KR100521845B1 - Growth hormone secretagogue containing dioscin - Google Patents

Abstract

The present invention relates to a therapeutic agent and a functional food caused by a deficiency of growth hormone containing a dioxin, a derivative thereof, or an extract of a plant comprising the same, and more specifically Eosin and its derivatives, and extracts containing it, have the effect of promoting growth hormone secretion, and also have low toxicity and can be usefully used as a therapeutic agent and a functional food caused by the lack of growth hormone.

Description

Growth hormone secretagogue containing dioxin {GROWTH HORMONE SECRETAGOGUE CONTAINING DIOSCIN}

The present invention relates to a therapeutic agent and a functional food caused by a deficiency of growth hormone containing dioxin, a derivative thereof or a plant extract comprising the same.

Growth hormone is a protein secreted by the animal's pituitary gland 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. They have various effects because they inhibit protein loss and delay physiological aging in older adults or the elderly. It can be used for the treatment of diseases [Horber, FF and Haymond, MW, J. Clin. Invest. , 1990, 86, 265-272. In addition, continuous administration of growth hormone to the human body increases the weight, muscle, and fat weight, thereby preventing diseases caused by aging, and even when administered temporarily, insulin and IGF-1 (Insulin-like growth factor) in plasma -1) is known to increase protein concentration in urine [Kohrt, WM et al., Med. Sci. Sports Exerc. , 1992, 24, 832-833; Zachweija, JJ et al., Am. J. Physiol. , 1994, 266 ( Endocrinol. Metab . 29), E840-844], in addition to increasing liver weight and size and improving the synthesis of proteins in terminal tissues such as muscle, bone and connective tissue [Kaiser, FE et al. al., J. Am. Geriatr. Soc. , 1991, 39, 235-240; Marcus, R. et al., J. Clin. Endocrino. Metab. , 1990, 70, 519-527.

Until now, studies have shown that children and adults with innate pituitary dysfunction are given growth hormone releasing hormone (GHRH) and arginine at the same time. Growth hormone response showed a higher effect, growth hormone response was higher in growth hormone deficient patients than many types of pituitary hormone deficiency, and the remaining vessels of the pituitary gland than patients with a complete pituitary system More effective results were obtained in patients with residual vascular components. Growth hormone's responsiveness to GHRH and arginine tended to decrease with age [ J. Clin. Endocrinol. Metab ., 2001, 86 (4), 1574-1579]. Ghrelin, an endogenous ligand for growth hormone secretagogue, was effective when administered to growth hormone deficient mice [ Diabetes , 2001, 50 (2), 227-232], when MK-677, an active growth hormone secretagogue, and aendronate, a potent bone resorption inhibitor, are used together, the formation rate is higher than that of arendronate alone. Maintain bone mineral density in postmenopausal women with osteoporosis. The effect on the IGF-1 level of the MK-677 and arendronate co-administration increased the IGF-1 level, alleviated the decrease in bone formation, and both the MK-677 and arendronate co-administration, The bone mineral density of the femoral neck was increased while the bone mineral density of the lumbar spine and hip was not increased. The metabolic effect of growth hormone through MK-677 reduced the direct inhibitory effect of arendronate on bone formation [ J. Clin. Endocrinol. Metab. , 2001, 86 (3), 1116-25].

In the past, growth hormone was mainly extracted from the cadaveric pituitary gland, but in the process it was found to cause the disease Creutzfeldt-Jakob disease in patients receiving growth hormone. As such, there is a risk that other diseases may be introduced into the patient during the separation process, and growth hormone isolated from the carcass has been limited in its use. With the development of genetic engineering, recombinant growth hormone produced using microorganisms can be produced and supplied in large quantities, and the risk of infection of other diseases is low, so the protein preparation including recombinant growth hormone occupies most of the market for dwarfism. The method of administering the recombinant recombinant growth hormone into the body is made by intramuscular injection using a syringe because the growth hormone itself is a huge protein and cannot be orally administered. As such, although recombinant growth hormone can be mass produced, attempts to develop oral preparations have been actively conducted in recent years due to the high cost required for a certain treatment period and the inability to use injection because it is impossible to orally administer. It is becoming.

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

Originally it varies from animal to animal, but in humans there is 44 amino acids of GHRH. The regulation of growth hormone secretion in the human body is a hormone called GHRH which promotes the secretion of growth hormone and somatostatin which inhibits the growth hormone secretion. It has been known to act to secrete

The GHRP-6 is a useful synthetic peptide that is less effective than the GHRH in biological activity but is highly compressed in terms of molecular weight [ Journal of Endocrinology , 1995, 10 (1), 1-9]. Since the development of GHRP-6, many researchers have reported that GHRP-6 does not act on the same receptors as GHRH, and the possibility of the presence of new receptors via these synthetic growth hormone secretagogues. Has been raised [ Endocrinology , 1989, 124, 2791]. This possibility is demonstrated by the discovery of the cDNA sequence of growth hormone secretagogue receptors present in the pituitary archromediate and hypothalamus of pigs and humans by Merck researchers. Science , 1996, 273, 974-971. This fact proved the synergism between the previously found growth hormone secretagogue and the synthetic compound growth hormone secretagogue, and is more interested in the identification of substances that exist in the body and are expected to play the same role as GHRH. This situation is increasing.

L-692, 429, developed by Merck, USA, is known to selectively secrete growth hormone [ Science , 1993, 260, 1640-1643], and showed effective body absorption in the experimental stage of animals. However, the results of human experiments, unlike that of the animal had a problem that the absorption is not very high upon oral administration. Therefore, L-163, 191, which improved these problems, was developed, which is a compound that increased the oral absorption rate, which is the biggest problem of the growth hormone secretagogues that have been developed previously, and has been reported to have an absorption rate of more than 60% when orally administered to dogs. Clinical trials are underway [ PNAS , 1995, 92, 7001-7005; Endocrinology , 1996, 137, 5284-5289. The results of these studies suggest that substances that stimulate the pituitary gland to secrete growth hormone can be used clinically as a substitute for growth hormone, while developing new drugs to treat diseases caused by deficiency of new growth hormone. It is not easy to do.

As part of such research, researches on finding substances that promote growth hormone secretion using natural extracts that are harmless to the human body are being conducted. Examples include alcoholic extracts of Astragalus root and 7-hydroxy-4'-methoxy-isoflavone, formononetin and stigmas-4-en-6β-ol-3-one (stigmast-4-en-6β-ol-3-one) and the like have been confirmed by the present inventors to stimulate growth hormone secretion [Korean Patent Registration Nos. 257840 and 251519; Kim, JS and Kim, CS, Kor. J. Pharmacogn. , 1997, 28 (2), 75-79; Kim, JS et al., Kor. J. Pharmacogn. , 1996, 27 (4), 336 ~ 341], relates to growth hormone secretagogue composition containing one or more extracts selected from the group consisting of extracts from raw paper, filamentous extract, lily extract, and gil dynasty extract [Korean Patent Application No. 1998] -0053921], a method for extracting growth hormone secretion stimulating factor from natural herbal medicines and the growth of water-soluble fast extract [National Patent Application No. 1998-014589] and the growth of glycoalkaloids asperosaponin H1 (asperosaponin H1) A study on hormone secretagogue [Korean Patent Application No. 1998-014590] has been reported.

On the other hand, Tobokyeong (土 茯 笭) is a herbal medicine that refers to the root diameter of Smilax china ( Smilax china ). Cheongmirae vine is a lily-leaved vine, which grows in the mountains of the Yellow Sea and sub-Pyeongnam, and is distributed in Japan, Manchuria, China, Taiwan, and the Philippines. The roots of these blue vines are called the footsteps in China and Tobok-ryeong in Korea, and the decoction solution is used to treat joint pain, muscle paralysis, diarrhea, dysentery, edema, morbidity, nausea, seed poisoning, and dental plaque.

Maple horse ( Dioscorea quinqueloba , 丹楓 ) is a perennial vine plant of the monocotyledonous genus, which is distributed in Korea and Japan, mainly in the mountain family. These maples are used as a medicine called Cheonsanryong (한 山 龍) in oriental medicine, soaked in blood and immersed in blood, and used for coronary artery disorder caused by blood, and cough and asthma caused by lung heat. It is used for boils and skin rashes because it sinks and lowers the heat of the blood. However, the Tobok-Ryong and Maple Horse have not yet been published for use in treating diseases caused by deficiency of growth hormone.

In addition, studies on diocin include inhibitory effects on the growth of human leukemia cancer cell lines ( Biol. Pharm. Bull. 2001, 24 (2), 159-162), antifungal and cytotoxic activity ( Biosci. Biotechnol. Biochem. 1998, 62 (10), 1904-1911), activity as antineoplastic agents ( Planta Med . 1996, 62 (6), 573-575), immunomodulating effect ( Anticancer Res. 1992, 12 ( 5), 1475-1478) and anti-cancer effects ( Anticancer Res. 1991, 11 (5), 1911-1917), but have not been reported for the treatment of diseases caused by deficiency of growth hormone. none.

Therefore, while the present inventors are studying a disease treatment agent caused by a deficiency of growth hormone, diocin and its derivatives, and plant extracts containing the same promote growth secretion of growth hormone, and also have low toxicity, so that the growth hormone deficiency The present invention was completed by finding that it can be used as a therapeutic agent and a functional food caused by the disease.

It is an object of the present invention to provide a plant extract containing diosin.

It is another object of the present invention to provide a therapeutic agent and a functional food caused by deficiency of growth hormone comprising diocin, a derivative thereof or a plant extract comprising the same as an active ingredient.

In order to achieve the above object,

The present invention provides a disease treatment agent and a functional food caused by a deficiency of growth hormone containing dioxin and its derivatives as an active ingredient.

In addition, the present invention provides a Tobok-Ryong extract and Maple Leaf Extract comprising dioxins and derivatives thereof.

In addition, the present invention provides a disease treatment agent and a functional food caused by a deficiency of growth hormone comprising the Tobok-Ryung extract or maple leaf extract containing the dioxins and derivatives thereof.

Hereinafter, the present invention will be described in detail.

The present invention includes a disease treatment agent caused by the deficiency of growth hormone containing a diocin and a derivative thereof represented by the following formula (1) as an active ingredient.

The dioxins may also be used as derivatives thereof, and derivatives of diosins which may be used in the present invention include those in which a hydroxy group of diosgenin and diosgenin which are easily obtained by hydrolysis is an ester form, and the like. an ester containing an alkyl group of C ₁ ~C _4. In addition, three kinds of prosapogenin obtained by partial hydrolysis of diocin are also available as derivatives of diocin.

Diosin of the present invention can be obtained by conventional organic synthesis and extracts of plants, for example, can be prepared from the extract of Tobokyeong or maple leaf as follows.

Smilax china is dissolved in methanol or a mixed solvent of methanol and water, extracted and filtered, and the filtrate is concentrated under reduced pressure. The methanol extract thus obtained is added with distilled water, followed by addition of the same amount of butanol, followed by shaking at room temperature, followed by standing overnight to obtain a butanol fraction. The butanol fraction obtained is melted using a column and divided into 10 small fractions. Six fractions of these were again eluted using a column to prepare diosin.

Further, Dioscorea quinqueloba was dissolved in methanol or a mixed solvent of methanol and water, extracted and filtered, and the filtrate was concentrated under reduced pressure. The resulting methanol extract is divided into six fractions using a column. Distillation was prepared by column chromatography of fraction 3 of which phase was increased in polarity with a solvent of water-saturated EtOAc-MeOH.

In addition, the present invention includes the Tobok-Ryong extract and Maple Leaf Extract comprising diosin and its derivatives.

Specifically, the Tobok-Ryung pulverized product is extracted in succession several times using a mixed solvent of methanol, methanol and water at room temperature to obtain a methanol extract. Preferably it is extracted three times continuously. The obtained methanol extract was concentrated under reduced pressure to obtain a methanol extract of Tobok-ryeong. Distilled water was added to the Methanol extract obtained in this manner, followed by addition of almost the same amount of butanol, followed by shaking at room temperature. The obtained butanol fraction was purified using a column, and then distilled under reduced pressure to obtain a butanol fraction.

The maple crushed product was extracted several times in succession using methanol, a mixed solvent of methanol and water using a reflux condenser to obtain a methanol extract. Preferably it is extracted three times continuously. The methanol extract obtained was concentrated under reduced pressure to obtain a maple leaf methanol extract.

In addition, the present invention includes a disease treatment agent caused by the deficiency of growth hormone containing dioxin and derivatives thereof, and Tobokyeong or maple leaf extract containing the same as an active ingredient.

Diosine and its derivatives obtained in the present invention, and the Tobok-ryeong methanol extract, butanol fraction and maple leaf methanol extract thereof, have an effect of promoting growth hormone secretion. As shown in the following examples, when the diocin of the present invention is injected into the pituitary cells of rats, it exhibited more than 30-fold secretion-stimulating activity with respect to the control group, and growth hormone secretion promoting effect was increased as the concentration of diocin was increased. At this time, ED ₅₀ was 1.14 × 10 ⁻⁵ M.

In addition, 10 μg / kg of diosin was administered to SD rats and blood was collected by measurement of the growth hormone of the isolated plasma. After 10 minutes of administration, compared to the control group, growth hormone showed a threefold increase. After ˜30 minutes it increased by 2-6 times compared to the control (see Table 3 and FIG. 1 ).

Diosin, its derivatives, Tobok-Ryong extract and Maple horse extract as described above activate the secretion of growth hormone. The growth hormone thus promoted is used for various purposes, as known by those skilled in the art. Various uses of these growth hormones can be summarized as follows: promoting growth hormone secretion in the elderly; Treatment of adults lacking growth hormone; Inhibition of glucocorticoid side effect catabolism, treatment of osteoporosis, promotion of immune system, promotion of wound healing, promotion of bone fracture regeneration, treatment of growth inhibition, treatment of acute or chronic kidney failure or insufficiency, physiologically short stature Iv) treatment, treatment of short stature with chronic diseases, treatment of growth inhibition by obesity and obesity, including children with growth hormone deficiency, due to Prader-Willi syndrome and Turner syndrome Treatment of growth inhibition; Reduction in length of hospitalization to promote recovery after major surgery such as gastrointestinal surgery or patients with discomfort in part of the body; Treatment of intrauterine growth inhibition, skeletal dysfunction, hypercortisone and Cushing's syndrome; Replacement of growth hormone in stressed patients; Treatment of bone dysplasia, nunan syndrome, sleep disorders, Alzheimer's disease, wound healing delays and psychosocial loss; Treatment of respiratory disorders and ventilator dependence; Attenuation of protein catabolism after major function; Treatment of malabsorption syndrome, reduction of cachexia and protein loss due to chronic diseases such as cancer or AIDS; TPN (total parenteral nutrition) promotes patient weight gain and protein gain; Treatment of hyperinsulinemia, including Langerhans ischaemia; Adjuvant therapy to induce ovulation and to prevent and treat ulcers of the stomach and duodenum; Prevent stimulation of thymic function and age-dependent decrease in thymic function; Adjuvant therapy for hemodialysis patients; Treatment of immunosuppressed patients and enhancement of antibody responses after vaccination; Muscle strengthening, fluidity, maintenance of skin thickness, bioabnormality of metabolism, and enhancement of bioabnormality of kidneys in the elderly; Stimulation of osteoblasts, bone remodeling and cartilage growth; Treatment of neuropathic diseases such as vascular peripheral and drug-induced neurological disorders, Mulang-Barré syndrome, lateral sclerosis, multiple sclerosis, vascular seizures and dehydration; Treatment of aging disorders and stimulation of the immune system in homologous animals; Growth promoters and stimulation of fleece growth in sheep in livestock [US Patent No. 5,578,593; No. 5,767,085.

Diosin, its derivatives, Tobokyeong extract and maple leaf extract including the same can be administered orally or parenterally during clinical administration and can be provided in the form of a general pharmaceutical formulation.

Diosin and its derivatives of the present invention, Tobokyeong extract and maple leaf extract including the same can be administered in a variety of oral and parenteral formulations in the actual clinical administration, when formulated, commonly used fillers, extenders, binders, It is prepared using diluents or excipients such as wetting agents, disintegrating agents, surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, for example, in dioxins and derivatives thereof, pulmonary methanol extracts including the same, and butanol fractions thereof. Starch, calcium carbonate, sucrose or lactose, gelatin and the like are mixed and prepared. 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 can be appropriately selected depending on the absorbency, inactivation rate, excretion rate, age, sex and condition of the user in the body. In the present invention, it is 1 to 1000 mg / kg, preferably 1 to 100 mg / kg, in the case of the Tobok-Ryong extract and the maple leaf extract. Moreover, in the case of the said diosin, it is 0.01-100 mg / kg, Preferably it is 0.1-10 mg / kg. The diosin, its derivatives, Tobokyeong extract and maple leaf extract comprising the same may be administered 1-3 times a day.

The Tobok-Ryung extract of the present invention, the maple leaf extract and diocin did not show the toxicity change upon oral administration of 1000 mg / kg and 100 mg / kg in experimental mice, respectively, and the minimum lethal dose (LD ₅₀ ) for oral administration was Maple leaf extract is 10 g / kg, it can be seen that the bio stability is very high, such as diocin 100 mg / kg or more, therefore, the Tobokyeong extract, maple leaf extract and diocin of the present invention can be administered stably to the living body have.

In another aspect, the present invention provides a functional food, health supplement food or special nutrition products containing the dioxin and its derivatives, Tobokyeong extract or maple leaf extract comprising the same as an active ingredient.

As used herein, the term "functional food" refers to a food product having improved functionality of a general food product by adding the diosin and its derivatives, Tobokyeong extract or maple leaf extract comprising the same.

In the present specification, a 'health supplement' or 'special dietary supplement', as distinguished from a functional food, is added to, or encapsulated, or powdered dioxin and its derivatives, or Tobokyeong extract or maple leaf extract comprising the same. As a health food manufactured with a suspension, it means that it induces a specific effect on health.However, unlike general medicine, it has the advantage that there is no side effect that can occur when the long-term use of the drug is made from food as a raw material. .

The content of functional foods, dietary supplements, and specialty health foods containing dioxins and their derivatives according to the present invention, Tobokyeong extract or maple leaf extract comprising the same can be varied according to the food group used, the content Is performed within the range of toxicity determined for use with the pharmaceutical 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 Tobok Ryong Methanol Extract

After extracting 3.6 kg of bokbokyeong continuously with 6 L of methanol three times at room temperature, the methanol solution obtained was concentrated under reduced pressure to obtain 345 g of bokbokyeong methanol extract.

Example 2 Preparation of Tobok Ryong Butanol Fractions

Tobokyeong methanol extract of Example 1 was taken in a separatory filter, and then 1400 ml of distilled water was added, and almost the same amount of butanol was added thereto. The solution was shaken at room temperature and left to stand overnight to obtain 107 g of Tobokyeong butanol fraction.

Example 3 Preparation of Diosin 1

Tobokyeong butanol fraction of Example 2 using a silica gel column CHCl ₃ -MeOH-H ₂ O (8: 2: 0.5, lower layer) and CHCl ₃ -MeOH-H ₂ O (7: 3: 1, lower layer Elution was carried out sequentially into 10 small fractions. The small fraction 6 was again eluted with a solvent of CHCl ₃ -MeOH-H ₂ O (8: 2: 0.5, lower portion) using a silica gel column to obtain 2.1 g of diosine.

Example 4 Preparation of Maple Methanol Extract

2 kg of root diameters of maple leaf ( Dioscorea quinqueloba Thunb.) Were cut and extracted three times in succession with methanol using a reflux condenser. The obtained methanol extract was filtered and concentrated under reduced pressure to obtain 87.3 g of maple leaf methanol extract.

Example 5 Preparation of Diosin 2

20 g of the maple leaf methanol extract of Example 4 was divided into six fractions using a silica gel column with a solvent of CHCl ₃ -MeOH-H ₂ O (7: 3: 1, lower layer). Fraction 3 was separated from water-saturated EtOAc-MeOH (98: 2) to water-saturated EtOAc-MeOH (95: 5) in a stepwise manner, followed by silica gel column chromatography to separate the material, and recrystallized with methanol. Diosin, a colorless needle crystal, was obtained.

Melting Point: 289 ~ 292 ℃

{α] _D ²⁵ : -110.5 ° (c, 0.3 in MeOH)

IR (KBr, cm ⁻¹ ) 3420, 1645, 1100 to 1000, 920, 900, 863, 835 (900> 920, 25 (R) -spiroketal), 810

¹ H NMR (300 MHz, Pyridine-d ₅ ) δ 0.68 (d, J = 5.2 Hz), 0.82 (s), 1.05 (s), 1.12 (d, J = 6.9 Hz), 1.60 (d, J = 6.9 kV), 1.74 (d, J = 6.2 kV), 4.92 (d, J = 6.7 kV), 5.32 (br d, J = 4.6 kV), 5.80 (s), 6.34 (s).

13 C NMR (75.5 MHz, pyridine-d ₅ ) δ 15.01, 16.32, 17.30, 19.40, 21.11, 29.27, 30.16, 30.60, 31.71, 31.84, 32.22, 32.32, 37.14, 37.50, 38.99, 39.88, 40.47, 41.98, 50.33 , 56.66, 62.92, 66.88, 78.14, 81.11, 109.26, 121.78, 140.84

Experimental Example 1 Growth Hormone Secretion Promotion Activity

(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) Growth hormone secretion promoting activity experiment

The pituitary cells isolated in step 1 were cultured and then suspended in test solution. Drugs were added to pituitary cells at a concentration of 10 μg / ml, and rat growth hormone secretion factors (rat GRF, Bachem Co., Torrance, CA, USA), which are the reference substances for growth hormone secretion, were 300 nM and 500 nM. , 1,000 nM was added and the test solution was added to a total volume of 1 ml and incubated at 37 ° C. for 15 minutes. After incubation, the supernatant was separated by centrifugation and stored at -70 ° C to measure growth hormone activity.

(Step 3) Growth Hormone Measurement

Growth hormone measurement was performed using a rat growth hormone RIA kit (rat GH RIA kit, Amersham, Buckinghamshire, England). The growth hormone contained in a constant amount of ¹²⁵ I-labeled growth hormone and the test substance stored above was different from the growth hormone-antibody complex (GH) by a competitive response to an anti-GH antibody. -Ab complex) was formed and precipitated by addition of a second antibody, and radioactivity of ¹²⁵ I was measured from the precipitate. The results are shown in Table 1 below.

matter Rat Growth Hormone Concentration (ng / ml) Diosin 219.391 ± 10.443 GRF ^* (0 nM) 7.633 ± 3.551 GRF (100 nM) 20.448 ± 4.505 GRF (300 nM) 37.296 ± 9.773 GRF (500 nM) 79.014 ± 14.757 GRF (1000 nM) 130.174 ± 4.000   * Indicates growth hormone secretion factor.

As shown in Table 1 , rat growth hormone was found to be 219.391 ± 10.443 ng / ml by diosin, which indicates that it acts on pituitary cells to promote secretion of growth hormone more than 30 times of the control group. .

Experimental Example 2 Measurement of Growth Hormone Secretion Effect According to Dioxin Concentration

Growth hormone secretion effect was measured according to the concentration of diosin in the same manner as in Experimental Example 1. The results are shown in Table 2 below.

Dioxin concentration (mg / ml) Rat Growth Hormone Concentration (ng / ml) 10 124.129 ± 1.525 5 92.057 ± 4.082 2.5 32.936 ± 6.759 1.25 28.501 ± 4.050 GRF ^* (0 nM) 8.756 ± 0.514 GRF (100 nM) 19.093 ± 1.666 GRF (300 nM) 24.893 ± 0.621 GRF (500 nM) 37.348 ± 2.343 GRF (1000 nM) 82.016 ± 7.502   * Indicates growth hormone secretion factor.

As shown in Table 2 , when the concentration of diocin is 1.25 mg / ㎖, the concentration of rat growth hormone was measured 28.501 ± 4.050 ng / ㎖, the concentration of rat growth hormone gradually increased as the concentration of diocin When the concentration of diosin was 10 mg / ml, the concentration of rat growth hormone was 124.129 ± 1.525 ng / ml. This indicates that the concentration of rat growth hormone depends on the concentration of diosin, which is 1.5 times the concentration of rat growth hormone relative to GRF (1000 nM). The ED ₅₀ calculated at this time was 1.14 × 10 ^-5 M.

Experimental Example 3 Growth Hormone Secretion Activity Test Using Animal Model

8-week-old SD rats were anesthetized at a concentration of 50 mg / kg of pentobarbital, and then diocin was dissolved in physiological saline containing 0.1% DMSO at 10 ㎍ / ㎏. Saline solution containing 0.1% DMSO was administered. After bleeding from the tail jugular vein at regular intervals, the secreted growth hormone was measured using the RIA kit.

Minutes Control group (n = 27) (ng / ml) Diosin-administered group (n = 4) (ng / ml) 10 13.19 ± 2.51 46.18 14.64 11.06 41.10 20 12.88 ± 3.29 21.31 17.72 21.25 76.37 30 12.52 ± 2.94 6.35 17.45 30.99 21.75 45 9.49 ± 3.24 24.01 7.24 4.97 14.59 60 8.19 ± 10.64 3.88 8.18 4.64 38.77

As shown in Table 3 and FIG . 1 , which shows the graph, the diosin of the present invention was administered to the rat jugular vein at a concentration of 10 μg / kg, and the blood was separated from the tail vein at regular intervals. As a result of measuring plasma growth hormone, it increased about 3 times after 10 minutes of drug administration compared to the control group, and increased 2 to 6 times after 20 to 30 minutes. Was measured.

Experimental Example 4 Acute Toxicity Experiment

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 Tobokyeong extract or maple leaf extract and diocin of the present invention, and orally administered once at doses of 1000 mg / kg / ml and 100 mg / kg / ml, respectively. It was. 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 Tobok-Ryung extract or Maple Horse Extract and Diosin 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 (LD ₅₀ ) of the Tobok-Ryong extract or Maple leaf extract was 10 g / kg, diocin was determined to be a safe substance more than 100 mg / kg.

Preparation Example 1 Manufacturing Method of Capsule

5 mg of diosin, 10 mg of Tobok Rin extract or 10 mg of maple leaf extract were mixed with 14.8 mg of lactose, 10.0 mg of polyvinyl pyrrolidone, and 0.2 mg of 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.

Diosin 5.0 mg 5.0 mg

(Tobok-Ryong Extract or Maple Leaf Extract ... 10 mg)

Lactose ... 14.8 mg

10.0 mg of polyvinylpyrrolidone

Magnesium Stearate 0.2 mg

Preparation Example 2 Manufacturing Method of Injection Solution

Injections were prepared by containing 10 mg of diosin, 20 mg of Tobok-ryong extract or 20 mg of maple leaf extract, 180 mg of mannitol, 26 mg of Na ₂ HPO ₄ .12H ₂ O and 2974 mg of distilled water. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

Diosin · 10 mg ··········

(Tobok-Ryong Extract or Maple Leaf Extract 20 mg)

Mannitol 180 mg

Na ₂ HPO ₄ · 12H ₂ O ·············· 26

Distilled water 0.2 mg

Preparation Example 3 Manufacturing Method of Beverage

It was prepared by mixing 0.1 g of diosin, 0.2 g of Tobok-ryong extract or 0.2 g of maple leaf 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.

Diosin 0.1 g

(Tobok-Ryong Extract or Maple Leaf 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

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As described above, the diosin and its derivatives of the present invention, Tobokyeong extract and maple extract containing the same act on the pituitary cells to promote the secretion of growth hormone, and the growth hormone secretion is also promoted as the concentration is increased. In addition, animal experiments using mice promoted growth hormone secretion in the same way, and the toxicity test resulted in less toxicity, which could be used as a therapeutic agent for diseases caused by deficiency of new growth hormones. Can be used as food.

1 is a graph showing the peak concentration of plasma growth hormone induced after administration of diocin to rats of the present invention.

Claims (7)

A therapeutic agent for diseases caused by growth hormone deficiency selected from the group consisting of arthritis, aging, Alzheimer's disease, dwarfism, obesity, osteoporosis, and ulcers, using diosin represented by the following formula (1) as an active ingredient. Formula 1 delete The disease treatment agent according to claim 1, wherein the diosin is isolated from Smilax china . The method of claim 3, wherein the separation of diosin from the Tobokyeong, i) dissolving the Smilax china pulverized product in methanol or a mixed solvent of methanol and water, extracting the filtrate, and concentrating the obtained filtrate under reduced pressure to obtain a methanol extract of Tobokyeong; And ii) adding distilled water to the methanol extract of Tobokyeong of i), adding butanol, shaking at room temperature, extracting and filtration, and concentrating the obtained filtrate under reduced pressure to obtain a fraction of Tobokyeong butanol. . According to claim 1, wherein the diosin is a disease treatment, characterized in that isolated from Dioscorea quinqueloba Thunb. The disease treatment agent according to claim 5, wherein the diocin is separated from the maple leaf methanol extract obtained by dissolving the maple leaf pulverized product in methanol or a mixed solvent of methanol and water, followed by filtration and concentrating the obtained filtrate under reduced pressure. delete