WO2018105999A1 - Composition for preventing and treating male infertility, containing iridoid derivative compound as active ingredient, and use thereof - Google Patents

Composition for preventing and treating male infertility, containing iridoid derivative compound as active ingredient, and use thereof Download PDF

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WO2018105999A1
WO2018105999A1 PCT/KR2017/014180 KR2017014180W WO2018105999A1 WO 2018105999 A1 WO2018105999 A1 WO 2018105999A1 KR 2017014180 W KR2017014180 W KR 2017014180W WO 2018105999 A1 WO2018105999 A1 WO 2018105999A1
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hydrogen atom
male infertility
sperm
present
acid
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PCT/KR2017/014180
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French (fr)
Korean (ko)
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박종관
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전북대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health

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  • the present invention relates to a composition for the prevention and treatment of male infertility and use thereof containing an iridoid derivative compound as an active ingredient.
  • the present invention relates to a composition for the prevention and treatment of male infertility and use thereof containing an iridoid derivative compound as an active ingredient.
  • Varicocele 19%-41% of primary infertility males and secondary male infertility males, characterized by pampiniform plexus expansion of the spermatic cord It appears in more than 80% of patients and is a major factor leading to 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.
  • 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.).
  • 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.
  • 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.
  • the present inventors have found that in vitro to examine the effects of a subsequent study, as part yirido Id (Iridoid) sperm count and motility changes in the human and pig sperm targeting derivative compounds for the development of an effective therapeutic agent for male sterility (In vitro The sperm motility efficacy test method was confirmed that the samples of the present invention to increase the sperm motility in human and pig semen 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.
  • R 2 is a group of compounds which are hydrogen atoms
  • the compounds of the present invention can be prepared with pharmaceutically acceptable salts and solvates according to methods conventional in the art.
  • 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.
  • 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).
  • the compounds of the present invention are commercially available or can be isolated and obtained by the following preparation methods.
  • 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 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.
  • composition of the present invention comprises 0.01 to 99% by weight of the compound relative to the total weight of the composition.
  • 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 include, but are not limited to, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • IVF in vitro fertilization
  • the present invention also provides a treatment method for treating male infertility patients, comprising administering to the infertility patient an iridoid derivative compound represented by the general formula (I), a pharmaceutically acceptable salt thereof.
  • the present invention also provides the use of an iridoid derivative compound represented by formula (I), a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating male infertility disease.
  • the present invention provides a dietary supplement for the prevention and improvement of male infertility containing the iridoid derivative compound represented by the general formula (I), a pharmaceutically acceptable salt thereof as an active ingredient.
  • 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.
  • composition of the present invention can be used as a major component or additive and adjuvant in the manufacture of various dietary supplements and dietary supplements for the purpose of preventing or treating male infertility or enhancing testicular function.
  • 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.
  • the present invention also provides a dietary supplement for the prevention and improvement of male infertility containing the iridoid derivative compound represented by the above general formula (I) as a main component thereof.
  • 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 compound as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.
  • 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.
  • natural flavoring agents such as, tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • 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.
  • the compound according to the present invention when 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.
  • 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.
  • 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.
  • the compounds of the present invention may be added to food or beverages for the purpose of preventing male infertility.
  • 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 through an in vitro sperm motility efficacy test to examine the effects of iridoid derivative compounds of the present invention on the sperm count and motility changes of human and porcine sperm. As sperm motility is increased in the porcine sperm, it was confirmed that the composition is useful as a pharmaceutical composition, health functional food and dietary supplement for the prevention and treatment of male infertility.
  • Figure 1 shows the effect on the effect on sperm motility in the inpatient experimental model of the compound of the present invention (in the figure: MTP: Monotropein, DAA: Deacetyl asperulosidic acid, CIN4; means CINTM1);
  • Figure 2 shows the effect of the compound of the present invention on sperm motility in porcine umbilical varicose veins experimental model. (Means CTR; Control, OP; Operation)
  • the dried extract was dissolved in physiological saline and mixed vigorously for 2 minutes at room temperature.
  • 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.
  • the dried extract was dissolved in physiological saline and mixed vigorously for 2 minutes at room temperature.
  • the dried extract was dissolved in physiological saline and mixed vigorously for 2 minutes at room temperature.
  • CINTM1 10 mg of dried Pagokcheon extract (CINThera-1), 1 mg of Tosa seed extract (CINThera-2) and 10 mg of onion extract (CINThera-3) obtained in Reference Example 1 were weighed, respectively (Vortex genie-2, scientific industries, USA) to obtain a mixed herbal extract 21mg was used as a sample of the following experimental example (hereinafter referred to as CINTM1 ).
  • butanol soluble fraction in the most active fractions was subjected to HPLC-DAD (Agilent 1290 HPLC) system and Shiseido UG18 (5 ⁇ m, 4.6 x 250 mm) column chromatography under the following analytical conditions
  • HPLC-DAD Alignment 1290 HPLC
  • Shiseido UG18 5 ⁇ m, 4.6 x 250 mm
  • HPLC analysis conditions 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
  • the active substance was obtained a standard calibration curve for monotropein and deacetyl asperulosidic acid, and the compound content in the ethanol extract was 1.15% and 1.45% respectively.
  • Monotropein (Monotropein: Sigma-Aldrich, SMB00471) and diacetyl asperulosidic acid (Deacetyl asperulosidic acid: Chemfaces, CFN92358) were purchased from each company, dissolved in distilled water, and used as the following experimental sample.
  • 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.
  • the percentage of motile spermatozoa (mean number of motile spermatozoa / total number of spermatozoa) ⁇ 100%
  • the sperm motility increase rate after 3 hours was calculated according to the following equation (2).
  • the percentage of motile spermatozoa (mean number of motile spermatozoa / total number of spermatozoa) ⁇ 100%
  • the sperm motility increase rate after 3 hours was calculated according to the following 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%
  • the above ingredients are mixed and filled in an airtight cloth to prepare a powder.
  • tablets are prepared by tableting according to a conventional method for preparing tablets.
  • the above ingredients are mixed and filled into gelatin capsules to prepare capsules.
  • 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).
  • 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.
  • 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
  • Nicotinamide 1.7 mg
  • 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.
  • Citric Acid 1000 mg
  • Plum concentrate 2 g
  • 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.
  • the composition can be usefully used in pharmaceutical compositions, health functional foods and health supplements for the prevention and treatment of male infertility.

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Abstract

The present invention relates to a composition for preventing and treating male infertility, containing an iridoid derivative compound as an active ingredient. Through an in vitro sperm movement promoting effect evaluation method experiment for investigating the effect of the iridoid 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

[규칙 제26조에 의한 보정 15.12.2017]　이리도이드 유도체 화합물을 유효성분으로 함유하는 남성 불임증 예방 및 치료용 조성물 및 이의 용도[Revision according to Rule 26.15.12.2017] Composition for preventing and treating male infertility containing iridoid derivative compound as an active ingredient and use thereof
본 발명은 이리도이드 유도체 화합물을 유효성분으로 함유하는 남성 불임증 예방 및 치료용 조성물 및 이의 용도에 관한 것이다.The present invention relates to a composition for the prevention and treatment of male infertility and use thereof containing an iridoid derivative compound as an active ingredient.
본 발명은 이리도이드 유도체 화합물을 유효성분으로 함유하는 남성 불임증 예방 및 치료용 조성물 및 이의 용도에 관한 것이다.The present invention relates to a composition for the prevention and treatment of male infertility and use thereof containing an iridoid derivative compound as an active ingredient.
정삭(spermatic cord)의 망상정맥총 (pampiniform plexus) 확장현상을 특징으로 하는 정계정맥류 (Varicocele), 1차 불임증 (primary infertility) 남성 환자의 19% - 41% 및 2차 남성 불임증 (secondary male infertility) 남성 환자의 80% 이상에서 나타나고, 남성 불임증을 초래하는 주요 인자이다 (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.). 가장 흔하게 수행되는 정계정맥류에 의한 남성 불임증 치료를 위한 수술요법은 남성 불임증 영역에서 수세기 동안 비미세 수술적 (nonmicrosurgical) 접근법, 복강경 (laparoscopic) 또는 미세수술적 (microsurgical) 정계정맥류 결찰술 (varicocelectomy)을 포함한 정계정맥류 결찰술 (varicocelectomy)이 수행되어 왔다 (Mehta A & Goldstein M. (2012) Microsurgical varicocelectomy: a review. Asian J Androl 15, 56-60.). 정계정맥류 결찰술 (varicocelectomy)을 받은 불임증 남성의 환자의 50-80%에서 정자 매개변수(semen parameters) 상의 급격한 개선효과를 나타냄에도 불구하고, (임신율: 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.), 상당부분의 수술후 임신 (fertile postoperation) 실패율과 이 수술법이 가진 장점의 부정을 주장하는 다른 보고들이 있어 왔다 (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.). 정계정맥류 (Varicocele) 유도 불임증의 정확한 기전은 아직 명확하지 않다. 고환내 혈류 변화 (blood flow alterations in the testis), 음낭 온도 상승 (scrotal temperature elevations), 비정상적 남성호르몬수치 (abnormal hormone values), 과도한 반응성 산소종 (excessive reactive oxygen species, ROS)을 포함한 정계정맥류-관련 고환 기능부전 (varicocelerelated testicular dysfunction)을 연구하기 위하여 몇몇 가정된 기전이 제시된 바가 있다 (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.). Varicocele, 19%-41% of primary infertility males and secondary male infertility males, characterized by pampiniform plexus expansion of the spermatic cord It appears in more than 80% of patients and is a major factor leading to 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 its benefits (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. Vascular-related varieties, 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.).
ROS는 필수적 생리학적 세포간 경로 (vital physiological intracellular processes) 조절자로서 작용하는 산소 대사과정 (oxygen metabolism) 및 에너지 생성 (energy production)의 부산물이다. 소량의 ROS는 남성 생식 기관 (male reproductive tract)에서의 정자 기능을 조절하는데 중요한 역할을 수행한다. 비정상적 조건하에서, ROS 수준 증가 또는 항산화능 상의 감소현상이 발생시에 산화적 스트레스 (oxidative stress)가 뒤따른다. 구체적으로, 산화적 스트레스는 정자에서의 단백질 발현 수준의 변형을 초래하고, 결국, 분자 및 유전자 결함을 유발한다 (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.). 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.).
정상부부에서 임신 가능성은 정상적인 성생활 후 첫 달에 20-25%, 6개월에 75%, 1년에 85-90%에 이른다. 정상적인 부부관계에도 불구하고 1년 내에 임신이 안 되는 경우를 불임이라고 볼 때 임상적으로 불임 환자는 기혼부부의 13-20%정도를 차지하고 있으며, 대한민국에서는 140만 쌍의 불임부부가 있을 것으로 추정된다. 그 중 불임 원인이 남성에게 있는 경우가 35-50%를 차지하고, 이 중 30%정도가 정자의 운동성 감소로 인해 발생하며, 남성불임증은 비뇨기과 외래 남성 환자의 4%를 점유하는 중요한 질환이다. 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.
최근 생식 생물학과 보조생식기술의 발달로 in vitro fertilization (IVF), micromanipulation 등 assisted reproductive technique (ART) 기술이 매우 진전하였으나 그 성공률이 20-40% 정도이며, 과도한 비용이 들며, 여성에게 매우 힘들고, 합병증이 발생할 수 있는 문제점이 있다. 체외 수정은 유전자의 문제를 초래할 수 있어 최대한 자연 임신을 목표로 하고 있다. 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.
현재까지 불임의 치료에 많은 발전이 있었지만 정자의 운동성을 향상시키는 남성불임치료제는 부재 상태이며, 임신을 향상시킬 수 있는 약물의 개발도 매우 저조한 편이다. 또한 내과적, 외과적 치료에도 불구하고 원인을 알 수 없이 정액의 질이 나쁜 남성에서 정액의 질을 향상시킬 수 있는 약물로 현재 L-carnitine이 사용되고 있으나 뚜렷한 효능을 보이지 못하고 있다. (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)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)
본원 발명자들은 파극천, 토사자 및 양파로 구성된 생약 추출물을 유효성분으로 함유하는 남성 불임증의 예방 및 치료용 약학 조성물 (한국특허등록 제 10-1481569호 및 PCT/WO2015/174636 A1); 파극천, 토사자 및 양파로 구성된 생약 추출물을 유효성분으로 함유하는 포유동물, 어류 또는 가금류의 불임증의 예방 및 치료용 수의학적 조성물 (한국특허등록 제 10-1544783호); 토사자 추출물, 특히 에탄올로 추출한 토사자 조추출물의 남성 불임증에 대한 치료효과를 확인하여 특허출원한 바가 있다 (한국 공개특허 제 10-2014-0102848호). 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).
그러나, 상기 문헌의 어디에도 파극천, 토사자 및/또는 양파로 구성된 생약 추출물로부터 분리된 이리도이드 (Iridoid) 유도체 화합물의 남성 불임증의 치료효과에 대하여 개시되거나 교시된 바가 없다. However, none of this document discloses or teaches the therapeutic effect of male infertility of Iridoid derivative compounds isolated from herbal extracts consisting of pagokcheon, earth and sand and / or onion.
이에 본 발명자들은 남성 불임증에 효과적인 치료제를 개발하기 위한 후속 연구의 일환으로 이리도이드 (Iridoid) 유도체 화합물을 대상으로 사람과 돼지정액의 정자수와 운동성 변화에 미치는 영향을 알아보기 위한 시험관내 (In vitro) 정자 운동 증진 효능 평가법 실험을 통하여 본 발명의 시료들이 사람과 돼지 정액에서 정자 운동성이 증가함을 확인하고 본 발명을 완성하였다. The present inventors have found that in vitro to examine the effects of a subsequent study, as part yirido Id (Iridoid) sperm count and motility changes in the human and pig sperm targeting derivative compounds for the development of an effective therapeutic agent for male sterility (In vitro The sperm motility efficacy test method was confirmed that the samples of the present invention to increase the sperm motility in human and pig semen and completed the present invention.
본 발명의 과제는 정자운동 증진에 의한 수정률 향상의 새로운 불임 극복기술개발로, 본 발명을 통해 종국적으로 정자운동 증진 및 출산율 회복을 위한 것이다.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.
따라서 본 발명은 하기 일반식 (I)로 표기되는 이리도이드 유도체 화합물, 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 남성 불임증 예방 및 치료용 약학 조성물을 제공한다.Therefore, the present invention provides a pharmaceutical composition for preventing and treating male infertility, which comprises an iridoid derivative compound represented by the following general formula (I), and a pharmaceutically acceptable salt thereof as an active ingredient.
Figure PCTKR2017014180-appb-C000001
Figure PCTKR2017014180-appb-C000001
상기 식에서In the above formula
R1은 수소원자, 히드록시(hydroxy)기 또는 C1 내지 C3 알킬기로 치환된 알콕시기이며;R 1 is a hydrogen atom, a hydroxy group or an alkoxy group substituted with a C1 to C3 alkyl group;
R2은 수소원자, 또는 C1 내지 C3 알킬기이다.R 2 is a hydrogen atom or a C1 to C3 alkyl group.
본 발명의 상기 일반식 화합물은The general formula compound of the present invention
바람직하기로는, 상기 식에서Preferably, in the above formula
R1은 수소원자, 히드록시(hydroxy)기 또는 메톡시기이며;R 1 is a hydrogen atom, a hydroxy group or a methoxy group;
R2은 수소원자인 화합물군; R 2 is a group of compounds which are hydrogen atoms;
보다 바람직하기로는, 상기 식에서More preferably, in the above formula
R1은 수소원자, 또는 히드록시(hydroxy)기 이며;R 1 is a hydrogen atom or a hydroxy group;
R2은 수소원자인 화합물군; R 2 is a group of compounds which are hydrogen atoms;
가장 바람직하기로는, 모노트로페인 (Monotropein) 및 디아세틸아스페룰로시딕 산 (Deacetyl asperulosidic acid)으로 구성된 군으로부터 선택된 화합물이다.Most preferably, the compound is selected from the group consisting of Monotropein and Deacetyl asperulosidic acid.
또한 본 발명은 상기 일반식 (I)로 표기되는 이리도이드 유도체 화합물, 이의 이성체, 약학적으로 허용가능한 염을 유효성분으로 함유하는 남성 불임증 예방 및 개선용 건강기능식품을 제공한다.In another aspect, the present invention provides a dietary supplement for preventing and improving male infertility containing an iridoid derivative compound represented by the general formula (I), an isomer thereof, and a pharmaceutically acceptable salt thereof as an active ingredient.
본원에서 정의되는 남성불임증은 씨스플라틴 (cisplatin), 빈블라스틴 (vinblastin), 블레오마이신 (bleomycin) 등의 항암제 부작용에 의한 불임증, 무정자증 (정액에 정자가 없는 경우), 감정자증 (정자의 수가 2000만마리/ml 이하인 경우), 약정자증(정자의 운동성이 있는 비율이 40%이하), 기형정자 (정상 정자의 형태가 40% 이하) 등의 정자와 관련된 모든 불임증을 포함한다. 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%).
본 발명의 화합물은 당해 기술분야에서 통상적인 방법에 따라 약학적으로 허용 가능한 염 및 용매화물로 제조될 수 있다.The compounds of the present invention can be prepared with pharmaceutically acceptable salts and solvates according to methods conventional in the art.
약학적으로 허용 가능한 염으로는 유리산(free acid)에 의해 형성된 산부가염이 유용하다. 산부가염은 통상의 방법, 예를 들면 화합물을 과량의 산 수용액에 용해시키고, 이 염을 메탄올, 에탄올, 아세톤 또는 아세토니트릴과 같은 수혼화성 유기 용매를 사용하여 침전시켜서 제조한다. 동일한 몰량의 화합물 및 물 중의 산 또는 알코올(예, 글리콜 모노메틸에테르)을 가열하고 이어서 상기 혼합물을 증발시켜서 건조시키거나, 또는 석출된 염을 흡인 여과시킬 수 있다.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.
이 때, 유리산으로는 유기산과 무기산을 사용할 수 있으며, 무기산으로는 염산, 인산, 황산, 질산, 주석산 등을 사용할 수 있고 유기산으로는 메탄술폰산, p-톨루엔술폰산, 아세트산, 트리플루오로아 세트산, 시트르산, 말레인산(maleic acid), 숙신산, 옥살산, 벤조산, 타르타르산, 푸마르산, 만데르산, 프로피온산(propionic acid), 구연산(citric acid), 젖산(lactic acid), 글리콜산(glycollic acid), 글루콘산(gluconic acid), 갈락투론산, 글루탐산, 글루타르산(glutaric acid), 글루쿠론산(glucuronic acid), 아스파르트산, 아스코르빈산, 카본산, 바닐릭산 및 히드로 아이오딕산 등을 사용할 수 있다.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.
또한, 염기를 사용하여 약학적으로 허용 가능한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리토 금속염은, 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리토 금속 수산화물 용액 중에 용해하고, 비 용해 화합물염을 여과한 후 여액을 증발, 건조시켜 얻는다. 이때, 금속염으로서는 특히 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하며, 또한 이에 대응하는 은염은 알칼리 금속 또는 알칼리토 금속염을 적당한 은염(예, 질산은)과 반응시켜 얻는다.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).
본 발명의 화합물의 약학적으로 허용 가능한 염은, 달리 지시되지 않는 한, 본 발명의 화합물에 존재할 수 있는 산성 또는 염기성기의 염을 포함한다. 예를 들면, 약학적으로 허용 가능한 염으로는 히드록시기의 나트륨, 칼슘 및 칼륨염이 포함되며, 아미노기의 기타 약학적으로 허용 가능한 염으로는 하이드로브로마이드, 황산염, 수소 황산염, 인산염, 수소 인산염, 이수소 인산염, 아세테이트, 숙시네이트, 시트레이트, 타르트레이트, 락테이트, 만델레이트, 메탄설포 네이트(메실레이트) 및 p-톨루엔설포네이트(토실레이트) 염이 있으며, 당업계에서 알려진 염의 제조 방법이나 제조과정을 통하여 제조될 수 있다. 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.
이하, 본 발명을 더욱 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명의 화합물들은 시중 구입가능하거나 또는 하기와 같은 제조방법으로 분리 및 수득될 수 있다. The compounds of the present invention are commercially available or can be isolated and obtained by the following preparation methods.
예를 들어, 이하, 본 발명을 상세히 설명한다.For example, the present invention will be described in detail below.
본 발명의 화합물들은 하기와 같이 제조될 수 있다. 건조된 파극천, 토사자 및/또는 양파로 구성된 생약 추출물을 세척 및 세절 후 정제수를 포함한 물, 메탄올, 에탄올, 부탄올 등의 탄소수 1 내지 4의 저급알코올 또는 이들의 혼합용매로부터 선택된 용매, 바람직하게는 물 및 메탄올 혼합용매, 보다 바람직하게는 메탄올을 수회 섞은 다음에 30℃ 내지 150℃, 바람직하게는 50℃ 내지 100℃의 온도에서 30분 내지 48시간, 바람직하게는 1시간 내지 12시간 동안 초음파 추출법, 열수 추출법, 상온 추출법 또는 환류추출법, 바람직하게는 초음파 추출법을 약 1 내지 20회, 바람직하게는 2 내지 10회 반복 수행하여 얻은 추출액을 여과, 감압 농축, 및 건조하여 본 발명의 조추출물을 얻을 수 있다.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.
또한, 본 발명의 극성용매 또는 비극성용매 가용 추출물은 상기에서 얻은 조추출물, 바람직하게는 10 내지 90% 에탄올 조추출물 중량의 약 0.0005 내지 0.005배, 바람직하게는 0.05 내지 0.5배 부피 (v/w%)의 물을 가한 후, 에틸 아세테이트 및 부탄올을 이용한 통상적인 분획과정을 수행하여 n-헥산, 메틸렌 클로라이드, 에틸 아세테이트 등의 비극성 용매에 가용한 비극성 용매 가용 추출 분획물; 및 부탄올, 물 등의 극성용매에 가용한 극성용매 가용 추출 분획물을 수득할 수 있다.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.
상기에서 수득한 n-헥산, 메틸렌 클로라이드, 에틸 아세테이트 등의 비극성 용매에 가용한 비극성 용매 가용 추출 분획물, 바람직하게는 에틸 아세테이트 가용분획물을 헥산 및 메틸렌클로라이드 혼합용매를 극성을 증가시키는 방법을 이용하여 플래쉬 컬럼크로마토그래피, RP C18 컬럼크로마토그래피 또는 실리카겔 오픈 컬럼크로마토그래피, Diaion HP-20 컬럼크로마토그래피등의 크로마토그래피를 이용한 정제방법을 선택적으로 수회 반복 수행하여 본 발명의 화합물들을 각각 정제 및 분리하여 수득할 수 있다. 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.
본 발명자들은 상기 이리도이드(Iridoid) 유도체 화합물을 대상으로 사람과 돼지정액의 정자수와 운동성 변화에 미치는 영향을 알아보기 위한 시험관내 (In vitro) 정자 운동 증진 효능 평가법 실험을 알아보기 위하여 시험관내 (In vitro) 정자 운동 증진 효능 평가법을 통하여 본 발명의 시료들이 사람과 돼지정액에서 정자 운동성이 증가함을 확인함으로서, 상기 조성물을 남성불임증의 예방 및 치료용 약학조성물, 건강기능식품 및 건강보조제로 유용함을 확인하였다.The inventors of the present invention to examine the in vitro sperm motility efficacy test experiment to determine the effect on the sperm count and motility changes of human and porcine sperm in the Iridoid derivative compounds ( In vitro ) In vitro ) sperm motility efficacy test method by confirming that the samples of the present invention to increase the sperm motility in human and pig sperm, the composition is useful as a pharmaceutical composition, health functional food and health supplements for the prevention and treatment of male infertility It was confirmed.
따라서, 본 발명은 상기의 제조방법 및 상기 제조공정으로 얻어진 화합물을 유효성분으로 함유하는 남성 불임증의 예방 및 치료를 위한 약학 조성물 및 건강기능식품을 제공한다. 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.
본 발명의 조성물은, 조성물 총 중량에 대하여 상기 화합물을 0.01 내지 99% 중량으로 포함한다.The composition of the present invention comprises 0.01 to 99% by weight of the compound relative to the total weight of the composition.
그러나 상기와 같은 조성은 반드시 이에 한정되는 것은 아니고, 환자의 상태 및 질환의 종류 및 진행 정도에 따라 변할 수 있다.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.
본 발명의 화합물을 포함하는 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.Compositions comprising a compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
본 발명에 따른 화합물을 포함하는 조성물은, 이에 제한되지는 않으나, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으며, 이에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리이케트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 화합물에 적어도 하나 이상의 부형제 적어도 면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로스 (sucrose) 또는 락토오스 (lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔 (witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.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 include, but are not limited to, 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.
본 발명의 화합물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직한 효과를 위해서, 화합물은 1일 0.01 mg/kg 내지 10 g/kg으로, 바람직하게는 1 mg/kg 내지 1 g/kg으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수 있다. 그러므로 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.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.
본 발명의 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구 및 직장, 또는 정맥 등의 방법을 통하여 투여할 수 있다. 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.
본 발명은 또한 상기 화합물을 유효성분으로 함유하는 정자 운동 증진제를 제공하며, 이러한 정자 운동 증진제는 체외수정시 수정율을 향상시키기 위한 것이다. 본 발명은 또한 상기 화합물을 포함하는 정자 운동 증진의 남성 불임 치료제로서 여성용 질정 및 질세정제, 그리고 질삽입제를 제공한다. 본 발명은 또한 체외수정시 수정율을 향상시키기 위하여 상기 화합물을 포함하는 체외수정용 정자보관용(IVF: in vitro fertilization) 첨가제를 제공한다.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.
또한 본 발명은 상기 일반식 (I)로 표기되는 이리도이드 유도체 화합물, 이의 약학적으로 허용가능한 염을 남성 불임증 환자에게 투여함을 포함하는, 남성 불임증 환자를 치료하기 위한 치료방법을 제공한다. The present invention also provides a treatment method for treating male infertility patients, comprising administering to the infertility patient an iridoid derivative compound represented by the general formula (I), a pharmaceutically acceptable salt thereof.
또한 본 발명은 남성 불임증 질환 치료용 약제를 제조하기 위한 상기 일반식 (I)로 표기되는 이리도이드 유도체 화합물, 이의 약학적으로 허용가능한 염의 용도를 제공한다. The present invention also provides the use of an iridoid derivative compound represented by formula (I), a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating male infertility disease.
또한 본 발명은 상기 일반식 (I)로 표기되는 이리도이드 유도체 화합물, 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 남성 불임증의 예방 및 개선을 위한 건강기능식품을 제공한다.In another aspect, the present invention provides a dietary supplement for the prevention and improvement of male infertility containing the iridoid derivative compound represented by the general formula (I), a pharmaceutically acceptable salt thereof as an active ingredient.
본원에서 정의되는 "건강기능식품"은 대한민국 건강기능식품에 관한 법률 제6727호에 따른 인체 또는 포유동물에서 목적질환을 예방 또는 개선하기 위한 종래 시품에 본 발명의 화합물을 첨가함으로서 물리학적 또는 정신적 기능성과 같은 기능성을 향상시키는 기능성 식품을 포함한다. 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.
본 발명의 남성 불임증의 예방 및 개선을 위한 건강기능식품은, 조성물 총 중량에 대하여 상기 화합물을 0.01 내지 95%, 바람직하게는 1 내지 80% 중량백분율로 포함한다.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.
더욱이, 본 발명의 조성물은 남성 불임증의 예방 또는 치료 또는 고환 기능증진을 목적으로 한 다양한 건강기능식품 및 건강보조식품 제조에 있어서 주요 구성요소 또는 첨가제 및 보조제로서 사용가능하다. 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 dietary supplements for the purpose of preventing or treating male infertility or enhancing testicular function.
또한, 남성 불임증의 예방 및 개선을 위한 목적으로 산제, 과립제, 정제, 캡슐제, 환제, 현탁액, 에멀젼, 시럽 등의 약학 투여형태 또는 티백제, 침출차, 건강 음료 등의 형태인 건강기능식품으로 제조 및 가공이 가능하다.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.
또한, 본 발명은 상기 일반식 (I)로 표기되는 이리도이드 유도체 화합물, 이의 약학적으로 허용가능한 염을 주성분으로 함유하는 남성 불임증의 예방 및 개선용 건강보조식품을 제공한다.The present invention also provides a dietary supplement for the prevention and improvement of male infertility containing the iridoid derivative compound represented by the above general formula (I) as a main component thereof.
상기한 “주성분”은 건강보조식품 전체 조성물 중량 대비 본 발명의 화합물을 약 30 내지 99(w/w%), 바람직하게는 50 내지 99(w/w%), 보다 바람직하게는 70 내지 99(w/w%)을 포함함을 의미한다.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%)).
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 화합물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제 (타우마틴, 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진 등)) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖ 당 일반적으로 약 1~20 g, 바람직하게는 약 5~12 g이다.The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound as an essential ingredient in the indicated ratios, 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.
본 발명에 따른 화합물을 식품첨가물로 사용할 경우, 상기 화합물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다. 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.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다. 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.
또한, 본 발명의 화합물은 남성 불임증 예방 효과를 목적으로 식품 또는 음료에 첨가될 수 있다. 이 때, 식품 또는 음료 중의 상기 화합물의 양은 전체 식품 중량의 0.01 내지 15 중량%로 가할 수 있으며, 건강 음료 조성물은 100 ㎖을 기준으로 0.02 내지 5 g, 바람직하게는 0.3 내지 1 g의 비율로 가할 수 있다.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.
본 발명의 이리도이드(iridoid) 유도체 화합물을 대상으로 사람과 돼지정액의 정자수와 운동성 변화에 미치는 영향을 알아보기 위한 시험관내 (In vitro) 정자 운동 증진 효능 평가법 실험을 통하여 본 발명의 시료들이 사람과 돼지정액에서 정자 운동성이 증가함으로서, 상기 조성물을 남성불임증의 예방 및 치료용 약학조성물, 건강기능식품 및 건강보조식품으로 유용함을 확인하였다.Samples of the present invention were tested through an in vitro sperm motility efficacy test to examine the effects of iridoid derivative compounds of the present invention on the sperm count and motility changes of human and porcine sperm. As sperm motility is increased in the porcine sperm, it was confirmed that the composition is useful as a pharmaceutical composition, health functional food and dietary supplement for the prevention and treatment of male infertility.
도 1은 본 발명의 화합물의 내원 환자 실험 모델에서의 정자 운동성에 미치는 영향을 에 대한 효과를 나타낸 것이며 (상기 도에서 : MTP: Monotropein이며, DAA: Deacetyl asperulosidic acid, CIN4; CINTM1을 의미함);Figure 1 shows the effect on the effect on sperm motility in the inpatient experimental model of the compound of the present invention (in the figure: MTP: Monotropein, DAA: Deacetyl asperulosidic acid, CIN4; means CINTM1);
도 2는 본 발명의 화합물의 돼지 정계 정맥류 실험 모델에서의 정자 운동성에 미치는 영향을 나타내는 것이다. (CTR; Control, OP; Operation을 의미함)Figure 2 shows the effect of the compound of the present invention on sperm motility in porcine umbilical varicose veins experimental model. (Means CTR; Control, OP; Operation)
당업자라면 다양한 변형 및 변이가 본 발명의 정신 또는 범위를 이탈하지 않는 범위에서 본 발명의 조성물, 용도 및 제조에 사용 가능함이 자명할 것이다.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.
본 발명의 하기 실시예에 의해 보다 상세히 설명하나, 본 발명의 어떠한 방법으로도 하기 실시예로 제한하고자 하지 않는 것으로 이해되어야 한다.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.
..
단, 하기 실시예 및 실험예는 본 발명의 범위를 제한하지 않는 범위에서 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다.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.
참고예Reference Example 1. 단일생약 추출물의  1. Single Herb Extract 제조예Production Example
(1) 파극천 추출물의 제조 (CINThera-1)(1) Preparation of Pageokcheon Extract (CINThera-1)
건조된 파극천 (덕현당, 대한민국: Morinda officinalis , Deok Hyeon Dang, South Korea) 3,000g에 95% 에탄올 6ℓ를 넣고 90분씩 3회 초음파 추출한 후, 여과하고 감압 조건하에서 농축하였다. 농축된 여과물은 감압 조건하에서 냉동 건조기 (ScanVac, Labogene)로 동결 건조하여 139.4g (4.6%)의 추출물 분말을 얻어 4℃에서 보관하였다.Dried Pajukcheon (Dukhyeondang, South Korea: Morinda officinalis , Deok Hyeon Dang, South Korea) 6 liters of 95% ethanol was added to 3,000 g three times for 90 minutes, followed by filtration and concentration 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.
실험에 사용하기 전, 건조된 추출물을 생리식염수에 녹이고 실온에서 2분간 격렬하게 섞었다.Before use in the experiment, the dried extract was dissolved in physiological saline and mixed vigorously for 2 minutes at room temperature.
(2) 토사자 추출물의 제조 (CINThera-2)(2) Preparation of Tosa Extract (CINThera-2)
건조된 토사자 (백장생, 대한민국, Cuscuta chinensis , Baek Jang Sang, Sounth Korea) 5.2Kg에 95% 에탄올 5ℓ를 넣고 90분씩 2회 초음파 추출한 후, 여과하고 감압 조건하에서 농축하였다. 농축된 여과물은 감압 조건하에서 냉동 건조기 (ScanVac, Labogene)로 동결 건조하여 264.3g(4.36%)의 추출물 분말을 얻어 4℃에서 보관하였다.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.
실험에 사용하기 전, 건조된 추출물을 생리식염수에 녹이고 실온에서 2분간 격렬하게 섞었다.Before use in the experiment, the dried extract was dissolved in physiological saline and mixed vigorously for 2 minutes at room temperature.
(3) 양파추출물의 제조 (CINThera-3)(3) Preparation of Onion Extract (CINThera-3)
양파 (창녕, 대한민국: Allium cepa , Changyeong, South Korea)를 잘 씻고 건조한 후 겉껍질을 포함한 3겹 부위를 1,000g에 70% 에탄올 5ℓ를 넣고 70℃ 에서 2시간 추출한 후, 여과하고 감압 조건하에서 농축하였다. 농축된 여과물은 감압 조건하에서 냉동 건조기 (ScanVac, Labogene)로 동결 건조하여 13.8g (1.38%)의 추출물 분말을 얻어 4℃에서 보관하였다.Onion (Changnyeong, South Korea: Allium cepa , Changyeong, South Korea) was well washed and dried, and then the three-ply portion including the outer shell was added 5L of 70% ethanol to 1,000g, extracted at 70 ℃ 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.
실험에 사용하기 전, 건조된 추출물을 생리식염수에 녹이고 실온에서 2분간 격렬하게 섞었다.Before use in the experiment, the dried extract was dissolved in physiological saline and mixed vigorously for 2 minutes at room temperature.
참고예Reference Example 2.  2. 혼합생약Mixed Herbal Medicine 추출물의  Of extract 제조예Production Example ( ( CINTheraCINThera -1: -One: CINTheraCINThera -2: -2: CINTheraCINThera -3=-3 = 10: 1: 1010: 1: 10 ))
- 상기 참고예 1에서 얻은 건조 상태의 파극천 추출물 (CINThera-1) 10mg, 토사자 추출물 (CINThera-2) 1mg 및 양파추출물 (CINThera-3) 10mg을 각각 칭량하여 혼합기(Vortex genie-2, scientific industries, USA)로 혼합하여 혼합생약 추출물 21mg을 얻어 이를 하기 실험예의 시료로 사용하였다 (이하, CINTM1이라 함).10 mg of dried Pagokcheon extract (CINThera-1), 1 mg of Tosa seed extract (CINThera-2) and 10 mg of onion extract (CINThera-3) obtained in Reference Example 1 were weighed, respectively (Vortex genie-2, scientific industries, USA) to obtain a mixed herbal extract 21mg was used as a sample of the following experimental example (hereinafter referred to as CINTM1 ).
실시예 1. 활성 화합물의 분리 Example 1. Isolation of Active Compounds
1-One- 1. 조추출물의1. Crude extracts 제조 Produce
- 한국특허공개 제 10-2014-0102848호에 개시된 바와 같은 방법으로, 건조된 파극천 (덕현당) 2.4 kg에 95% 에탄올 6ℓ를 넣고 90분씩 2회 열탕 추출 후, 여과하고 감압 조건하에서 농축하였다. 농축된 여과물은 감압 조건하에서 냉동 건조기 (ScanVac, Labogene)로 동결 건조하여 248.03g (10.3 %)의 파극천 에탄올 추출물 분말(이하, MOE라 함)을 얻어 4℃에서 보관하였다. 실험에 사용하기 전, 건조된 추출물을 생리식염수에 녹이고 실온에서 2분간 격렬하게 섞었다. In a method as disclosed in Korean Patent Publication No. 10-2014-0102848, 6 liters of 95% ethanol was added to 2.4 kg of dried Pagokcheon (Dukhyundang), extracted twice with boiling water 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 248.03 g (10.3%) of Pakcheoncheon ethanol extract powder (hereinafter referred to as MOE) 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.
1-2. 정제물의 제조예1-2. Example of preparation
비교예 1에서 얻은 건조된 파극천 에탄올 추출물중 80 g을 증류수 1,000 ml에 현탁(suspension)하고, 당업계에 통상적인 분획방법에 따라 n-헥산, 에틸아세테이트(ethyl acetate) 및 n-부탄올(n-BuOH)로 차례대로 분획하고 감압 건조하여 헥산 가용분획물 3.47g, 에틸아세테이트 가용분획물 2.33g, 부탄올 가용분획물 13.12g 및 물 가용분획물 59.15g을 각각 얻어 동결건조하였다(이하, 각각, MOHE", MOEA", MOBU" 및 "MO-WA"라 함). 80 g of the dried Papocheon ethanol extract obtained in Comparative Example 1 was suspended in 1,000 ml of distilled water, and n-hexane, ethyl acetate and n-butanol (n- BuOH), and dried under reduced pressure, lyophilized to obtain 3.47 g of hexane soluble fraction, 2.33 g of ethyl acetate soluble fraction, 13.12 g of butanol soluble fraction and 59.15 g of water soluble fraction (hereinafter referred to as MOHE ", MOEA"). , MOBU "and" MO-WA ").
활성물질의 동정을 위하여, 하기 실험에서, 가장 활성이 높은 분획 중 부탄올 가용분획물을 HPLC-DAD (Agilent 1290 HPLC) 시스템 및 Shiseido UG18 (5μm, 4.6 x 250 mm) 컬럼 크로마토그래피를 하기 분석 조건으로 수행하여 부탄올 가용분획물중 활성성분인 monotripein 과 Deacetyl asperulosidic acid을 표준품과 비교하여 확인하였다.For identification of actives, in the following experiments, butanol soluble fraction in the most active fractions was subjected to HPLC-DAD (Agilent 1290 HPLC) system and Shiseido UG18 (5μm, 4.6 x 250 mm) column chromatography under the following analytical conditions The active ingredients, monotripein and Deacetyl asperulosidic acid, were identified in the butanol soluble fraction by comparison with the standard.
HPLC 분석조건HPLC analysis conditions
referencereference
UV DetectorUV Detector 254nm254nm
columncolumn Shiseido UG18 (4.6 x 250 mm, 5um)Shiseido UG18 (4.6 x 250 mm, 5um)
mobile phasemobile 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)(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 rateflow rate 1 mL/min1 mL / min
또한 활성물질은, 모노트로페인 (Monotropein) 및 디아세틸아스페룰로시딕 산 (Deacetyl asperulosidic acid)에 대한 표준 정량 곡선(standard calibration curve)을 구하였고, 에탄올추출물에서의 화합물 함량은 각각 1.15% 및 1.45%로 각각 나타났다. In addition, the active substance was obtained a standard calibration curve for monotropein and deacetyl asperulosidic acid, and the compound content in the ethanol extract was 1.15% and 1.45% respectively.
1-One- 3. 화합물의3. Compound 분리 Separation
상기 실시예 1-2에서 수득한 부탄올 가용 추출물을 20 ml 메탄올 용매에 재용해 시킨 후에 HPLC 컬럼 크로마토그래피를 수행하여 (Gilson HPLC System, 321 pump, 317 UV detector, Inno C18 column, 20 x 250mm, flow rate=10 ml/min, mobile phase: 95% H2O) 하기 물성치를 갖는 모노트로페인 (Monotropein) 및 디아세틸아스페룰로시딕 산 (Deacetyl asperulosidic acid)을 각각 분리하였고, 분리된 화합물들의 순도는 HPLC-DAD (Agilent Series 1260, Waldbronn, Germany) 및 1H NMR, 13C NMR spectra (400 MHz Bruker NMR AVANCE III system, Bruker, Germany)로 측정하였으며 (>95%), 이들의 물리화학적 데이타를 기존 문헌과 비교하여 동정하였다.After dissolving the butanol soluble extract obtained in Example 1-2 in 20 ml methanol solvent, HPLC column chromatography was performed (Gilson HPLC System, 321 pump, 317 UV detector, Inno C18 column, 20 x 250 mm, flow). rate = 10 ml / min, mobile phase: 95% H2O) Monotropein and Deacetyl asperulosidic acid having the following physical properties were separated, respectively, and the purity of the separated compounds was HPLC. -DAD (Agilent Series 1260, Waldbronn, Germany) and 1 H NMR, 13C NMR spectra (400 MHz Bruker NMR AVANCE III system, Bruker, Germany) (> 95%), and their physical and chemical data By comparison.
a. a. 모노트로페인Monotropine ( ( MonotropeinMonotropein ) :):
참고문헌: JENSEN, Heidi D., et al. Hydrophilic carboxylic acids and iridoid glycosides in the juice of American and European cranberries (Vaccinium macrocarpon and V. oxycoccos), lingonberries (V. vitis-idaea), and blueberries (V. myrtillus). Journal of agricultural and food chemistry, 2002, 50.23: 6871-6874.References: JENSEN, Heidi D., et al. Hydrophilic carboxylic acids and iridoid glycosides in the juice of American and European cranberries (Vaccinium macrocarpon and V. oxycoccos), lingonberries (V. vitis-idaea), and blueberries (V. myrtillus). Journal of agricultural and food chemistry , 2002, 50.23: 6871-6874.
1H NMR: (400 MHz, Bruker model digital AVANCE III 400, Germany): δ 7.42 (1H, d, J=1.3Hz,H-3), 6.24 (1H,dd,J=5.7,2.7Hz,H-6), 5.69(1H,dd,J= 5.7, 1.7 Hz, H-7), 5.63 (1H, d, J= 1.8 Hz, H-1), 4.77 (1H, s, H-1), 3.89 (1H, dd, J = 12.4, 2.1 Hz, H-63.69 (1H, dd, J= 12.4, 6.0 Hz, H-63.67 (1H, d, J= 11.5 Hz, H-10a), 3.62 (1H, d, J= 11.5 Hz, H-10b), 3.54 (ddt, J= 8.8 Hz, J= 2.9, 1.5 Hz, H-53.50 3.45 (1H, m, H-5), 3.46 (1H, t, J= 9.2 Hz, H-33.36 (1H, dd, J= 9.8,9.1 Hz, H-43.24 (1H, dd, J= 9.4, 8.1 Hz, H-22.69 (1H, dd, J= 8.8, 1.8 Hz, H-9), 1 H NMR: (400 MHz, Bruker model digital AVANCE III 400, Germany): δ 7.42 (1H, d, J = 1.3 Hz, H-3), 6.24 (1H, dd, J = 5.7,2.7 Hz, H- 6), 5.69 (1H, dd, J = 5.7, 1.7 Hz, H-7), 5.63 (1H, d, J = 1.8 Hz, H-1), 4.77 (1H, s, H-1), 3.89 ( 1H, dd, J = 12.4, 2 .1 Hz, H-63.69 (1H, dd, J = 12.4, 6.0 Hz, H-63.67 (1H, d, J = 11.5 Hz, H-10a), 3.62 (1H, d, J = 11.5 Hz, H-10b), 3.54 (ddt, J = 8.8 Hz, J = 2.9, 1.5 Hz, H-53.50 3.45 (1H, m, H-5), 3.46 (1H, t, J = 9.2 Hz, H-33.36 (1H, dd, J = 9.8,9.1 Hz, H-43.24 (1H, dd, J = 9.4, 8.1 Hz, H-22.69 (1H, dd, J = 8.8, 1.8 Hz, H- 9),
13C NMR spectra(100 MHz Bruker model digital AVANCE III 400, Germany): δ 170.59 (C-11), 151.43 (C-3), 137.19 (C-7), 131.83 (C-6), 110.19 (C-4), 98.14 (C-194.18 (C-1), 84.67 (C-8), 76.20 (C-575.52 (C-372.55 (C-269.47 (C-10), 66.32 (C-460.58 (C-643.87 (C-9), 36.92 (C-5).13C NMR spectra (100 MHz Bruker model digital AVANCE III 400, Germany): δ 170.59 (C-11), 151.43 (C-3), 137.19 (C-7), 131.83 (C-6), 110.19 (C-4 ), 98.14 (C-194.18 (C-1), 84.67 (C-8), 76.20 (C-575.52 (C-372.55 (C-269.47 (C-10)), 66.32 (C-460.58 (C-643.87 (C -9), 36.92 (C-5).
b. b. 디아세틸아스페룰로시딕Diacetylasperulosidic 산 ( Mountain ( DeacetylDeacetyl asperulosidicasperulosidic acid):  acid):
참고문헌: Hideaki OTSUKA, et al. Isolation of 10-O-Acyl Iridoid Glucosides from a Philippine Medicinal Plant, Oldenlandia corymbosa L.(Rubiaceae). Chemical and Pharmaceutical bulletin, 1991, 39.8: 2049-205; GUZuet al. Chemical constituents of Galium tortumense. Turkish Journal of Chemistry, 2006, 30.4: 515-523.Reference: Hideaki OTSUKA, et al. Isolation of 10-O-Acyl Iridoid Glucosides from a Philippine Medicinal Plant, Oldenlandia corymbosa L. (Rubiaceae). Chemical and Pharmaceutical bulletin , 1991, 39.8: 2049-205; GUZue et al. Chemical constituents of Galium tortumense. Turkish Journal of Chemistry , 2006, 30.4: 515-523.
1H - NMR: (400 MHz, Bruker model digital AVANCE III 400, Germany): d 7.63 (1H, s, H-3), 5.99 (1H, s, H-7), 5.02 (1H, d, J= 9.0 Hz, H-1), 4.78 (1H, d, J= 5.3 Hz, H-6), 4.70 (1H, d, J= 7.8 Hz, H-1'), 4.42 (1H, d, J= 15.8 Hz, H-10b), 4.18 (1H, d, J= 15.6 Hz, H-10a), 3.59 (1H, dd, J= 12.0, 5.3 Hz, H-6'a), 3.38 (1H, t, J= 8.6 Hz, H-4'), 3.28 -3.19 (3H, m, H-2', H-3', H-5'), 3.28 (1H, d, J= 11.8 Hz, H-6'b), 2.97 (1H, t, J= 6.5 Hz, H-5), 2.53 (1H, t, J= 8.1 Hz, H-9); 1 H-NMR: (400 MHz, Bruker model digital AVANCE III 400, Germany): d 7.63 (1H, s, H-3), 5.99 (1H, s, H-7), 5.02 (1H, d, J = 9.0 Hz, H-1), 4.78 (1H, d, J = 5.3 Hz, H-6), 4.70 (1H, d, J = 7.8 Hz, H-1 '), 4.42 (1H, d, J = 15.8 Hz, H-10b), 4.18 (1H, d, J = 15.6 Hz, H-10a), 3.59 (1H, dd, J = 12.0, 5.3 Hz, H-6'a), 3.38 (1H, t, J = 8.6 Hz, H-4 '), 3.28 -3.19 (3H, m, H-2', H-3 ', H-5'), 3.28 (1H, d, J = 11.8 Hz, H-6'b ), 2.97 (1H, t, J = 6.5 Hz, H-5), 2.53 (1H, t, J = 8.1 Hz, H-9);
13C - NMR spectra (100 MHz Vruker model digital AVANCE III 400, Germany): δ 170.76 (C-11), 155.51 (C-3), 151.40 (C-8), 129.83 (C-7), 108.30 (C-4), 101.44 (C-1), 100.35 (C-178.36 (C-377.70 (C-575.34 (C-6), 74.88 (C-271.53 (C-462.75 (C-661.65 (C-10), 45.75 (C-9), 42.63 (C-5),13C-NMR spectra (100 MHz Vruker model digital AVANCE III 400, Germany): δ 170.76 (C-11), 155.51 (C-3), 151.40 (C-8), 129.83 (C-7), 108.30 (C- 4), 101.44 (C-1), 100.35 (C-178.36 (C-377.70 (C-575.34 (C-6), 74.88 (C-271.53 (C-462.75 (C-661.65 (C-10)), 45.75 ( C-9), 42.63 (C-5),
실시예 2Example 2 . 활성 화합물의 준비. Preparation of Active Compounds
회사로부터 모노트로페인 (Monotropein: Sigma-Aldrich, SMB00471) 및 디아세틸아스페룰로시딕 산 (Deacetyl asperulosidic acid: Chemfaces, CFN92358)을 각각 구입하여 증류수에 용해시켜 하기 실험예 시료로 사용하였다.Monotropein (Monotropein: Sigma-Aldrich, SMB00471) and diacetyl asperulosidic acid (Deacetyl asperulosidic acid: Chemfaces, CFN92358) were purchased from each company, dissolved in distilled water, and used as the following experimental sample.
실험예Experimental Example 1.  One. In vitroIn vitro 정자 운동 증진 효능 평가 Evaluation of Efficacy of Sperm Exercise
상기 실시예의 시료들의 사람정액의 정자수와 운동성 변화에 미치는 영향을 알아보기 위하여 문헌에 기재된 시험관내 (In vitro) 정자 운동 증진 효능 평가법을 이용하여 하기와 같이 실험을 수행하였다 (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). 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. 실험 과정1-1. Experiment process
불임으로 내원한 환자 지원자(전북대학병원 비뇨기과, 20~50대, 약 85명)로부터 얻은 정액에서 정자의 수, 운동성을 측정하고 한 시간 동안 37oC의 인큐베이터에 넣은 후, 정액이 충분히 액화되면 200μl의 정액에 20μl의 시료 추출물을 넣어 최종 농도를 2mg/ml 되게 한 후에 3시간 후의 정자의 운동성을 챔버 (MAKLER counting chamber; MAKLER counting chamber; self-Medical Instruments, Haifa, Israel)를 사용하여 현미경(Axiovert25, ZEISS, USA)으로 측정하였다.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).
대조군으로는 3H 완충액 Hams F-10(N6635,Sigma Chemical Co., USA) + 0.4% HAS(A1653,Sigma Chemical Co., USA) + 12mM HEPES (H4034,Sigma Chemical Co., USA) [3H, pH 7.4]을 사용하였으며, 시료들도 3H 완충액을 사용하여 녹인 후에 pH를 7.4로 맞춘 후, 원심분리기(centrifuge; A32010(1), LABOGENE)를 사용하여 원심분리 (13,000rpm, 2min, 4℃)하여 상등액을 수득하여 사용하였다.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.
정자 운동성은 하기 수학식 1에 따라 계산되었다.Sperm motility was calculated according to the following equation (1).
[수학식 1][Equation 1]
The percentage of motile spermatozoa = (mean number of motile spermatozoa/total number of spermatozoa) ×100%The percentage of motile spermatozoa = (mean number of motile spermatozoa / total number of spermatozoa) × 100%
3시간 후의 정자 운동성증가 비율은 하기 수학식 2에 따라 계산되었다.The sperm motility increase rate after 3 hours was calculated according to the following equation (2).
[수학식 2][Equation 2]
The percentage of motile spermatozoa (3 hours after incubation) =The percentage of motile spermatozoa (3 hours after incubation) =
(sperm mobility 3 hours after incubation on control group - sperm mobility 3 hours after incubation at test group/ sperm mobility 3 hours after incubation on control group ) ×100% (sperm mobility 3 hours after incubation on control group-sperm mobility 3 hours after incubation at test group / sperm mobility 3 hours after incubation on control group) × 100%
1-2. 실험 결과1-2. Experiment result
- 상기에서 불임으로 내원한 환자로부터 얻은 정액에서 정자의 수, 운동성을 측정한 결과, 모노트로페인 (Monotropein) 및 디아세틸아스페룰로시딕 산 (Deacetyl asperulosidic acid)에서는 10μM 농도에서 평균(n=3) 74.7%, 및 73.0% 로 정자 운동성 증가가 가장 높게 나타남을 확인하였다. (표 2)-The sperm count and motility of semen obtained from infertility patients were measured. As a result, monotropein and deacetyl asperulosidic acid were averaged at a concentration of 10 μM (n = 3) The highest increase of sperm motility was found in 74.7% and 73.0%. Table 2
treatmenttreatment 3H*(pH 7.4)3H * (pH 7.4) MTP*(10μ M)MTP * (10 μ M) DAA*(10μ M)DAA * (10 μ M) CIN4*(0.05mg/ml)CIN4 * (0.05 mg / ml)
n=1n = 1 zerozero Total count(x106/ml)Total count (x106 / ml) 6464 6767 6565 6161
Motility (%)Motility (%) 71.871.8 70.570.5 71.571.5 73.773.7
3hrs3hrs Total count(x106/ml)Total count (x106 / ml) 6363 6363 5555 5252
Motility (%)Motility (%) 72.572.5 7575 74.774.7 7979
n=2n = 2 zerozero Total count(x106/ml)Total count (x106 / ml) 7171 7373 7070 7373
Motility (%)Motility (%) 5050 48.348.3 4747 50.850.8
3hrs3hrs Total count(x106/ml)Total count (x106 / ml) 6363 5555 5555 5454
Motility (%)Motility (%) 50.750.7 52.852.8 5252 5757
n=3n = 3 zerozero Total count(x106/ml)Total count (x106 / ml) 7474 7979 6565 7575
Motility (%)Motility (%) 38.838.8 39.239.2 4040 38.238.2
3hrs3hrs Total count(x106/ml)Total count (x106 / ml) 6363 6161 5656 5959
Motility (%)Motility (%) 38.538.5 39.639.6 4040 4646
*: 3H: Hams F-10(Sigma-Aldrich, N6635,) + 0.4% HAS (Sigma-Aldrich, A1653) + 12mM HEPES(Sigma-Aldrich, H4034), MTP= Monotropein, DAA= Deacetyl asperulosidic acid, CIN4= CINTM1*: 3H: Hams F-10 (Sigma-Aldrich, N6635,) + 0.4% HAS (Sigma-Aldrich, A1653) + 12 mM HEPES (Sigma-Aldrich, H4034), MTP = Monotropein, DAA = Deacetyl asperulosidic acid, CIN4 = CINTM1
1-3. 실험 결과1-3. Experiment result
- 상기에서 불임으로 내원한 환자로부터 얻은 정액에서 정자의 수, 운동성을 측정한 뒤 3시간 후의 정자 운동성증가 비율을 측정한 결과, 모노트로페인 (Monotropein) 및 디아세틸아스페룰로시딕 산 (Deacetyl asperulosidic acid) 에서는 10μM 농도에서 평균(n=3) 3.48%, 및 3.16% 로 3시간후의 대조군 정자 운동성에 비해 증가가 됐음을 확인하였다. (도 1) -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, monotropein (Monotropein) and diacetyl asperulosidic acid (Deacetyl) asperulosidic acid) In the 10 μM concentration, the average (n = 3) 3.48%, and 3.16% was confirmed that the increase compared to the control sperm motility after 3 hours. (Figure 1)
실험예Experimental Example 2.  2. In vitroIn vitro 정자 운동 증진 효능 평가 Evaluation of Efficacy of Sperm Exercise
상기 실시예의 시료들의 돼지정액의 정자수와 운동성 변화에 미치는 영향을 알아보기 위하여 문헌에 기재된 시험관내 (In vitro) 정자 운동 증진 효능 평가법을 이용하여 하기와 같이 실험을 수행하였다 (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). 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. 실험 과정2-1. Experiment process
KPG 한국돼지유전자 (김제시, 전라북도)로부터 얻은 정액에서 정자의 수, 운동성을 측정하고 200μl의 정액에 20μl의 시료 추출물을 넣어 최종 농도를 2mg/ml 되게 한 후에 3시간 후의 정자의 운동성을 챔버 (MAKLER counting chamber; MAKLER counting chamber; self-Medical Instruments, Haifa, Israel)를 사용하여 현미경(Axiovert25, ZEISS, USA)으로 측정하였다.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.
대조군으로는 3H 완충액 Hams F-10(N6635,Sigma Chemical Co., USA) + 0.4% HAS(A1653,Sigma Chemical Co., USA) + 12mM HEPES (H4034,Sigma Chemical Co., USA) [3H, pH 7.4]을 사용하였으며, 시료들도 3H 완충액을 사용하여 녹인 후에 pH를 7.4로 맞춘 후, 원심분리기(centrifuge; A32010(1), LABOGENE)를 사용하여 원심분리 (13,000rpm, 2min, 4℃)하여 상등액을 수득하여 사용하였다.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.
정자 운동성은 하기 수학식 3에 따라 계산되었다.Sperm motility was calculated according to the following equation (3).
[수학식 3][Equation 3]
The percentage of motile spermatozoa = (mean number of motile spermatozoa/total number of spermatozoa) ×100%The percentage of motile spermatozoa = (mean number of motile spermatozoa / total number of spermatozoa) × 100%
3시간 후의 정자 운동성증가 비율은 하기 수학식 4에 따라 계산되었다.The sperm motility increase rate after 3 hours was calculated according to the following equation (4).
[수학식 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%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. 실험 결과2-2. Experiment result
- 투여 후 정자의 수를 측정한 결과, 돼지 정계 정맥류 유발군 중 모노트로페인 (Monotropein) 및 디아세틸아스페룰로시딕 산 (Deacetyl asperulosidic acid) 화합물에서는 10μM 농도에서 평균(n=3) 74.7%, 및 73.0% 로 정자 운동성 증가가 가장 높게 나타나 정자운동 증진 효능이 탁월한 것으로 확인되었다 (표 3 및 도 2 참조). -After the administration, the number of sperm was measured, and the average (n = 3) at the concentration of 10 μM in the monotropein and deacetyl asperulosidic acid compounds in the swine umbilical vein inducing group was 74.7% , And 73.0% showed the highest increase in sperm motility, indicating that sperm motility promoting effect was excellent (see Table 3 and FIG. 2).
treatmenttreatment 3H*(pH 7.4)3H * (pH 7.4) MTP*(10μ M)MTP * (10 μ M) DAA*(10μ M)DAA * (10 μ M)
n=1n = 1 zerozero Total count(x106/ml)Total count (x10 6 / ml) 5252 5454 4949
Motility (%)Motility (%) 5757 54.754.7 58.658.6
3hrs3hrs Total count(x106/ml)Total count (x10 6 / ml) 5252 5151 4545
Motility (%)Motility (%) 62.562.5 62.662.6 5656
n=2n = 2 zerozero Total count(x106/ml)Total count (x10 6 / ml) 5656 5151 5151
Motility (%)Motility (%) 53.853.8 47.647.6 4747
3hrs3hrs Total count(x106/ml)Total count (x10 6 / ml) 5050 5555 5353
Motility (%)Motility (%) 5555 6464 4949
n=3n = 3 zerozero Total count(x106/ml)Total count (x10 6 / ml) 4444 4949 4242
Motility (%)Motility (%) 5353 53.853.8 46.846.8
3hrs3hrs Total count(x106/ml)Total count (x10 6 / ml) 4040 4646 4646
Motility(%)Motility (%) 47.847.8 55.555.5 4646
*: 3H: Hams F-10(Sigma-Aldrich, N6635,) + 0.4% HAS (Sigma-Aldrich, A1653) + 12mM HEPES(Sigma-Aldrich, H4034), MTP= Monotropein, DAA= Deacetyl asperulosidic acid, *: 3H: Hams F-10 (Sigma-Aldrich, N6635,) + 0.4% HAS (Sigma-Aldrich, A1653) + 12 mM HEPES (Sigma-Aldrich, H4034), MTP = Monotropein, DAA = Deacetyl asperulosidic acid,
하기에 본 발명의 제형화 방법 및 담체의 종류를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 대표적인 제제예를 하기에 설명하고자 함이다.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.
제제예 1. 산제의 제조Formulation Example 1 Preparation of Powder
화합물 (MTP) : 20 mgCompound (MTP): 20 mg
유당 : 100 mgLactose: 100 mg
탈크 : 10 mgTalc: 10 mg
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.The above ingredients are mixed and filled in an airtight cloth to prepare a powder.
제제예 2. 정제의 제조Formulation Example 2 Preparation of Tablet
화합물 (DAA) : 10 mgCompound (DAA): 10 mg
옥수수전분 : 100 mgCorn starch: 100 mg
유당 : 100 mgLactose: 100 mg
스테아린산 마그네슘 : 2 mgMagnesium Stearate: 2 mg
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.
제제예 3. 캅셀제의 제조 Formulation Example 3 Preparation of Capsule
화합물 (MTP) : 10 mgCompound (MTP): 10 mg
결정성 셀룰로오스 : 3 mgCrystalline Cellulose: 3 mg
락토오스 : 14.8 mgLactose: 14.8 mg
마그네슘 스테아레이트 : 0.2 mgMagnesium Stearate: 0.2 mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.
제제예 4. 주사제의 제조Formulation Example 4 Preparation of Injection
화합물 (DAA) : 10 mgCompound (DAA): 10 mg
만니톨 : 180 mgMannitol: 180 mg
주사용 멸균 증류수 : 2974 mgSterile Distilled Water For Injection: 2974 mg
Na2HPO4,12H2O : 26 mgNa 2 HPO 4 , 12H2O: 26 mg
통상의 주사제의 제조방법에 따라 활성성분을 용해시키고, 모든 성분을 1 앰플당(2 ㎖) 상기의 성분 함량으로 제조한다.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).
제제예 5. 액제의 제조Formulation Example 5 Preparation of Liquid
화합물 (MTP) : 20 mgCompound (MTP): 20 mg
이성화당 : 10 gIsomerized sugar: 10 g
만니톨 : 5 gMannitol: 5 g
정제수 : 적량Purified water: appropriate amount
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100 ㎖으로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조한다.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.
제제예 6. 건강 식품의 제조Formulation Example 6 Preparation of Healthy Food
화합물 (DAA) : 1000 ㎎ Compound (DAA): 1000 mg
비타민 혼합물 : 적량Vitamin Mixture: Proper
비타민 A 아세테이트 : 70 mg Vitamin A Acetate: 70 mg
비타민 E : 1.0 ㎎Vitamin E: 1.0 mg
비타민 B1 : 0.13 ㎎Vitamin B1: 0.13 mg
비타민 B2 : 0.15 ㎎ Vitamin B2: 0.15 mg
비타민 B6 : 0.5 ㎎Vitamin B6: 0.5 mg
비타민 B12 : 0.2 mgVitamin B12: 0.2 mg
비타민 C : 10 ㎎ Vitamin C: 10 mg
비오틴 : 10 mgBiotin: 10 mg
니코틴산아미드 : 1.7 ㎎ Nicotinamide: 1.7 mg
엽산 : 50 mgFolic acid: 50 mg
판토텐산 칼슘 : 0.5 ㎎ Calcium Pantothenate: 0.5 mg
무기질 혼합물 : 적량Mineral Mixtures: Proper
황산제1철 : 1.75 ㎎ Ferrous Sulfate: 1.75 mg
산화아연 : 0.82 ㎎ Zinc Oxide: 0.82 mg
탄산마그네슘 : 25.3 ㎎ Magnesium Carbonate: 25.3 mg
제1인산칼륨 : 15 ㎎ Potassium phosphate monobasic: 15 mg
제2인산칼슘 : 55 ㎎ Dibasic calcium phosphate: 55 mg
구연산칼륨 : 90 ㎎ Potassium citrate: 90 mg
탄산칼슘 : 100 ㎎ Calcium Carbonate: 100 mg
염화마그네슘 : 24.8 ㎎ Magnesium Chloride: 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 본 발명의 정신 및 범위를 벗어나지 않는다고 간주되는 임의로 변형 실시하여도 무방하다. 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.
제제예 7. 건강 음료의 제조Formulation Example 7 Preparation of Healthy Drink
화합물 (MTP) : 1000㎎Compound (MTP): 1000 mg
구연산 : 1000 ㎎ Citric Acid: 1000 mg
올리고당 : 100 gOligosaccharide: 100 g
매실농축액 : 2 gPlum concentrate: 2 g
타우린 : 1 gTaurine: 1 g
정제수를 가하여 : 전체 900 ㎖Add purified water: 900 ml total
통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간 동안 85℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2ℓ 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 건강음료 조성물 제조에 사용한다. 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.
상술한 바와 같이, 다양한 방법으로 본 발명은 변형가능하고, 이러한 변형은 본 발명의 정신 및 범위를 벗어나지 않는 것으로 간주되며, 이러한 모든 변형은 당업자에게는 하기한 본 발명의 청구범위 이내 포함되는 의도임이 자명할 것이다. 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.
상술한 바와 같이, 본 발명의 화합물을 대상으로 사람과 돼지정액의 정자수와 운동성 변화에 미치는 영향을 알아보기 위한 시험관내 (In vitro) 정자 운동 증진 효능 평가법 실험을 통하여 본 발명의 시료들이 사람과 돼지정액의 in vitro 실험에서 정자 운동성이 증가함을 확인함으로서, 상기 조성물을 남성불임증의 예방 및 치료용 약학조성물, 건강기능식품 및 건강보조식품에 유용하게 이용될 수 있다.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 (10)

  1. 하기 일반식 (I)로 표기되는 이리도이드 유도체 화합물, 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 남성 불임증 예방 및 치료용 약학 조성물:Iridoid derivative compound represented by the following general formula (I), a pharmaceutical composition for preventing and treating male infertility containing a pharmaceutically acceptable salt thereof as an active ingredient:
    Figure PCTKR2017014180-appb-I000001
    Figure PCTKR2017014180-appb-I000001
    상기 식에서In the above formula
    R1은 수소원자, 히드록시(hydroxy)기 또는 C1 내지 C3 알킬기로 치환된 알콕시기이며;R 1 is a hydrogen atom, a hydroxy group or an alkoxy group substituted with a C1 to C3 alkyl group;
    R2은 수소원자, 또는 C1 내지 C3 알킬기이다.R 2 is a hydrogen atom or a C1 to C3 alkyl group.
  2. 제 1항에 있어서, 상기 식에서The compound of claim 1 wherein
    R1은 수소원자, 히드록시(hydroxy)기 또는 메톡시기이며;R 1 is a hydrogen atom, a hydroxy group or a methoxy group;
    R2은 수소원자인 화합물, 또는 이의 약학적으로 허용가능한 염임을 특징으로 하는 약학 조성물.R 2 is a compound which is a hydrogen atom, or a pharmaceutically acceptable salt thereof.
  3. 제 2항에 있어서, 상기 식에서The compound of claim 2 wherein
    R1은 수소원자, 또는 히드록시(hydroxy)기이며;R 1 is a hydrogen atom or a hydroxy group;
    R2은 수소원자인 화합물, 또는 이의 약학적으로 허용가능한 염임을 특징으로 하는 약학 조성물.R 2 is a compound which is a hydrogen atom, or a pharmaceutically acceptable salt thereof.
  4. 제 3항에 있어서, 모노트로페인 (Monotropein) 및 디아세틸아스페룰로시딕 산 (Deacetyl asperulosidic acid)으로 구성된 군으로부터 선택된 화합물, 또는 이의 약학적으로 허용가능한 염임을 특징으로 하는 약학 조성물.4. The pharmaceutical composition according to claim 3, which is a compound selected from the group consisting of monotropein and deacetyl asperulosidic acid, or a pharmaceutically acceptable salt thereof.
  5. 제 1항에 있어서, 상기 남성 불임증은 항암제 부작용에 의한 불임증, 무정자증, 감정자증, 약정자증, 또는 기형정자임을 특징으로 하는 약학 조성물. 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.
  6. 하기 일반식 (I)로 표기되는 이리도이드 유도체 화합물을 유효성분으로 함유하는 남성 불임증 예방 및 개선용 건강기능식품:Functional food for the prevention and improvement of male infertility containing an iridoid derivative compound represented by the following general formula (I) as an active ingredient:
    Figure PCTKR2017014180-appb-I000002
    Figure PCTKR2017014180-appb-I000002
    상기 식에서In the above formula
    R1은 수소원자, 히드록시(hydroxy)기 또는 C1 내지 C3 알킬기로 치환된 알콕시기이며;R 1 is a hydrogen atom, a hydroxy group or an alkoxy group substituted with a C1 to C3 alkyl group;
    R2은 수소원자, 또는 C1 내지 C3 알킬기이다.R 2 is a hydrogen atom or a C1 to C3 alkyl group.
  7. 제 6항에 있어서, 상기 건강기능식품은 산제, 과립제, 정제, 캡슐제, 환제, 현탁액, 에멀젼, 시럽제, 티백제, 침출차, 또는 건강 음료 형태인 건강기능식품.The health functional food according to claim 6, 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.
  8. 하기 일반식 (I)로 표기되는 이리도이드 유도체 화합물을 유효성분으로 함유하는 남성 불임증 예방 및 개선용 건강보조식품:A male dietary supplement for preventing and improving infertility of males containing an iridoid derivative compound represented by the following general formula (I) as an active ingredient:
    Figure PCTKR2017014180-appb-I000003
    Figure PCTKR2017014180-appb-I000003
    상기 식에서In the above formula
    R1은 수소원자, 히드록시(hydroxy)기 또는 C1 내지 C3 알킬기로 치환된 알콕시기이며;R 1 is a hydrogen atom, a hydroxy group or an alkoxy group substituted with a C1 to C3 alkyl group;
    R2은 수소원자, 또는 C1 내지 C3 알킬기이다.R 2 is a hydrogen atom or a C1 to C3 alkyl group.
  9. 제 1항의 일반식 (I)로 표기되는 이리도이드 유도체 화합물, 이의 약학적으로 허용가능한 염을 남성 불임증 환자에게 투여함을 포함하는, 남성 불임증 환자를 치료하기 위한 치료방법.A method for treating male infertility patients, comprising administering to the male infertility patients an iridoid derivative compound represented by the general formula (I) of claim 1, and a pharmaceutically acceptable salt thereof.
  10. 남성 불임증 질환 치료용 약제를 제조하기 위한 제 1항의 일반식 (I)로 표기되는 이리도이드 유도체 화합물, 이의 약학적으로 허용가능한 염의 용도.Use of an iridoid derivative compound represented by the general formula (I) of claim 1 for the preparation of a medicament for treating male infertility disease, and a pharmaceutically acceptable salt thereof.
PCT/KR2017/014180 2016-12-08 2017-12-06 Composition for preventing and treating male infertility, containing iridoid derivative compound as active ingredient, and use thereof WO2018105999A1 (en)

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