KR100702183B1 - Promotor of release of Luteinizing hormone comprising Liriope spicata extract or Ophiopogonin D - Google Patents

Abstract

The present invention relates to a luteinizing hormone secretagogue comprising the opiopogonin D, Macmundong extract or fraction as an active ingredient.

Opiopogonin D and Macmundong extract of the present invention act on the pituitary cells to promote the secretion of luteinizing hormone, it can be useful for the prevention and treatment of infertility, breast cancer, prostate cancer and the like.

Luteinizing hormone

Description

Promoter of luteinizing hormone comprising Liriope spicata extract or Ophiopogonin D}

1 is a diagram showing the effect of inducing rat luteinizing hormone secretion according to the concentration of opiopogonin D.

The present invention relates to a luteinizing hormone secretagogue comprising the opiopogonin D, Macmundong extract or fraction as an active ingredient.

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).

Limoope spicata is a perennial herb of the monocotyledonous liliaceae, distributed in Korea and mainly in the shade of mountains and fields. These pulses are used for the treatment of physical weakness, pulmonary tuberculosis, diabetes, chronic bronchitis, constipation, fever and cold in Korean medicine.

Existing studies on pharmaceuticals containing the extract of Macmundong extract show that the effect of promoting growth hormone secretion (Korean patent applications 2002-35767 and 2002-9752) and the treatment of neurological diseases (Korean Patent No. 449245) Is known.

In addition, it may be mixed with other herbal medicines to be used for the treatment of various diseases, for example, it may be included in the herbal composition of the new composition of the sulfur-free cheongsimwon similar composition that exerts an excellent effect on the circulatory system symptoms such as blood pressure (Korean patent) 234494), Cordyceps sinensis, cow sulfur, etc. may be included in the composition for treating diabetes comprising 17 herbal ingredients (Korean Patent No. 195886).

However, it is not known that opiopogonin D included in the mammundong extract and the mammundong extract has an effect of promoting luteinizing hormone secretion.

Therefore, the present inventors while studying the pharmacological activity of the extract, the medicinal extract and opiopogonin D (Ophiopogonin D) contained in it has an effect of promoting the secretion of luteinizing hormone is useful in the treatment of infertility, prostate cancer, breast cancer, etc. It was found that the present invention can be used to complete the present invention.

It is an object of the present invention to provide a luteinizing hormone secretagogue comprising the opiopogonin D as an active ingredient.

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

In order to achieve the above object, the present invention provides a luteinizing hormone secretagogue which contains opiopogonin D as an active ingredient.

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

In addition, the present invention provides a luteinizing hormone secretagogue comprising the butanol fraction obtained by extracting the aqueous layer obtained by adding distilled water and ether to the methanol extract of Macmundong with 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 opiopogonin D represented by the following formula (1) as an active ingredient.

Opiopogonin D 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 opiopogonin D of the present invention was used by extraction, separation and purification from wortmundong, and can be obtained by all conventional methods, and commercially available reagents can be used.

In addition, the present invention includes opiopogonin D, and includes the McMoon-dong methanol extract extracted by dissolving McMoon-Dong in methanol or a mixed solvent of methanol and water, and the McMoon-Dong butanol fraction obtained by extracting the obtained McMoon-Dong methanol extract using butanol.

In order to obtain opiopogonin D, first, Liriope spicata was dissolved in methanol or a mixed solvent of methanol and water, extracted, filtered, and the filtrate was concentrated under reduced pressure. Distilled water and ether were added to the obtained methanol extract to obtain an aqueous layer, followed by addition of butanol to obtain a butanol fraction. The butanol fraction obtained is purified using a column to yield opiopogonin D.

The present invention includes opiopogonin D, a lutein dong methanol extract comprising the same, and a progesterone-releasing hormone comprising the butanol fraction thereof as an active ingredient.

Opiopogonin D obtained in the present invention, Megmundong methanol extract comprising the same and butanol fraction thereof has the effect of promoting the secretion of luteinizing hormone. As shown in the following examples, when the opiopogonin D, Macmundong methanol extract, and butanol fraction thereof of the present invention were injected into the pituitary cells of rats, they showed more than 9-fold progesterone secretion-promoting activity compared to the control group. As concentration increased, luteinizing hormone secretion promoting effect increased.

As described above, opiopogonin D, mackmundong methanol extract including the same, and the 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

Opiopogonin D of the present invention, the pulse extract Methanol extract and the butanol fraction thereof including the same can be administered orally or parenterally during clinical administration and can be provided in the form of a general pharmaceutical formulation.

Opiopogonin D of the present invention, Macmundong methanol extract and its butanol fraction thereof may be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents. And diluents or excipients such as disintegrants and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as opiopogonin D, pulmonary dong methanol extract containing it, and the butanol fraction thereof, for example, starch. , Calcium carbonate, sucrose or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrene talc are also used. Oral liquid preparations include suspensions, solvents, 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 oral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilizers, 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, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The content in the preparation of the active ingredient according to the present invention may be appropriately selected depending on the absorbency, inactivation rate, excretion rate, age, gender and condition of the active ingredient in the body. In the present invention, in the case of the methanol extract of Macmundong and its butanol extract, it is 1 to 1000 mg / kg, and preferably 1 to 100 mg / kg. In addition, in the case of opiopogonin D, it is 0.01 to 100 mg / kg, and preferably 0.1 to 10 mg / kg. The Macmundong extract and opiopogonin D may be administered 1-3 times a day.

Macmundong extract and opiopogonin D of the present invention did not show toxicity change when orally administered 1000 mg / kg and 100 mg / kg in experimental mice, respectively, and the minimum lethal dose (LD50) was 20.61 ± 7.08g / Kg and opiopogonin D is 100 mg / kg, so all of them are safe substances of 0.1g / kg or more, and the minimum lethal dose is 0.1g / kg or more. Opogonin D can be administered stably to a living body.

In addition, opiopogonin D, Macmundong methanol extract or butanol fraction thereof of the present invention may also be provided in the form of a functional food, dietary supplement or special nutrition.

The above-mentioned functional food means the food which improved the functionality of a general foodstuff by adding the said opiopogonin D, macmoon-dong methanol extract, or its butanol fraction to a general foodstuff.

Distinguishing from functional foods, 'health supplement' or 'special dietary supplement' means the opiopogonin D, macmundong methanol extract or a butanol fraction thereof added to a general food, or a health prepared by encapsulation, powdering or suspension. As a food, it means that when ingested to bring a specific effect on health, unlike general medicine has the advantage that there is no side effect that can occur during long-term use of the drug as a food raw material.

The content of functional foods, dietary supplements and specialty health foods containing opiopogonin D, Macmundong methanol extract or butanol fraction thereof according to the present invention can be varied according to the food group used, the content of the pharmaceutical It is performed within the range of toxicity measured for use with the composition.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention.

Example 1 Preparation of Methanol Extract of Macmundong

6Kg of dry leaflet leaf Ligape spicata tubers were cut into small pieces and extracted three times with methanol using a reflux condenser for 3 hours. The extract was filtered and concentrated under reduced pressure to obtain 1056 g of methanol extract.

Example 2 Preparation of Macmundong Butanol Fraction

Distilled water and ether were added to the methanol extract obtained in Example 1, and the ether soluble part was removed after fractionation in a separatory funnel. The aqueous layer was extracted three more times with n-butanol saturated with water again to obtain 92 g of Macmundong butanol fraction.

Example 3 Preparation of Opiopogonin D

The n -butanol fraction obtained in Example 2 was eluted with a solvent of water-saturated ethyl acetate-gradient using silica gel chromatography, and divided into eight fractions. Fraction 5 was eluted with a solvent of chloroform-methanol-water (52: 28: 8, lower portion) to obtain a crude fraction of opiopogonin D which was then RP-18 (MeOH: H2O = 8: 2) coulmn. Was carried out to obtain the pure compound.

1) Liebermann-Burchard test: positive.

2) Melting Point: 263-265 ℃

1 H-NMR (250 MHz, pridine- d5 ) δ 0.66 (3H, d, J = 4.1 Hz, 27-CH3), 0.84 (3H, s, 18-CH3), 1.05 (3H, d, J = 6.6 Hz, 21-CH3), 1.40 (3H, s, 19-CH3), 1.50 (3H, d, J = 6.3 Hz, fucose CH3), 1.7 (3H, d, J = 6.0 Hz, rhamnose CH3), 4.65 (1H, d, J = 8.6 Hz, anomeric H of fucose), 4.98 (1H, d, J = 7.6 Hz, anomeric H of xylose), 5.57 (1H, d, J = 7.9 Hz, H-6), 6.36 (1H, brs, anomeric H of rhamnose

13C-NMR (62.9 MHz, pridine- d5 ) δ84.5 (C-1), 38.0 (C-2), 68.2 (C-3), 43.8 (C-4), 139.5 (C-5), 124.7 ( C-6), 32.0 (C-7), 33.0 (C-8), 50.5 (C-9), 50.5 (C-10), 23.9 (C-11), 40.4 (C-12), 40.1 (C -13), 57.1 (C-14), 31.7 (C-15), 81.0 (C-16), 63.0 (C-17), 16.8 (C-18), 14.9 (C-19), 42.8 (C- 20), 15.0 (C-21), 109.2 (C-22), 32.4 (C-23), 29.2 (C-24), 30.5 (C-25), 66.7 (C-26), 17.1 (C-27 ), 100.5 (fucose C-1), 74.6 (fucose C-2), 85.5 (fucose C-3), 72.5 (fucose C-4), 71.0 (fucose C-5), 17.3 (fucose C-6), 101.7 (rhamnose C-1), 72.7 (rhamnose C-2), 72.5 (rhamnose C-3), 74.2 (rhamnose C-4), 69.3 (rhamnose C-5), 19.1 (rhamnose C-6), 106.6 ( xylose C-1), 73.4 (xylose C-2), 78.3 (xylose C-3), 70.8 (xylose C-4), 67.0 (xylose C-5)

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 aseptic manipulation, followed by collagenase type (Gibco co., Grand Island, NY) and hyaluronidase (hyaluronidase). Sigma co., St. Louis, Mo.) rat rat pituitary cells (rat pituitary cells) were isolated. The isolated pituitary cells were suspended in DMEMbe'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 culture was carried out in a humidified condition of.

(Step 2) luteinizing hormone secretion promoting activity experiment

The pituitary cells isolated in step 1 were cultured and then suspended in test solution. Opiopogonin D (100 µg / ml), Methanol extract (1 mg / ml) of Macmundong and butanol fraction (20 µg / ml) of Macmundong were added to pituitary cells 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 in the above-mentioned substance was produced by a competitive reaction to the anti-LH antibody. Formation hormone-antibody complex (LH-Ab complex) was formed and precipitated by addition of a secondary antibody (second antibody), and then the concentration was calculated using a standard curve by measuring the radioactivity of ¹²⁵ I from the precipitation.

As a result, in the control group, luteinizing hormone concentration was 1.94 ng / ml, whereas in the experimental group treated with Methanol extract (1 mg / ml) and butanol fraction (20 μg / ml) of Macmundong, 2.36 ng / ml and 2.56 ng / ml, In the experimental group treated with opiopogonin D (100 µg / ml), it was 17.63 ng / ml, indicating that the secretion of luteinizing hormone was significantly increased by the butanol fraction of opiopogonin D and opiopogonin D treatment.

TABLE 1

Control Opiopogonin D treated group (100 µg / ml) Macmundong methanol extract treatment group (1mg / ml) Macmundong butanol fraction treatment group (20μg / ml) Rat luteinizing hormone concentration (ng / ml) 1.94 17.63 2.36 2.56

In addition, as a result of measuring the concentration of luteinizing hormone secreted according to the concentration of opiopogonin D, the concentration of luteinizing hormone secreted from when the concentration of opiopogonin D is 1 μg / ml or more rapidly increased to 100 ㎍ / ml showed the maximum effect (see FIG. 1).

Experimental Example 2  : Acute Toxicity of Oral Administration in Rats

Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats. Two animals per group were suspended in 0.5% methylcellulose solution of Macmundong extract and opiopogonin D of the present invention, respectively, and administered orally at a dose of 1000 mg / kg / ml and 100 mg / kg / ml, 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, the pulmonary tract extract and opiopogonin D of the present invention did not show toxicological changes in rats up to 1000 mg / kg and 100 mg / kg, respectively, and the minimum lethal dose (LD50) was 20.61 ± 7.08g / Kg and opiopogonin D is 100 mg / kg, all were determined to be a safe substance with a minimum lethal dose of 0.1 g / kg or more.

Preparation Example 1 Manufacturing Method of Capsule

5 mg opiopogonin D or 10 mg ganmundong extract was mixed with 14.8 mg lactose, 10.0 mg polyvinyl pyrrolidone and 0.2 mg magnesium stearate. No. solid the mixture using a suitable device. Filled in 5 gelatin capsules.

The components of the powder and capsules are as follows.

Opiopogonin D · 5.0 mg 5.0 mg

(Mangmun-dong Extract ... 10 mg) ·············

Lactose ... 14.8 mg

10.0 mg of polyvinylpyrrolidone

Magnesium Stearate 0.2 mg

Preparation Example 2 Preparation of Beverages

It was prepared by mixing 0.1 g of opiopogonin D or 0.2 g of Macmundong extract, vitamin C, powdered vitamin E, ferric nitrate, zinc oxide, nicotinic acid amide, vitamin A, vitamin B ₁ and vitamin B ₂ .

The components of the beverage are as follows.

Opiopogonin D 0.1 g

(Mangmun-dong extract ... 0.2 g)

15 g of vitamin C

7.5 g of powdered vitamin E ··················

Ferrous iron ·························· 19.75 g

Zinc Oxide ······ 3.5 g

Nicotinic acid amide 3.5 g

0.2 g of vitamin A

Vitamin B ₁ 0.25 g

0.3 g of vitamin B ₂

* Water ···························

As described above, opiopogonin D of the present invention and the pulsed Methanol extract and its butanol fraction containing the same act on the pituitary cells to promote the secretion of luteinizing hormone, and as a result of the toxicity test, it is useful as a luteinizing hormone secretagogue. Can be used. In addition, the opiopogonin D of the present invention and the pulse extract Methanol extract and its butanol fraction comprising the same can be used as a prophylactic or therapeutic agent for infertility, prostate cancer or breast cancer by promoting the secretion of luteinizing hormone.

Claims (5)

Progesterone-releasing hormone for the treatment of infertility, prostate cancer, breast cancer, polycystic ovary syndrome, chronic kidney failure or Klinefelter syndrome, comprising opiopogonin D represented by the following formula (1) as an active ingredient: [Formula 1]

The luteinizing hormone secretagogue according to claim 1, wherein the opiopogonin D is extracted, separated and purified from pulmonary sinus. The method of claim 1, wherein the opiopogonin D is Extracting Methanol with methanol or a mixed solvent of methanol and water to prepare a methanol extract of Methanol; Extracting the aqueous layer obtained by distilling the methanol extract of mackmundong with distilled water and ether to prepare n-butanol fraction of mackmundong; Chromatographing the Macmundong n-butanol fractions with ethyl acetate and methanol solvent to prepare eight fractions; Chromatographing the fifth of the eight fractions with a mixed solvent of chloroform-methanol-water (chloroform: methanol: water = 52: 28: 8) to prepare a crude fraction of opiopogonin D; And Progesterone-forming hormone secretagogue, characterized in that the crude fraction of opiopogonin D prepared by chromatography with a mixed solvent of methanol and water (methanol: water = 8: 2). delete delete

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