KR100702184B1 - Promotor of release of Luteinizing hormone comprising Anemarrhena asphodeloides extract or Timosaponin A-III - Google Patents

Abstract

The present invention relates to a luteinizing hormone secretagogue comprising as an active ingredient thymosaponin A-III, hair extract or fraction.

Timothy saponin A-III and hair extract of the present invention acts on the pituitary cells to promote the secretion of luteinizing hormone, and thus can be useful for the prevention and treatment of infertility, breast cancer, prostate cancer and the like.

Progesterone

Description

Promoters of release of Luteinizing hormone comprising Anemarrhena asphodeloides extract or Timosaponin A-III}

1 is a diagram showing the effect of inducing rat luteinizing hormone secretion according to the concentration of thymosaponin A-III.

The present invention relates to a luteinizing hormone secretagogue comprising as an active ingredient thymosaponin A-III, hair extract or fraction.

Luteinizing hormone is a glycoprotein hormone secreted from the anterior pituitary gland of vertebrates and is one of the gonad stimulating hormones and is also known as hepatocellular stimulating hormone (ICSH). Progesterone is secreted from basophil cells of the anterior pituitary gland, and the secretion of luteinizing hormone by the pituitary gland is promoted by the luteinizing hormone releasing factor synthesized in the hypothalamus.

It has a molecular weight of about 30,000, and dissociates into two different units having a molecular weight of about 15,000. One of the units has a structure similar to that of follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH). have.

Progesterone is a hormone that stimulates testicular androgen, ovarian estrogen and progesterone production. Specifically, in females, it acts on the follicles of mature ovaries, causing ovulation and luteinization, and in males, acts on hepatic cells of testes, promoting the release of male hormones. Sperm formation is complete. Therefore, luteinizing hormone is widely used to treat infertility in men as well as women.

The relationship between luteinizing hormone secretion and various diseases has been extensively studied. Promoting the secretion of luteinizing hormone is infertile (Ma X et al, Proc Natl Acad Sci US A. 2004 Dec 7; 101 (49): 17294- 17299.Epub 2004 Nov 29, McGovern PG et al, Fertil Steril. 2004 Nov; 82 (5): 1273), prostate cancer (Bencini S et al, Ann NY Acad Sci. 2003 Dec; 1010: 789-92, Mulligan T et al, Drugs Today (Barc) .1998 May; 34 (5): 455-61, Letsch M et al, Clin Cancer Res. 2003 Oct 1; 9 (12): 4505-13, Sugimura Y et al, Rinsho Byori 2004 Jul; 52 (7): 604-10), Jones KL et al, Endocr Relat Cancer. 2004 Sep; 11 (3): 391-4, Miyoshi Y et al, Breast Cancer. 2003; 10 (2 It is known that it can be used for the treatment of 105-11).

In addition, it promotes the secretion of luteinizing hormones, thereby increasing polycystic ovary syndrome (Aruna J et al, Int J Gynaecol Obstet. 2004 Dec; 87 (3): 237-41), chronic renal failure (Holly JL et al, Adv Chronic Kidney Dis. 2004 Oct; 11 (4): 337-41) and Klinefeld syndrome (Zitzmann M et al, J Clin Endocrinol Metab. 2004 Dec; 89 (12): 6208-17).

On the other hand, Anemarrhena asphodeloides Bunge is a perennial herb of the monocotyledonous lily tree, which uses the rhizome of the genus, and is native to China and distributed evenly in the Hwanghae Province in Korea. The leaves are gathered from the root and grow slowly, and are divided into exogenous, normal and endogenous leaves from the outer part of the leaf. The normal leaf of the middle part is the largest, and the shape of the leaf is lanceolate lanceolate, and the tip is quite pointed. The leaves are also solid and the outer parts are shiny and shiny. These roots and stems contain timomosaponin, chimonin, prococathechi acid, pantothenic acid, and antipyretic, cardiovascular, diuretic, It has antibacterial, expectorant, sedative, hypoglycemic and anticancer effects and is used as an antipyretic.

Existing studies on pharmaceuticals containing hair extracts have been described in Korean Patent Application No. 2002-34716, which includes ground mulberry, cheonhwabun, red ginseng, jihwang, jimo, rhubarb, corn whiskers, and in addition to Angelica, Schisandra chinensis, Astragalus or Vitamin E. It provides a composition for the prevention or treatment of diabetes mellitus, C, B, etc., and the Korean Patent Application No. 1996-30941 describes that the hair extract is effective in controlling rejection and autoimmune diseases caused by organ transplantation. In the Republic of Korea Patent Application No. 1998-14591, the extract is shown to stimulate the growth hormone secretion activity similar to the growth hormone secretagogue hormone can be useful as a therapeutic agent, anti-aging agent, etc. for growth hormone deficiency diseases such as dwarfism It is starting. However, it has not been known that thymosaponin A-III contained in hair extract and hair extract has an effect of promoting luteinizing hormone secretion.

Thus, the present inventors while studying the pharmacological activity of the hair extract, the hair extract and thymosaponin A-III contained in it has an effect of promoting the secretion of luteinizing hormone can be useful in the treatment of infertility, prostate cancer, breast cancer, etc. It was found that the present invention was completed.

An object of the present invention is to provide a luteinizing hormone secretagogue comprising thymosaponin A-III as an active ingredient.

Still another object of the present invention is to provide a progesterone-releasing hormone comprising the methanol extract or butanol fraction of the hair as an active ingredient.

In order to achieve the above object, the present invention provides a luteinizing hormone secretagogue containing thymosaponin A-III as an active ingredient.

In addition, the present invention provides a luteinizing hormone secretagogue which contains methanol extract of hair as an active ingredient.

In addition, the present invention provides a progesterone hormone secretagogue containing n-butanol fraction obtained by the stepwise fractionation of methanol extract of gimo with ether and n-butanol as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention includes a luteinizing hormone secretagogue which contains thymosaponin A-III represented by the following formula (1) as an active ingredient.

[Formula 1]

Timosafonin A-III of Formula 1 may be used in the form of a pharmaceutically acceptable salt, and includes all salts, hydrates, and solvates prepared by conventional methods. The thymosaponin A-III of the present invention was used by extraction, separation and purification from hair, and can be obtained by all conventional methods, and commercially available reagents can be used. In addition, the composition of the present invention may contain one or more active ingredients exhibiting the same or similar function in addition to the thymosaponin A-III.

In addition, the present invention comprises thymosaponin A-III, which is obtained by dissolving the extract of methanol by dissolving it with methanol or a mixed solvent of methanol and water, and the obtained extract of methanol, which is fractionated in the order of ether and n-butanol. N-butanol fraction of.

Extraction, separation and purification of thymosaponin A-III from gimo according to the present invention are as follows.

Wash gimos thoroughly with water, dry it in the shade, and cut it. The fine hair root rhizome is extracted with hot methanol three times for 6 hours and concentrated to obtain a methanol concentrate. The methanol concentrate is fractionated in sequence of ether, n-butanol to obtain an ether fraction and n-butanol fraction.

The butanol fraction obtained above was separated by column chromatography using a mixed solvent of ethyl acetate and methanol to obtain five small fractions. At this time, it is preferable to use a solvent of ethyl acetate: methanol = 100: 0 to 92: 8 (v / v).

The second of the five subfractions is subjected to column chromatography again using a mixed solvent of chloroform-methanol-water to separate thymosaponin A-III. At this time, it is preferable to use a solvent having chloroform: methanol: water = 7: 3: 1 (v / v).

The thymosaponin A-III obtained in the present invention, the ethanol methanol extract including the same, and the n-butanol fraction thereof have an effect of promoting the secretion of luteinizing hormone. As shown in the following examples, when the thymosaponin A-III of the present invention, the thymo methanol extract containing the same, and n-butanol fraction thereof were injected into the pituitary cells of rats, three times or more luteinizing hormone secretion was promoted compared to the control group. Activity was increased, and luteinizing hormone secretion promoting effect increased with increasing concentration.

As described above, thymosaponin A-III, a methanol extract containing it, and an n-butanol fraction thereof promote the secretion of luteinizing hormone. Promoted luteinizing hormone is used for various purposes, as known by those skilled in the art. The various uses of these luteinizing hormones can be summarized as follows: Treatment of infertility, prostate cancer, breast cancer, polycystic ovary syndrome, chronic renal failure and Klinefeldt syndrome

The thymosaponin A-III can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation. That is, thymosaponin A-III of the present invention can be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrating agents and surfactants, etc. It is prepared using diluent or excipient. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, and thymosaponin A-III. And gelatin etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, 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. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and the suspension solvent, 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, glycerol and gelatin may be used.

Dosage units may contain, for example, one, two, three or four times the individual dosage, or they may contain 1/2, 1/3 or 1/4 times. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose. The effective dose of thymosaponin A-III is 0.001 to 100 mg / kg, preferably 0.1 to 10 mg / kg, and may be administered 1 to 6 times a day.

As a result of oral administration of thymosaponin A-III of the present invention to rats, 50% lethal dose (LD50) by oral administration toxicity test was determined to be a safe substance of at least 100 mg / kg or more.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormonal therapy, chemotherapy and biological response modifiers to promote luteinizing hormone secretion.

The composition of the present invention can be added to health food for the purpose of promoting the secretion of luteinizing hormone. When thymosaponin A-III of the present invention is used as a food additive, the thymosaponin A-III may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in preparing foods or beverages, thymosaponin A-III of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw materials. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. .

There is no particular limitation on the kind of food. 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, and the like and include all of the health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents and the like used in beverages. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

Example 1 Extraction, Separation and Purification of Timothy Saponin A-III from Gemini

1. Extracting from Jimmy

Jimmy was washed with water, dried in the shade, and then chopped. 1 kg of fine hair root diameter was extracted with hot methanol three times for 6 hours, and concentrated to obtain 260 g of methanol concentrate.

The methanol concentrate was fractionated in the order of ether, n-butanol to obtain an ether fraction (11 g) and n-butanol fraction (50 g).

2. Isolation and Purification from Butanol Extract

The butanol fraction obtained in 1 was separated by column chromatography using a mixed solvent of ethyl acetate and methanol (ethyl acetate: methanol = 100: 0 to 92: 8 (v / v)) to obtain five small fractions.

The second of the five subfractions was again subjected to column chromatography using a mixed solvent of chloroform-methanol-water (chloroform: methanol: water = 7: 3: 1 (v / v) at the bottom) to give 1.2 g of Timosaponin A-III was isolated.

1) colorless needle shape

2) Melting Point:> 300 ℃

3) Radiance: [α] D25: 42.5 ° (c = 0.7, pyridine)

4) Liebermann-Burchard test: positive

5) Molish test: positive

6) ¹ H-NMR (300 MHz, pyridine-d ₅ ) δ: 0.80 (3H, s, 18-CH3), 0.96 (3H, s, 19-CH3), 1.06 (3H, d, J = 7.0 Hz, 21-CH3), 1.14 (3H, d, J = 6.8 Hz, 27-CH3), 3.35 (1H, d, J = 10.9 Hz, 26β-H), 4.90 (1H, d, J = 7.6 Hz, galactose H -1), 5.26 (1H, d, J = 7.6 kPa, glucose H-1)

7) ¹³ C-NMR (75.5 MHz, pyridine-d ₅ ) δ: 30.96 (C-1), 27.01 (C-2), 75.22 (C-3), 30.96 (C-4), 36.96 (C-5 ), 26.80 (C-6), 26.44 (C-7), 35.56 (C-8), 40.29 (C-9), 35.26 (C-10), 21.17 (C-11), 40.34 (C-12) , 40.90 (C-13), 56.50 (C-14), 32.16 (C-15), 81.35 (C-16), 63.00 (C-17), 16.58 (C-18), 24.01 (C-19), 42.50 (C-20), 14.89 (C-21), 109.69 (C-22), 26.21 (C-23), 26.21 (C-24), 27.55 (C-25), 65.12 (C-26), 16.29 (C-27), 102.53 (gal C-1), 81.87 (gal C-2), 76.89 (gal C-3), 69.86 (gal C-4), 76.59 (gal C-5), 62.20 (gal C -6), 106.10 (glc C-1), 75.49 (glc C-2), 78.03 (glc C-3), 71.80 (glc C-4), 78.34 (glc C-5), 62.86 (glc C-6) )

Experimental Example 1: luteinizing hormone secretion promoting activity test

(Step 1) Pituitary Cell Culture

The pituitary gland was removed from Sprague-Dawley rats at 4 to 5 weeks of age under sterile manipulation, followed by collagenase (collagenase, Gibco co., Grand Island, NY) and hyaluronidase (hyaluronidase, Sigma co). , St. Louis, Mo.) rat rat pituitary cells were isolated.

The isolated pituitary cells were suspended in Dulbecco's Modified Eagle's medium containing 10% horse serum, 2.5% fetal bovine serum (FBS) and antibiotics, followed by 37 ° C. and 5% CO _2. The cells were cultured in a humidified condition.

(Step 2) luteinizing hormone secretion promoting activity experiment

The pituitary cells isolated in step 1 were cultured and then suspended in test solution. Timothy saponin A-III (10 μg / ml), Geum methanol extract (1 mg / ml), ether and butanol fractions (1 mg / ml) were added to pituitary cells in 1 ml each, and as a negative control medium (1% DMSO Pituitary cells were used. After incubation at 37 ° C. for 15 minutes, the supernatant was separated by centrifugation and stored at −70 ° C. to measure luteinizing hormone activity.

The luteinizing hormone measurement was performed using a rat LH RIA kit (rat LH RIA kit, Amersham, Buckinghamshire, England).

The luteinizing hormone contained in a constant amount of ¹²⁵ I-labeled luteinizing hormone and the test substance stored above was produced by different reactions of luteinizing hormone by a competitive reaction to an anti-LH antibody. An antibody complex (LH-Ab complex) was formed and precipitated by addition of a second antibody, and the corresponding concentration was calculated using a standard curve by measuring the radioactivity of ¹²⁵ I from the precipitation.

As a result, the luteinizing hormone concentration was 1.94ng / ml in the control group, whereas the luteinizing hormone concentration was shown in Table 1 in the experimental group treated with thymosaponin A-III, methanol extract and butanol fraction, respectively, as shown in Table 1 The secretion of luteinizing hormone was significantly increased by the treatment of A-III and extracts and fractions of the hair.

TABLE 1

Control Timothy saponin A-III treatment group (10 µg / ml) Jimo methanol extract treatment group (1 mg / ml) Jimmy Butanol fraction treatment group (1 mg / ml) Rat luteinizing hormone concentration (ng / ml) 1.94 5.94 21.63 4.17

In addition, as a result of measuring the concentration of luteinizing hormone secreted according to the concentration of thymosaponin A-III, the concentration of luteinizing hormone secreted since the concentration of thymosaponin A-III is 0.1 μg / ml or more rapidly increases Maximum effect was shown at 2 μg / ml (see FIG. 1).

Experimental Example 2 Oral Acute Toxicity in Rats

Acute toxicity experiments were performed using 6-week-old specific pathogen-free (SPF) SD rats. Two animals per group were suspended orally administered at a dose of 1000 mg / kg / ml and 100 mg / kg / ml, respectively, with the hair extract of the present invention and thymosaponin A-III suspended in 0.5% methylcellulose solution, respectively. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, hematological and halal biochemical tests were performed, and necropsy was performed to observe abdominal and thoracic organ abnormalities.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. As a result, both the extract and thymosaponin A-III of the present invention did not show toxicological changes in rats up to 1000 mg / kg and 100 mg / kg, respectively, and both were determined to be safe substances having a minimum lethal dose of 0.1 g / kg or more.

Formulation Example 1 Preparation of Injection Solution

Injection solutions containing 10 mg of the active ingredient were prepared in the following manner.

1 g of thymosaponin A-III, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

Timothy saponin A-III 1 g

0.6 g sodium chloride

0.1 g of ascorbic acid

Distilled Water Determination

Formulation Example 2: Preparation of Syrup

A syrup containing thymosaponin A-III of the present invention as an active ingredient 2% (weight / volume) was prepared by the following method.

Timothy saponin A-III, saccharin, and sugar were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, spices, ethanol, sorbic acid and distilled water was prepared and mixed therein. Water was added to this mixture to 100 ml.

The components of the syrup are as follows.

Timothy saponin A-III 2 g

Saccharin 0.8 g

25.4 g per

Glycerin 8.0 g

0.04 g of spices

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled Water Determination

Formulation Example 3 Preparation of Tablet

Tablets containing 15 mg of active ingredient were prepared by the following method.

250 g of thymosaponin A-III, 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid were mixed. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was prepared as a tablet.

The components of the tablet are as follows.

250 g of thymosaponin A-III

Lactose 175.9 g

180 g potato starch

32 g of colloidal silicic acid

10% gelatin solution

Potato Starch 160 g

50 g of talc

5 g of magnesium stearate

Formulation Example 4 Preparation of Health Drink

Instant sterilization by homogeneously mixing thymosaponin A-III with subsidiary ingredients such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%). Healthy drinks were prepared by packaging in small packaging containers such as glass bottles and plastic bottles.

As described above, the thymosaponin A-III of the present invention and the methanol extract, and the ether and butanol fractions thereof of the present invention act on the pituitary cells to promote the secretion of luteinizing hormone, and as a result of the toxicity test, the luteinizing hormone secretion accelerator is less toxic. It can be usefully used as. In addition, the thymosaponin A-III of the present invention and the ethanol extract including butyol fraction thereof may be used as a prophylactic or therapeutic agent for infertility, prostate cancer or breast cancer by promoting the secretion of luteinizing hormone.

Claims (5)

A luteinizing hormone secretagogue for the treatment of infertility, prostate cancer, breast cancer, polycystic ovary syndrome, chronic renal failure or Klinefelter syndrome, comprising thymosaponin A-III represented by the following formula (1): [Formula 1]

The luteinizing hormone secretagogue according to claim 1, wherein the thymosaponin A-III is extracted, separated and purified from the hair. The method of claim 1, wherein the thymosaponin A-III is Extracting the hairs with methanol or a mixed solvent of methanol and water to prepare the hair concentrate of methanol; Preparing the n-butanol fraction by fractionating the methanol concentrate of the timothy in the order of ether and n-butanol; Chromatographing the n-butanol fraction with a mixed solvent of ethyl acetate and methanol (ethyl acetate: methanol = 100: 0 to 92: 8 (v / v)) to obtain five fractions; and A second body of the five fractions prepared by chromatography with a mixed solvent of chloroform-methanol-water (chloroform: methanol: water = 7: 3: 1 (v / v / v)) Hormones that form hormones. delete delete

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