphingomonas paucimobilis, and Proteus mirabilis were found to be the largest at 11.1 mm, 13.4 mm, 13.1 mm, and 13.6 mm, respectively.

Next, PDE-3 in Comparative Example 3, PDE-2 in Comparative Example 2, and PDE-1 in Comparative Example 1 were analyzed with the same tendency as the experimental results of antioxidant activity.

In addition, it was confirmed that the samples of Example 1 and Comparative Examples 1 to 3 did not have antibacterial properties in Lactobacillus coleohominis , a vaginal bacteria.

Therefore, it was confirmed that the samples of Example 1 and Comparative Examples 1 to 3 did not affect vaginal bacteria and selectively exhibited antibacterial properties against vaginitis-causing bacteria.

<Experimental Example 3> Vaginal epithelial cell line cytotoxicity evaluation

MTS analysis was performed to confirm the cytotoxicity of the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3. The concentration of the samples of Example 1 and Comparative Examples 1 to 3 was set in the range of 100 to 1,000 μg/mL, where DPPH and ABTS radical scavenging activities were confirmed in Example 1, and VK2/E6E7 (ATCC CRL), a female vaginal epithelial cell line. 2616) was used as a medium to determine whether the test substance affects vaginal epithelial cell lines.

Vaginal epithelial cell line culture

VK2/E6E7 (ATCC CRL 2616), a vaginal epithelial cell line, was purchased from ATCC and grown by adding growth factor 5 μg/L, bovine pituitary extract 50 mg/L, 1% antibiotics, and 0.4 mM calcium chloride to keratinocyte serum free medium. It was used as a medium and cultured under conditions of 37°C and 5% CO ₂ .

Specifically, MTS assay, a cytotoxicity test, was used as a safety test for the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3. Quantification was measured by modifying the Mosmann method.

VK2/E6E7 (ATCC CRL 2616), a vaginal epithelial cell line, was dispensed at 1Х10 ⁴ cell/ml and cultured for 24 hours, and the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3 were mixed at 100 to 1,000 μg/ml. After dilution to a concentration of , it was added and cultured again for 24 hours. Afterwards, 20 μL of MTS reagent was added and incubated for 2 hours, and the absorbance was measured at 570 nm using a microplate reader.

Cell viability was calculated using Equation 1 above, and the results are shown in Figure 2 below.

As shown in Figure 2 below, as a result of the cytotoxicity of the samples of Example 1 and Comparative Examples 1 to 3 in vaginal epithelial cell lines, it was confirmed that the cell viability was more than 95% at all concentrations.

Therefore, it was confirmed that the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3 were not cytotoxic to female vaginal epithelial cell lines.

<Experimental Example 4> LPS inflammation-induced vaginal epithelial cell line cytokine evaluation test

Based on the results of the cytotoxicity evaluation test in Experimental Example 3 described above, 0 μg of the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3 were added to VK2/E6E7 (ATCC CRL 2616), a female vaginal epithelial cell line. After treatment at concentrations of /mL, 100 μg/mL, 250 μg/mL, and 500 μg/mL, the concentration of TNF-α, an inflammatory cytokine indicator expressed by treatment with LPS, an inflammation-inducing substance, was evaluated.

Specifically, to evaluate the production of inflammation-related cytokine (TNF-α) in the cell culture medium, it was measured using an ELISA kit. Vaginal epithelial cell line VK2/E6E7 (ATCC CRL 2616) was dispensed into a 96-well plate at a concentration of 1x10 ⁵ cells/well, and then cultured in a 5% carbon dioxide incubator at 37°C for 12 hours to completely attach the cells. Afterwards, they were treated with test substances (PDE-1 to 4) at concentrations of 0, 100, 250, and 500 μg/mL, and then treated with 1 μg/mL of LPS, an inflammation-inducing substance, and re-cultured for 24 hours. Afterwards, TNF-α was quantified using the Human TNF-α quantikine ELISA kit (R&D systems®, USA), and the results are shown in Figure 3 below.

As shown in Figure 3 below, as a result of comparing the expression level of inflammatory cytokines after administration of inflammation-inducing substances, the concentration of inflammatory cytokine TNF-α decreased depending on the concentration of the samples of Example 1 and Comparative Examples 1 to 3. Confirmed.

Among the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3, the largest decrease in concentration of TNF-α was analyzed as PDE-4 of Example 1, and Example 1 and Comparative Examples 1 to 3 When the sample was not processed, TNF-α was analyzed as 1069.6 pg/mL.

In addition, as the concentration of the samples of Example 1 and Comparative Examples 1 to 3 increased, the concentration of TNF-α decreased, and when treated at 500 μg/mL, TNF-α was 396.4 pg/mL, which is the expression level of inflammatory cytokines. It was confirmed to reduce . The inhibition of TNF-α expression was confirmed to be greater in the following order: PDE-3 in Comparative Example 3, PDE-2 in Comparative Example 2, and PDE-1 in Comparative Example 1.

<Experimental Example 5> Cervical cancer cell proliferation inhibition evaluation test

In order to confirm the effectiveness of the samples of Example 1 and Comparative Examples 1 to 3 in cervical cancer, the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3 were added to HeLa cells, a cervical cancer cell line, at 0 μg/mL. , cell proliferation inhibition was evaluated by treatment at concentrations of 100 μg/mL, 250 μg/mL, and 500 μg/mL.

Cervical cancer cell culture

Cervical cancer HeLa cells (Korea Cell Line Bank) were used in DMEM medium with 10% FBS (fetal bovine serum), 100 unit/mL penicillin, and 100 mg/mL streptomycin, and cultured at 37°C and 5% CO ₂ conditions. did.

Specifically, cultured HeLa cells are dispensed into a 96 well plate at a concentration of 1Х10 ⁵ cells/mL and then cultured in an incubator at 37°C and 5% CO ₂ for 24 hours. Thereafter, the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3 were diluted in PBS (phosphate buffered saline), treated at concentrations of 0, 100, 250, and 500 μg/mL, and re-cultured for 24 hours. Afterwards, 20 μL of MTS reagent was added and incubated for 2 hours, and the absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated using Equation 1 above, and the results are shown in Figure 4 below.

As shown in Figure 4 below, when the results of treating cervical cancer cells with the samples (PDE-1 to 4) of Example 1 and Comparative Examples 1 to 3 at 0 μg/mL were used as a control, Example 1 and Comparative Example 1 As the treatment concentration of samples from 3 to 3 (PDE-1 to 4) increased, cell proliferation inhibition tended to increase in cervical cancer cells.

This tendency is similar to the antibacterial evaluation and cytokine results of vaginitis-causing bacteria in vaginal epithelial cell lines, and the PED-4 concentration of 500 μg/mL in Example 1 showed the highest inhibition of cervical cancer cell proliferation at 52.2%. indicated.

The next substance with high inhibition of cervical cancer cell proliferation was PDE-3 of Comparative Example 3, which was confirmed to be 59.9% at a concentration of 500 μg/mL. In addition, PDE-2 of Comparative Example 2 and PDE-1 of Comparative Example 1 showed cervical cancer cell proliferation inhibition rates of 66.5% and 69.1%, respectively, at a concentration of 500 μg/mL.

In conclusion, when powder or extract of steamed dried product using Punarnava herb , wild yam ( Dioscorea ) and Echinacea is included as an active ingredient, the obtained composition is effective in treating inflammation or carcinoma of the vagina or cervix. It provides a vaginal cleansing effect by inhibiting cell proliferation, an antibacterial effect against vaginitis-causing bacteria, or a cell proliferation inhibition effect and product safety against vaginal epithelial cell lines or cervical cancer cells, and is used as a cleaning composition, pharmaceutical composition, or other vaginal or It was confirmed that it is suitable for applications that require the effect of simultaneously preventing or treating inflammation or carcinoma of the cervix and vaginal cleansing.

Claims (6)

A composition for preventing or treating inflammation or carcinoma of the vagina or cervix, comprising as an active ingredient the powder or extract of steamed and dried products using Punarnava herb, Dioscorea and Echinacea . According to paragraph 1,
The Punarnava herb, wild yam ( Dioscorea ) and Echinacea ( Echinacea ) are a composition for preventing or treating inflammation or carcinoma of the vagina or cervix satisfying a weight ratio of 1: 0.5 to 1.5: 0.5 to 1.5. According to paragraph 1,
The steamed dried product is first steamed at 90 to 110°C for 1 to 24 hours, the first steamed dried product is secondarily steamed at 90 to 110°C for 1 to 24 hours, and the secondarily steamed dried product is steamed at 90 to 110°C for 1 hour. The steamed product that has been steamed for a third time for 24 hours is dried at 60 to 92°C for 1 to 8 hours and then dried at -30°C or lower to a moisture content of 10% or less to prevent inflammation or carcinoma of the vagina or cervix. Therapeutic composition. According to paragraph 1,
A composition for preventing or treating inflammation or carcinoma of the vagina or cervix, wherein the steamed dried product is extracted with alcohol. According to clause 4,
A composition for preventing or treating inflammation or carcinoma of the vagina or cervix, wherein the alcohol is extracted at 60 to 90° C. for 2 to 5 hours under reflux. delete