KR20020084871A - Composition comprising an extract of cassia obtusifolia for hormone replacement theraphy - Google Patents

Abstract

PURPOSE: A composition containing a Cassia tora L. extract which is estrogen-like material and effective in hormone activity is provided. Therefore, the composition can be used as a therapeutic agent or health food for hormone replacement therapy of a menopausal woman and orally or parenterally administered for a clinical application use. CONSTITUTION: Cassia tora L. is extracted 1 to 20 times with water or 5 to 20 times with low alcohol selected from the group consisting of methanol or a methanol aqueous solution, ethanol or an ethanol aqueous solution, normal propanol, iso-propanol, normal butanol and a mixture thereof at 60 to 100deg.C for 1 to 6hr, filtered or centrifuged and then concentrated under reduced pressure. An effective amount of the extract for treatment of hormone replacement therapy is in the range of 0.001 to 100mg/kg per a day and a food product and beverage contains 0.1 to 50% by weight of the extract.

Description

COMPOSITION COMPRISING AN EXTRACT OF CASSIA OBTUSIFOLIA FOR HORMONE REPLACEMENT THERAPHY}

The present invention relates to a composition for treating hormone replacement containing an extract of Cassia obtusifolia , specifically, isolated and purified from water by using water or a lower alcohol and exhibiting binding activity to human estrogen receptor. It relates to a composition comprising the extract 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, Cassiae semen is the seed of superseeds (Cassia obtusifolia L.) or Cassia tora L .. I dry it and collect a seed. The taste is sweet and bitter and the nature is slightly cold. Lowers and clears the liver, brightens the eyes, has a diuretic effect, and dilutes the stool to act as a stool. It is used when the eyes are red and swollen due to liver fever or windy fever, or when there are many tears, and it contains hachocho, gardenia and mulberry leaves. . In addition, constipation caused by heat or constipation caused by drying the constipation can be boiled or eaten powdered to eat, lowering blood pressure or lowering cholesterol in the blood.

In the meantime, anthraquinones such as chrysophanol, emodin, and physcion, obtusifolin, obtusin, chryso-obtusin, nord Anthraquinone glycosides, such as nor-rubrofusarin and rubrofusarin, have been isolated and identified. However, to date, it is not clear what component of the defect is active.

Accordingly, the present inventors have made efforts to find a natural product that can be used for estrogen replacement therapy. As a result, the presenter has revealed that the extract of the C. vulgaris exhibits binding activity to the human estrogen receptor, and thus can be effectively used for hormone replacement treatment of postmenopausal women as an 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 extract of the defect using a recombinant yeast expressing the human estrogen receptor.

C; No treatment group E2; 17β-estradiol

1-1; Cassia extract 1 mg / ml 1-2; Cassia extract 100 ㎍ / ml

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

In another aspect, the present invention provides a health food composition for hormone replacement therapy comprising the extract of the fault 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 pharmaceutical composition for hormone replacement therapy comprising as an active ingredient a deficiency extract to produce an estrogen-like substance that binds 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, in the method of extracting hot water, the cultivator is dried and pulverized, 1 to 20 times of water is added in weight ratio, and then extracted at 60 to 100 ° C. for 1 to 6 hours, filtered or centrifuged, and the supernatant is concentrated under reduced pressure. Manufactures

In addition, the lower alcohol extraction method is added to 5 to 20 times lower alcohol to the tuber, 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 tuber 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 defector by the extraction method exhibits hormonal activity, the ethanol extract of the defector 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. High estrogenicity was observed in yeast to which the extract was applied.

Through the above experimental results, it was confirmed that the ethanol extract of the present invention binds to the estrogen receptor and can 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 extract of the present invention as an active ingredient, the pharmaceutical composition of the present invention can be usefully used as a preventive and therapeutic agent for osteoporosis.

The pharmaceutical composition 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 form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form extracts may contain 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 Cassiae extract of the present invention range from 0.001 to 100 mg / kg body weight in humans, and may be administered once or in several 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 another aspect, the present invention provides a health food composition for hormone replacement therapy comprising the extract of the fault to produce an estrogen-like substance showing the binding activity to the estrogen receptor.

The health food composition of the present invention can be usefully used as a prophylactic and therapeutic agent for osteoporosis because the same effect as the administration of estrogen can be expected by ingesting the health food composition containing the extract of the present invention. In order to act as a hormone-like substance, foods to which the extract of the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, and health supplements.

The food extract may be prepared by adding the extract of the present invention to the raw material during food preparation or by appropriately mixing the cooked food, in which case the content of the extract from the final 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] 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 lower by adding 20% glycerol (glycerol, Sigma, USA) to the growth medium until it was used for hormonal activity experiments with the extract.

Example 1 Preparation of Clarifier Extract

2 L of 70% ethanol was added per 100 g, refluxed for 3 hours while heating to 90 DEG C, cooled and filtered, and concentrated under reduced pressure using a rotary evaporator to obtain a clarifier ethanol extract.

Example 2 Determination of Hormone Activity (Estrogenicity)

In order to confirm that the ethanol extract of the isolated isolate from Example 1 exhibited hormonal activity, the extract was applied to ER + LYS 8127 (YER) in 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 tuber extract of the present invention was treated with 1 mg / ml and 100 μg / ml, respectively, in the tube, and treated with 17β-estradiol (E2, ^10-9 M) as a positive control, and 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, there was a significant difference in the experimental group treated with 1 mg / ㎖, 100 ㎍ / ㎖ concentration extract of the present invention, respectively compared to the positive control treated with 17β- estradiol (E2). The positive control group treated with 10 ⁻⁹ M of 17β-estradiol (E2) showed an absorbance of about 2.7, whereas the experimental group treated with 100 μg / ml and 1 mg / ml of the deflector extract showed absorbances of 5.1 and 4.6, respectively. It showed relatively higher estrogen-generating ability than 17β-estradiol, which is conventionally used for hormone replacement therapy.

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

As discussed above, the extract of the present invention, because it produces an estrogen-like substance that binds to the estrogen receptor can be useful as a pharmaceutical composition for hormone replacement therapy in postmenopausal women.

Claims (5)

A pharmaceutical composition for treating hormone replacement in postmenopausal women using the 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 extract of Cultivator 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 using the extract as an active ingredient.