KR20020084875A - Composition comprising an extract of dyospyros kaki leaves for hormone replacement theraphy - Google Patents

Abstract

PURPOSE: A composition for hormone replacement therapy containing a natural extract such as persimmon leaves extract which produces an estrogen-like material is provided which can be used as a health food product or a therapeutic agent for hormone replacement therapy of a menopausal woman. CONSTITUTION: Persimmon leaves are extracted 1 to 20 times with water, extracted at 60 to 100deg.C for 1 to 6hr, filtered or centrifuged and then concentrated under reduced pressure, or they are extracted 5 to 20 times with lower alcohol, preferably 60 to 80% ethanol at 60 to 100deg.C while flowing backward for 1 to 6hr, filtered and then concentrated. For example, 100g persimmon leaves are added to 2L ethanol, flowed backward while heating at 90deg.C for 3hr, filtered and then concentrated under reduced pressure. Thereafter, the extract is applied to Saccharomyces cerevisiae to identify estrogenicity.

Description

COMPOSITION COMPRISING AN EXTRACT OF DYOSPYROS KAKI LEAVES FOR HORMONE REPLACEMENT THERAPHY}

The present invention relates to a composition for the treatment of hormone replacement using persimmon ( Dyospyros Kaki , Thunb) leaf extract as an active ingredient, specifically, it is isolated and purified from persimmon leaves using water or a lower alcohol, and is applied to the human estrogen receptor. It relates to a composition comprising the persimmon leaf extract showing an binding activity as an active ingredient.

In the United States alone, more than 4000 million women enter menopause each year. The average age of women who reached the last menstrual age was about 28 years, of which about 75 to 85 percent would develop estrogen deficiency (Hammond et al., Fertil. Steril. , 37 (1)). : 5-25, 1982). One of the most common diseases following the onset of ovarian failure is 'hot flash' or vasomotor complexes, which are suddenly occurring and generally last for several minutes, frequently observed red flashes and often Accompanying dizziness, nausea, headache, palpitations, and sweating, more than 90% of these symptoms can be alleviated by providing adequate estrogen.

There are many postmenopausal symptoms caused by chronic low estrogen, the most serious of which are osteoporosis and ischemic heart disease. It has been reported that 25% of women older than 60 years have developed vertebral compression fractures as a result of osteoporosis associated with estrogen deficiency, and 50% have developed vertebral fractures at age 75. The very high rate of hip fractures in old age is also attributable to this osteoporosis.

Hormone replacement therapy (HRT) with estrogens or estrogen / gestagen combinations is now known as the standard method for treating symptoms associated with menopause (Ernster, VL et al., Prev. Med ., 17: 201-223, 1988).

Estrogens protect the cardiovascular system, bone (reduced risk of osteoporosis) and central nervous system (avoid hot flashes). However, chronic use of estrogen in hormone replacement therapy increases the risk of endometrial cancer (Ernster, VL et al., Prev. Med ., 17: 201-223, 1988). Simultaneous use of gestagen to reduce this risk inhibits the stimulating effect of estrogen on the endometrium (Gibbson, WE, Am. J. Obstet. Gynecol. , 154: 46-61, 1986), but in contrast, estrogen and In the case of the combination treatment of gestagens , there is a problem of deteriorating the protective effect of the estrogen component on plasma lipids (Lobo, R. Am. J. Obstet. Gynecol ., 166: 1997-2004, 1992).

Moreover, with estrogen / gestagen based on lower hormonal dosages than using oral contraceptives, undesirable intra-menstrual bleeding can occur (Hillard, TC et al., Am. J. Obstet. Gynecol. , 167: 1-7, 1992). Finally, recent papers show that many gestagens may increase the risk of breast cancer (Staffa, JA et al., An Epidemiologic Review , 57: 473-491, 1992; King, RJB, J. Ster. Biochem Molec. Bio ., 39: 8111-8118, 1991).

In particular, estrogen replacement therapy is a more complex issue for women with the uterus. Estrogen treatment is associated with an increased incidence of endometrial cancer due to proliferation of the endometrium induced by continuous 'competitive' estrogens. Regular administration of progestin seems to reduce the rate of endometrial cancer in postmenopausal women who receive estrogen by several folds through the antiproliferative effect (Barbieri et al., Menopause Management , July / August, 12-24, 1992). However, the combination of estrogen and progestin frequently causes undesirable uterine bleeding and has the disadvantage that the cardiovascular effect of estrogen can be minimized by progestin. Nevertheless, estrogen's regimen, plus a series of concurrent progestins, is now used as a common form of hormone replacement therapy in postmenopausal women.

In addition, estrogens have been reported to be effective in treating dementia (Black SE, Patterson C., Feightner J., Can. J. Neurol. Sci., Feb: 28 Suppl 1: S56-66, 2001), neuroprotective action (Dubal DB., Wise PM., Endocrinology, Jan; 142 (1): 43-8, 2001) and improvement effects on neurodegenerative diseases (Kim H., Xia H., Li L., Gewin J., Biofactors, 12 (1-4): 243-50, 2000).

On the other hand, persimmon (Dyospyros Kaki, Thunb) is wild throughout the country, persimmon leaves contain flavonoid glycosides, tannins, phenols, resins, carcumin compounds, reducing sugars, polysaccharides, essential oils, organic acids, chlorophyll. Flavonoid glycosides contained in persimmon leaves include astragarine and myristic, and are particularly rich in vitamin C. Some persimmon leaves in April to June contain about 100 mg of vitamin C in 100 g. Persimmon leaves are said to be good for bronchial protection according to our traditional folk remedies, and because they contain a lot of vitamin C, they help to absorb minerals, such as iron, which can easily be lost after excessive drinking.

In addition, persimmon bogam is known that persimmon leaves are effective in purpura, along with the prevention and treatment of vascular disease, has been used for a long time pharmacological action, and recent research has been recently recognized persimmon leaf tea because the physiological activity of persimmon leaf is proven. Persimmon leaves contain a variety of phenolic compounds, including tannins, these components are known to have a physiological effect through a number of pathways. Flavonoids in persimmon leaves are known to inhibit high blood pressure-causing enzymes or gallate among tannins to inhibit blood pressure rise.

Accordingly, the present inventors have diligently tried to find a natural product that can be used for estrogen replacement therapy. As a result, the present inventors have found that the persimmon leaf extract exhibits binding activity to the human estrogen receptor and thus can be effectively used for hormone replacement therapy in postmenopausal women as estrogen-like substance Was completed.

It is an object of the present invention to provide a hormone replacement therapeutic composition containing a natural extract as an active ingredient as a hormone analogous substance such as androgen or estrogen.

1 is a result of examining the estrogen generating ability (estrogenicity) of the persimmon leaf extract using a recombinant yeast expressing the human estrogen receptor.

C; No treatment group E2; 17β-estradiol

1-1; Persimmon leaf extract 100 μg / ml 1-2; Persimmon Leaf Extract 1mg / ml

In order to achieve the above object, the present invention provides a hormone replacement therapy pharmaceutical composition comprising the persimmon leaf extract to produce an estrogen-like substance showing binding activity to the estrogen receptor.

In another aspect, the present invention provides a health food composition for hormone replacement therapy using the persimmon leaf extract to produce an estrogen-like substance showing the binding activity to the estrogen receptor.

Hereinafter, the present invention will be described in detail.

The present invention provides a hormone replacement therapeutic pharmaceutical composition comprising the persimmon leaf extract that produces an estrogen-like substance showing binding activity to the estrogen receptor.

Clarifier extract contained as an active ingredient in the pharmaceutical composition of the present invention can be obtained by extracting from the Clarifier by a conventional method using water or a lower alcohol.

For example, the method of extracting hot water is dried persimmon leaf, pulverized and added 1 to 20 times of water by weight ratio, followed by extraction for 1 to 6 hours at 60 to 100 ° C., filtration or centrifugation, and the supernatant concentrated under reduced pressure to extract persimmon leaf. Manufactures

In addition, the lower alcohol extraction method is added 5 to 20 times lower alcohol to persimmon leaves, refluxed for 1 to 6 hours while heating to 60 to 100 ℃, cooled and filtered and concentrated under reduced pressure using a rotary evaporator to obtain a persimmon leaf extract, In this case, the filtration process may be repeated two or more times with respect to the residue remaining after filtration. In this case, as the lower alcohol, methanol or methanol aqueous solution, ethanol or ethanol aqueous solution, normal propanol, iso-propanol, normal butanol and mixtures thereof may be used, and ethanol of 60 to 80% is most preferred.

In order to investigate whether the ethanol extract extracted from the persimmon leaf by the extraction method exhibits hormonal activity, the ethanol extract of the persimmon leaf is applied to a recombinant yeast expressing the human estrogen receptor. The reaction was measured.

Treatment of the test substance with 1 mg / ml and 100 μg / ml showed a significant difference compared to 17β-estradiol (E2), which was used as a positive control. Persimmon leaf compared to E2, a positive control. High estrogenicity was observed in yeast to which the extract was applied.

Through the above experimental results, it was confirmed that the persimmon leaf ethanol extract of the present invention can bind to the estrogen receptor and act as an analogue of the human estrogen hormone.

Since the same effect as the administration of estrogen can be expected by administering a pharmaceutical composition comprising the persimmon leaf extract of the present invention exhibiting such an effect, the pharmaceutical composition of the present invention can be usefully used as a prophylactic and therapeutic agent for osteoporosis.

Persimmon leaf extract of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general drug replacement therapy.

That is, the pharmaceutical composition may be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used Is prepared using. 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 starch, calcium carbonate, sucrose ( Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and 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 parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized replacement treatments, 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 a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular.

Typical daily dosages of the persimmon leaf extract of the present invention range from 0.001 to 100 mg / kg body weight, and may be administered once or in divided doses. However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, and the severity of the patient, and therefore the dosage may be determined in any aspect of the invention. It does not limit the scope.

In addition, the persimmon leaf extract of the present invention may be provided as a food or beverage additive for treating hormone replacement. In order to act as a hormone-like substance, foods to which the persimmon leaf extract of the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, and health supplements.

The persimmon leaf extract of the present invention can be added to the raw material during the manufacture of food, or properly mixed with the cooked food to prepare a food, in which case the content of the persimmon leaf extract in the finally prepared food or beverage ranges from 0.1 to 50% by weight. .

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

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Reference Example 1 Recombinant Yeast

Dr. Donald C. McDonnell of Saccharomyces cerevisiae ER + LYS 8127 (YER) (Duck University [Chemical Industry Institute of Toxicology, USA]) used to investigate the hormonal activity of the persimmon leaf extract of the present invention. Donor) is a hER, hAR gene and reporter system whose expression is regulated by the CUP1 metallothionine promoter, which stably expresses the estrogen response element and β-gal gene. It is a recombinant yeast containing a vector. ER + LYS 8217 is a saccharomyces cerevisiae 3.35 g / ml yeast nitrogen base (yeast nitrogen base, Difco, USA), 2% dextrose (dextrose, Gibco, USA), 30 μg / ml L-lysine Incubated in growth media containing -HCl (L-lysine-HCl, Sigma, USA), 35 μg / ml L-histidine-HCl (L-histidine-HCl, Gibco, USA) It was. Recombinant yeast was stored at −80 ° C. or less by adding 20% glycerol (glycerol, Sigma, USA) to the growth medium until it was used for hormonal activity experiments with persimmon leaf extract.

Example 1 Preparation of Persimmon Leaf Extract

The persimmon leaf was added 2 L of 70% ethanol per 100 g and refluxed for 3 hours while heating to 90 ℃, cooled and filtered, and concentrated under reduced pressure using a rotary evaporator to obtain an ethanol extract of the persimmon leaf.

Example 2 Measurement of Hormone Activity of Persimmon Leaf Extract

In order to confirm that the ethanol extract of persimmon leaves isolated and purified from Example 1 exhibited hormonal activity, the persimmon leaf extract was applied to ER + LYS 8127 (YER) to recombinant yeast Sakaromyces cerevisiae expressing human estrogen receptor. The estrogen production ability was investigated.

Specifically, the recombinant yeast stored at −80 ° C. according to Reference Example 1 was dissolved, and then placed in the same growth medium, and then grown at 30 ° C. and 300 rpm in a shaking incubator. Proliferated recombinant yeast was diluted to the appropriate concentration, 500 μM of CuSO ₄ (Sigma, USA) was added and the appropriate amount was dispensed into 50 ml cornical tubes. The tube was treated with persimmon leaf extract of the present invention in the experimental group 1 mg / ㎖ and 100 ㎍ / ㎖, respectively, treated with 17β- estradiol (E2, 10 ^-9 M) as a positive control, 100% ethanol as a solvent control (absolute ethanol) was used.

The experimental group and the control group treated with the respective materials were incubated for 18 hours at 30 ° C. and 300 rpm in a shake incubator, and then the culture solution of each tube was diluted to the same concentration and a 96 well microtiter plate (96 well microtiter plate, Costar, USA) was dispensed into three wells of 100 μl in each group. 2 mg / ml o-nitrophenyl-β-D-galactopyranoside (o-nitrophenyl-β-D-galactopyranoside, Sigma, USA), 0.1% lauryl sulfate (Sigma, USA), 50 mM β-mercaptoethanol (β-mercaptoethanol, BDH chemical, England), and 200 U / mL Z buffer solution (oxalyticase, Enzogenetics, USA) were dispensed and mixed After 20 minutes, the color development was measured at a wavelength of 420 nm using a microplate reader.

As a result, the experimental group treated with persimmon leaf extract of the present invention at a concentration of 1 mg / ml and 100 µg / ml, respectively, showed a significant difference compared to the positive control treated with 17β-estradiol (E2). The positive control group treated with 10 ⁻⁹ M of 17β-estradiol (E2) showed absorbance of about 2.7, whereas the experimental group treated with persimmon leaf extract at 100 μg / ml and 1 mg / ml showed absorbances of 1.25 and 1.75, respectively. It showed relatively high estrogen generating ability.

Therefore, the persimmon leaf extract of the present invention is an estrogen-like substance showing estrogen receptor binding activity, the composition comprising it can be usefully used for the hormone replacement treatment of diseases such as osteoporosis.

As described above, the persimmon leaf extract of the present invention generates an estrogen-like substance that binds to the estrogen receptor, so it can be usefully used as a pharmaceutical composition for treating hormone replacement in postmenopausal women.

Claims (5)

A pharmaceutical composition for treating hormone replacement in postmenopausal women using persimmon leaf extract as an active ingredient. The pharmaceutical composition of claim 1, wherein the hormone is estrogen. The pharmaceutical composition according to claim 1, wherein the persimmon leaf extract is prepared by extracting with water or lower alcohol. 4. A pharmaceutical composition according to claim 3, wherein the lower alcohol is selected from the group consisting of methanol or methanol aqueous solution, ethanol or ethanol aqueous solution, normal propanol, iso-propanol, normal butanol and mixtures thereof. Health food composition for hormone replacement treatment of postmenopausal women with persimmon leaf extract as an active ingredient.