WO2018105998A1 - Composition for preventing and treating male infertility, containing flavonoid derivative compound as active ingredient, and use thereof - Google Patents

Abstract

The present invention relates to a composition for preventing and treating male infertility, containing a flavonoid compound as an active ingredient. Through an in vitro sperm movement promoting effect evaluation method experiment for investigating the effect of the flavonoid derivative compound of the present invention on changes in the sperm count and mobility of human and porcine semen, it was confirmed that samples of the present invention show increased sperm mobility in the in vitro experiment with the human and porcine semen. Accordingly, the composition can be useful for a pharmaceutical composition, a health functional food and a health supplemental food for preventing and treating male infertility.

Description

[Revision according to Rule 26.15.12.2017] Composition for the prevention and treatment of male infertility containing 하는 flavonoid derivative compound as an active ingredient and use thereof

The present invention relates to a composition for the prevention and treatment of male infertility containing the flavonoid derivative compound as an active ingredient and its use.

The present invention relates to a composition for the prevention and treatment of male infertility containing the flavonoid derivative compound as an active ingredient and its use.

Varicocele, 19%-41% of primary infertility males and secondary male infertility males, characterized by pampiniform plexus expansion of the spermatic cord Appears in more than 80% of patients and is the leading cause of male infertility (Agarwal A, Deepinder F, Cocuzza M, Agarwal R, Short RA, Sabanegh E, et al.(2007) Efficacy of varicocelectomy in improving semen parameters: new meta-analytical approach.Urology 70, 532-538; Agarwal A, Sharma RK, Desai NR, Prabakaran S, Tavares A & Sabanegh E. (2009) Role of Oxidative Stress in Pathogenesis of Varicocele and Infertility.Urology 73, 461-469.). Surgical therapies for treating male infertility with the most commonly performed varicose veins include centuries of nonmicrosurgical approaches, laparoscopic or microsurgical varicose vein ligation in the male infertility area. Varicocelectomy has been performed (Mehta A & Goldstein M. (2012) Microsurgical varicocelectomy: a review.Asian J Androl 15, 56-60.). Despite a dramatic improvement in semen parameters in 50-80% of infertile men who underwent varicocelectomy (pregnancy rates: pregnancy rates, 31-74%) (Cheng D, Zheng XM, Li SW, Yang ZW & Hu LQ. (2006) Effects of epidermal growth factor on sperm content and motility of rats with surgically induced varicoceles.Asian J Androl 8, 713-717. Ding H, Tian JQ, Du W, Zhang LY, Wang HZ & Wang ZP. (2012) Open non-microsurgical, laparoscopic or open microsurgical varicocelectomy for male infertility: a meta-analysis of randomized controlled trials.BJU Int 110, 1536-1542; Fisch H & Hyun G. (2012) Varicocele repair for low testosterone.Curr Opin Urol 22, 495-498; Fretz PC & Sandlow JI. (2002) Varicocele: current concepts in pathophysiology, diagnosis, and treatment.Urol Clin North am 29, 921-937; Mehta A & Goldstein M. (2013) Microsurgical varicocelectomy: a review.Asian J Androl 15, 56-60; Tanriverdi O, Miroglu C, Horasanli K, Altay B, Caliskan KC & Gumus E. (2006) Testicular blood flow measurements and mean resistive index values after microsurgical and high ligation varicocelectomy.Urology 67, 1262-1265.), And other reports have argued for the majority of failures in fertile postoperation and the negation of the benefits of this procedure (Evers JL & Collins JA. (2003) Assessment of efficacy of varicocele repair for male subfertility: a systematic review.The lancet 361, 1849-1852; Ficarra V, Cerruto MA, Liguori G, Mazzoni G, Minucci S, Tracia A, et al. (2006) Treatment of varicocele in subfertile men: the Cochrane reviewa contrary opinion.Eur Urol 49, 258-263.). The exact mechanism of varicocele-induced infertility is not yet clear. Varicose veins-related, including blood flow alterations in the testis, scrotal temperature elevations, abnormal hormone values, and excessive reactive oxygen species (ROS) Several hypothesized mechanisms have been suggested for the study of variable testicular dysfunction (Agarwal A, Sharma RK, Desai NR, Prabakaran S, Tavares A & Sabanegh E. (2009) Role of Oxidative Stress in Pathogenesis of Varicocele and Infertility.Urology 73, 461-469; Fretz PC & Sandlow JI. (2002) Varicocele: current concepts in pathophysiology, diagnosis, and treatment.Urol Clin North am 29, 921-937.).

ROS is a byproduct of oxygen metabolism and energy production that acts as a regulator of essential physiological intracellular processes. Small amounts of ROS play an important role in regulating sperm function in the male reproductive tract. Under abnormal conditions, oxidative stress is followed when an increase in ROS levels or a decrease in antioxidant capacity occurs. Specifically, oxidative stress results in alteration of protein expression levels in sperm, eventually leading to molecular and genetic defects (Sharma R, Agarwal A, Mohanty G, Hamada AJ, Gopalan B, Willard B , et al. (2013) Proteomic analysis of human spermatozoa proteins with oxidative stress.Reprod Biol Endocrinol 11, 48.).

Pregnancy in normal couples ranges from 20-25% in the first month after normal sex, 75% in 6 months, and 85-90% in 1 year. Infertility is considered to be infertility in one year despite the normal marital relationship. Infertility patients account for 13-20% of married couples, and there are an estimated 1.4 million pairs of infertile couples in Korea. . Among them, infertility causes 35-50% of men, and 30% of them are caused by decreased sperm motility, and male infertility is an important disease occupying 4% of urological outpatient male patients.

Recent advances in reproductive biology and assisted reproductive technology have made progress in assisted reproductive techniques (ART), including in vitro fertilization (IVF) and micromanipulation, but their success rate is 20-40%, excessively expensive, very difficult for women, and complications. There is a problem that can occur. In vitro fertilization can lead to genetic problems, aiming at natural pregnancy as much as possible.

To date, there have been many developments in the treatment of infertility, but male infertility drugs that improve sperm motility are absent, and the development of drugs that can improve pregnancy is very low. In addition, despite medical and surgical treatment, L-carnitine is currently used as a drug to improve semen quality in men with poor semen quality. (Mark sigman, Stacy Glass, Janice Campagnone, John L. Reyor, Carnitine for the treatment of idiopathic asthenospermia: a randomized, double-blind, placebo-controlled trial, Fertility and Sterility Vol 85, No 5, 2006)

The inventors of the present invention provides a pharmaceutical composition for the prevention and treatment of male infertility containing a herbal extract consisting of pageokcheon, earth and sand and onion (Korean Patent Registration No. 10-1481569 and PCT / WO2015 / 174636 A1); Veterinary composition for the prevention and treatment of infertility of mammals, fish or poultry containing a herbal extract consisting of pageukcheon, earth and sand and onion (Korean Patent Registration No. 10-1544783); There was a patent application for confirming the treatment effect on the male infertility of the earth and sand extract, in particular, the crude earth and sand extract extracted with ethanol (Korean Patent No. 10-2014-0102848).

However, none of this document discloses or teaches the therapeutic effect of male infertility of flavonoid derivative compounds isolated from herbal extracts consisting of pagokcheon, earth and sand and / or onion.

The present inventors, as part promote in vitro to investigate the effect of targeting a flavonoid derivative compound on sperm count and motility changes in the human and pig sperm (In vitro) sperm motility in follow-up studies to develop an effective treatment for male infertility It was confirmed that the samples of the present invention through efficacy evaluation experiment to increase the sperm motility in human and porcine sperm and completed the present invention.

An object of the present invention is to develop a new fertility overcoming technology to improve fertilization rate by improving sperm movement, and ultimately through the present invention to improve sperm movement and recovery of fertility rate.

Accordingly, the present invention provides quercetin 4'-glucoside (Spiraeoside), kaempferol 3-glucoside; Quercetin-3'-galactoside (Hyperoside) and 3-methoxyquercetin (3-methoxyquercetin; isorhamnetin), flavonoid compounds, isomers thereof, pharmaceutically acceptable thereof It provides a pharmaceutical composition for preventing and treating male infertility containing a possible salt as an active ingredient.

In addition, the present invention is Quercetin (Quercetin) 4'- glucoside (glucoside) (Spiraeoside: Spiraeoside, kaempferol) 3-glucoside (glucoside); Quercetin-3'-galactoside (Hyperoside) and 3-methoxyquercetin (3-methoxyquercetin; isorhamnetin), flavonoid compounds selected from the group consisting of isomers, pharmaceutically acceptable It provides a health functional food for preventing and improving male infertility containing salt as an active ingredient.

Male infertility as defined herein includes infertility caused by side effects of anticancer drugs such as cisplatin, vinblastin, bleomycin, spermatosis (if sperm is not present in sperm), spermosis (number of sperm) All sterility related to sperm, such as less than 20 million / ml), spermosis (less than 40% of sperm motility), malformed sperm (typically less than 40%).

The compounds of the present invention can be prepared with pharmaceutically acceptable salts and solvates according to methods conventional in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equal molar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, and the like may be used as the inorganic acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, and trifluoroacetic acid may be used as the organic acid. , Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid (gluconic acid), galacturonic acid, glutamic acid, glutaric acid (glutaric acid), glucuronic acid (glucuronic acid), aspartic acid, ascorbic acid, carbonic acid, vanic acid and hydroiodic acid may be used.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the invention include salts of acidic or basic groups which may be present in the compounds of the invention, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts. It can be prepared through.

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

The compounds of the present invention are commercially available or can be isolated and obtained by the following preparation methods.

For example, the present invention will be described in detail below.

The compounds of the present invention can be prepared as follows. After washing and rinsing the herbal extract consisting of dried rupture cloth, earth and sand and / or onion, water, including purified water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol and butanol, or a solvent selected from a mixed solvent thereof, preferably water And an ultrasonic extraction method for 30 minutes to 48 hours, preferably 1 hour to 12 hours at a temperature of 30 ° C. to 150 ° C., preferably 50 ° C. to 100 ° C., after mixing the methanol mixed solvent, more preferably methanol several times. The crude extract of the present invention can be obtained by filtration, concentration under reduced pressure, and drying of the extract obtained by performing hot water extraction, room temperature extraction or reflux extraction, preferably ultrasonic extraction, about 1 to 20 times, preferably 2 to 10 times. have.

In addition, the polar or non-polar solvent soluble extract of the present invention is about 0.0005 to 0.005 times the weight of the crude extract, preferably 10 to 90% ethanol crude extract, preferably 0.05 to 0.5 times the volume (v / w% Non-polar solvent soluble extract fractions which were added to non-polar solvents such as n-hexane, methylene chloride, ethyl acetate, etc. by performing normal fractionation process with ethyl acetate and butanol after addition of water; And polar solvent soluble extract fractions soluble in polar solvents such as butanol, water and the like.

Flash the non-polar solvent soluble extract fractions, preferably ethyl acetate soluble fractions, which are used in non-polar solvents such as n-hexane, methylene chloride, ethyl acetate, and the like, by increasing the polarity of the mixed solvent of hexane and methylene chloride. Purification using chromatography, such as column chromatography, RP C18 column chromatography or silica gel open column chromatography, and Diaion HP-20 column chromatography, may be performed several times selectively to obtain and purify the compounds of the present invention, respectively. Can be.

The inventors of the present invention, through the in vitro sperm motility efficacy test method to determine the effect on the sperm count and motility changes of human and porcine sperm in the flavonoid derivative compounds, By confirming the increase in sperm motility, the composition was found to be useful as a pharmaceutical composition, health functional food and health supplements for the prevention and treatment of male infertility.

Accordingly, the present invention provides a pharmaceutical composition and health functional food for the prevention and treatment of male infertility containing the above method and the compound obtained by the manufacturing process as an active ingredient.

The composition of the present invention comprises 0.01 to 99% by weight of the compound relative to the total weight of the composition.

However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

Compositions comprising a compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising the compounds according to the invention are, but are not limited to, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterilization, respectively, according to conventional methods. Carriers, excipients, and diluents that may be formulated in the form of injectable solutions may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one or more excipients in the compound, at least cotton, starch, calcium carbonate, sucrose. Or lactose, gelatin or the like is mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound is preferably administered at 0.01 mg / kg to 10 g / kg per day, preferably at 1 mg / kg to 1 g / kg. Administration may be administered once a day or may be divided several times. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be anticipated, for example, by oral and rectal or intravenous methods.

The present invention also provides a sperm motility enhancer containing the compound as an active ingredient, the sperm motility enhancer is to improve the fertilization rate in vitro fertilization. The present invention also provides female vaginal and vaginal cleansing agents and vaginal intercalating agents for treating male infertility of sperm motility enhancement comprising the compound. The present invention also provides an in vitro fertilization (IVF) additive for in vitro fertilization comprising the compound to improve fertilization rate in vitro fertilization.

In addition, the present invention is Quercetin (Quercetin) 4'- glucoside (glucoside) (Spiraeoside: Spiraeoside, kaempferol) 3-glucoside (glucoside); A flavonoid compound selected from the group consisting of Quercetin-3'-galactoside (Hyperside) and 3-methoxyquercetin (isorhamnetin), a pharmaceutically acceptable salt thereof Provided is a treatment method for treating male infertility patients, comprising administering to male infertility patients.

The present invention also provides a quercetin 4'-glucoside (Spiraeoside), kaempferol 3-glucoside (glucoside) for the manufacture of a medicament for treating male infertility disease; Use of flavonoid compounds selected from the group consisting of Quercetin-3'-galactoside (Hyperside) and 3-methoxyquercetin (, isorhamnetin), pharmaceutically acceptable salts thereof To provide.

In addition, the present invention is Quercetin (Quercetin) 4'- glucoside (glucoside) (Spiraeoside: Spiraeoside, kaempferol) 3-glucoside (glucoside); A flavonoid compound selected from the group consisting of Quercetin-3'-galactoside (Hyperside) and 3-methoxyquercetin (isorhamnetin), a pharmaceutically acceptable salt thereof To provide a health functional food for the prevention and improvement of male infertility containing as an active ingredient.

As defined herein, "health functional food" refers to physical or mental functionality by adding a compound of the present invention to a conventional specimen for preventing or ameliorating a target disease in a human body or a mammal according to Korean Health Functional Food Act No. 6723. It includes a functional food to improve the functionality such as.

The dietary supplement for the prevention and improvement of male infertility of the present invention comprises the compound in an amount of 0.01 to 95%, preferably 1 to 80% by weight, based on the total weight of the composition.

Moreover, the composition of the present invention can be used as a major component or additive and adjuvant in the manufacture of various dietary supplements and supplements for the purpose of preventing or treating male infertility or enhancing testicular function.

In addition, for the purpose of preventing and improving male infertility, it is prepared as a pharmaceutical dosage form such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, or health functional foods in the form of tea bags, leach teas, and health drinks. And processing is possible.

In addition, the present invention is Quercetin (Quercetin) 4'- glucoside (glucoside) (Spiraeoside: Spiraeoside, kaempferol) 3-glucoside (glucoside); A flavonoid compound selected from the group consisting of Quercetin-3'-galactoside (Hyperside) and 3-methoxyquercetin (isorhamnetin), a pharmaceutically acceptable salt thereof It provides a health supplement for the prevention and improvement of male infertility containing the main ingredient.

The "main ingredient" is about 30 to 99 (w / w%), preferably 50 to 99 (w / w%), more preferably 70 to 99 (compound) of the compound of the present invention based on the total weight of the dietary supplement. w / w%)).

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The proportion of the natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

When the compound according to the present invention is used as a food additive, the compound may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, etc., includes all of the health food in the conventional sense.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the compounds of the present invention may be added to food or beverages for the purpose of preventing male infertility. At this time, the amount of the compound in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition is added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be.

Samples of the present invention were tested in human and swine sperm through an in vitro sperm motility test to evaluate the effects on the sperm count and motility changes of human and swine sperm in the flavonoid compounds of the present invention. By increasing the mobility, it was confirmed that the composition is useful as a pharmaceutical composition, health functional food and health supplement food for the prevention and treatment of male infertility.

Figure 1 shows the effect on sperm motility in human sperm by the compound of the present invention (in the figure: KOG: kaempferol 3-O-glucoside, QG: quercetin-4-glucoside (spiraeoside) ,, CIN4; Meaning CINTM1);

Figure 2 shows the effect of the compound of the present invention on sperm motility in porcine umbilical varicose veins experimental model (HYP: Hyperoside, IG = Isorhamnetin-3-O-glucoside, KOG: Kaempherol-3-O-glucoside, QC: quercetin, QG: means quercetin-4-glucoside (spiraeoside).

Those skilled in the art will appreciate that various modifications and variations can be used in the compositions, uses and preparations of the invention without departing from the spirit or scope of the invention.

Although described in more detail by the following examples of the present invention, it should be understood that they are not intended to be limited to the following examples in any way of the present invention.

.

However, the following Examples and Experimental Examples are only illustrative in a range not limiting the scope of the present invention, the contents of the present invention is not limited by the following Examples and Experimental Examples.

Reference Example 1. Preparation Example of Single Herb Extract

(1) Preparation of Pageokcheon Extract (CINThera-1)

Dried Pajukcheon (Dukhyeondang, South Korea: Morinda officinalis , Deok Hyeon Dang, Sounth Korea) 6 L of 95% ethanol was added to 3,000 g, ultrasonically extracted three times for 90 minutes, filtered and concentrated under reduced pressure. The concentrated filtrate was lyophilized with a freeze dryer (ScanVac, Labogene) under reduced pressure to obtain 139.4 g (4.6%) of extract powder and stored at 4 ° C.

Before use in the experiment, the dried extract was dissolved in physiological saline and mixed vigorously for 2 minutes at room temperature.

(2) Preparation of Tosa Extract (CINThera-2)

Dried earthenware (baekjangsaeng, Korea, Cuscuta) chinensis , Baek Jang Sang, Sounth Korea) 5L of 95% ethanol was added to 5.2Kg, and ultrasonically extracted twice for 90 minutes, filtered and concentrated under reduced pressure. The concentrated filtrate was lyophilized with a freeze dryer (ScanVac, Labogene) under reduced pressure to obtain 264.3 g (4.36%) of extract powder and stored at 4 ° C.

Before use in the experiment, the dried extract was dissolved in physiological saline and mixed vigorously for 2 minutes at room temperature.

(3) Preparation of Onion Extract (CINThera-3)

Onion (Changnyeong, South Korea: Allium cepa , Changyeong, Sounth Korea) was well washed and dried, and then, three layers including the outer shell were put in 5 g of 70% ethanol in 1,000 g, extracted at 70 ° C. for 2 hours, filtered and concentrated under reduced pressure. The concentrated filtrate was lyophilized with a freeze dryer (ScanVac, Labogene) under reduced pressure to obtain 13.8 g (1.38%) of extract powder and stored at 4 ° C.

Before use in the experiment, the dried extract was dissolved in physiological saline and mixed vigorously for 2 minutes at room temperature.

Reference Example 2. Preparation Example of Mixed Herbal Extract (CINThera-1: CINThera-2: CINThera-3 = 10: 1: 10)

Weighing the dried paepukcheon extract (CINThera-1) obtained in the Reference Example 1, 1mg of Tosa seed extract (CINThera-2) and 10mg of onion extract (CINThera-3), respectively, by weighing a mixer (Vortex genie-2, scientific industries, USA ) were mixed with a catalyst to this takes the mixed herbal extract 21mg experimental sample (hereinafter, referred to as CINTM1).

Example 1. Isolation of Active Compounds

1-1. Preparation of Crude Extracts

Tosa extract: In a manner similar to that disclosed in Korean Patent Publication No. 1014815690000, 5 L of 95% ethanol was added to 20.4 Kg of dried Tosa (baekjangsaeng), ultrasonically extracted twice for 90 minutes, filtered and concentrated under reduced pressure. The concentrated filtrate was lyophilized with a freeze dryer (ScanVac, Labogene) under reduced pressure to obtain 264.3 g (1.32%) of Tosa ethanol extract powder (hereinafter referred to as CJC) and stored at 4 ° C. Before use in the experiment, the dried extract was dissolved in physiological saline and mixed vigorously for 2 minutes at room temperature.

Onion extract: After washing and drying onion (Changnyeong) well, three layers including the outer skin was added to 217g of 4L ethanol and extracted for 3 hours at 70 ℃, filtered and concentrated under reduced pressure. The concentrated filtrate was lyophilized with a freeze dryer (ScanVac, Labogene) under reduced pressure to obtain 37.79 g (17.41%) of extract powder and stored at 4 ° C.

1-2. Example of preparation

102 g of the dried earthworm ethanol extract obtained in Reference Example 1 was suspended in 2000 ml of distilled water, and then n-hexane, ethylacetate and n-butanol (BuOH) were used in accordance with a conventional fractionation method in the art. Fractions were dried under reduced pressure and lyophilized to obtain 2.2 g of hexane soluble fraction, 7.76 g of ethyl acetate soluble fraction, 28.4 g of butanol soluble fraction and 60.5 g of water soluble fraction (hereinafter, CJHE ", CJEA", and CJBU ", respectively). And "CJ-WA".

For identification of the active substance, in the following experiment, the compound was isolated and purified from the ethyl acetate fraction of the most active fraction, and the content of kaempferol 3-glucoside in 1 g of crude extract was determined by HPLC-. Agilent 1290 HPLC (DAD) system and Shiseido UG18 (5μm, 4.6 x 250mm ID) column chromatography were performed under the following analytical conditions to quantitatively analyze the content of the active ingredient kaempferol 3-glucoside; As a result, it contained 8.49 mg.

In addition, the onion ethanol extract was eluted with water and acetonitrile gradient elution using a Gilson HPLC system (Gilson 321 pump, 157 UV / Vis detector) (0 min, 15% actonitrile, 35 min 68% acetonitrile, movement speed: 10 ml / min) , Rt = 22 minutes, 32 minutes) was used to try to separate the compound was purified by separation.

reference UV Detector 254nm column Shiseido UG18 (4.6 x 250 mm, 5um) mobile phase (A) acetonitrile and (B) water with 0.1% trifluoroactic acid 0 min-10min (100% B) 10 min-40 min (100% -70% B) 40 min 50 min (70% -50% B) flow rate 1 mL / min

One- 3. Compound  Separation

3 g of the crude onion extract obtained in Example 1-2 was re-dissolved in 100 ml of methanol solvent, and then HPLC column chromatography was performed to provide quercetin 4'-glucoside (spira) having the following physical properties. Eoside: Spiraeoside) was isolated and the purity of the isolated compound was measured by HPLC-DAD (Agilent Series 1260, Waldbronn, Germany) and ¹ H NMR, 13C NMR spectra (400 MHz Bruker AVANCE III NMR system, Germany). (> 95%), their physicochemical data were identified by comparison with existing literature.

a. Quercetin 4'-glucoside (Spiraeoside)

¹ H NMR: (400 MHz, DMSO- d _6, Bruker model digital AVANCE III 400, Germany): d 12.43 (1H, s, OH-5), 10.84 (1H, s, OH-3), 9.56 (1H, s, OH-7), 8.98 (1H, s, OH-3 '), 7.70 (1H, d, J = 2.2 Hz, H-2'), 7.62 (1H, d, J = 8.7, 2.2 Hz, H -6 '), 7.24 (1H, d, J = 8.8 Hz, H-5'), 6.45 (1H, d, J = 2.0 Hz, H-8), 6.20 (1H, d, J = 2.0 Hz, H -6), 4.85 (1H, d, J = 7.4 Hz, H-1``), 3.40-3.26 (4H, m, H-2 '', H-3 '', H-4 '', H- 5 ''), 4.11 (1H, br s, H-6``b), 3.73 (1H, d, J = 11.0 Hz, H-6''a).

13C NMR: (100 MHz, DMSO- d _6, Bruker model digital AVANCE III 400, Germany): d 176.09 (C-4), 164.10 (C-7), 160.76 (C-5), 156.27 (C-9) , 146.82 (C-2), 146.38 (C-4 '), 145.94 (C-3'), 136.46 (C-3), 125.15 (C-1 '), 119.57 (C-6'), 115.84 (C -5 '), 115.19 (C-2'), 103.14 (C-10), 101.39 (C-1 ''), 98.30 (C-6), 93.56 (C-8), 77.33 (C-5 '' ), 75.99 (C-3``), 73.32 (C-2 ''), 69.81 (C-4 ''), 60.73 (C-6 '').

Example 2 . Preparation of Active Compounds

Quercetin 4'-glucoside (spiraeoside: Sigma-Aldrich, 91802), kaempferol 3-glucoside (Sigma-Aldrich, catalog No. 68437-5MG), quercetin from the company ) -3'-galactoside (hyperoxide: Hyperoside (Wako, 087-08651) and 3-methoxyquercetin (, 3-methoxyquercetin;, isorhamnetin (Sigma-Aldrich, 17794)) were purchased respectively in distilled water It was dissolved and used as the following Experimental sample.

Experimental Example  One. In vitro  Evaluation of Efficacy of Sperm Exercise

In order to determine the effect of the sperm count and motility changes of human sperm of the samples of the Example was performed as follows using the in vitro sperm motility efficacy evaluation method described in the literature (Martin Blomberg Jensen, Poul J. Bjerrum, Torben E. Jessen, John E. Nielsen, Ulla N.Joensen, et al. (2011) Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa.Human Reproduction, Vol. 26, No. 6 pp. 1307-1317).

1-1. Experiment process

After measuring the number and motility of semen from semen obtained from infertility patient volunteers (Chonbuk National University Urology Department, 20-50 years old, about 85 patients), and put in a 37 ^o C incubator for an hour, After 200 μl of semen was added 20 μl of sample extract to a final concentration of 2 mg / ml, the sperm motility after 3 hours was measured using a microscope (MAKLER counting chamber; MAKLER counting chamber; self-Medical Instruments, Haifa, Israel). Axiovert25, ZEISS, USA).

Controls include 3H buffer Hams F-10 (N6635, Sigma Chemical Co., USA) + 0.4% HAS (A1653, Sigma Chemical Co., USA) + 12 mM HEPES (H4034, Sigma Chemical Co., USA) [3H, pH 7.4], and the samples were also dissolved in 3H buffer, the pH was adjusted to 7.4, and then centrifuged (13,000 rpm, 2 min, 4 ° C.) using a centrifuge (A32010 (1), LABOGENE). Supernatant was obtained and used.

Sperm motility was calculated according to the following equation (1).

[Equation 1]

The percentage of motile spermatozoa = (A: mean number of motile spermatozoa / B: total number of spermatozoa) × 100%

The sperm motility increase rate after 3 hours was calculated according to the following equation (2).

[Equation 2]

The percentage of motile spermatozoa 3 hours after incubation = (sperm mobility 3 hours after incubation on control group-sperm mobility 3 hours after incubation in test group / sperm mobility 3 hours after incubation in control group) × 100%

1-2. Experiment result

As a result of measuring the number of sperm and motility in semen obtained from infertility patients, quercetin 4'-glucoside (spiraeoside), and kaempferol 3-glucoside compound The average increase in sperm motility was found to be 74.7% and 73.0% at a concentration of 10 μM. Table 2

treatment 3H * (pH 7.4) KOG * (10 μ M) QG * (10 μ M) CIN4 * (0.05 mg / ml) n = 1 zero Total count (x10 ⁶ / ml) 79 66 67 66 Motility (%) 55 53 52.1 56.7 3hrs Total count (x10 ⁶ / ml) 60 57 57 58 Motility (%) 40 43.3 42.3 56 n = 2 zero Total count (x10 ⁶ / ml) 71 78 70 73 Motility (%) 50 48 47 50.8 3hrs Total count (x10 ⁶ / ml) 63 50 55 54 Motility (%) 50.7 51 52 57 n = 3 zero Total count (x10 ⁶ / ml) 24 16 22 25 Motility (%) 25 26.3 21.8 24.7 3hrs Total count (x10 ⁶ / ml) 19 12 11 13 Motility (%) 21 25 22 30.5 *: 3H: Hams F-10 (Sigma-Aldrich, N6635,) + 0.4% HAS (Sigma-Aldrich, A1653) + 12 mM HEPES (Sigma-Aldrich, H4034) [pH7.4] KOG * = Kaempherol-3-O -glucoside, QG * = quercetin-4-glucoside (spiraeoside), CIN4 = CINTM1

1-3. Experiment result

As a result of measuring the number of sperm and motility in semen obtained from infertility patients, the rate of sperm motility increased after 3 hours, quercetin 4'-glucoside (spiraeoside), and carmferol ( In the case of kaempferol) 3-glucoside compound, it was confirmed that the mean (n = 3) was 4.36% and 9.30% at 10 μM concentration compared to the control sperm motility after 3 hours. (Figure 1 and Table 3)

treatment 3H * (pH 7.4) CIN3 * (0.005mg / ml) n = 1 zero Total count (x10 ⁶ / ml) 59 56 Motility (%) 54.6 55 3hrs Total count (x10 ⁶ / ml) 56 56 Motility (%) 57.1 58.6 n = 2 zero Total count (x10 ⁶ / ml) 65 61 Motility (%) 68 62.9 3hrs Total count (x10 ⁶ / ml) 59 60 Motility (%) 65.5 68.3 *: 3H: Hams F-10 (Sigma-Aldrich, N6635,) + 0.4% HAS (Sigma-Aldrich, A1653) + 12 mM HEPES (Sigma-Aldrich, H4034) [pH7.4], CIN3 = CINThera-3

Experimental Example 2. In vitro  Evaluation of Efficacy of Sperm Exercise

In order to determine the effect on the sperm count and motility change of the porcine sperm of the samples of the above example, the experiment was performed as follows using the in vitro sperm motility efficacy evaluation method described in the literature (Tardif S, Laforest JP , Cormier N, Bailey JL. (1999) The importance of porcine sperm parameters on fertility in vivo. Theriogenology.Vol. 52, No. 3 pp. 447-59).

2-1. Experiment process

Measure the number and motility of sperm from semen from KPG Korea Pork Gene (Gimje, Jeollabuk-do), and add 20 μl of sample extract to 200 μl of semen to achieve a final concentration of 2 mg / ml. It was measured under a microscope (Axiovert25, ZEISS, USA) using a counting chamber; MAKLER counting chamber; self-Medical Instruments, Haifa, Israel.

Controls include 3H buffer Hams F-10 (N6635, Sigma Chemical Co., USA) + 0.4% HAS (A1653, Sigma Chemical Co., USA) + 12 mM HEPES (H4034, Sigma Chemical Co., USA) [3H, pH 7.4], and the samples were also dissolved in 3H buffer, the pH was adjusted to 7.4, and then centrifuged (13,000 rpm, 2 min, 4 ° C.) using a centrifuge (A32010 (1), LABOGENE). Supernatant was obtained and used.

Sperm motility was calculated according to the following equation (3).

[Equation 3]

The percentage of motile spermatozoa = (A: mean number of motile spermatozoa / B: total number of spermatozoa) × 100%

The sperm motility increase rate after 3 hours was calculated according to the following equation (4).

[Equation 4]

The percentage of motile spermatozoa 3 hours after incubation = (sperm mobility 3 hours after incubation on control group-sperm mobility 3 hours after incubation in test group / sperm mobility 3 hours after incubation in control group) × 100%

2-2. Experiment result

As a result of measuring the number and motility of the sperm in the swine sperm obtained from KPG Korean swine gene, the quercetin 4'-glucoside (spiraeoside), and the kaempferol 3-glucoside compound at 10 μM concentration The average (n = 3) was 74.7%, and 73.0%, which showed the highest increase in sperm motility.

treatment 3H * (pH 7.4) HYP * (10 μ M) IG * (10 μ M) KOG * (10 μ M) QG * (10 μ M) n = 1 zero Total count (x10 ⁶ / ml) 52 53 49 55 51 Motility (%) 57 57.8 57 53.3 56.7 3hrs Total count (x10 ⁶ / ml) 52 44 45 43 49 Motility (%) 62.5 51.7 60 49 68.4 n = 2 zero Total count (x10 ⁶ / ml) 56 49 49 43 50 Motility (%) 53.8 46.8 47.4 46.5 50 3hrs Total count (x10 ⁶ / ml) 50 49 47 49 43 Motility (%) 55 47.4 47 55.5 58.8 n = 3 zero Total count (x10 ⁶ / ml) 44 46 47 46 48 Motility (%) 53 48.5 53 53.8 50 3hrs Total count (x10 ⁶ / ml) 40 43 45 40 45 Motility (%) 47.8 46.2 49 55 53.8 *: 3H: Hams F-10 (Sigma-Aldrich, N6635,) + 0.4% HAS (Sigma-Aldrich, A1653) + 12 mM HEPES (Sigma-Aldrich, H4034), HYP = Hyperoside, IG = Isorhamnetin-3-O- glucoside, KOG * = Kaempherol-3-O-glucoside, QG * = quercetin-4-glucoside (spiraeoside)

Hereinafter, the formulation method and the type of carrier of the present invention will be described, but the present invention is not intended to be limited thereto, but specifically, a representative formulation example will be described below.

Formulation Example 1 Preparation of Powder

Compound (IG): 20 mg

Lactose: 100 mg

Talc: 10 mg

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

Formulation Example 2 Preparation of Tablet

Compound (HYP): 10 mg

Corn starch: 100 mg

Lactose: 100 mg

Magnesium Stearate: 2 mg

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation example  3. Manufacture of capsule

Compound (KOG): 10 mg

Crystalline Cellulose: 3 mg

Lactose: 14.8 mg

Magnesium Stearate: 0.2 mg

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

Formulation Example 4 Preparation of Injection

Compound (DAA): 10 mg

Mannitol: 180 mg

Sterile Distilled Water For Injection: 2974 mg

Na ₂ HPO ₄ , 12H2O: 26 mg

The active ingredient is dissolved in accordance with a conventional method for preparing an injection, and all ingredients are prepared in the above-described ingredient content per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Compound (KOG): 20 mg

Isomerized sugar: 10 g

Mannitol: 5 g

Purified water: appropriate amount

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

Compound (HYP): 1000 mg

Vitamin Mixture: Proper

Vitamin A Acetate: 70 mg

Vitamin E: 1.0 mg

Vitamin B1: 0.13 mg

Vitamin B2: 0.15 mg

Vitamin B6: 0.5 mg

Vitamin B12: 0.2 mg

Vitamin C: 10 mg

Biotin: 10 mg

Nicotinamide: 1.7 mg

Folic acid: 50 mg

Calcium Pantothenate: 0.5 mg

Mineral Mixtures: Proper

Ferrous Sulfate: 1.75 mg

Zinc Oxide: 0.82 mg

Magnesium Carbonate: 25.3 mg

Potassium phosphate monobasic: 15 mg

Dibasic calcium phosphate: 55 mg

Potassium citrate: 90 mg

Calcium Carbonate: 100 mg

Magnesium Chloride: 24.8 mg

Although the composition ratio of the said vitamin and mineral mixture was mixed and comprised in the preferable embodiment the component suitable for a healthy food, you may change arbitrarily the compounding ratio considered that it does not deviate from the mind and range of this invention.

Formulation Example 7 Preparation of Healthy Drink

Compound (KOG): 1000 mg

Citric Acid: 1000 mg

Oligosaccharide: 100 g

Plum concentrate: 2 g

Taurine: 1 g

Add purified water: 900 ml total

After mixing the above components according to a conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated and then stored in the present invention For the preparation of healthy beverage compositions.

As described above, the present invention may be modified in various ways, and such modifications are deemed without departing from the spirit and scope of the present invention, and all such modifications are obvious to those skilled in the art to be included within the scope of the following claims. something to do.

As described above, the samples of the present invention were tested through an in vitro sperm exercise enhancing efficacy test to examine the effects of sperm count and motility changes of human and swine sperm on the compound of the present invention. By confirming that sperm motility increases in vitro experiments of porcine sperm, the composition can be usefully used in pharmaceutical compositions, health functional foods and health supplements for the prevention and treatment of male infertility.

Claims (7)

Quercetin 4'-glucoside (Spiraeoside), kaempferol 3-glucoside, Quercetin-3'-galactoside ( Hyperside: Hyperoside) and 3-methoxyquercetin (3-methoxyquercetin; isorhamnetin) for the prevention and treatment of male infertility, which contains a flavonoid compound, an isomer thereof, and a pharmaceutically acceptable salt thereof as an active ingredient. Pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein the male infertility is infertility due to side effects of an anticancer drug, amorphism, emotion, spermatozoa, or malformed sperm. Quercetin 4'-glucoside (Spiraeoside), kaempferol 3-glucoside, Quercetin-3'-galactoside ( Hyperside: Hyperoside) and 3-methoxyquercetin (3-methoxyquercetin; isorhamnetin) for preventing and ameliorating male infertility, which contains a flavonoid compound, an isomer thereof, and a pharmaceutically acceptable salt thereof as an active ingredient. Health functional food. The health functional food according to claim 3, wherein the health functional food is in the form of powder, granule, tablet, capsule, pill, suspension, emulsion, syrup, tea bag, leach tea, or health beverage. Quercetin 4'-glucoside (Spiraeoside), kaempferol 3-glucoside, Quercetin-3'-galactoside ( Hyperside: Hyperoside) and 3-methoxyquercetin (3-methoxyquercetin; isorhamnetin) for the prevention and improvement of male infertility, which contains a flavonoid compound, an isomer thereof, and a pharmaceutically acceptable salt thereof as an active ingredient. Health supplements. Quercetin 4'-glucoside (Spiraeoside), kaempferol 3-glucoside, Quercetin-3'-galactoside ( Male, comprising administering to a male infertility a flavonoid compound, an isomer thereof, or a pharmaceutically acceptable salt thereof, selected from the group consisting of hyperside: Hyperoside) and 3-methoxyquercetin (, isorhamnetin) Treatment for Infertility Patients. Quercetin 4'-glucoside (Spiraeoside), kaempferol 3-glucoside, Quercetin, for the manufacture of a medicament for treating male infertility disease Use of a flavonoid compound, an isomer thereof, or a pharmaceutically acceptable salt thereof selected from the group consisting of -3'-galactoside (hyperoside: Hyperoside) and 3-methoxyquercetin (, isorhamnetin) .

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