KR100542479B1 - Phytoestrogen compositions for hormon substitute therapy containing phytoestrogens derived from pomegranate - Google Patents

Abstract

The present invention relates to a phytoestrogen composition for hormone replacement therapy containing phytoestrogens derived from pomegranate, and more particularly, it can effectively prevent and treat various symptoms that can be caused by estrogen deficiency in menopausal or postmenopausal women. A phytoestrogens composition for hormone replacement therapy containing a pomegranate derived phytoestrogen, which is expected to be present,

According to the present invention, catechin 1.0-2.0ppm, dyedzein 20-30ppm, genistein 0.1-0.5ppm, quercetin 8.0-14.0ppm, 17β-estradiol 0.05-0.3ppm and 2,3-di-MeO-estra A pomegranate-derived phytoestrogen-containing hormone replacement therapy phytoestrogen composition comprising 0.01 to 0.10 ppm of diol is provided.

Pomegranate-derived phytoestrogen-containing phytoestrogens composition for hormone replacement therapy according to the present invention contains a large amount of safe phytoestrogens with excellent estrogen properties and no side effects, and thus may be caused by estrogen deficiency in menopausal or postmenopausal women. It is expected to be able to effectively prevent and treat all kinds of symptoms. Especially, it has an excellent effect on hot flashes, osteoporosis, and atherosclerosis, and it is useful as a composition for hormone replacement therapy because it has a very low body fat increase as a side effect. It is expected.

Phytoestrogens, pomegranates

Description

PHYTOESTROGEN COMPOSITIONS FOR HORMON SUBSTITUTE THERAPY CONTAINING PHYTOESTROGENS DERIVED FROM POMEGRANATE}

The present invention relates to a phytoestrogens composition containing a pomegranate-derived phytoestrogens, and more particularly, to a phytoestrogens composition for hormone replacement therapy for the prevention and treatment of menopausal or menopausal symptoms containing a large amount of phytoestrogens derived from pomegranate will be.

In general, the endocrine system is an organ system that produces and secretes various hormones involved in maintaining homeostasis, reproduction, development, and behavior of a living body, and is composed of gland, hormones, and target cells. Through the body circulates through the information and commands to each target cell and tissue involved in growth and development, cancer and differentiation.

Estrogen, one of these endocrine hormones, is synthesized from cholesterol in the body. The secretory sources are mainly follicles and corpus luteum of the ovary, and fetal placenta, adrenal gland during pregnancy. It is also secreted from the testis. Estrogen secretion in the ovary is follicle stimulating hormone, a hormone that regulates the menstrual cycle and enables pregnancy when the gonadotropin secreted by the anterior pituitary gland transmits signals to the reproductive organs. Follicle stimulating hormone (FSH) and luteenizing hormone (LH) are secreted, which in turn act on the ovary, releasing estrogen and progesterone. Estrogen stimulates the endometrium in the first half of the menstrual cycle to thicken the endometrium and increase blood supply so that the fertilized egg can be implanted, and progesterone is secreted in the second half to make the endometrium better. In other words, estrogen is involved in the growth of the endometrium, the development of the uterine muscle, the development of secondary sexual characteristics, the mediation of the menstrual cycle, the maternal change during pregnancy, and the promotion of the proliferation of the mammary gland. Representative female hormones, or other parts of the body have a wide range of effects.

The estrogens are estradiol, estrone, estriol and the like generically, among which estradiol is known to be the strongest. Estrogens function through receptors and are primarily metabolized in the liver and are excreted in the urine as synthetic estrogens.

In premenopausal women, the ovary is a major source of estrogen. The main secretion product is estradiol, synthesized by granulosa cells from the androgen precursor supplied by the follicular membrane cells. Secreted estradiol can be reversibly oxidized to estrone and estrogen can be converted to estriol. Interconversion between estrone and estradiol occurs mainly in the liver catalyzed by 17-hydroxysteroid dehydrogenase. In men and postmenopausal women, the main source of estrogen is adipose tissue. In these tissues, estrone is synthesized from dehydroepiandrosterone secreted by the adrenal cortex, so it is known that the secretion of estrogen by adipose tissue can be partially regulated by using androgen precursors (Mendelson, CR and Simpson, ER, 52: 169-176, (1987)).

In premenopausal women, 17β-estradiol produced by the ovary is the major circulating estrogen. Serum estradiol levels are low in puberty girls and high in tungsten girls. In women, the concentration of estradiol ranges from about 100 pg / ml (367 pmol / l) of the follicular phase to about 600 pg / ml (2200 pmol / l) of the ovulatory phase. During gestation, it will rise to nearly 20,000 pg / ml (70,000 pmol / l). After menopause, serum estradiol concentrations have been reported to drop to levels comparable to those seen in men of comparable age (5-20 pg / ml) (Yen, SSC and Jaffe, RB, 3rd ed. Philadelphia: WB Saunders, ( 1991).

As a woman enters menopause, as the ovary ages, the response to pituitary gonadotropin (follicle-stimulating hormone (FSH) and luteinizing hormone (LH)) decreases, resulting in early follicular Further shortening results in shorter menstrual cycles, shorter ovulation phases, reduced progesterone production, and more irregular cycles. Eventually, the follicles will not react and will not produce estrogens. Reduced secretion of estrogens affects not only the female genitourinary system but also the entire body. Menstruation is called menopause when ovulation is stopped and menopausal symptoms due to hormone reduction before or after menopause appear. .

In addition, immature menopause occurring before the age of 40 is commonly referred to as unidentified ovarian failure, which is believed to be associated with a large amount of long-term smoking, high altitude residence, and malnutrition. Menopause can result from ovarian extraction, chemotherapy, radiation to the pelvis, or any process that lowers the blood supply to the ovaries.

In postmenopausal women, the rapid decrease in estrogens significantly increases the risk of developing psychological and emotional signs such as fatigue, excitement, insomnia, poor concentration, depression, memory loss, headache, anxiety, and nervousness or timidity, and recurrence. Fatigue and excitement due to sleep disturbance, intermittent dizziness, paresthesia, palpitations and tachycardia, nausea, constipation, diarrhea, arthralgia, myalgia, cold hands and legs, and weight gain are also significantly increased. In addition, osteoporosis is not only easily induced, but also mortality due to cardiovascular diseases such as heart disease, high blood pressure and stroke is increased (Kalin MF & Zumoff B. Steroids 55: 330-352, 1990). In addition, it causes changes in the skin and urogenital system and increases the likelihood of developing autoimmune diseases, cataracts and colorectal cancer.

Significant decreases in estrogen as women enter menopause, such as thinning of the vaginal mucosa and vulva skin, changing normal bacterial colonies, and decreasing the size of labia minora, clitoris, uterus and ovaries It causes a change. Thus, inflammation of the vaginal mucosa (atrophic vaginitis), frequent urination and urgency, vaginal dryness and sexual dysphoria can be caused. In addition, there is a tendency to develop hue loss of the pelvic muscles, incontinence, cystitis and vaginitis.

Hot flashes and sweating are known to occur in about 75% of postmenopausal women. Flushing typically occurs during or after menopause and is directly related to estrogen deficiency. Most of them persist for more than 1 year and 25 to 50% of them last more than 5 years. Hot flashes begin with a sudden hot flash that concentrates on the face, neck, and upper chest, the skin turns red, and this hot flash lasts 30 seconds to 5 minutes, often involves a lot of sweating, sometimes accompanied by palpitations, Usually accompanied by sweating and chills (Casper, RF, Yen, SSC. Neuroendocrinology of menopausal flushes: An hypothesis of flushmechanism. Clin Endocrinol 1985; 22: 293).

Such hot flashes may range from once or twice daily, or in many cases once daily during the day, usually several times a day. Hot flashes can cause sleep arousal and disturb sleep, and many women experience a lot of sweating, which can lead to quite embarrassing problems in social life. Hot flashes are believed to be due to dysfunction of the hypothalamus due to estrogen withdrawal. Temperature changes are reported to be mediated by the occurrence of hot flashes at the same time as the pulse of luteinizing hormone (Casper, RF, Yen, SSC, Wilkes, MM. Menopausal flushes: ANeuroendocrine link with pulsatile luteinizing hormone secretion.Science 1979 205: 823 and Tataryn, IV.Meldrum, DR, Lu, KH, et al. LH, FSH and skin temperature during the menopausal hot flash.J Clin Endocrinol Metab 1979; 49: 152). The initiation mechanism of hot flashes is endogenous opioid peptide withdrawal, with estrogens being found to increase central opioid peptide activity, while menopause is associated with a decrease or loss of endogenous central opioid activity (Reid, RL, Quigley, ME). , Yen, SS.The disappearance of opiodidergic regulation of gonadotropin secretionin postmenopausal women.J Clin Endocrinol Metab 1983; 57: 1107).

Currently, the most effective known methods for preventing or treating the hot flashes are known to supplement estrogens by administering estrogens.

In addition, it has been reported that cardiovascular susceptibility to atherosclerosis, which is a frequent occurrence in postmenopausal women, can be effectively reduced by increasing blood estrogen levels (Stout D.W., 1982). On the other hand, as a result of experiments on rabbits with atherosclerosis, it was confirmed that estrogen lowers blood lipid content and shows antiatherosclerotic effect (Ivanova L.V., 1996; Numano., 1971). In addition, examples of using estrogen as a cholesterin synthesis inhibitor have been reported (Ekzoesterol, etinilestradiol, premarin; P.A. Nussbaumer, Hunkler F., 1967).

Atherosclerosis with complications is more common in postmenopausal women due to decreased blood estrogen levels and decreased or lost ovarian function. In the West, long-term hormonal therapy with estrogen is widely used, and many clinical trials Studies also report the positive effects of estrogen (Myasnikov L.A., 1961; Kraznai I., 1963; Patrono V., 1972). In addition, it has been reported that the above good results have been obtained in the treatment of 50 to 70 year old males with myocardial infarction, which is a complication of atherosclerosis of the heart (Statut R.U., 1985).

In addition, estrogen is known to have a palliative effect against autoimmune diseases such as rheumatoid arthritis (Chander &Spector; 50: 139). Autoimmune diseases such as rheumatoid arthritis are caused by abnormal control in cell-mediated and humoral-mediated immunity and are often associated with abnormal or enhanced T cell, B cell and macrophage effector function against autologous antigens. It is believed that the activity of these cellular components on autoantigens is associated with disruption of the feedback mechanism associated with self tolerance. For example, autoimmune diseases such as Hashimoto's thyroiditis prevail in women of childbearing age, with a female to male incidence of about 50: 1 (Ahmed et al., 121: 531 (1985)).

Estrogens have been shown to have an inhibitory role on T cell function but have an immune stimulating effect on B cells. Thus, estrogen is expected to be useful for the prevention and treatment of diseases associated with activated T cells, including rheumatoid arthritis, polysclerosis, Guillan-Barre syndrome, Hashimoto's thyroiditis, through inhibition of T cell function. Holmadahl, J. 2: 651 (1989).

In addition to the inhibitory effect of estrogens on T cells, estrogens are believed to play a protective role against autoimmune diseases. Marui, j. 92: 1866 (1993) reports that antioxidants inhibit endothelial expression of VCAM-1. In other words, VCAM-1 is a ligand for VLA4, an integrin of T cells and macrophages involved in trafficking outside the vascular system, around blood vessels and into target organs, and because estrogen is an antioxidant, 4 It is known to inhibit dependent trafficking and consequently to interfere with the cascade of immunity associated with autoimmune-mediated diseases.

Estrogens are also reported to be inhibitors of colorectal cancer. Cohort's findings in this study suggest that estrogen supplementation reduces the risk of colorectal cancer in women by bile acid synthesis. Recently, the reduction in mortality in colorectal cancer in the West is believed to be the result of extensive use of postmenopausal estrogen supplementation (Mayer, Chapter 92, in, 14th ed., 1998).

In addition to the above, estrogens have been reported to accelerate the growth of endothelial cells in vitro and in vivo (Morales, DE, 91: 755-63 (1995); Krasinski, K., 95: 1768-72 (1997). )). Estrogen induces rapid reendothelialization after vascular injury, which may be due to increased local expression of vascular endothelial growth factor. In addition, estrogens inhibit apoptosis of human endothelial cells cultured in an estrogen receptor-dependent manner (Spyridopoulos, I., J., 95: 1505-14 (1997)). Endothelial initiation by estrogens has been reported to decrease the response to injury by increasing the availability of nitric oxide and directly inhibit the proliferation of smooth muscle cells (Cornwell, TL, J., 267: C1405-C1413 (1994).

In addition, estrogen has been suggested to have a prophylactic effect against cataracts by epidemiological proof. Although women are at higher risk of developing cataracts than men, this increased risk is known to occur when postmenopausal estrogens are reduced (Livingston, PM, J., 26: 1-6, (1994) Klein, BE, J., 116: 219-225, (1998). In a study of 544 women, early menopause reported a 2.9-fold increase in the risk of developing cataracts (Shibata, T., J., 26: 25-33, (1994) ). In addition, small-scale epidemiologic studies have reported that postmenopausal estrogen supplementation reduces the incidence of cataracts (Klein., J., 112: 85-91, (1994); Cumming, RG and Mitchell, P., J., 145: 242-249, (1997); Benitez del Castillo, JM, J., 104: 970-973, (1997)). In vivo rat models of age-related cataracts suggest that the prophylactic effect of estrogens is due to genomes (Bigsby, R.M., J ,. 96: 9328-9332, (1999)).

As described above, estrogen is excellent for protecting the functions of various tissues such as bone tissue, heart, blood vessels, central nervous system, skin tissue, as well as urogenital system of women. Hormone replacement therapy (HRT) has been widely used as a treatment method (Lobo RA & Speroff L. Fertil Steril 61: 592-595, 1994), which is attributed to the reduced estrogen secretion. Synthetic estrogen or a mixture of synthetic estrogen and synthetic progesterone. Clinically, it is applied for treatment of amenorrhea or dysmenorrhea, artificial movement of menstruation, menopausal disorders, hormone therapy for prostate or breast cancer, and osteoporosis. Oral contraceptives also consist of progesterone and estrogen. In addition, clomphene, an ovulatory inducer, and tamoxifen, which are used to treat breast cancer, are nonsteroidal antiestrogens. Administration of such synthetic estrogen hormone preparations has been applied in the form of oral administration in the form of tablets, or in the form of a patch for topical application or a cream for topical application.

However, many studies have reported that long-term administration of synthetic estrogens can lead to breast and uterine cancer (eg, Hunt K et al, Br J Obstet Gynaecol 94: 620-635, 1987; Ravnikar). VA.Women's Health Issues 2: 75-82, 1992). In recent years, controversy has been heating up among scholars and across society, with a number of credible claims that long-term synthetic estrogen replacement therapy is likely to cause breast cancer. Although not yet proven, it is believed that the risk of developing breast cancer in women receiving synthetic estrogen replacement therapy is associated with the dose of estrogen exposure over time.

Therefore, there is a recent trend to actively search for phytoestrogen, which is a phytoestrogen, as a safer substitute for the synthetic estrogen used in the hormone replacement therapy.

The phytoestrogens described above refer to nonsteroidal compounds present in plants that exert an estrogenous effect in the body of animals, and most of the grains, fruits and vegetables known to be effective in anticancer activity and heart disease are phytoestrogens. It contains a small amount, especially soybean processed foods including soybeans (soy milk, tofu, miso, etc.), and various grains, fruits, alfalfa, and clover. It is known that isoflavones are mainly contained in legumes, and lignans are mainly contained in cereals.

The activating effect of the above-mentioned phytoestrogens is only 1/100 to 1/1000 of the endogenous estrogen produced in the body, but since the content is much larger, a sufficient estrogen effect can be obtained as a result. Representative activities of phytoestrogens are known to prevent bone loss, increase bone density, reduce FSH and LH when administered to postmenopausal women, and alleviate menopausal symptoms. Genistein, one of isoflavones, It has been reported that there is an anticancer effect of inhibiting the growth of cancer cells by inhibiting protein tyrosine kinase of the cells.

Therefore, it can be used for the prevention and treatment of osteoporosis in postmenopausal women, lowers LDL (low density lipid) and high HDL (high density lipid), and acts as an estrogen agonist or antagonist with anticancer effect in endometrium and breast. In addition, there is a request for an effective phytoestrogens that can be used as a hormone replacement therapy for the prevention and treatment of menopausal symptoms that can be used effectively and safely for skin improvement, prevention of cataracts and colon cancer, and prevention of autoimmune diseases. It is rapidly increasing.

In response to this request, the food and pharmaceutical industry is now seeking to find safe natural hormone replacement plants that have the physiological activity of estrogen in natural resources, do not cause breast cancer or uterine cancer, and do not act as endocrine disruptors. Efforts are ongoing.

Accordingly, it is an object of the present invention to provide a phytoestrogen composition for hormone replacement therapy that can effectively prevent and treat menopausal or menopausal symptoms in menopausal or postmenopausal women.

In particular, the present invention is to provide a phytoestrogen composition for hormone replacement therapy that can effectively prevent and treat facial flushing or osteoporosis in menopausal or postmenopausal women.

Hereinafter, the present invention will be described in detail.

The present invention is to provide a phytoestrogen composition for hormone replacement therapy containing a large amount of phytoestrogens derived from pomegranate as a natural estrogen source for menopausal or postmenopausal women.

Pomegranate is used as a main ingredient in the production of the phytoestrogens composition for hormone replacement therapy of the present invention, and the pomegranate is a native plant in southwestern Asia and northwestern India, centered on Iran, and is now widely spread in subtropical and tropical regions. Pomegranate, especially black pomegranate, has been known as a tonic for a long time, and it is known to have a good effect on prevention of hypertension and atherosclerosis. In addition, it contains a large amount of water-soluble sugars (38-47%), and contains a variety of vitamins and minerals.

Although there is no special restriction | limiting in the kind of pomegranate in this invention, Black pomegranate is preferable, A specific example is Iranian black pomegranate.

According to the present inventors, pomegranate concentrate has a slight difference depending on the pomegranate origin and harvesting time, in particular, the black pomegranate concentrate contains a significant amount of phytoestrogens, specifically catechin 1.0 ~ 2.0ppm, 20 to 30 ppm of diyzedine, 0.1 to 0.5 ppm of genistein, 8.0 to 14.0 ppm of quercetin, 0.05 to 0.3 ppm of 17β-estradiol, and 0.01 to 0.10 ppm of 2,3-di-MeO-estradiol.

Pomegranate concentrate prepared according to the present invention contains a water content of 35 to 42% by weight, a lipid content of 0.3 to 0.6% by weight, protein content of 0.7 to 1.2% by weight, ash 1.2 to 1.6% by weight, carbohydrates 38 to 47% by weight. The pomegranate concentrate in this invention is used by the meaning containing the concentrate to the flesh, the concentrate of the flesh and seeds, and the concentrate from the raw material containing a peel.

Amino acids include eight essential amino acids of threonine, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan, lysine, asphaltic acid, serine, glutamic acid, proline, glycine, alanine, cysteine, tyrosine, histidine, alginine.

The water-soluble vitamins include thiamine (vitamin B ₁ ), riboflavin (vitamin B ₂ ), ascorbic acid (vitamin C), niacin, vitamin B ₆ , and the fatty acids include myristic acid, palmitic acid, stearic acid, oleic acid, Linoleic acid and the like, and the weak acid component includes glycolic acid and acetic acid slightly.

The phytoestrogen-containing hormone replacement therapy composition according to the present invention comprises 0.1 to 99% by weight of the pomegranate concentrate, based on the total weight of the composition, preferably 3 to 70% by weight, more preferably 5 to 50% by weight. Include. Most preferably, it is 35 to 45% by weight in the form of tablets or granules, and 5 to 15% by weight in the form of a liquid.

In addition, optionally, the phytoestrogens-containing hormone replacement therapy composition according to the present invention may include 0.1 to 5.0% by weight of isoflavones, which are phytoestrogens derived from 칡, soybean, or 칡 and soybeans, based on the total weight of the composition.

The phytoestrogen-containing hormone replacement therapy composition according to the present invention is not strictly limited, but may contain 0.1 to 8.0% by weight of chitooligosaccharide or cartilage derived from cartilage, which is helpful for skin improvement and activation of bone and cartilage tissue. .

In addition, if necessary, vitamins such as vitamin E, vitamin C, nicotinic acid amide, riboflavin and the like may contain 0.01 to 4% by weight.

The phytoestrogen-containing hormone replacement therapy composition according to the present invention may include collagen and / or mucopolysaccharide in the form of a solid preparation such as tablets, granules, or capsules, in which case the content is based on the total weight of the composition. And 2.5 to 5.0% by weight and 5.0 to 10.0% by weight, respectively.

The phytoestrogen-containing hormone replacement therapy composition according to the present invention is formulated into granules or conventional tablet forms by a compacting method or a smashing method using a roller compactor by a wet granulation method or a dry granulation method.

In general, in the manufacture of tablets, all additives other than the main component are collectively referred to as additives or excipients, and in more detail, may be divided into excipients, binders, disintegrants, lubricants and the like.

Excipients are selected in consideration of tablet experience, cost, compatibility with the main ingredient, etc., and are usually used for the purpose of giving a tablet a certain volume and good tabletting ability. Examples of suitable excipients include lactose, raolin, mannitol, starch, powdered white sugar, calcium phosphate, microcrystalline cellulose, colloidal silica and the like, but particularly preferred excipients in the present invention include lactose. The content is in the range of 10-25% by weight, based on the total weight of the composition.

In addition to improving adhesion between the granules, the binder acts to maintain the shape of the tablet after being compressed. The amount of the binder is determined according to the experience of the person to be added, the nature of the other ingredients, etc. If the amount is too small, the binding capacity is insufficient, if too large it will become too hard to slow down the disintegration and dissolution rate of the tablet. Referring to the type and amount of the preferred binder, corn starch suspension, glucose solution, maltose, natural gum, cellulose derivatives (methylcellulose, carboxymethylcellulose, microcrystalline cellulose), gelatin, povidone, collagen, mucopolysaccharide, etc. Preferred binders in the present invention include collagen or mucopolysaccharide having a skin improving effect and cartilage strengthening effect, and the ratio thereof is as described above.

Glidants have effects such as improved flow from the hopper to the die, prevention of sticking to punches or dies, reduced resistance when taking tablets out of the tablet press, glossing of the tablets, and the like. Examples of preferred lubricants include talc, magnesium stearate, stearic acid, calcium stearate, fumed silicon dioxide, and the like. Particularly preferred glidants include sun-activated iron and sodium alginate, and if the amount of the glidant is too high, the disintegration and elution of the tablets is delayed. In the present invention, at least one of the glidants is added to the total tablet content. It is good to use in the ratio of 0.1 to 0.5 weight% with respect to.

Disintegrants have the effect of swelling upon contact with moisture to disrupt tablets in the gastrointestinal tract, and examples of preferred disintegrants include those mentioned above as binders and excipients.

Excipients, binders and disintegrants are generally appropriately combined in consideration of the interrelationships between components, and the like may be added as a binder to act as a disintegrant by interaction with other excipients, and vice versa. In some cases, the strict distinction between them is not absolute, but rather relative.

In the case of the tablets, granules, and capsules in the form of solid preparations, the pomegranate concentrate can be prepared in 500 mg, 750 mg, or 1000 mg formulations based on the weight of the concentrated fruit juice. .

On the other hand, when the phytoestrogens-containing hormone replacement therapy composition according to the present invention is in the form of a liquid, it is preferable to add 3 to 10% by weight of polydextrose based on the total weight of the composition to form a stabilized colloidal solution.

Glucoseamine and the aforementioned vitamins can of course be included in the amounts described above.

The rest of the rest is mostly purified water.

In the case of the above-mentioned liquid form, one or two packs of vacuum-packed packs in a volume of 50 ml or 100 ml may be taken orally per day.

Hereinafter, a method for preparing a composition for phytoestrogen-containing hormone replacement therapy according to the present invention will be described.

First, the manufacturing step by the solvent extraction method by liquid liquid separation is as follows.

(A) a solvent mixing step of adding water, methanol, ethanol, n-hexane, ethyl acetate, or any mixture thereof to the pulverized pomegranate

(B) reflux step for 0.5 to 2 hours under temperature conditions between 60 and boiling point

(C) upper separation step

(D) solvent removal step

There is no particular limitation on the extraction solvent, preferred is methanol and ethanol, and n-hexane and / or ethyl acetate may be added if necessary.

The reflux step and the phase separation step are preferably repeated two to three times.

The concentrate via the solvent removal step is about 30% concentrate, with a total phytoestrogen content of about 1300-1700 pg / ml.

Next, the manufacturing step by the solvent extraction method by hydrochloric acid treatment is as follows.

(A) a solvent mixing step of adding water, methanol, ethanol, n-hexane, ethyl acetate, or any mixture thereof to the dried and pulverized pomegranate

(B) reflux step for 0.5-2 hours

(C) upper separation step

(D) HCl addition step

(E) stirring step

(F) glass filter filtration step

(G) a stationary step of standing in a hot water bath at 60 to 100 ° C. for 1 to 3 hours after adding HCl.

(H) cooling stage

(I) acetonitrile addition step

(J) solvent removal step

The reflux step and the phase separation step are preferably repeated two to three times. HCl is combined with sulfuric acid or glucuronic acid by using 0.1 ~ 1.0M concentration and stirring thoroughly to make it homogeneous, then using a glass filter to remove fine pulverized particles of skin, pulp and / or seeds and standing in a hot water bath. Estrogens present in the form are hydrolyzed to non-conjugated estrogens. Subsequently, acetonitrile is added to extract intracellular components, and the solvent is removed using distillation under reduced pressure or the like.

The concentrate via the solvent removal step is about 40% concentrate, with a total phytoestrogens content of about 1420-1830 pg / ml.

Following the acetonitrile addition step described above, (K) an enzyme hydrolysis step at 37 ° C., (L) ethyl acetate addition extraction step (preferably repeated two or three times), and finally (M) Solvent removal may also be performed.

In many cases, estrogen is present in the form of a conjugate with sulfuric acid or glucuronic acid, so that the enzymatic hydrolysis step may be performed independently or in addition to the hydrolysis by HCl to make an estrogen in a non-conjugated form. The decomposition step is performed by adding an acetate buffer solution adjusted to pH 5.2 with 0.2 M acetic acid and sodium acetate, followed by the addition of glucuronidase / arylsulfatase derived from Helix pomatia (manufactured by Boehringer Mannheim, Germany).

The concentrate in this case is about 50% concentrate, with a total phytoestrogen content of about 1540-1980 pg / ml.

Next, the manufacturing method by the solid phase extraction method is comprised of the following steps.

(A) a solvent mixing step of adding water, methanol, ethanol, n-hexane, ethyl acetate, or any mixture thereof to the dried and pulverized pomegranate

(B) reflux step for 0.5-2 hours

(C) upper separation step

(D) Sedolit AD-2 ion exchange resin pass step

(E) concentration step

(F) enzymatic hydrolysis step at 37 ° C. as described above

(G) ethyl acetate addition extraction step

(H) solvent removal step

It may be preferable to repeat the steps of (B) and (C) and the step of (G) 2-3 times. The column pass-through solution in step (D) is collected and stored separately, and the eluate using an acetone: methanol (1: 9 concentration gradient) solution is performed after steps (D) to (G), and then the column in step (D). Combine with the pass-through to perform the solvent removal step of (H).

The concentrate in this case is about 50% concentrate, with a total phytoestrogen content of about 1560-1960 pg / ml.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended only to illustrate the present invention and are not intended to limit the present invention.

Example 1 Preparation of Black Pomegranate Concentrate

After harvesting, 1 kg of iranian black stone flesh 3 kg after harvesting was pulverized, 8 liters of ethyl alcohol was poured and refluxed at a temperature of 70 ° C. for 1 hour. The upper layer was separated after reflux. The remainder was again refluxed under the same conditions and after the reflux the upper layer was separated and the combined upper layers were combined. The solvent was removed from the upper layer using a rotary vacuum evaporator at a temperature of 65 ° C.

* As a result, 214 g of a viscous residue was obtained.

Example 2 Preparation of Black Pomegranate Concentrate

After harvesting, 1 kg of iranian black stone flesh 3 kg after harvesting was pulverized, 8 liters of ethyl alcohol was poured and refluxed at a temperature of 70 ° C. for 1 hour. The upper layer was separated after reflux. The remainder was again refluxed under the same conditions and after the reflux the upper layer was separated and the combined upper layers were combined. The solvent was removed from the upper layer using a rotary vacuum evaporator at a temperature of 65 ° C.

As a result, 214 g of a viscous residue was obtained.

Example 2 Preparation of Black Pomegranate Concentrate

After harvesting, 1 kg of Iranian black stone (pulp + seed) 3 months after harvesting was ground, 9 liters of methyl alcohol was poured and refluxed at a temperature of 60 ° C. for 1 hour. The upper layer was separated after reflux. The remainder was again refluxed under the same conditions and after the reflux the upper layer was separated and the combined upper layers were combined. 100 ml of 0.5 M HCl was added to the combined upper layers, followed by stirring for 30 minutes, followed by filtration through a glass filter.

100 ml of 0.5 M HCl was further added to the filtrate, and it was allowed to stand in a hot water bath at 65 DEG C for 2 hours, and then cooled to room temperature. Then 100 ml of acetonitrile were added and then the solvent was removed using a rotary vacuum evaporator at a temperature of 70 ° C.

As a result, 267 g of a viscous residue was obtained.

Example 3 Preparation of Black Pomegranate Concentrate

After adding 200 ml of 0.2 M acetic acid buffer solution (pH 5.2) to 100 g of the residue obtained in Example 2, glucuronidase / arylsulfatase of Helix pomatia (manufactured by Boehringer Mannheim, Germany) (glucuronida) The estrogen conjugate was hydrolyzed at 37 ° C. by adding 5.5 U / ml (38 ° C.); arylsulfatase 2.6 U / ml (38 ° C.) in an amount of 50 μL. Then, 100 ml of ethyl acetate was added and extracted twice. Thereafter, the solvent was removed using a rotary vacuum evaporator at a temperature of 80 ° C.

As a result, 87 g of a viscous residue was obtained.

Example 4 Preparation of Black Pomegranate Concentrate

After harvesting, 1 kg of Iranian black stone (pulp + seed + pepper) three months after harvesting was ground, 4 L of methyl alcohol, 2 L of N-hexane, and 4 L of water were poured and refluxed at a temperature of 85 ° C. for 1 hour. The upper layer was separated after reflux. The remainder was again refluxed under the same conditions and after the reflux the upper layer was separated and the combined upper layers were combined.

The combined upper layers were loaded into an ion exchange resin Serdolit AD-2 resin column (particles of acetone / methanol / distilled water, then purified by reflux with acetone / methanol / distilled water) and stored in distilled water. H.L. The column was passed according to the method described in Steroids, 1977, 30, 581 and the flow through was collected separately. The column was again eluted with a solution having a concentration gradient of acetone: methanol 1: 9, and then the eluate was concentrated using a rotary vacuum evaporator at a temperature of 65 ° C.

300 ml of 0.2 M acetic acid buffer solution (pH 5.2) was added to the concentrated solution, followed by addition of glucuronidase / arylsulfatase to hydrolyze the estrogen conjugate for 2 hours at 37 ° C. Then 200 ml of ethyl acetate was added and extracted twice.

Thereafter, the extract and the column passing solution were combined and the solvent was removed using a rotary vacuum evaporator at a temperature of 85 ° C.

As a result, 135 g of a viscous residue was obtained.

Example 5: Preparation of Tablets

Using the concentrate obtained in Example 4, a tablet formulated at the following compounding ratio (wt%) was prepared:

Black Pomegranate Concentrate 40.0

Glucoseamine 5.0

Collagen 3.5

Mucodose 7.5

Vitamin E (powder) 2.5

Riboflavin 0.5

Sodium Alginate 0.3

Sun Active Iron 0.2

Lactose 20.0

Isoflavones 2.5

Carboxymethylcellulose 15.0

Glucose 3.3

------------------------------------

                            100.0

Example 6 Preparation of Liquid

The concentrate obtained in Example 1 was used to prepare a 50 ml dose of the formulation formulated in the following blending ratios (wt%):

Black Pomegranate Concentrate 10.0

Glucoseamine 0.7

Polydextrose 6.3

Vitamin C 0.1

Sorbitol 1.0

Nicotinic Acid 0.01

Purified water balance

----------------------------------

                            100.0

Example 7: General Component Analysis

General ingredient analysis was performed on the black pomegranate concentrate obtained in Example 1 and the purification of Example 5.

The moisture content of the sample was 105 ° C atmospheric pressure drying, the crude fat content was Soxhlet extraction method (Auto Soxtec System HT 1043, Tecator, Sweden), and the crude protein content was measured by nitrogen method (Kjeltec 1030 Auto Analyzer, Tecator, Sweden). It was calculated by multiplying the coefficient 6.25, and the content of crude fiber was measured by H ₂ SO ₄ -NaOH decomposition (Dosi-Fiver 6 units, JP Selecta, Spain), and crude ash was measured by direct ashing. Carbohydrate content is expressed as 100% minus the amount of protein, fat, fiber and ash.

The results are shown in Table 1 below.

Furtherance Example 1 Example 5 moisture 39.3 2.6 Geology 0.4 1.5 protein 0.9 3.8 Ash 1.4 7.6 carbohydrate 42.0 84.5 synthesis 100.0 100.0

Example 8 Amino Acid Analysis

Amino acid analysis was performed on the black pomegranate concentrate obtained in Example 3 and the purification of Example 5.

The digestion solution obtained by acid hydrolysis of 1 g of the sample with 20 ml of 6N HCl in a test tube for 24 hours at 105 ° C. was filtered through a 0.2 μm filter and analyzed by an amino acid autoanalyzer using the ninhydrin method. The analytical instrument used a 6300 amino acid analyzer (Beckmann Inc., Fullerton, Calif., USA) and detection was at 460 nm and 530 nm. The column used was a sodium column (4.6 mm x 15 cm).

The results are shown in Table 2 below (unit is ppm).

amino acid Example 3 Example 5 Asphalt Mountain 896.2 1523.7 Threonine 88.7 311.8 Serine 459.5 545.4 Glutamic acid 1310.2 1735.7 Proline 174.0 2384.9 Glycine 106.5 505.3 Alanine 270.3 1095.1 Cysteine 7.6 152.3 Valine 97.5 518.8 Methionine 92.7 Not detected Isoleucine 52.7 368.1 Leucine 64.8 442.2 Tyrosine 59.4 111.2 Phenylalanine 57.5 138.8 Histidine 158.3 295.4 Tryptophan 74.5 440.6 Lee Sin 82.1 510.5 Arginine 187.7 1237.9

Example 9 Mineral and Weak Acid Analysis

Mineral analysis was performed on the black pomegranate concentrate obtained in Example 4 and the purification of Example 5.

Mineral pretreatment was performed by dry method and used for inductively coupled plasma-atomic emission spectrophotometer (Spectro flame Modula E, Fitchburg, MA, USA). The instrument operating conditions are as follows:

-------------------------------------------------- ---

1.2Kw for power reception

3.5bar for atomizer pressure mainhard type C

Aerosol Flow Rate 0.3ℓ / min

Sheath gas flow 0.3ℓ / min

Cooling gas flow 12ℓ / mim

Wavelength (nm) Fe 275.574

                       Cu 324.754

                       K 766.491

                       Zn 213.856

                       Mn 257.610

                       P 178.290

                       Na 588.995

                       Ca 373.690

--------------------------------------------------

The results are shown in Table 3 below (unit is ppm).

Furtherance Example 4 Example 5 K 2550.8 2908.0 Ca 80.0 143.9 Na 10.1 14.7 P 150.0 167.8 Fe 6640.0 7311.0 Cl 3464.0 898.0 Mn 0.59 5.3 Cr 0.02 0.06 Zn 1.85 2.09 Cu 0.9 0.9 Pb 1.0 0.9 CD Not detected Not detected NO ₂ 14972.0 2285 HPO ₄ 33980.0 8243 SO ₄ 4060.0 955 Glycolic acid 40680.0 406.7 Formic acid Not detected 1.6 Acetic acid 6800.0 33.3

Example 10 Vitamin Analysis

Vitamin analysis was performed on the black pomegranate concentrate obtained in Example 4 and the tablets of Example 5.

Vitamins A and E were extracted with a mixed solvent of chloroform / water and analyzed by HPLC. The column was μ-Bondapak C ₁₈ (4.6mmx15cm), the solvent was 100% methanol, and the moving speed of the solvent was 1 ml / min. Detection was performed at 325 nm for vitamin A and 295 nm for C. Vitamin C was quickly extracted with 5% metaphosphophosphate solution, and HPLC method μ-Bondapak C ₁₈ (4.6mm inner diameter x 15cm), solvent was 100% H ₂ O and the flow rate of solvent flowed by 1ml per minute and detection was 270nm. At. Other water soluble vitamins were extracted with a 1: 1 mixed solvent of methanol and water, followed by HPLC gradient concentration over 20 minutes in a column of μ-Bondapak C ₁₈ (4.6mmx15cm) with a solvent of 60% methanol at 100% H ₂ O. 1 ml per minute and detection was performed at 270 nm.

The results are shown in Table 4 below (unit: mg / 100g).

Furtherance Example 4 Example 5 Water soluble vitamins Thiamine (vitamin B ₁ ) 0.12 0.37 Riboflavin (vitamin B ₂ ) 0.02 0.06 Ascorbic acid (vitamin C) 20.00 25.41 Niacin 0.80 0.99 Vitamin B ₆ 0.02 0.06 Fat-soluble vitamins Tocopherol (vitamin E) Not detected Not detected

Example 11 Fatty Acid Analysis

Fatty acid analysis was performed on the black pomegranate concentrate obtained in Example 2 and the purification of Example 5.

Fatty acid composition was analyzed according to the Bligh and Byer method, the n-hexane layer was methyl esterified, dehydrated with anhydrous Na ₂ SO ₄ , filtered and analyzed by GC. For analysis, GC used Hewlett Packard 6890A (Palo Alto, CA, USA), and the column was HP FFAP.The injection port was 260 ° C, the detection port was 270 ° C, and the oven was 220 ° C for 2 ° C per minute. Raised up. The gas used was helium, the sample injection amount was 0.5 µl, the split race was 50: 1, and the detection was a flame ionization detector.

The results are shown in Table 5 below (unit is peak area%).

Furtherance Example 2 Example 5 Myristic acid 13.1 2.5 Palmitic acid (16: 0) 8.3 30.1 Stearic acid (18: 0) 69.40 51.6 Oleic acid (18: 1) 6.8 3.9 Linoleic acid (18: 2) Not detected 3.3 Linoleic acid (18: 3) Not detected Not detected Etc 2.4 8.6

Example 12 and Comparative Examples 1 and 2: Phytoestrogen Analysis

The phytoestrogens analysis was carried out on the soybean and the root root concentrates as the black pomegranate concentrates obtained in Example 4 and Comparative Examples 1 and 2.

The phytoestrogens standards used were flavones, hexaestrols, DES, equols, chrysins, enterolactones, α-giralanol, giralanone, 17-β-estradiol, biocanin-A, catechins, β-giral 20 kinds of enol, α-giralenol, dydzein, genistein, kaempferol, epigenim, quercetin, and estrogens are estrone, 17-β-estradiol, 2-hydroxyestron, 2- Hydroxyestradiol, 6-α-dihydroestrone, 6-α-hydroescistradiol, 4-methoxyestradiol, estriol, 16-epitriol, 16,17-epitriol, 16- 16 species of α-2-hydroxyestrone, 17-epitriol, 6-ketoestriol, 2-methoxyestriol, 6-hydroxyestriol and 16-ketoestradiol, internal standard The material was all d4-17β-estradiol using Sigma, USA.

The standard solution was used by weighing 0.01 g accurately, making 1000 ppm with methanol, and diluting 10 times with refrigeration.

The derivatization reagent was purchased from MSTFA (N-methyl-N-trimethylsilyltrizluoacetamide) and trimethylchlorosilane (TMCS), and a mixed solution of STRA and TMCS in a ratio of 100: 1 (v / v) was prepared. Used.

Serdolit AD-2 resin (100-200 microns) was purchased from Serve (Germany).

For the pretreatment and analysis of samples, see Chung et al. In other words, 10 mg / ml of internal standard d4-17β-estradiol was added to 25 mg of the sample extracted with methanol, and hydrolyzed at 55 ° C. for 3 hours, and then 50 μl of a 100: 1 mixture of MSTFA / TMSC was added and reacted at 60 ° C. for 30 minutes. TMS ester derivative was prepared, and then 2 µl of the TMS ester derivative was injected into the GC / MS. Derivatized and injected into GC / MS. The analytical instrument used used a 5970 Mass Selective Detector (MSD) connected by direct interface to the Hwelette-Packard 5890 PIUS gas chromatography. The separator was Ultra-2 (length 25mm, inner diameter 0.20mm, film thickness 0.33㎛), the temperature of the separator was raised from 20 ℃ / min to 20 ℃ / min from 180 ℃ to 260 ℃ was maintained for 10 minutes. The temperature of the detector was set at 300 ° C., the detector temperature at 300 ° C., and the flow rate of the mobile gas helium was 0.85 ml / min, and the injection method was set in the split mode (ratio 1:12). Selected ion monitoring (SIM) method was used to select and detect only characteristic ions on the mass spectrum.

The results are shown in Table 6 below (unit is ppm).

Furtherance Example 4 (black pomegranate) Comparative Example 1 (soybeans) Comparative Example 2 (root) Equol Not detected Not detected 0.63 Biocanine-A Not detected 0.66 0.65 Catechin 1.48 Not detected Not detected Dyed Jane 23.72 745.46 51.15 Genistein 0.29 3.48 0.31 Apigenin Not detected 35.33 Not detected Quercetin 9.75 Not detected Not detected 17β-estradiol 0.15 Not detected Not detected 2,3-di-MeO-estradiol 0.04 Not detected Not detected

Example 13:

Thirty-five women aged 55 to 65 who are experiencing hot flashes were randomly divided into two groups of 15, one group taking nine tablets (750 mg / dog) prepared in Example 5 for one month and the other. The group was asked to take nine tablets of the same shape three times a day for three months instead of the above-mentioned tablets instead of the above-mentioned tablets as a flashbo, and to investigate the relief of hot flashes, and the results are shown in Table 7 below.

3 days 1 week 2 weeks 3 weeks 1 month Example 5 Administration Group 6 (1) 12 (5) 14 (8) 15 (10) 15 (14) Flashbo group 1 (0) 2 (0) 1 (0) 3 (0) 2 (0)

In the above table, the number is the number of people who responded that it was alleviated and the number in parentheses was the number of people who answered that the flushing phenomenon disappeared.

From the above results, it could be confirmed that the tablet according to the present invention had an excellent effect on the treatment and prevention of hot flashes due to menopause.

Example 14

ROS 17 / 2.8, which is an osteoblast cell line related to bone formation, was used to evaluate whether the composition for hormone replacement therapy containing the black pomegranate extract according to the present invention was promoted.

Dulbecco's modification of ROS 17 / 2.8 cells with 10% fetal bovine serum without phenol red and 1% antibiotic-antibacterial mixture (100 IU penicillin, 100 mg streptomycin, 250 ng amphotericin) Inoculated in Dulbecco's Modified EagleMedium and incubated in 37 ° C., 5% CO ₂ incubator. Cell proliferation was performed by MTT assay and radiolabeled thyme through metabolic reduction principle of MTT (3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazolin bromide). It was measured by a thymidine incorporation assay through din ( ³ H-Thymidine) radioactivity.

The extract in Example 3 was evaluated, and the growth rate was compared by setting the genistein isolated from soybean as a comparative control and the sample-free group as a control.

First, ROS 17 / 2.8 cells were seeded in 96-wells with cell count 2 × 10 ³ with 8% estrogen deficient-FBS medium and cultured with 0.8% estrogen deficient-transmitted serum for 24 hours to rule out the effects of the deceased serum. The composition was changed and stimulated for 48 hours with phytoestrogens mixture (Example 12) and genistein (10 ^-9 and 10 ^-12 molar concentrations) from the Black Pomegranate extract under 2.5% estrogen deficiency-transmitted serum conditions. A 75 μl empty (1.6 mg / mL) solution was added to each well, followed by reaction at 37 ° C. for 3 hours, followed by addition of 75 μl of dimethylsulfoxide to formazan crystals formed on cells. ) Was dissolved. Absorbance was measured at 540 nm using a microplate reader. Subsequently, the same cells were seeded in a 24-well cell number 1 × 10 ⁴ and stimulated as described above. Four hours before the end of stimulation, radiolabeled thymidine solution was added at 1 μc per well and incubated at 37 ° C. The cultured cells were precipitated with 3% trichloroacetate solution and lysed by adding 200 μl of 2% disodium carbonate solution. Radioactivity was measured on the beta counter. The ability to promote bone formation was expressed relative to the control.

The results are shown in Table 8 below.

Concentration (m) Control 10 ^{-12 1-9} Phytoestrogen Mixture in Example 3 100 117 126 Soybean Genistein 100 113 111

As can be seen from Table 8 above, the phytoestrogens mixture in Example 3 already exhibited 117% cell proliferation at low concentrations of 10 ^-12 M and up to about 126% at physiological concentrations of 10 ^-9 M It increased and promoted the proliferation of osteoblasts in a dose-dependent manner. Genistein in soybean also increased osteoblast proliferation up to 113%, but was found to have a much lower effect than the phytoestrogens mixture of Example 3.

Example 15:

In order to evaluate the bone formation of the composition for hormone replacement therapy containing the black pomegranate extract according to the present invention, after inoculating KS483 cells, which are undifferentiated osteoblasts of mouse fetus, with a cell number of 2.4 × 10 ⁴ in 36-well plates, The cells were stimulated under the same conditions as in Example 14 under 8% S. aureus serum when grown and reached a dense state. Genistein was set as a control group and the sample-free group was set as a control group.

Cell differentiation-inducing substances ascorbic acid (43㎍ / mL), beta-glycerophosphate 3mM and dexamethasone 5nM were added every 2 days, and cultured for 3 weeks, and cell staining at each time point was compared to promote differentiation. .

At 3 weeks of induction of differentiation, the number of nodules formed by Boncosa staining with silver nitrate was compared. Differentiation promoting action is shown relative to the control.

Concentration (m) Control 10 ^{-12 1-9} Phytoestrogen Mixture in Example 3 1.0 2.12 ± 0.00 3.38 ± 0.01 Soybean Genistein 1.0 ND 0.92 ± 0.01

From the results of Table 9 above, the phytoestrogens mixture in Example 3 already showed a two-fold cell differentiation promoting effect at a low concentration of 10 ^-12 M, and increased to 3.4 times at a physiological concentration of 10 ^-9 M It can be seen that it shows a dose-dependent differentiation promoting effect. On the other hand, genistein of soybean showed about 0.9-fold differentiation at physiological concentrations.

Therefore, the composition for hormone replacement therapy containing the black pomegranate extract of the present invention was determined to have a very good osteoblast differentiation effect.

Example 16:

In order to evaluate the osteoporosis inhibitory effect of the composition for hormone replacement therapy containing the black pomegranate extract, 12-week-old Sprague-Dawley female mice (average weight 240 g) were placed under both an ovary under pentobarbital anesthesia. Was extracted and control mice were sham. After removing the ovaries, one week later the mice were divided into three groups:

The group fed the diet of Example 6 (5 mL / kg / day), the subcutaneous injection of estrogen twice a week (50 μg / 100 μl soybean oil), and the control group fed the control diet. Four weeks after the ovary removal, the rats were sacrificed to separate the hind tibia.

The tibia was immediately stored in formalin fixative and demineralized, and the proximal metaphysis site was traversed to 5 μm. The degree of osteoporosis was determined by trabecular bone density, cortical bone thickness, and adipocyte density in the bone marrow cavity. Each result was expressed as a relative value to the singer group, and the difference between groups was tested by univariate variance analysis.

Suzhou Bone Mineral Density Cortical bone thickness Fat cell density Singer 100 ± 1.1 100.0 ± 8.6 100.0 ± 5.7 Ovarian removal group No treatment group 13.1 ± 0.3 105.3 ± 14.1 283.1 ± 3.8 Diet of Example 3 62.1 ± 3.6 112.5 ± 11.2 116.8 ± 17.4 Estrogen treatment 82.7 ± 4.2 107.1 ± 11.3 135.7 ± 18.2

As can be seen from Table 10 above, after 4 weeks of ovarian removal, the bone mineral density of the tibia in the untreated group decreased to 13% of the control group, indicating severe osteoporosis, and bone density of the estrogen-treated rats after ovarian removal. Was about 83% of the control group was confirmed that the progress of osteoporosis was effectively suppressed. On the other hand, the bone density of the mice fed the diet of Example 3 after ovarian removal was 62% of the control group. This value is lower than estrogen but more than five times higher than the bone mineral density of osteoporosis resulting from ovarian removal, which strongly suggests the possibility that the progress of osteoporosis can be significantly inhibited upon oral administration of the hormone replacement therapy composition according to the present invention. It became.

As osteoporosis progressed after the ovarian removal, the bone marrow fat cell density increased by 2.8 times in the untreated group, whereas in the case of artificially subcutaneous administration of estrogen after ovarian removal, the fat accumulation in the bone marrow was about 36 % Increased. In contrast, the increase in bone marrow fat accumulation in the mice fed the diet of Example 3 after ovarian removal was only 16.8% of the control group. Therefore, these results indicate that the increase in fat, which is a side effect of the administration of synthetic estrogen, is much less when the composition for hormone replacement therapy according to the present invention is ingested.

Example 17

In order to investigate the effects on atherosclerosis, male mice that were fasted for one day to cause hyperlipidemia were selected and tested. Vitamin D2 and cholesterol were administered 80,000 ed / 100 g and 20 mg / 100g for 3 days, respectively, and vitamin-cholesterol hyperlipidemia was induced and intraperitoneal administration of Triton WP-1339 225 mg / kg was used. The amount of lipid was then measured 20 hours after Triton and 20 hours after the administration of the vitamin and cholesterol mixture. The extract of Example 4 was administered four times in an amount of 3 mg / kg 3 days before hyperlipidemia induction and 3 hours before sacrifice.

In the case of endogenous hyperlipidemia, the initial cholesterol level was 0.83 ± 0.07mmol / l and the amount of triglyceride was 0.28 ± 0.003mmol / l. It increased to 0.04 mmol / L, but the extract of Example 4 significantly decreased to 0.65 ± 0.02 mmol / L and 0.26 ± 0.021 mmol / L, respectively. In addition, the amount of triglyceride in blood increased by 29 times to 8.2 ± 0.41 mmol / l and the amount of cholesterol increased by 6.7 times to 5.45 ± 0.13 mmol / l. On the other hand, when the extract of Example 4 was administered, the amount of cholesterol decreased to 2.76 ± 0.24 mmol / L, but there was no change in the amount of triglycerides in the blood.

On the other hand, in the case of vitamin-cholesterol hyperlipidemia, the blood concentrations of cholesterol and triglyceride increased 3.2 times compared to the initial concentration, but when the extract of Example 4 was administered, the amount of cholesterol was normalized and the amount of triglyceride was increased.

From the above results, it was confirmed that the composition for hormone replacement therapy according to the present invention effectively reduced the concentrations of cholesterol and triglycerides.

Example 18

In order to investigate the effect of the extract on the central nervous system, the acute toxicity test was performed by intravenously administering 3 to 10 mg / kg of the extract of Example 4 to the urine coma and the morphine-urethane coma. There was a slight increase in arterial blood pressure (3-7%) for 3 minutes and a slight increase in the arterial stimulation effect of adrenaline, but no change was observed in the arterial pressure with the administration of histamine and acetylcholine.

From these results, it was confirmed that the composition for hormone replacement therapy according to the present invention did not cause any acute toxicity, and also had no effect on the function of the midline nervous system.

As described above, the composition for hormone replacement therapy according to the present invention contains a large amount of safe phytoestrogens showing good estrogenity and little side effects, and thus can be caused by estrogen deficiency in menopausal or postmenopausal women. It is expected to effectively prevent and improve the symptoms. In particular, the composition for hormone replacement therapy according to the present invention has excellent effects on hot flashes, osteoporosis and atherosclerosis, and is very low in body fat as a side effect. Is a new useful invention.

Claims (5)

At least one solvent extract selected from the group consisting of water, methanol, ethanol and n-hexane, 1.0-2.0 ppm of catechin as a phytoestrogen hydrolyzed in an unconjugated form with sulfuric acid or glucuronic acid, 20 -30 ppm, Genistein 0.1-0.5 ppm, Quercetin 8.0-14.0 ppm, Pomegranate extract containing 17--estradiol 0.05-0.3 ppm and 2,3-di-MeO-estradiol 0.01-0.10 ppm, based on the total weight of the composition Hormone replacement therapy phytoestrogens composition for the treatment of osteoporosis or hot flashes prevention of menopausal or postmenopausal women containing 0.1 to 99% by weight. The menopause according to claim 1, wherein the pomegranate extract has a water content of 35 to 42% by weight, a lipid content of 0.3 to 0.6% by weight, a protein content of 0.7 to 1.2% by weight, an ash content of 1.2 to 1.6%, and a carbohydrate 38 to 47% by weight. Or phytoestrogens for hormone replacement therapy for the treatment of osteoporosis or hot flashes in postmenopausal women. The phytoestrogen composition for hormone replacement therapy for treating osteoporosis or hot flashes of menopausal or postmenopausal women according to claim 1, wherein the phytoestrogen is hydrolyzed with HCl. The phytoestrogen composition for hormone replacement therapy according to claim 1, wherein the phytoestrogens are enzymatic hydrolyzed by glucuronidase / arylsulfatase to treat osteoporosis or hot flashes in menopausal or postmenopausal women. According to claim 1, wherein the composition is 0.1 to 5.0% by weight based on the total weight of the composition of isoflavones, which are phytoestrogens derived from mussels, soybeans or murine soybeans, and 0.1 to 8.0% by weight of glucoseamine %, And phytoestrogens for hormone replacement therapy for the treatment of osteoporosis or hot flashes in menopausal or postmenopausal women further comprising 2.5 to 5.0% by weight and 5.0 to 10.0% by weight of collagen and mucopolysaccharide, respectively.