KR100257840B1 - Use of stigmast-4-en-6beta-ol-3-on and its derivatives as growth hormone releasing factor - Google Patents

Abstract

PURPOSE: A stigmast-4-en-6β-ol-3-one(formula 1) and derivatives thereof, and Astragali Radix extract containing the same are provided, which can be used as growth hormone secretion stimulant. CONSTITUTION: A process for the preparation of Astragali Radix extract comprises the steps of: pulverizing the Astragali Radix into powder, adding 80% ethanol, and extracting four times per a week at room temperature(A); adding distilled water to the ethanol extract, and processing systematic separation using n-hexane(B), ethyl acetate(C), and n-butanol(D); and separating into each fractions and water layer(E); extracting for 2.5hrs at 90deg.C with boiling water(F); adding distilled water to the residue, and leaving for 2days at room temperature and extracting again; processing primary silicagel column chromatography(chloroform:acetone = 9:1) the extract to separate into 25 small fractions; processing secondary silicagel column chromatography(chloroform:methanol = 9:1) the 15th fraction to get refined fraction, and concentrating under decompression in a water bath of 40deg.C; adding chloroform to dissolve, and leaving at room temperature for 24hrs to get crystal; and refining the crystal to get stigmast-4-en-6β-ol-3-one compound.

Description

Use of Stigmas-4-en-6β-ol-3-one and its derivatives as growth hormone secretion stimulating factor

The present invention relates to the use of a compound of formula 1 (Stigmast-4-ene-6β-ol-3-one), a derivative thereof, and an Astragali Radix extract comprising the same, as a growth hormone secretion factor. .

Formula 1

Growth hormone (GH) is a protein secreted by the pituitary gland of animals and is a peptide hormone consisting of 191 amino acids. These growth hormones have been isolated from animals for a long time and have been used mainly for the treatment of dwarfism patients, and have a variety of functions because they inhibit protein loss and delay physiological aging in older adults or the elderly. It has been reported that it can be used for diseases (Horber, FF and Haymond, MW, 1990, J. Clin. Invest., 86, 265-272). In other words, continuous administration of growth hormone increases the weight, weight of muscles and fats, thereby preventing diseases caused by aging, and even when administered temporarily, insulin and IGF-1 (Insulin-like growth factor-1) in plasma ), It is known to decrease protein concentration in urine (Kohrt, WM et al., 1992, Med. Sci. Sports Exerc., 24, 832-837; Zachweija, JJ et al., 1994, Am. J. Physiol., 266 (Endocrinol. Metab. 29), E840-844). In addition, growth hormone has been reported to increase the weight and size of the liver and improve the synthesis of proteins in terminal tissues such as muscle, bone and connective tissue (Kaiser, FE et al., 1991, J. Am. Geriatr. Soc., 39, 235-240; Marcus, R. et al., 1990, J. Clin. Endocrinol. Metab., 70, 519-527).

Until now, the growth hormone is mainly extracted from the pituitary gland of the carcass, there is a risk that it can enter the patient from the causative organism of other diseases in the separation process, its use has been very limited. However, with the recent development of genetic engineering, it is possible to mass produce and supply growth hormones using microorganisms and to reduce the risk of infection of other diseases, and protein preparations containing recombinant growth hormones occupy most of the market for dwarfism. Recombinant growth hormone is so popular that self-treatment using a syringe is usually administered by intramuscular injection. As such, although recombinant growth hormone can be mass produced, attempts have recently been made to develop oral formulations due to the considerable cost required for a certain treatment period and the inconvenience of using injections.

The basic requirement for growth hormone preparations for oral administration is to have high absorption rate in the body and to have biological activity that can selectively stimulate growth hormone secretion. In order to have a high absorption rate in the body by oral administration, the smaller the molecular weight is advantageous, and to perform the physiological activity in the body smoothly, it must have a special physical and chemical properties.

Representatives of these basic requirements that have been recently studied and developed up to clinical trials include organically synthesized L-692,429 ( Horm. Res., 40: 109-115, 1993) and L-163,191 ( PNAS, 92). : 7001-7005, 1995), and the peptide compounds GHRP-2 ( Endocrinology, 140: R9-R13, 1994), GHRP-1 ( J. Neuroendocrinology, 6: 185, 1994) and hexarelin (Hexarelin; His- D-2-methyl-Trp-Ala-Trp-D-Phe-Lys-NH2). The materials are newly researched and developed using the basic structure of GHRP-6 (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2), a peptide compound commonly known in the early 1980s.

Recently, as an approach for developing a new material, a method for mass screening of candidate materials using a chemical library has been in the spotlight. In addition, a peptide library composed of short peptides is also used, and a natural library composed of various substances in nature can also be used to develop such a new substance. Developing new drugs using these diverse and extensive libraries is considerably more efficient than traditional approaches in terms of the time and cost of development, so developed pharmaceutical companies have long been active in developing new drugs using their libraries. come. Specifically, L-163,191 and hexarelin may be referred to as substances developed by this approach.

Originally it varies from animal to animal, but in humans there is a growth hormone releasing hormone (GHRH) consisting of 44 amino acids. GHRP-6 is a useful synthetic peptide that is less effective than GHRH in biological activity but highly compressed in terms of molecular weight size (Korean Journal of Endocrinology, 10 (1): 1-9, 1995). Although much research has been conducted on the detailed mechanism of action of GHRP-6, no clear conclusions have been made yet. Recently, however, it has been reported that receptors of GHRP are present and found in the hypothalamus and pituitary gland ( Science, 273 (5277): 974, 1996). In addition, it is known that protein kinase C is partially involved in the action of the substance or calcium ions are introduced during signaling to induce growth hormone secretion.

L-692,429 ( Science, 260: 1640-1643, 1993), developed by Merck, USA, has been shown to selectively secrete growth hormone, and has shown effective body absorption even in the experimental phase of animals. However, human test results were different from those of animals. Therefore, L-163,191 has been developed to improve this problem, which is characterized by high physiological activity during oral administration and is currently in clinical trials based on the results of animal experiments using dogs ( PNAS, 92: 7001-7005, 1995). Research on these substances suggests that substances that stimulate the pituitary gland to secrete growth hormone may be useful clinically as growth hormone replacements, while developing new growth hormone secretion factors is not easy. Shows.

Astragali Radix ( Astragalus membranaceus Bunge) is a herb that belongs to the legume (Leguminosae), and according to the explanation of Ishijin of the New Farm, the color is yellow and is named after the long term to protect the human body. In Korea, Astragalus roots have been used a lot as a medicine after ginseng in the past, and Astragalus has been used as a tonic, diuretic, and antiperspirant. In particular, oriental medicine is used for hundreds of prescriptions such as Bogiikgitang, Hwanggigunjungtang, Sipjeondaebotang, Kamimidaebotang, Palbohoechuntang, and Cheongseoikgitang.

In addition to Korea, A. chinensis L .; A complanatus R. Br .; A. sinicus ; A. chrysopterus ), Inner Mongolia ( A. membranaceus var. Mongholicus), Japan ( A. aitosensis; Hedysarum iwawoki Hara; A. adsurgens Pallas subsp fujisanensis Kita;.. A. polybotrys Hands-Mazz), Egypt (A. spinosus Vahl; A. trigonus) , Bulgaria (A. onobrychis) distribution, in our country, Astragalus quality in the Central province of Gangwon Jeongseon, etc. It is known that a lot is produced.

To date, triterpenoid glycoside strains and isoflavonoid strains among flavonoids have been mainly reported as components. Specifically, polysaccharides isolated from Astragalus have been reported to improve the function of T cells in cancer patients, and triterpenoids and flavonoids are known to have antioxidant, anti-inflammatory and blood pressure lowering activities (Huaping , L., Yau, ZO and Bo, G., 1994, Chin.H. Plast.Surg . And Burns, 3 (102), 138-141; Geng, CS, 1986, Chin.J. Mod. Dev.Trad Med., 6 (1), 62-64); Zhang, YD et al., 1992, Acta Pharm. Sin., 27 (6), 401-406; Harborne, JB and Baxter, H., 1993, Phytochemistry Dictionary, Taylor & Francis, London, Washington DC., Pp 673; Tang, W. and Eisenbrand, G., 1992, Chinese Drugs of Plant Origin, Springer-Verlag, Berlin, Heidelberg, New York, pp. 191).

In addition, the present inventors have already described the 2-benzendicarboxylic acid diisononylester, β-sitosterol, 3, in the fraction obtained by partially purifying the alcohol extract of the Astragalus root with n-hexane. -o-β-D-galactopyranosyl-β-sitosterol (3-o-β-D-galactopyranosyl-β-sitosterol), stigmas-4-en-6β-ol-3-one (stigmast-4 en-6β-ol-3-one), 7-hydroxy-4'-methoxy-isoflavone and 9Z, 12Z-octadecadienoic acid (9Z, 12Z -octadecadienoic acid) has been reported separately (Kim, JS and Kim, CS, 1997, Kor. J. Pharmacogn., 28 (2): 75-79; Kim, JS et al., 1996, Kor. J. Pharmacogn., 27 (4): 336-341).

Therefore, the present inventors studied their physiological activity by using various components isolated from Astragali Radix, and thus, the stigmas-4-ene compound, which has been identified only in structure and whose use is unknown, has been known so far. The present invention was completed by confirming that -6β-ol-3-one and an extract containing the same act as growth hormone secretory stimulating factors on pituitary cells to increase the production of growth hormone.

It is an object of the present invention to provide a use of stigmas-4-ene-6β-ol-3-one and derivatives thereof, and astragalus extract containing them as growth hormone secretion stimulating factors.

In order to achieve the above object, the present invention is a pharmaceutical composition for growth hormone secretion stimulating factor using the stigmas-4-en-6β-ol-3-one and its derivatives as an active ingredient, and extracted with a lower alcohol alcohol It is added and then fractionated with n-hexane to provide a horticultural extract for growth hormone secretion stimulating factor comprising stigmat-4-ene-6β-ol-3-one and derivatives thereof.

Hereinafter, the present invention will be described in detail.

The present invention provides the use of stigmas-4-ene-6β-ol-3-one and its derivatives of the following formula (1) as growth hormone secretion stimulating factors.

Formula 1

The present invention isolates the stigmas-4-en-6β-ol-3-one from Astragali Radix extract, and is a growth hormone of various astragalus extracts and stigmas-4-ene-6β-ol-3-one. The activity as a secretory stimulatory factor is confirmed.

Specifically, sulfuric acid is extracted with a lower alcohol, suspended with distilled water, and then extracted with the addition of an organic solvent to produce the sulfuric acid extract. At this time, ethanol is preferably used as the lower alcohol, and n-hexane, ethyl acetate, n-butanol, or the like is preferably used as the organic solvent.

In addition, by extracting the sulfur group with a lower alcohol, suspended in distilled water and then extracted by adding n-hexane as an organic solvent and repeated adsorption chromatography stigmat-4- of the formula (1) has not been reported so far En-6β-ol-3-one is isolated ( J. Nat. Prod., 53: 1430-1435, 1990; Phytochemistry, 28: 2859-2861, 1989). At this time, ethanol is preferably used as the lower alcohol, and silica gel column chromatography is preferably used as the adsorption chromatography.

The present invention is measured by the growth hormone secretion stimulation activity such as the Astragalus extract and stigmas-4-en-6β-ol-3-one by a double antibody RIA method using pituitary cells ( Tanaka, T. et al., 1983, J. Clin.Endocrinol., 56 (1), 18-20; Wroblewski, VJ et al., 1991, Endocrinology, 129 (1), 465-474).

As a result, all of the yellow ethanol extract was separated from the organic ethanol extract of n-hexane, ethyl acetate, n-butanol and the like, and the growth hormone secretion activity was excellent (see Table 3). In addition, the stigmat-4-ene-6β-ol-3-one and derivatives thereof of the present invention showed excellent growth hormone secretion stimulating activity even at low concentrations (see Table 4).

In order to use the compound of the present invention prepared by the above process as a therapeutic agent, it may be prepared by a known method in the pharmaceutical field, and may be a carrier or excipient (pharmaceutically), itself or pharmaceutically acceptable. It may be prepared and used in the form of a powder, granules, tablets, capsules or injections by mixing with diluents and the like.

The human dose of the active ingredient according to the present invention is appropriately selected according to the absorption of the active ingredient in the body, the rate of water activation and excretion, the age, sex and condition of the patient, the severity of the disease to be treated, but generally 1 About 5 to 55 μg per day, but about 10 to 30 μg is preferred. Therefore, the unit dosage form of the present invention is prepared to have a content of 1-10 μg of the active substance of the present invention in consideration of the above-mentioned effective amount range. Preferably it is formulated to contain 2-5 μg. The unit dosage form thus formulated may be administered several times at regular intervals or by using a specialized dosing method according to the judgment of an expert who monitors or observes the administration of the drug as needed and the needs of the individual. It may be administered once to three times a day.

Hereinafter, the present invention will be described in detail by examples.

The following examples illustrate the invention, but the scope of the invention is not limited by the examples.

Example 1 Preparation of Astragalus Extract

Astragalus roots grown in Sindong-eup, Jeongseon-gun, Gangwon-do, South Korea were directly purchased and evaluated. Powdered yellow roots were extracted and 80% aqueous ethanol was added and extracted four times at room temperature one week apart (A). Distilled water was slightly added to the ethanol extract, and the system was separated in the order of n-hexane (B), ethyl acetate (C), and n-butanol (D), and these were divided into a fraction layer and a water layer (E), respectively. In addition, it was extracted for 2.5 hours at 90 ℃ with water to cut it again (F), or distilled water was added to the residue was left at room temperature for 2 days and then extracted again. Double n-hexane fractions (40 g) were subjected to primary silica gel column chromatography (fixed phase: silica gel 60 (Merck No. 7734); size 1300 * 5.0 cm; mobile phase: chloroform: acetone = 9: 1 → 1: 1 → 100% Ethanol; flow rate 3.8 ml / min) and pre-separated into 25 subfractions. The 15th subfraction was subjected to secondary silica gel column chromatography (fixed phase: silica gel 60 (Merck No. 9385); size 63.0 * 2.0 cm; mobile phase: chloroform: methanol = 9: 1; flow rate 3.0 ml / min → 1.0 ml /Min→3.0 ml / min) to obtain a somewhat purified fraction, which was collected and concentrated under reduced pressure in a water bath of about 40 ℃. To the mixture was added 2-3 drops of chloroform, which were dissolved and stored at room temperature and 4 ° C for 24 hours to precipitate white crystals. This was washed with methanol to separate the mother liquor from the yellow liquid, and the white crystals were dissolved in chloroform to repeat the above process to obtain an amorphous powder. This was placed on a TLC plate (mobile phase: methylene chloride: ethyl acetate = 7: 3) and sprayed with an anisealdehyde-sulfuric acid reagent and heated to yellow color, but turned to scarlet. 4.4 mg of -en-6β-ol-3-one was obtained.

Example 2 Physicochemical Characteristics of Compounds of Formula 1

(1) Name of the compound: stigmast-4-en-6β-ol-3-one

(2) the appearance of the substance: white amorphous powder

(3) Melting Point: 180 ℃

(4) Molecular weight: 428 (C ₂₉ H ₄₈ O ₂ )

EIMS: m / z (rel. Int.) 428 [M] (15), 410 [MH ₂ O] ⁺ , 385, 287 [M-side chain] ⁺ (16), 245 [287-ring D] ⁺ ( 26), 227 [245-H ₂ O] ⁺ (15), 152, 121, 107, 95, 81, 69, 57 (59), 55 (93)

(5) hydrogen nuclear magnetic resonance spectrum

Hydrogen nuclear magnetic resonance spectra measured using chloroform-d (CDCl ₃ ) as a solvent and tetramethylsilane as a standard are shown in Table 1.

Hydrogen number δ (ppm) Redundancy J (Hz) ¹ H- ¹ H-cosy 4 5.75 s Of Of 6 4.28 t 1.92 H7 OH (disappears when water is added) 4.60 s Of Of 18 0.65 s Of Of 9 1.31 s Of Of 21 0.75 d 6.4 H20 26 0.86 d 6.4 H25 27 0.77 d 6.4 H25 29 0.78 t 7.11 H28

(6) carbon nuclear magnetic resonance spectrum

The results of carbon nuclear magnetic resonance spectra measured using chloroform-d (CDCl ₃ ) as a solvent and tetramethylsilane as a standard are shown in Table 2.

Carbon number δ (ppm) DEPT 13C-1H-cosy δ (ppm) Carbon number One 37.09 CH2 2 34.24 CH2 3 200.41 C 4 126.31 CH 5.75 H4 5 168.47 C 6 73.16 CH 4.28 H6 7 38.52 CH2 8 29.72 CH 9 53.63 CH 10 37.98 C 11 20.97 CH2 12 39.60 CH2 13 42.51 C 14 55.89 CH 15 24.14 CH2 16 28.15 CH2 17 56.07 CH 18 11.98 CH3 0.65 H18 19 19.48 CH3 1.31 H19 20 36.10 CH 21 18.74 CH3 0.75 H21 22 34.10 CH2 23 26.20 CH2 24 45.85 CH 25 29.18 CH 26 19.78 CH3 0.86 H26 27 19.02 CH3 0.77 H27 28 23.08 CH2 29 11.95 CH3 0.78 H29

<Example 3> Growth hormone secretion stimulation activity analysis using Astragalus extract

In order to examine the growth hormone secretion stimulation activity of each Astragalus extract fraction obtained in Example 1, rat pituitary cells were cultured in a 24-well plate at 1.5 × 10 cells / ml for 5 days. Next, 100 μl of each fraction was dissolved in 1 ml of dimethylsulfooxide (DMSO), added to each well, incubated for 15 minutes, and the growth hormone concentration in the medium was measured by a double antibody RIA method (Tanaka, T. et al., 1983, J. Clin.Endocrinol., 56 (1), 18-20; Wroblewski, VJ et al., 1991, Endocrinology, 129 (1), 465-474). At this time, the standard curve for growth hormone was examined at a concentration ranging from 10 ng / ml to 121 pg / ml, and rat growth hormone including ¹²⁵ I labeled growth hormone, rat growth hormone monkey antiserum, and rat growth hormone as a kit. Assay kits (NIDDK, Harbor-UCLA Medical Center, CA, USA) were used. Next, monkey antibody IgG (CAPPEL) to goat antibody IgG and polyethylene glycol (PEG) at a final concentration of 6% were added as a secondary antibody to precipitate an immunocomplex and measure radioactivity contained in the precipitate.

The activity of releasing growth hormone from each fraction of the Astragalus extract investigated by the above process is shown in Table 3 as the radioactivity contained in the growth hormone, from which fraction B, fraction C and fraction D are compared with Bonferonni multiple (Bonferonni multiplex) comparison; P <0.01), the growth hormone secretion stimulation activity is superior to the reference.

Growth Hormone Secretion Stimulation Activity Using Astragalus Extract Fraction Concentration (mg / ml) Growth hormone (ng / ml: mean ± SE; n = 4) standard - 4.75 ± 0.55 A 2.7 6.19 ± 0.98 B 33.5 119.40 ± 27.16 C 21.5 107.90 ± 28.23 D 9.3 112.86 ± 7.39 E 3.8 18.22 ± 11.48 F - 4.52 ± 0.62

Example 4 Growth Hormone Secretion Stimulation Activity of the Compound of Formula 1

According to the present invention, the n-hexane fraction is separated into 25 subfractions by performing silica gel column chromatography in the process of Example 1, and the compounds contained in each fraction are identified and the growth hormone secretion stimulating activity is performed using the same. It measured by the process of Example 3. Specifically, the following B18 fraction was identified as formmononetin, and the B22 fraction was identified as stigmas-4-en-6β-ol-3-one, and these compounds were not soluble in the medium and DMSO and added to the medium at a concentration of 10 μg / ml. In addition, the remaining fraction of the compound was added at a concentration of 20 μg / ml to investigate the effect of growth hormone secretion stimulation activity by the method of the above practical example. The results are shown in Table 4.

Fraction Growth hormone (ng / ml) Fraction Growth hormone (ng / ml) standard 15.64161 B13 52.10497 B1 23.52482 B14 26.64114 B2 18.43854 B15 23.46763 B3 20.29841 B16 20.93033 B4 17.87618 B17 24.59034 B5 20.52722 B18 17.78540 (35.6) B6 18.16321 B19 13.81224 B7 22.44635 B20 26.36741 B8 21.00092 B21 20.05441 B9 18.1914 B22 26.89108 (53.8) B10 19.13619 B23 22.90420 B11 18.12996 B24 24.13575 B12 49.1624 B25 21.23102

As described above, the compound stigmat-4-en-6β-ol-3-one and derivatives thereof of the present invention and the hexane extract, ethyl acetate extract and n-butanol extract of Astragalus, including growth hormone secretion stimulating factor As it has excellent activity, it can be usefully used for the prevention of aging and disease.

Claims (2)

A pharmaceutical composition for growth hormone secretion stimulating factor comprising the compound of formula 1 (Stigast-4-ene-6β-ol-3-one) and derivatives thereof as an active ingredient. Formula 1 Astragali Radix, a growth hormone secretion stimulating factor containing stigmag-4-en-6β-ol-3-one and derivatives thereof, obtained by extracting with lower alcohol and adding water and then fractionating with n-hexane. extract.