WO2014126269A1 - Pharmaceutical composition for improving male sexual function and for treating infertility - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for improving male sexual function and for improving infertility, more specifically relates to a pharmaceutical composition for improving male sexual function and treating infertility which includes as an active ingredient at least one extract selected from among a corni extract, a boxthorn extract, a raspberry extract, a cuscuta seed extract, an Omija extract and a plantago seed extract, and a food composition for improving male sexual function and preventing infertility. The composition has an effect in improving male sexual function or on infertility, through the NO concentration in HUVEC, an ACE (angiotensin converting enzyme) reducing effect, a PDE (phosphodiesterase)-5 lowering effect, a CREM expression, an increase in the number and activity of sperms, an increase in the testosterone content and the like.

Description

Pharmaceutical composition for improving male sexual function and treating infertility

The present invention relates to a pharmaceutical composition for improving male sexual function and infertility, and more particularly, comprising at least one extract selected from Cornus extract, Goji extract, Bokbunja extract, Tosa extract, Schisandra extract and Cha-cha extract as active ingredients. It relates to a pharmaceutical composition for improving male sexual function and infertility, and to a food composition for improving male sexual function and infertility. The composition improves male sexual function through NO concentration, angiotensin converting enzyme (ACE) inhibitory effect, PDE (Phosphodiesterase) -5 inhibitory effect, CREM expression, sperm count, sperm activity, and testosterone content in HUVEC. It is effective against infertility.

Modern living environment is confused with biorhythms due to overwork, drinking, stress, environmental pollution, etc. and the frequency of exposure to lifestyle diseases such as hypertension, diabetes, obesity, etc. In many cases, aging and sexual dysfunction occur, and more than 40 years of age are more severe. During infertility, infertility is a major social problem. It is also important to solve the problem of erectile dysfunction through improved blood flow, but more fundamentally, it is more important to resolve infertility by improving sex hormones, improving sperm count and activity.

Infertility refers to the fact that a couple cannot live in pregnancy for more than a year without contraception. Infertile couples account for 10 to 20% of the total couple. The incidence of infertility is infertility in 15% of normal women in the United States and 8% of outpatients of obstetrics and gynecology in Korea. The cause of infertility is about 40% in women only, about 40% in men only, about 10% in both men and women, and about 10% in case of unknown causes. Half of them are male factors (Korean society for Sexual Medicine and Andrology.Namseonggwahak.Seoul, Gunjachulpansa. 5-54, 161-163. (2003); The Korean Urological Association.Textbook of Urology.3rd.Seoul Goryeouihak.507 -22. (2001)).

Male infertility leads to infertility if any part of the sperm is produced, transported, discharged by ejaculation, and then combined with the egg, resulting in infertility.In modern men, sperm production and condition, spermatogenic disorders, and sperm motility Is most problematic (Jouannet P, et. Al., APMIS. 109 (3), 333-44. (2001)). Disorders of spermatogenesis account for the largest proportion of male infertility, mainly due to abnormalities in hormone secretion leading to the hypothalamus, pituitary gland, and testes, genetic factors, testicular dysplasia, latent testicles, varicose veins, and exposure to high temperatures. Sperm formation is not normal due to the cause, or a high rate of malformed sperm production (The Korean Urological Association.Textbook of Urology.3rd.Seoul Goryeouihak.507-22. (2001)). Sperm motility is essential in the process of fertilization in combination with eggs (Tienthai P. Anim Reprod Sci. 80, 131-46. (2004)). Sperm motility begins to acquire fertility as it passes through the woman's womb and induces hyperactivation motility and acrosome reactions (Guzick DS et. Al., N Engl J Med. 345, 1388-93. (2001).

Recently, male infertility is increasing due to frequent drinking, smoking, environmental pollution, nutrition, excessive drug use, exposure to environmental toxins, excessive sexual intercourse, fatigue, and stress. Decreases in numbers (Saleh RA and Agarwal A. J Androl. 23, 737-52. (2002)).

As a cause of male infertility, there are major causes of infertility, sperm disorders, spermatogenesis disorders and sperm motility (The Korean Urological Association.Textbook of Urology. 3rd. Seoul Goryeouihak. 507-22. (2001)). Spermatogenic disorders include oligozoospermia, which are produced in the testicles, and then produce less sperm (asalnozoospermia). (Saleh RA and Agarwal A. J Androl. 23, 737-52. 2002)).

The condition of sperm that can be conceived includes 60 million to 100 million sperm in 1 ml of semen, the number of abnormal sperm should be 20-30% or less, and 50% or more activity for 4-8 hours after ejaculation. The amount of semen should be 2 to 5 ml, but sperm deficiency is a symptom that causes infertility because the number of sperm in 1 ml of semen is less than 30 million.

Various studies have been patented or registered for compositions for preventing or treating male hypogonadism. In the prior art, oral administration of cadmium chloride to experimental animals to induce sperm damage artificially to induce male reproductive function (JP-A-2002-0085401), or tetrachlorodibenzo-pi-dioxin to experimental animals (Korean Patent 10-0508539) and the like. However, efficacy screening studies for the prevention or treatment of all male reproductive dysfunctions were not reasonable, not considering the actual duration of sperm production. In this example, the three hundred seconds fermentation stock solution (Korean Patent 10-0508539) that enhances the reproductive function of men has not been described in detail the experimental method in the efficacy screening method using experimental animals, preventing or treating male reproductive function or hematopoiesis In the promoting composition and its preparation method (JP-A-2002-0085401), testis were extracted one or two weeks after oral administration of a liquid solution to an experimental animal, and sperm count and sperm activity were observed. In addition, in the composition for the prevention and treatment of male reproductive function (Publication Patent Publication No. 10-2005-0028475) intraperitoneally administered the composition to the experimental animals twice a day for l8 days 1 day after the end of the autopsy, sperm count, sperm Activity and sperm activity persistence were measured.

Research on male infertility is largely dependent on Western medical procedures, so the study of herbal medicines using herbal medicines has been withdrawn. Nevertheless, in Korea, the treatment of male sexual function improvement relies on Korean medicine for social and customary and ethical reasons, and there are a number of successful cases. In most cases, there is no basic and systematic study on the exact mechanism of action and each prescription. Most of the health foods on the market are mostly mixed with ingredients that are known to improve sexual function, and are manufactured and marketed by disregarding scientific research such as proper amount of active ingredients, synergistic effects, and duration of administration.

Accordingly, the present inventors are trying to develop a male infertility treatment, one or more extracts selected from cornus extract, goji berry extract, bokbunja extract, earthworm extract, Schizandra extract and chaja extract is cytotoxic in TM3 cells, NO in HUVEC In vitro tests such as concentration, ACE (Angiotensin converting enzyme) inhibitory effect, PDE (Phosphodiesterase) -5 inhibitory effect, CREM expression, serum cGMP changes, serum testosterone changes, organ weight (testis, spermatozoa, seminal vesicles) The present invention was completed by confirming that the in vivo test of measurement, sperm count, and sperm motility is effective in improving male sexual function and infertility treatment.

An object of the present invention is to provide a pharmaceutical composition for improving male sexual function and infertility using a cornus extract, goji berry extract, bokbunja extract, earthenware extract, Schizandra extract and chacha extract.

Another object of the present invention to provide a food composition for improving male sexual function and infertility using a cornus extract, goji berry extract, bokbunja extract, tosa extract, Schizandra extract and chacha extract.

In order to achieve the above object, the present invention, male sexual function improvement and infertility treatment pharmaceutical composition comprising at least one extract selected from Cornus extract, Goji berry extract, Bokbunja extract, Tosa extract, Schisandra extract and cha-cha extract as an active ingredient To provide.

In addition, the present invention improves male sexual function comprising at least one extract selected from cornus extract, goji berry extract, bokbunja extract, earthenza extract, schisandra extract and chaja extract including a food supplement acceptable as a active ingredient It provides a food composition for preventing infertility.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for improving male sexual function and infertility, comprising at least one extract selected from Cornus extract, Goji berry extract, Bokbunja extract, Tosa extract, Schizandra extract and chacha extract as an active ingredient.

In the pharmaceutical composition for improving male sexual function and infertility treatment of the present invention, the extract further comprises a pharmaceutically acceptable carrier, wherein the cornus extract, wolfberry extract, bokbunja extract, earthworm extract and Schizandra extract Extract consisting of (preferably named the five extracts KH-204 in the present invention) is preferred, based on 100 parts by weight of cornus extract 50-150 parts by weight, bokbunja 20-100 parts by weight, earthenware 20-100 weight It is more preferable that it is 10-50 weight part of parts and Schizandra chinensis. Or the composition is preferably containing the chajeon extract as an active ingredient.

In addition, in the pharmaceutical composition for improving male sexual function and infertility treatment of the present invention, the extract is i) adding water or ethanol to the medicine and extracting for 1 to 4 hours at 80 to 150 ℃; ii) removing the precipitated impurities by centrifugation at 1000-5000 rpm and filtering the extract solution with a 0.3-0.6 μm filter; And iii) concentrating the filtrate with a rotary vacuum concentrator.

Specifically, in the pharmaceutical composition for improving male sexual function and infertility treatment of the present invention, 1 to 20 times of water is added to the cornus, goji berry, bokbunja, earth and sand, schisandra and chacha, and at 1 to 24 hours at 80 to 150 ° C. It is preferably obtained by filtration after extraction, or by adding 1 to 20 times of 10 to 50% ethanol, extracting at 40 to 100 ° C. for 1 to 24 hours, and then filtering.

Among them, the composition consisting of Cornus, Wolfberry, Bokbunja, Tosa and Schisandra chinensis extract was named as KH-204, and their ethanol extract and hot water extract were measured.

Specifically, the extract may be obtained by extracting each medicinal herb with water or an organic solvent, and examples of the organic solvent may include lower alcohol, acetone, chloroform, methylene chloride, ether, ethyl acetate, hexane and the like. Lower alcohols include methanol, ethanol, propanol and butanol, with ethanol being most preferred.

Or 1-20 times, preferably 2-15 times, more preferably 10 times water, and 80-150 ° C., preferably 100, to the cornus oil, wolfberry, bokbunja, earth and sand, schisandra chinensis and dried tea powder or powder. 1 to 24 hours, preferably 3 to 5 hours, and more preferably 4 hours at a temperature of 120 to 120 ℃ and then filtered to produce a hydrothermal extract of each medicinal herbs, the filtrate obtained by filtration is concentrated under reduced pressure It can manufacture. In the above extraction methods, the extraction process may be repeated two or more times as necessary, and the extract obtained after filtration may be freeze-dried or reduced-pressure drying to form a powder.

Specifically, 1 to 20 times, preferably 2 to 15 times, more preferably 10 times of 10 to 50% ethanol aqueous solution is added to the cornus oil, wolfberry, bokbunja, earth and sand, schisandra chinensis and powdered tea or dried powder or 40 to The ethanol extract of each medicinal herb may be prepared by extracting the mixture at 100, preferably 50 to 60 hours for 1 to 24 hours, preferably for 3 to 5 hours, more preferably for 4 hours, and then filtering the same. The filtrate can be prepared by concentration under reduced pressure. In the above extraction methods, the extraction process may be repeated two or more times as necessary, and the extract obtained after filtration may be freeze-dried or reduced-pressure drying to form a powder.

In addition, the pharmaceutical composition of the present invention may further include other pharmaceutically acceptable herbal medicines or extracts thereof to enhance the pharmacological effect. In this case, after preparing the extract of the herbal medicine according to the extraction method and added to the pharmaceutical composition or by mixing the one or more selected herbs and herbal medicine selected from the cornus oil, wolfberry, bokbunja, earth and sand, Schisandra chinensis and chason and extracted by the method The obtained extract may be included in the composition. In the above, pharmaceutically acceptable means a physiologically acceptable and, when administered to a human, usually does not cause an allergic reaction or the like.

In addition, the pharmaceutical compositions according to the invention may further contain one or more pharmaceutically acceptable carriers, excipients or diluents.

Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration.

Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like.

In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition for improving male sexual function and infertility of the present invention may be administered to any mammal, including humans. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration. Can be. Preferably the pharmaceutical composition of the present invention may be administered transdermally. In the above, transdermal administration 'means that the active ingredient contained in the pharmaceutical composition for improving male sexual function and infertility is administered into the skin by administering the pharmaceutical composition of the present invention to cells or skin. For example, the pharmaceutical composition of the present invention may be prepared in an injectable formulation and administered by lightly pricking the skin with a 30 gauge thin injection needle, or directly applying to the skin.

The pharmaceutical composition of the present invention may be formulated into a preparation for oral or parenteral administration according to the route of administration as described above.

In the case of preparations for oral administration, the compositions of the present invention may be formulated using methods known in the art as powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions and the like. Can be. For example, oral formulations can be obtained by tablets or dragees by combining the active ingredients with solid excipients, milling them, adding suitable auxiliaries and then processing them into granule mixtures. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol and starch, cellulose, including starch, corn starch, wheat starch, rice starch and potato starch, etc. Fillers such as celluloses, gelatin, polyvinylpyrrolidone, and the like, including methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate and the like may optionally be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

Formulations for parenteral administration may be formulated by methods known in the art in the form of injections, creams, lotions, external ointments, oils, humectants, gels, aerosols and nasal inhalants. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a prescription generally known in all pharmaceutical chemistries.

The total effective amount of one or more extracts selected from cornus extract, goji berry extract, bokbunja extract, earthworm extract, schizandra extract, and chacha extract, which are the active ingredients of the composition of the present invention, may be administered to a patient in a single dose. It may be administered by a fractionated treatment protocol which is administered for a long time in multiple doses. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the extent of the disease. Preferably the preferred total dose of one or more extracts selected from Cornus extract, Goji berry extract, Bokbunja extract, Tosa extract, Schizandra extract and Chacha extract of the present invention is about 100 μg to 5 mg per kg of patient body weight per day, most preferably Preferably from 500 μg to 1 mg. However, the dosage of the extract is determined in consideration of various factors such as the age, weight, health status, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the pharmaceutical composition. In view of this, one of ordinary skill in the art will be able to determine the appropriate effective dosage for the particular use of the extract as an agent for improving male sexual function and infertility. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, route of administration and method of administration as long as the effect of the present invention is shown.

In the present invention, 'male sexual improvement and infertility treatment' generally means normalization or enhancement of sperm count, sperm activity, testosterone hormone, sexual behavior, and sexual function that can cause infertility.

In addition, the present invention improves male sexual function comprising at least one extract selected from cornus extract, goji berry extract, bokbunja extract, earthenza extract, schisandra extract and chaja extract including a food supplement acceptable as a active ingredient It provides a food composition for preventing infertility.

The food composition of the present invention includes all forms such as functional foods, nutritional supplements, health foods and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the cornus extract, Goji berry extract, Bokbunja extract, Tosa extract, Schisandra chinensis extract and chasen extract themselves are prepared in the form of tea, juice and drink, or granulated, encapsulated and Can be ingested powdered. In addition, the cornus extract, goji berry extract, Bokbunja extract, Tosa fruit extract, Schisandra chinensis extract and chacha extract and mixed with a known substance or active ingredient known to improve male sexual function and infertility effect to be prepared in the form of a composition Can be.

In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. ham, sausage cornebipe) Etc.), breads and noodles (e.g. udon, soba noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, syrups, dairy products (e.g. butter, cheese, etc.), edible vegetable oils, margarine, vegetable protein, The retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) may be prepared by adding the cornus extract, wolfberry extract, bokbunja extract, earthworm extract, schisandra extract and chasen extract of the present invention.

In addition, in order to use the Cornus extract, Goji berry extract, Bokbunja extract, Tosa extract, Schisandra chinensis extract and chacha extract of the present invention in the form of food additives can be prepared in powder or concentrate form.

味地

湯)이 있는데, 이 처방에서 산수유는 가장 중요한 약재로 사용된다.Cornus is a deciduous arborescent belonging to the dogwood family. According to "Agreement Gagam" and "Hyangjakbangbang", etc., it is effective in strengthening sound tone, Shinjeong and Shingi, and converging. Headache, tinnitus, seawater disease, fever, excessive menstruation, etc. It is said that it can also be used for cold weather. It is the most widely known Chinese medicine for the general public such as Jayang Yangjiang.

味 地

湯), cornus oil is used as the most important medicine in this prescription.

)의 대노(大

)와 허흡(

吸)을 다스리고, 근골(筋骨)을 굳세게 하며, 음 (陰)을 강하게 하고 오노(五

)와 칠상(七

)을 다스리며 정기를 보하고 안색을 바꿔서 희게 하고 명목(明目), 안신(安神)하고 장수한다"고 전한다. 또한 본초 강목에서는 독성이 없고, 열을 식히고, 체내에 쌓인 사기, 흉부의 염증 소갈, 당뇨병, 관절, 류마티스, 신경통 등에 좋으며 오래 복용하면 근육을 건전하게 하고 전신이 상쾌하고 더위와 추위를 모르는 젊음을 찾는다고 하였고 차, 술 등 다양한 방법으로 오랫동안 이용되었다.The wolfberry is a deciduous shrub reaching 2.5 m in height, the tip of the branch hangs downward, the short branch is thorny, and the leaves are 2 to 3 single-footed under the long branch. From the old days, the agreement was written as "Homan (미), beautiful", "non-toxic"

Great oar

) And breath (

Reigns, strengthens the musculature, strengthens yin, and ono

) And Seven Statues

), And to maintain regular, change the complexion to whiten, nominal, anxin and longevity. ”Also, in the herbal wood, it is not toxic, cools, accumulates in the body, and inflammation of the chest. It is good for diabetes, joints, rheumatism, neuralgia, etc. It is said that if you take a long time to keep the muscles healthy and refresh the young, do not know the heat and cold youth, it has been used for a long time in various ways such as tea and alcohol.

喝)과 여자의 무자(無子)를 주치하고 남자의 음기를 굳세고 길게 하며 간을 도우며, 눈을 맑게 하며, 신장의 기운을 가득하게 하여 몸을 가볍게 하고, 머리털이 희어지지 않게 한다"라 기록되어 있다. 또한, 최근 연구에서 신장기능, 불임증, 음위증, 유정몽설, 강장제, 혈액 등을 맑게 해주며 간을 보하고 눈을 맑게 하는 효능이 증명되었으며 복분자 딸기의 덜 익은 열매의 가수분해성 탄닌으로서 1종의 갈로탄닌(gallotannin)과 3종의 엘라기탄닌(ellagitannin)을 분리하였으며, 이화학적 성상과 스펙트럼 데이터를 토대로 갈릭산, 2,3-(S)-HHDP-D-글루코피라노즈, 산귄(sanguiin) H-4 및 산귄 H-6를 보고하였다. 복분자의 경우 효소적 지질과 산화에 대해서도 강한 억제 활성을 나타내며 그 성분으로는 갈릭산 등의 페놀 화합물이 확인되었다. 페놀성 화합물은 식품계에 널리 분포되어 있는 2차 대사산물의 하나로서 다양한 구조와 분자량을 가지며, 이들은 페놀성 히드록실기를 가지고 있기 때문에 단백질 등의 거대분자들과 결합하는 성질을 가지며, 항산화 효과, 항균성, 아질산염 소거능 등의 생리활성 기능도 가진다.Bokbunja is a dry, deciduous shrub belonging to the fruit of the ripe Bokbunja strawberry dried and tastes warm and has the effect of protecting the liver and kidneys. According to the agreement, the bokbunja are flat, taste sweet, sour, and have no poison.

주) and the woman's ignorance, to strengthen and lengthen the man's tone, to help the liver, to clear the eyes, to fill the energy of the kidneys, to lighten the body and not to whiten the hair. " In addition, recent studies have demonstrated the effects of clearing the kidneys, infertility, vulgaris, well-being dreams, tonics, blood, and protecting the liver and clearing the eyes, as a hydrolyzable tannin of the unripe fruit of bokbunja strawberry. One gallotannin and three ellagitannins were isolated, and based on physicochemical properties and spectral data, gallic acid, 2,3- (S) -HHDP-D-glucopyranose, and wild penguin (sanguiin) H-4 and mountain horn H-6 were reported, and bokbunja showed strong inhibitory activity against enzymatic lipids and oxidation, and phenolic compounds such as gallic acid were identified. On board As a secondary metabolite that is distributed, it has various structures and molecular weights, and because they have phenolic hydroxyl groups, they have a property of binding to macromolecules such as proteins, and have physiological properties such as antioxidant effect, antimicrobial activity, and nitrite scavenging ability. It also has an active function.

Earthenware dried seeds of perennial herbaceous ginseng and perennial plants belonging to the ume flower family. Tosa is a sweet, sweet and plain nature, and is effective in protecting cancer, heart, blood pressure and blood sugar, kidney and spleen. In Dongbobogam, "enhancing energy and invigorating, low back pain and knee ache symptomatic effect is good if you drink from time to time, it is effective for diabetes." Longer doses can make your eyes brighter and longer. "

), 구갈, 번열, 해소를 고친다고 전한다. 이러한 오미자는 차, 술, 화채 등으로 다양하게 이용되어지고 있다.Schisandra chinensis is a dried fruit of Schisandra chinensis, belonging to the Magnolia family, and has five flavors, and is effective in protecting liver function, anti-aging, antibacterial action, metabolism and immune function. In Dongbogam, protect lungs and kidneys

), It is said that it corrects the mouth, the heat, the dissolution. These schizandra are used in various ways such as tea, liquor, and flower.

水), 청열(淸熱), 명목(明目), 거담(祛痰)의 효능이 있고, 소변불통, 대하, 혈뇨, 해수, 목적(目赤)을 치료하는데 주로 쓰인다. <동의보감>에 따르면 차전자는 기운이 허약하여 소변이 잘나오지 않는 것을 주로 치료하고, 다섯가지 종류의 임질을 다스리며, 소변의 막히고 체함을 통하게 하고, 수분이 잘 배설되게 하며, 눈을 밝게 하고 충혈을 없애주며, 간장의 열독을 다스린다고 한다. 따라서 차전자는 수독형의 부종으로 인해 붓는 증상을 개선해 준다.Chason refers to the seed of Plantago asiatica L., a plant belonging to the Plantaginaceae, which is a Korean medicine

Efficacy in water, clear heat, nominal, expectorant, and is effective in treating urinary irritation, lobster, hematuria, seawater, and the purpose. According to <Agreement Bom>, Cha is mainly for treating weakness of urine due to weak energy, controlling five kinds of gonorrhea, clogging and urinating urine, draining water well, brightening eyes and congestion Eliminates, and is said to rule the soy sauce. Therefore, the tea can improve the symptoms of swelling due to toxic edema.

The present inventors measured the cytotoxicity of TM3 cells, which are testicular cells of male mice, by MTT assay, using hydrothermal and 30% ethanol extracts and mixed extracts of Cornus, Goji, Bokbunja, Tosa, Schisandra chinensis and Cha-chan. (See Table 1 and FIGS. 1-8). As a result, it was confirmed that there is no toxicity in KH-204 ethanol, KH-204 hydrothermal water, cornus oil, Schisandra chinensis, and wolfberry, and the samples of earth and sand, tea and bokbunja were found to be slightly toxic.

In addition, HUVEC cells were used to measure the NO production efficacy involved in eNOS and closely involved in erection (see Table 3 and FIGS. 9 and 10). As a result, it was confirmed that KH-204 30% ethanol extract and hot water extract group increased the concentration of nitrate. In other words, the samples improve the function of the penis by increasing the production of vasodilator NO.

In addition, ACE (Angiotensin converting enzyme) inhibitory effect of each extract of the present invention was measured (see Table 5 and Figure 11). As a result, wolfberry (49.1%), KH-204 hydrothermal extraction (47.0%), Schisandra (45.9%), chason (36.3%), KH-204 30% ethanol extraction (34.2%), Bokbunja (29.9%) The inhibition rate was high. Therefore, these samples may help maintain an erection by indirectly increasing blood flow to the penis through relaxation of blood vessels.

In addition, PDE (Phosphodiesterase) -5 inhibition rate was measured (see Table 8 and FIG. 14). The inhibitory effects of PDE-5 were favorable in the order of Bokbunja (34.5%), Schisandra chinensis (35.6%), Goji (32.8%), and KH-204 (30% Ethanol Extract, 25.6%).

In addition, long-term weight measurement and sperm function were measured through animal experiments. KH-204 ethanol extract and the positive control red ginseng was measured, first, the mouse weight (see Table 10 and Figure 15) showed a weight reduction than red ginseng. However, the true weight showed a tendency to increase in the group administered with high concentration of KH-204 (ethanol extraction) (see Table 11 and Figure 16). Testis weight was similar between the group administered KH-204 (ethanol extraction) and the group administered red ginseng (see Table 12 and FIG. 17). In general, the weight of the intestine was similar between the group administered KH-204 (ethanol extraction) and the group administered red ginseng (see Table 13 and FIG. 18). Sperm water tended to increase as the concentration of KH-204 (ethanol extract) increased, and more sperm counts were found in all groups treated with KH-204 (ethanol extract) than the red ginseng 400 group used as a positive control (Table 14). And FIG. 19). The sperm motility of the mice tended to increase as the concentration of KH-204 (ethanol extract) increased, and higher sperm motility was observed in all groups treated with KH-204 (ethanol extract) than the red ginseng 400 group used as a positive control. (See Table 15 and FIGS. 20-26).

The present invention constituted as described above is a pharmaceutical composition for improving male sexual function and infertility, and has the effect of increasing sperm count, sperm motility, testosterone hormone content, sexual behavior, sexual function, etc. It can help improve male reproductive and sexual function, as well as patients suffering from sexual dysfunction and male infertility, without the side effects and ethical and social problems.

1 is a graph showing the cytotoxicity test of KH-204 ethanol extract.

Figure 2 is a graph showing the cytotoxicity test of KH-204 hydrothermal extract.

Figure 3 is a graph showing the cytotoxicity test of cornus extract.

Figure 4 is a graph showing the cytotoxicity test of the Schizandra chinensis extract.

5 is a graph showing the cytotoxicity test of the bokbunja extract.

Figure 6 is a graph showing the cytotoxicity test of the soil extract.

Figure 7 is a graph showing the cytotoxicity test of the tea extract.

8 is a graph showing the cytotoxicity test experiment of goji berry extract.

9 is a graph showing the change of nitrate in HUVEC of KH-204 ethanol extract.

10 is a graph showing the change of nitrate in HUVEC of KH-204 hydrothermal extract.

11 is a graph showing the results of measuring the ACE inhibition rate of each sample (%), it shows the inhibition rate of angiotensin-converting enzyme by each sample (concentration: 500 ppm).

12 is a view illustrating the principle of measuring the PDE (Phosphodiesterase) -5 inhibitory effect used in the present invention.

13 and 14 are graphs showing measurement results of standard curve (FIG. 13) and phosphodiesterase-5 inhibition rate for each sample (500 ppm) for obtaining PDE-5 inhibition rate (%), respectively (FIG. 14).

Figure 15 is a graph showing the final weight of the mouse after oral administration of KH-204 30% ethanol extract samples by concentration for 8 days.

Figure 16 is a graph showing the left mouse vesicle weight after oral administration of KH-204 30% ethanol extract samples by concentration for 8 days.

Figure 17 is a graph showing the left testis weight of the mouse after oral administration of KH-204 30% ethanol extract samples by concentration for 8 days.

FIG. 18 is a graph showing left mouse anaerobic weights after oral administration of KH-204 30% ethanol extract samples by concentration for 8 days.

19 is a graph showing the number of mouse sperm after oral administration of KH-204 30% ethanol extract sample for 8 days by concentration.

20 is a graph showing mouse sperm motility (%) after oral administration of KH-204 30% ethanol extract sample for 8 days at different concentrations.

21 to 26 are micrographs showing the motility of sperm, the sperm photograph of the control group (optical microscope 200x magnification) (Fig. 21), the sperm photograph of the group administered with 50 mg / kg of KH-204 30% ethanol extract ( Optical microscope 200x magnification) (FIG. 22), KH-204 30% ethanol extract 100 mg / kg sperm photograph (Optical microscope 200x magnification) (FIG. 23), KH-204 30% ethanol extract 200mg / kg Sperm picture (optical microscope 200x) (FIG. 24), group sperm picture (optical microscope 200x) (FIG. 25) administered with KH-204 30% ethanol extract 400 mg / kg, and positive control group This is a sperm photograph (optical microscope 200x magnification) of the group administered red ginseng 400 mg / kg.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Preparation Example Preparation of Male Sexual Function Improvement and Infertility Treatment Composition

Preparation Example 1 Preparation of 30% Ethanol Extract

<1-1> Preparation of Cornus 30% Ethanol Extract

First, impurities were removed, and 22 g of dried cornus oil was prepared, and 4-8 times 30% aqueous ethanol solution was added thereto, and the mixture was extracted under reflux for 3 to 5 hours at 60 to 70 hours. The extract was filtered and the filtrate was concentrated under reduced pressure at 50 to 60 or less.

<1-2> Preparation of wolfberry 30% ethanol extract

After removing impurities, 42 g of dried wolfberry was prepared, and 4-8 times 30% aqueous ethanol solution was added thereto, followed by extraction under reflux for 3 to 5 hours at 60 to 70 ° C. The extract was filtered and the filtrate was concentrated under reduced pressure at 50 ~ 60 ℃.

<1-3> Preparation of Bokbunja 30% Ethanol Extract

After removing impurities, 26 g of dried bokbunja was prepared, and 4-8 times of 30% aqueous ethanol solution was added thereto, and the mixture was extracted under reflux for 3 to 5 hours at 60 to 70 ° C. The extract was filtered and the filtrate was concentrated under reduced pressure at 50 ~ 60 ℃.

<1-4> Preparation of Tosa 30% Ethanol Extract

After removing impurities, 26 g of well-dried earthworms were prepared, and 4-8 times 30% aqueous ethanol solution was added thereto, followed by extraction under reflux for 3 to 5 hours at 60 to 70 ° C. The extract was filtered and the filtrate was concentrated under reduced pressure at 50 ~ 60 ℃.

<1-5> Preparation of Schizandra chinensis Extract 30% Ethanol

6 g of Omija, which had been freed of impurities and dried, was prepared, and 4-8 times 30% aqueous ethanol solution was added thereto, and the mixture was refluxed at 60 to 70 ° C. for 3 to 5 hours. The extract was filtered and the filtrate was concentrated under reduced pressure at 50 ~ 60 ℃.

<1-6> Preparation of KH-204 30% ethanol extract

Prepare the concentrated cornus oil, gojijaja, bokbunja, earthenware, Schisandra chinensis concentrate into powder, add 10 times the amount of purified water and boil it for 2 hours in a water bath (Daeung, Korea) to take a solution, centrifuged at 3,000 rpm and supernatant Filtered using a 0.45 ㎛ filter, concentrated and concentrated in a rotary vacuum concentrator (Eyela A-1000S, Tokyo Rikakikai Co., Tokyo, Japan) to recover the sample and freeze-dried (ILSHIN, Korea) to recover 23.5 g It was used for the experiment while storing at -20 ℃.

<1-7> Preparation of Chason 30% Ethanol Extract

After removing impurities, 32 g of well-dried charcoal was prepared, and 4-8 times of 30% ethanol aqueous solution was added thereto, and the mixture was extracted under reflux for 3 to 5 hours at 60 to 70 ° C. The extract was filtered and the filtrate was concentrated under reduced pressure at 50 ~ 60 ℃. Finally, 30 ml of distilled water was added, suspended, and freeze-dried to obtain a final brown powder.

Preparation Example 2 Preparation of KH-204 Hot Water Extract

After removing impurities, 32 g of dried cornus oil, 32 g of Gojijaja, 16 g of Bokbunja, 16 g of Tosa and 16 g of Schisandra chinensis were prepared, and 4 to 8 times of water was added thereto and refluxed at 100 to 120 ° C. for 3 to 5 hours. . The extract was filtered and the filtrate was concentrated under reduced pressure at 50 ~ 60 ℃. To this, 10-fold purified water was added and boiled in a medicine bath (Daewoong, Korea) for 2 hours, the solution was collected and centrifuged at 3,000 rpm. The supernatant was collected and filtered using a 0.45 µm filter, followed by a rotary vacuum concentrator (Eyela A-1000S). , Tokyo Rikakikai Co., Tokyo, Japan), the sample was recovered and lyophilized (ILSHIN, Korea) to recover 23.5 g and stored at -20 ℃ was used in the experiment.

Example 1 Measurement of Cytotoxicity in TM3 Cells

Mouse testicular cell TM3 was analyzed for cytotoxicity of the sample by MTT analysis. TM3 cells (KCLB21714) were distributed by the Korea Cell Line Bank. Fetal bovine serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), Sigma (Sigma Chem. Co., USA) ). ELISA microplate reader was used VersaMax (Molecular Device, USA).

Specifically, in order to determine the cytotoxicity of the sample, using the testis cells of male mice, TM3 cells using the method of Sladowski et al. (Sladowski D, et. Al. J Immunol Methods 157, 203-207. (1993)) Analysis was performed. 180 μl of TM3 cells (2 X 10 ⁵ cells) in 96 well plates were treated by 20 concentrations of each sample, and then treated to 62.5 ppm, 125 ppm, 250 ppm, 500 ppm, 1000 ppm, 2000 ppm, and 4000 ppm, respectively. After incubation at 37 for 24 hours, 20 μl of MTT solution (5 mg / ml) was added and incubated at 37 ° C. for 3 hours. Thereafter, the supernatant was removed, and 200 μl of DMSO was added per well, followed by reaction for 20 minutes, and the survival rate was calculated by measuring absorbance at an ELISA reader 570 nm. The value of the normal group (group treated with PBS instead of sample treatment) was used as a control, and the OD (Optical density) value at this time was defined as 100% of cell viability, and the measured OD value of the remaining group was converted to relative value. Is as follows.

Cell viability (%) = (experimental / control) X 100

The cytotoxicity results of each sample are shown in Table 1 below, FIGS. 1 to 8.

Table 1 Control 62.5 125 250 500 1000 2000 4000 (ppm) KH-204 Ethanol Extract 100.00 ± 0.09 89.40 ± 0.016 89.86 ± 0.036 90.02 ± 0.020 85.41 ± 0.041 86.26 ± 0.014 86.22 ± 0.061 73.58 ± 0.025 (%) KH-204Hot Water Extraction 100.00 ± 0.09 86.36 ± 0.009 87.07 ± 0.041 86.74 ± 0.039 87.79 ± 0.031 88.60 ± 0.011 87.55 ± 0.008 78.25 ± 0.060 (%) Cornus 100.00 ± 0.09 86.64 ± 0.051 85.75 ± 0.007 85.12 ± 0.003 85.20 ± 0.014 84.16 ± 0.025 68.62 ± 0.019 61.71 ± 0.057 (%) Schisandra 100.00 ± 0.09 96.47 ± 0.075 92.01 ± 0.144 88.49 ± 0.095 83.94 ± 0.080 82.39 ± 0.195 79.49 ± 0.019 78.70 ± 0.003 (%) Bokbunja 100.00 ± 0.09 92.23 ± 0.033 69.98 ± 0.034 61.88 ± 0.035 62.07 ± 0.013 52.20 ± 0.081 47.98 ± 0.105 35.32 ± 0.113 (%) Tosa 100.00 ± 0.09 102.44 ± 0.006 99.13 ± 0.059 80.00 ± 0.001 65.58 ± 0.077 59.96 ± 0.110 57.48 ± 0.004 52.83 ± 0.058 (%) Difference 100.00 ± 0.09 99.29 ± 0.073 90.84 ± 0.010 81.16 ± 0.087 75.35 ± 0.048 71.32 ± 0.018 69.10 ± 0.065 67.87 ± 0.056 (%) Wolfberry 100.00 ± 0.09 94.41 ± 0.982 85.20 ± 0.874 85.16 ± 0.892 82.03 ± 0.840 80.21 ± 0.822 79.22 ± 0.812 77.30 ± 0.793 (%)

Cell viability was measured by treating the samples with concentrations of TM3 cells, and the cell viability of the control group without the sample was compared to 100%. When the cell count of 20% or more decreased, it was determined to be cytotoxic.

As a result, as shown in Table 1, the KH-204 ethanol extract sample had a survival rate of 73.58% at 4000 ppm concentration, and the KH-204 hydrothermal extract sample had a slight toxicity from 4000 ppm with a survival rate of 78.25% at 4000 ppm concentration. Was observed. Cornus, Schisandra chinensis, and Goji were viable at 2000 ppm, with viability of 68.2%, 79.49%, and 79.22%, respectively. Tosa and char were found to be 65.58% and 75.35% at 500 ppm, respectively. Bokbunja was cytotoxic at 69.98% at 125 ppm.

In general, it is judged to be toxic when the cell viability is below 80% at 1000 ppm concentration. It is confirmed that it is not toxic in KH-204 ethanol, KH-204 hydrothermal water, cornus oil, Schisandra chinensis, and wolfberry. It was confirmed to be toxic. However, since the toxicity was not confirmed in the mixture of KH-204, the following in virto experiment and in vivo experiment were performed.

Example 2 Measurement of NO Concentration in HUVEC

In order for the penis to erect, first the complete relaxation of the cavernous artery and the smooth muscle and the accumulation of blood in the sinusoidal vascular space, and the relaxation of the penile smooth muscle is achieved by neurotransmitters and non-neurotransmitters. Recently, the regulation of smooth muscle in the corpus cavernosum by prostaglandin and EDRF (endothelium derived relaxing factor) has been found to play an important role in erection. Palmer et al. Have found that nitric oxide (NO) is one of the strongest EDRFs, and studies on the role of NO in the relaxation of the corpus cavernosum smooth muscle have been actively conducted. NO is biosynthesized by the biochemical action of nitric oxide synthase (NOS) in the body. Immunohistochemistry revealed NOS-containing neurons and their organs, and their distributions were similar in neuroanatomy in rats and humans. NO, a factor that promotes vascular smooth muscle relaxation in penile corpus cavernosum tissue and improves erectile function, is an incomplete and instable substance and cannot be directly quantified because its half-life is very short. By measuring, the content of NO can be expected. HUVEC cells were used to measure the NO production efficacy involved in eNOS and closely involved in erection.

Total nitric oxide assay kit was used to measure NO concentration in HUVEC. HUVEC 900 (110 ⁴ cells) per well was added to a 12 well plate and incubated in a 37, 5% CO ₂ incubator for 24 hours, then the medium was discarded and the cultured cell surface was washed with 1 × PBS. Samples dissolved in 1X PBS were mixed with EGM-2 so that the final concentration was 0, 50, 100, 200, 400 ppm and incubated in 37, 5% CO ₂ incubator for 48 hours. After incubation, NADH 25 and nitrate reductase 25 were mixed in the culture medium 50, and then reacted for 37 to 30 minutes. Griess reagent 50 and Griess reagent 50 were added thereto, and the mixture was allowed to stand for 10 minutes at room temperature. After the reaction, the absorbance was measured at 550 to calculate the concentration of nitrate, and the nitrate was measured as an index of NO production.

The concentrations of KH-204 ethanol and hot water extracts used and the concentration of control red ginseng are shown in Table 2 below.

TABLE 2 group Experimental concentration KH-204 30% Ethanol Extract 0, 50, 100, 200, 400 ppm KH-204 hydrothermal extraction 0, 50, 100, 200, 400 ppm Red Ginseng (positive control) 400 ppm

As a result, for each concentration of each KH-204 ethanol and hot water extract, the results of NO production in the control red ginseng are shown in Table 3, Figure 9 and Figure 10 below.

TABLE 3 0 (voice control) 50 100 200 400 Red Ginseng 400 (Yangseong Control Group) (ppm) KH-204 Ethanol Extract 0.002 ± 0.092 3.877 ± 0.783 6.038 ± 0.468 7.508 ± 0.065 16.369 ± 0.609 8.362 ± 0.020 (%) KH-204 Hot Water Extract 0.002 ± 0.092 0.031 ± 0.109 0.454 ± 0.403 2.438 ± 0.555 7.438 ± 0.533 8.362 ± 0.020 (%)

As a result, the concentration of nitrate was about 3.877, 6.038, 7.508, 16.369 μmol / ml as the KH-204 30% ethanol extract sample was treated with 50, 100, 200, and 400 ppm. The negative control resulted in 0.002 μmol / ml, and compared with this, the concentration of nitrate was increased in the KH-204 30% ethanol extract group (FIG. 9).

The concentration of nitrate was about 0.031, 0.454, 2.438, 7.438 μmol / ml when the KH-204 hydrothermal extract sample was treated with 50, 100, 200, 400 ppm, compared with the negative control treated with the medium instead of the sample. When KH-204 was shown to increase as 30% ethanol extract (Fig. 10).

In particular, the concentration of 400 ppm of KH-204 30% ethanol extract was 16.369 μmol / ml, and the concentration of nitrate was increased more than the value of 8.362 μmol / ml of 400 ppm of red ginseng used as a positive control.

The KH-204 hydrothermal extract samples showed higher concentrations of nitrate than the negative control, but were lower than the nitrate concentration of the 400 ppm group of red ginseng, the positive control, so that the KH-204 30% ethanol extract was higher than the KH-204 hydrothermal extract. In comparison, it was found that nitrate was produced more effectively. The test results suggest that the sample improves penile function by increasing the production of vasodilator NO.

Example 3 Measurement of Inhibitory Effect of Angiotensin Converting Enzyme (ACE)

Inhibiting the action of ACE involved in the synthesis of angiotensin, a potent vasoconstrictor, to maintain vascular muscle relaxation, the key to erectile maintenance, which is closely related to male sexual function enhancement and strengthening, Indeed, some ACE inhibitors have been proposed as candidates for improving and enhancing sexual function. Angiotensin-converting enzyme (ACE; peptodyldipeptide hydrolase EC 3.4.15.1) is an enzyme that plays an important role in controlling blood pressure. The enzyme increases blood pressure by hydrolyzing angiotensin, which consists of 10 amino acids, to angiotensin. Angiotensin, a potent vasoconstrictor, stimulates the release of aldosterone. As a result, aldosterone increases the retention of Na ⁺ and water in the kidney and increases arterial blood pressure. Inhibiting the production of angiotensin by inhibiting ACE can lower blood pressure. Relaxing the blood vessels also increases the blood flow in the penis, which is significant in that penile erection is induced. In this test, N-Hippuryl-His-Leu was used in place of ACE substrate Angiotensin and ACE was extracted from rabbit lung tissue powder and used as enzyme solution. Inhibition rate was confirmed by measuring hippuric acid produced through enzymatic reaction at 228 nm (I., M., Y., Shon and S., H., Na., J Korean Soc Food Sci Nutr. 36, 1511-1516. (2007); E., K., Cho et. Al, J. Life Sci. 21, 811-818. (2011); C. Liang et. Al., Natural product sciences, 17 ( 4), 363-366. (2011).

The principle is summarized as follows.

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu ( Angiotensin ) + ACE (Enzyme) : Reactant

↓

Asp-Arg- Val-Tyr-Ile-His-Pro-Phe ( Angiotensin ) + His-Leu: product

Hippuric acid-His-Leu (N-Hippuryl-His-Leu) + ACE (Enzyme) : reactant

↓

Hippuric acid + His-Leu: product

Boric acid, rabbit lung acetone powder Sigma and HHL (hippuryl-histidyl-leucine) used in this test were purchased from Sigma-Aldrich and ultrapure water produced from PURELAB Plus was used. Other reagents used were extra pure grade reagents. UV-VIS absorbance was used Spectronic Genesys 5 (Milton Roy Co., Ivyland, USA).

Table 4 summarizes the samples, extraction conditions and concentrations used in this test.

Table 4 sample Extraction condition Manufacturing concentration ppm Final concentration ppm KH-204 30% ethanol 2500 500 KH-204 Hydrothermal 2500 500 Schisandra 30% ethanol 2500 500 Wolfberry 30% ethanol 2500 500 Difference 30% ethanol 2500 500 Bokbunja 30% ethanol 2500 500 Tosa 30% ethanol 2500 500 Cornus 30% ethanol 2500 500 Enalapril maleate salt (positive control) 2500 25

Samples were dissolved in distilled water to 2500 ppm and stored at 4 ° C. until use. This experiment was performed by modifying the method of Cho et al. (E., K., Cho et. Al., J. Life Sci. 21, 811-818. (2011)). Rabbit lung acetone powder (Sigma) was extracted with 0.1 M Sodium borate buffer (pH 8.3) containing 0.3 M NaCl at a concentration of 0.1 g / ml (w / v) and extracted at 4 ° C. for 2 hours. After centrifugation at 4 ℃, 4000 rpm for 40 minutes, the supernatant was used as ACE coenzyme solution. The substrate was used after dissolving HHL (hippuryl-histidyl-leucine) in 0.1 M Sodium borate buffer (pH 8.3) containing 0.3 M NaCl at a concentration of 5 mg / ml (w / v). The ACE inhibitory activity was added to 50 μl of ACE coenzyme solution, followed by preliminary reaction at 37 ° C. for 5 minutes, followed by the addition of a substrate solution, followed by reaction at 37 ° C. for 30 minutes. 150 μl of 0.1 M HCl was added to stop the reaction. After adding 750 μl of ethyl acetate, the mixture was stirred for 15 seconds and centrifuged at 4, 3000 rpm for 5 minutes. The supernatant was completely dried and dissolved in 3 ml of distilled water, and the absorbance was measured at 228 nm.

Distilled water was used as a negative control instead of coenzyme solution and Enalapril maleate salt was used as a positive control. ACE inhibition rate was calculated using the following formula.

% Inhibition of ACE = [1- (ACE test / ACE control)] X 100

As a result, the ACE inhibition rate of each sample is shown in the following 5, FIG.

Table 5 Sample Name Extraction conditions density ACE inhibition rate (%) KH-204 Hydrothermal 500 ppm 47.0 ± 8.2 KH-204 30% ethanol 34.2 ± 2.7 Schisandra 30% ethanol 45.9 ± 6.8 Wolfberry 30% ethanol 49.1 ± 8.2 Difference 30% ethanol 36.3 ± 8.2 Bokbunja 30% ethanol 29.9 ± 2.7 Tosa 30% ethanol 12.8 ± 0.0 Cornus 30% ethanol 16.0 ± 1.4 Enalapril maleate salt (positive control) 25 ppm 98.3 ± 5.4

The inhibition rate was 5 mg / ml HHL (hippuryl-histidyl-leucine) was used as a substrate and the absorbance was measured at 228 nm by reacting 0.1 g / ml ACE coenzyme solution at 37 ℃ for 30 minutes. When comparing the inhibition rate by sample, Gojija (49.1%), KH-204 hydrothermal extraction (47.0%), Schisandra chinensis (45.9%), Cha-chan (36.3%), KH-204 30% ethanol extract (34.2%), Bokbunja ( 29.9%), the highest inhibition rate. Therefore, the six samples are thought to help maintain erection by indirectly increasing blood flow to the penis through the relaxation of blood vessels.

Example 4 Measurement of PDE (Phosphodiesterase) -5 Inhibition Rate

An erection of the penis occurs due to the complex interaction of physiological processes including the central nervous system, peripheral nervous system and smooth muscle. In particular, nitric oxide (NO) release from nonadrenergic, noncholinergic nerves and endothelium activates guanylate cyclase in the corpus cavernosum and increases intracellular cGMP (cyclic GMP) levels. Increasing intracellular cGMP decreases intracellular calcium levels, leading to columnar smooth muscle relaxation, which in turn leads to penile erection through cavernous volume expansion. Since cGMP is broken down by PDE (phosphodiesterase) -5, its activity must be inhibited to increase the concentration of cGMP, thereby improving erectile function. In this test, 5-GMP is produced by PDE-5 using 3,5-cGMP as a substrate, and phosphoric acid is produced by 5-Nucleotidase. The amount of phosphate produced was measured to determine how much PDE-5 activity was inhibited, and based on this, it was intended to be used as a data for improving male infertility (FIG. 12).

The Cyclic nucleotide phosphodiesterase assay kit (BML-AK800, Enzo life science) was purchased and used for measuring the absorbance. The UV-VIS spectrophotometer is a Spectronic Genesys 5 (Milton Roy Co., Ivyland, USA) model and an ELISA microplate reader VersaMax (Molecular Device, Sunnyvale, USA) was used.

Table 6 summarizes the medicinal samples, extraction conditions and concentrations, including KH-204 ethanol and hot water extract used in this test.

Table 6 sample Extraction conditions Concentration (ppm) Final concentration (ppm) KH-204 30% ethanol 2500 500 KH-204 Hydrothermal 2500 500 Wolfberry 30% ethanol 2500 500 Bokbunja 30% ethanol 2500 500 Cornus 30% ethanol 2500 500 Schisandra 30% ethanol 2500 500 Tosa 30% ethanol 2500 500 Difference 30% ethanol 2500 500 IBMX (positive control) 9

The samples were dissolved in distilled water to 2500 ppm and stored at 4 ° C until use, and PDE inhibition effect was according to Jang et al. (JD Corbin and SH Francis. J. Andrology. 24, S38-S41. (2003)) It was measured using a phosphodiesterase assay kit. 5-GMP was treated with 5-nucleotidase, and then treated with BIOMOL GREEN reagent to measure absorbance at 620 nm to obtain a standard curve, and reacted by adding each sample with PDE using 3,5-cGMP as a substrate. Treatment with -nucleotidase and treatment with BIOMOL GREEN reagent measured absorbance at 620 nm, and the amount of 5-GMP produced was compared with the control group to calculate PDE inhibition rate. The negative control group excluded substrate only and the positive control group used 3-isobutyl-1-methylxanthine (IBMX) as a positive control group. The IC ₅₀ value of IBMX was 5.5 ppm.

% Inhibition of PDE-5 = [1- (PDE test-sample blank / control-control blank)] X 100

As a result, it is as Table 7 and FIG. 13 of the obtained standard curve.

TABLE 7 5-GMP (n) OD 620 3.00 0.6640 2.00 0.5045 1.50 0.3844 1.00 0.2647 0.75 0.2427 0.50 0.1583 0.25 0.1121 0 0.0708

As a result, the PDE inhibition rate measurement results obtained are as shown in Table 8 and FIG.

Table 8 Sample Name Extraction conditions density Inhibition Rate (%) KH-204 Hydrothermal 500 ppm -3.0 ± 1.2 KH-204 30% ethanol 25.6 ± 1.4 Tosa 30% ethanol 13.5 ± 1.6 Wolfberry 30% ethanol 32.8 ± 0.5 Difference 30% ethanol -3.40 ± 1.3 Bokbunja 30% ethanol 34.5 ± 2.92 Schisandra 30% ethanol 35.6 ± 1.6 Cornus 30% ethanol -15.3 ± 1.8 IBMX (positive control) 9 ppm 94.7 ± 3.6

As a result, the IC ₅₀ value of Sildenafil is 3.5 nM, the IC ₅₀ value of Vardenafil is 6.6 nM and the IC ₅₀ value of Tadalafil is 0.2 nM. The inhibitory effects of PDE-5 were favorable in the order of Bokbunja (34.5%), Schisandra chinensis (35.6%), Goji (32.8%), and KH-204 (30% ethanol extract, 25.6%).

The in vitro test was completed as above, and in vivo test through animal experiment was performed below.

Example 5 Animal Experiment (Organic Weight Measurement and Sperm Function Measurement)

The effects of KH-204 on normal animal models were verified. The concentrations of KH-204 ethanol extract used and the concentration of control red ginseng are shown in Table 9 below.

Table 9 group Negative Control Sample processing group Positive control density 0 (/) 50 (/) 100 (/) 200 (/) 400 (/) Red Ginseng 400 (/)

ICR-based mice (Woongseong, 8 weeks old) were purchased from Orient Bio Co., Ltd. (Gapyeong, Gyeonggi-do), and were used for 1 week after the experiment. During the experiment, water and feed were freely ingested. The room temperature was 22 ± 2 ℃, the humidity was 55 ± 5%, and the contrast was automatically adjusted in 12 hour cycles.

Specifically, the samples were orally administered to the negative control group administered sterile water, the sample treatment group administered KH-204 (30% ethanol extract), and the red ginseng administered group to the positive control group once daily for 8 days. After the adaptation period, the male mice were divided into the control group and the sample administration group, and the sample administration group was divided into 50, 100, 200, and 400 mg / kg concentration groups, and 6 mice were used in each group. Oral administration was performed once a day for 8 days. After 24 hours of the last oral administration, the mice were weighed, and then sacrificed to separate left testes, left testes, and left testes. The mL was dispensed into 24 wells and 10 minutes later sperm count was measured on a microscope at 200 magnification.

<5-1> mouse weight

As a result, the final weight of the mouse is shown in Table 10 and FIG.

Table 10 Control 50 (mg / kg) 100 (mg / kg) 200 (mg / kg) 400 (mg / kg) Red Ginseng 400 (mg / kg) Weight (g) 37.01 ± 1.63 36.53 ± 1.09 36.73 ± 1.13 34.61 ± 1.85 29.29 ± 1.58 36.09 ± 1.08

As a result, when KH-204 (ethanol extract) was administered at 200 (mg / kg) and 400 (mg / kg), the weight of mice was decreased to 34.61 g and 29.29 g, respectively. In particular, the group that received the 400 (mg / kg) dose decreased about 7 g and had a weight loss of about 20%.

<5-2> weight of organ (vesicle)

As a result, the weight of the seminal vesicles of the mouse is shown in Table 11 and FIG.

Table 11 Control 50 (mg / kg) 100 (mg / kg) 200 (mg / kg) 400 (mg / kg) Red Ginseng 400 (mg / kg) Seminal vesicles (g) 0.075 ± 0.02 0.078 ± 0.01 0.084 ± 0.02 0.089 ± 0.02 0.091 ± 0.01 0.094 ± 0.01

As a result, the weight of the seminal vesicles of the mice tended to increase as the KH-204 (ethanol extract) concentration was higher.

<5-3> organ weight

As a result, the testis weight of the mouse is shown in Table 12 and FIG.

Table 12 Control 50 (mg / kg) 100 (mg / kg) 200 (mg / kg) 400 (mg / kg) Red Ginseng 400 (mg / kg) Testis (g) 0.11 ± 0.01 0.11 ± 0.02 0.11 ± 0.01 0.13 ± 0.01 0.12 ± 0.01 0.13 ± 0.00

As a result, the testes of mice had the highest weight in the group administered KH-204 (ethanol extract) 200 (mg / kg), and the same results as the group administered red ginseng 400 (mg / kg).

<5-4> long-term (negative) weight

As a result, the uterine weight of the mouse is shown in Table 13 and FIG.

Table 13 Control (Mg / kg) 0 (mg / kg) 0 (mg / kg) 0 (mg / kg) Hemp 400 (mg / kg) Negation (g) 0.040 ± 0.000 0.041 ± 0.002 0.042 ± 0.004 0.041 ± 0.002 0.037 ± 0.002 0.042 ± 0.004

As a result, there was little difference between the groups of mice, but in the group to which KH-204 (ethanol extraction) 400 (mg / kg) was administered, the weight of the cells was reduced.

<5-5> sperm number

As a result, the number of sperm of the mouse is shown in Table 14 and FIG.

Table 14 Control (Mg / kg) 0 (mg / kg) 0 (mg / kg) 0 (mg / kg) Hemp 400 (mg / kg) Sperm (Marie) 94.80 ± 7.19 106.33 ± 8.59 118.60 ± 18.66 123.00 ± 14.52 127.00 ± 16.31 103.80 ± 10.43

As a result, the sperm count of mice tended to increase as the concentration of KH-204 (ethanol extract) increased, and all KH-204 (ethanol extract) were administered to the red ginseng 400 (mg / kg) group as a positive control. Many sperm counts were identified in the group.

<5-6> sperm movement

As a result, the sperm movement of the mouse is shown in Table 15 and FIGS. 20 to 26.

Table 15 Control (Mg / kg) 0 (mg / kg) 0 (mg / kg) 0 (mg / kg) Hemp 400 (mg / kg) Sperm Motility (%) 53.73 ± 16.69 68.46 ± 3.52 70.88 ± 6.12 74.09 ± 8.20 91.12 ± 2.56 66.50 ± 8.56

As a result, the sperm motility of the mice tended to increase as the concentration of KH-204 (ethanol extract) increased, and all the KH-204 (ethanol extract) were administered to the red ginseng 400 (mg / kg) group as a positive control. High sperm motility was identified in the group.

<Other Preparation Example 1> Preparation of a male infertility treatment containing KH-204 ethanol extract as an active ingredient

The present inventors confirmed that the KH-204 ethanol extract of the male infertility treatment effect is excellent through the above embodiment to prepare a male infertility treatment containing KH-204 ethanol extract as an active ingredient as follows. In addition, the preparation of the following therapeutic agent can be used for the application of the health food as well as the therapeutic agent.

<1-1> Soft gelatin capsules containing KH-204 ethanol extract

KH-204 Ethanol Extract 20%, Vitamin C 4.5%, Vitamin D ₃ 0.001%, Manganese Sulfate 0.1%, Beeswax 15%, Palm Oil 30%, Safflower Seed Oil 30.399%

 <1-2> Preparation of Intravenous Formulation Containing KH-204 Ethanol Extract

KH-204 Ethanol Extract 2%, Mannitol 3%, Saline 95%

<1-3> Tablet containing KH-204 ethanol extract

KH-204 Ethanol Extract 35%, Vitamin C 15%, Vitamin D ₃ 0.001%, Manganese Sulfate 0.1%, Crystalline Cellulose 25.0%, Lactose 21.999%, Magnesium Stearate 2.9%

<Other Preparation Example 2> Preparation of functional food containing KH-204 ethanol extract as an active ingredient

The present inventors have confirmed that KH-204 ethanol extract is excellent in the male infertility treatment activity through the above embodiment to prepare a functional food containing it as an active ingredient as follows.

<2-1> Preparation of Drink

Honey 522 mg, thioctoamide amide 5 mg, nicotinic acid amide 10 mg, riboflavin sodium hydrochloride 3 mg, pyridoxine hydrochloride 2 mg, inositol 30 mg, orteic acid 50 mg, KH-204 ethanol extract 1.2 mg, water 200 ml, the composition And the contents were prepared using conventional methods.

<2-2> production of chewing gum

Chewing gum was prepared using conventional methods using 40% gum base, 56.36% sugar, 0.64% KH-204 ethanol extract, 1% fruit flavor, 2% water, and the composition and content.

<2-3> Preparation of Candy

Sugar 60%, starch syrup 39.26%, KH-204 ethanol extract 0.64%, orange flavor 0.1% The candy was prepared by the conventional method using the composition and content.

<2-4> Preparation of Biscuits

Force primary 88 kg, gravity primary 76.4 kg, white sugar 16.5 kg, salt 2.5 kg, glucose 2.7 kg, palm shortening 40.5 kg, ammo 5.3 kg, medium sodium 0.6 kg, sodium bisulfite 0.55 kg, rice flour 5.0 kg, vitamin B1 0.003 kg , Vitamin B2 0.003 kg, milk flavor 0.16 kg, water 71.1 kg, whole milk powder 4 kg, substitute milk powder 1 kg, calcium phosphate 0.1 kg, spray salt 1 kg, spray oil 25 kg, KH-204 ethanol extract 0.5 kg, Biscuits were prepared using conventional methods in terms of composition and content.

<2-5> Preparation of Ice Cream

Milk fat 10.0%, nonfat milk solids 10.4%, sugar 12.0%, starch syrup 3.0%, emulsifying stabilizer (span, span) 0.5%, fragrance (strawberry) 0.15%, water 63.31%, KH-204 ethanol extract 0.64%, the composition And ice cream was prepared using conventional methods.

<2-6> manufacturing of chocolate

Sugar 34.36%, cocoa butter 34%, cocoa mass 15%, cocoa powder 15%, lecithin 0.5%, vanilla flavor 0.5%, KH-204 ethanol extract 0.64%, the composition and content of the chocolate using conventional methods Prepared.

As mentioned above, although preferred embodiment of this invention was described in detail, this invention is not limited to the said embodiment, A various deformation | transformation by a person of ordinary skill in the art within the scope of the technical idea of this invention is carried out. This is possible.

Claims (7)

A pharmaceutical composition for improving male sexual function and infertility, comprising at least one extract selected from Cornus extract, Goji berry extract, Bokbunja extract, Tosa extract, Schizandra extract and Cha-cha extract. The pharmaceutical composition of claim 1, wherein the extract further comprises a pharmaceutically acceptable carrier. According to claim 1, wherein the composition is a pharmaceutical composition, characterized in that consisting of Cornus extract, Goji extract, Bokbunja extract, Tosa extract and Schizandra extract. The pharmaceutical composition according to claim 3, wherein the composition is 50 to 150 parts by weight of goji berry, 20 to 100 parts by weight of bokbunja, 20 to 100 parts by weight of earthenware and 10 to 50 parts by weight of Schisandra chinensis. . According to claim 1, wherein the composition is male sexual function improvement and infertility pharmaceutical composition comprising the chajeon extract as an active ingredient. The pharmaceutical composition according to any one of claims 1 to 5, wherein the extract is water or ethanol extract. Food composition for improving male sexual function and infertility comprising at least one extract selected from Cornus extract, Goji berry extract, Bokbunja extract, Tosa extract, Schisandra chinensis extract and Cha-cha extract as food ingredients .

Images