KR20210009075A - Health food containing phytoestrogen of pomegranate and manufacturing method - Google Patents

Abstract

The present invention relates to a healthy food containing phytoestrogen of pomegranate and a manufacturing method thereof and, more specifically, to a method for manufacturing healthy food including a pomegranate extract containing a large amount of phytoestrogen. According to the present invention, provided is a method for manufacturing healthy food, which contains 1 to 99 wt% of a phytoestrogen extract comprising 10 to 20 ppm of catechin, 20 to 30 ppm of daidzein, 1 to 5 ppm of genistein, 80 to 140 ppm of quercetin, 5 to 3 ppm of 17 β -estradiol, and 1 to 10 ppm of 2,3-di-MeO-estradiol with respect to the total weight, for preventing and mitigating osteoporosis or hot flashes in menopausal or postmenopausal women. Since the healthy food contains a large amount of safe phytoestrogen that has excellent estrogenic properties and have no side effects, the healthy food is expected to effectively prevent and treat all symptoms that may be caused by estrogen deficiency in menopausal or postmenopausal women and, in particular, provides an excellent effect on hot flashes, osteoporosis, and atherosclerosis while an increase in body fat as a side effect is very rare. The method comprises a solvent mixing step, a refluxing step, an HCl addition and stirring step, a filtering step, an HCl addition and hot water bath setting step, an acetonitrile addition extracting step, a solvent removal step, and a formulation step.

Description

Health food containing phytoestrogen of pomegranate and manufacturing method {Health food containing phytoestrogen of pomegranate and manufacturing method}

The present invention relates to a health food and a manufacturing method containing phytoestrogen of pomegranate, and more particularly, to a method of manufacturing a health functional food comprising a pomegranate extract containing a large amount of phytoestrogen.

In general, the endocrine system is an organ system that produces and secretes various hormones involved in the maintenance of homeostasis, reproduction, development, and behavior of a living body, and is composed of glands, hormones, and target cells, and the hormone produced here is blood. It circulates through the body and transmits information and commands to target cells and tissues to participate in growth and development, and the differentiation of cancer and water.

Estrogen, one of these endocrine hormones, is synthesized from cholesterol in the body, and its secretion sources are mainly follicles and corpus luteum of the ovary, and fetal placenta during pregnancy, adrenal glands. · It is also secreted from the testis (精巢). The secretion of estrogen in the ovaries is follicle-stimulating hormone, a hormone that regulates the menstrual cycle and enables pregnancy when gonadotropin, secreted from the anterior pituitary gland, transmits a signal to the reproductive system. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are secreted, which in turn act on the ovaries to secrete estrogen and progesterone. Estrogen stimulates the endometrium in the first half of the menstrual cycle to thicken the endometrium so that the fertilized egg can be implanted, and increase the blood supply, and in the latter part, progesterone is secreted to make the endometrium a better condition for implantation. In other words, estrogen is involved in proliferation of the endometrium, development of the uterine muscles, the expression of secondary sexual characteristics, the formation of the menstrual cycle, causing maternal changes during pregnancy, and promoting the proliferative and secretion of the mammary gland duct (乳腺管). It is a representative female hormone, but it has a wide range of effects on many other parts of the body.

The aforementioned estrogen is generically referred to as estradiol, estrone, and estriol, and among them, estradiol is known to be the most powerful. Estrogen functions by mediating receptors, and is mainly metabolized in the liver, becoming cosynthetic estrogen and excreted in the urine.

In premenopausal women, the ovaries are the main source of estrogen. The main secretion product is estradiol, which is synthesized by granulosa cells from androgen precursors supplied by follicular membrane cells. Secreted estradiol is reversibly oxidized to estrone and estrogen can be converted to estriol. Interconversion between estrone and estradiol occurs primarily 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, and thus 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 ovaries, is the major circulating estrogen. Serum estradiol levels are low in adolescent girls and high in menarche girls. The concentration of estradiol in women ranges from about 100 pg/ml (367 pmol/l) in the follicular phase to about 600 pg/ml (gett pmol/l) in the ovulatory phase. During pregnancy, it rises to almost 20,000 pg/ml (70,000 pmol/l). After menopause, serum estradiol concentrations have been reported to drop to levels similar to those found in men of the same age (5-20 pg/ml) (Yen, SSC and Jaffe, RB, 3rd ed Philadelphia: WB Saunders, (1991). ))

As women enter menopause, as the ovaries age, their response to pituitary gonadotropin (follicle-stimulating hormone (FSH) and luteinizing hormone (LH)) decreases, resulting in a decrease in the initial follicular phase. Shorter, shorter menstrual cycles, shorter ovulation periods, reduced progesterone production, and more irregular cycles. Eventually, the follicle becomes unresponsive and cannot produce estrogen. Decreased estrogen secretion has a great effect not only on the genitourinary system of women, but also throughout the body, and menopause is called menopause when ovulation is stopped and menstruation disappears, and menopause symptoms due to a decrease in hormones appear before or before menopause.

In addition, immature menopause occurring before age 40 is collectively referred to as ovarian failure of unknown cause, and it is believed to be related to large amounts of long-term smoking, living at high altitude, and malnutrition. Menopause may result from ovariectomy, chemotherapy, irradiation of the pelvis, or any process that lowers the blood supply to the ovaries.

In postmenopausal women, due to the rapid decrease in estrogen, psychological and emotional signs such as fatigue, excitement, insomnia, decreased concentration, depression, memory loss, headache, anxiety, nervousness, and fear of small heartache increase significantly, and relapse. Fatigue and excitement, intermittent dizziness, paresthesia, palpitations and tachycardia, nausea, constipation, diarrhea, joint pain, muscle pain, cold hands and feet, and weight gain due to sleep disturbance caused by sexual hot flashes are also significantly increased. In addition, not only osteoporosis is easily induced, but also the mortality rate from cardiovascular diseases such as heart disease, high blood pressure, and stroke increases (Kalin MF & Zumoff B Steroids 55:330-352, 1990). Also, changes in the skin and genitourinary system And the likelihood of developing autoimmune diseases, cataracts, and colon cancer is increased.

When a woman enters menopause and estrogen decreases significantly, for example, the vaginal mucosa and vulva skin become thinner, the usual bacterial colony changes, and the size of the labia minora, clitoris, uterus and ovaries decreases. It causes change. Therefore, it may cause inflammation of the vaginal mucosa (atrophic vaginitis), frequent urination and urination, vaginal dryness and sexual discomfort. In addition, there is a tendency to develop pelvic muscle tone loss, incontinence, cystitis and vaginitis.

It is known that about 75% of menopausal women experience hot flashes and sweating. Redness usually occurs during or after menopause, and is directly related to estrogen deficiency. Most of them last more than 1 year, and 25 to 50% last more than 5 years. Facial flushing begins with a sudden heat sensation concentrated on the face, neck and upper chest, and the skin turns red, and this heat sensation lasts for 30 seconds to 5 minutes, often accompanied by a large amount of sweating, sometimes with palpitations, It is usually accompanied by sweating and chills (Casper, RF, Yen, SSC Neuroendocrinology of menopausal flushes: Anhypothesis of flushmechanism Clin Endocrinol 1985; 22:293).

Such facial flushing may range from once or twice a day, or, in many cases, about once per hour, day or night, and usually occurs several times a day. The hot flush phenomenon causes sleep arousal, disrupts sleep, and can cause quite embarrassing problems in social life because numerous women experience a large amount of sweating. It is believed that hot flashes are caused by hypothalamic heat regulation dysfunction due to estrogen withdrawal. It has been reported that temperature changes are mediated from the fact that hot flushes occur simultaneously with luteinizing hormone pulses (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 initiating mechanism of hot flashes is an endogenous opioid peptide withdrawal phenomenon. , Estrogen increases central opioid peptide activity, while menopause has been shown to be 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, it is known that the most effective known method for preventing or treating the above-described hot flashes is to supplement the deficient estrogen by administering estrogen.

In addition, it has been reported that the susceptibility to cardiovascular atherosclerosis, which has a high incidence in menopausal women, can be effectively reduced by increasing the estrogen concentration in the blood (Stout DW, 1982). As a result of experiments on rabbits with sclerosis, estrogen was found to lower the lipid content in the blood and to exhibit antiatherosclerotic effects (Ivanova LV, 1996; Numano, 1971). In addition, estrogen was synthesized by cholesterol (cholesterin). Examples of its use as an inhibitor have also been reported (Ekzoesterol, etinilestradiol, premarin; PA Nussbaumer, Hunkler F, 1967).

Complications of atherosclerosis occur in menopausal women due to a decrease in blood estrogen concentration and a decrease or loss of ovarian function, and long-term hormone therapy using estrogen is widely used in the West, and many clinical studies Results also report the positive effects of estrogen (MyasnikovLA, 1961; Kraznai I, 1963; Patrono V, 1972) In addition, treatment of men aged 50-70 years with myocardial infarction, a complication of atherosclerosis of the heart. It has also been reported that the above-described good results were obtained (Statut RU, 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 known to be effective in cell-mediated immunity and humoral immunity. It is caused by abnormal regulation and is often associated with abnormal or enhanced T-cell, B-cell and macrophage effector cell function to autoantigens, and the activity of these cellular components to autoantigens is a factor in feedback mechanisms associated with autoresistance. It is believed to be related to destruction. For example, autoimmune diseases such as Hashimoto's thyroiditis predominate in women of childbearing age, and the incidence ratio of women to men is about 50:1 (Ahmed et al., 121:531 (1985)).

Estrogen has been demonstrated to have an inhibitory role on T cell function, but to have an immune stimulating effect on B cells. Therefore, estrogen is expected to be useful in the prevention and treatment of diseases related to activated T cells including rheumatoid arthritis, polysclerosis, Guillain-Barre syndrome, and Hashimoto's thyroiditis through inhibition of T cell function. Yes (Holmadahl, J 2:651 (1989))

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

In addition, estrogen has been reported to be an inhibitor of colon cancer. In this regard, the cohort study reported that estrogen supplementation therapy reduced the risk of colorectal cancer in women by synthesizing bile acids. In recent years, it is believed that the reduction in mortality of female colorectal cancer in the West is a result of the widespread use of postmenopausal estrogen supplementation (Mayer, Chapter 92, in, 14th ed, 1998).

In addition to the above, estrogen has 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, estrogen inhibits the death of human endothelial cells cultured in an estrogen receptor-dependent manner (Spyridopoulos, I, J, 95:1505-14 (1997)) The initial restoration of endothelial integrity by estrogen is the use of nitric oxide. It has been reported to cause a decrease in response to injury by increasing the likelihood and directly inhibit the proliferation of smooth muscle cells (Cornwell, TL, J, 267:C1405-C1413 (1994)).

In addition, it has been suggested that estrogen has a prophylactic effect against cataracts by epidemiologic evidence. Although women have a higher risk of developing cataracts than men, this increased risk is known to be more prone to postmenopausal estrogen depletion (Livingston, PM, J, 26:1-6, (1994); Klein, BE, J, 116:219-225, (1998)) In a study involving 544 women, early menopause was reported to increase the risk of developing cataracts by about 29 times (Shibata , T, J, 26:25-33, (1994)) In addition, according to the results of a small epidemiological study, it is reported that estrogen supplementation therapy after menopause 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)) Age-related cataract in vivo rat The (rat) model suggests that the prophylactic effect of estrogen is due to the genome (Bigsby, RM, J, 96:9328-9332, (1999)).

As described above, estrogen has an excellent function of protecting not only the urogenital system of women, but also various tissues such as bone tissue, heart, blood vessels, central nervous system, and skin tissue. Hormone replacement therapy (HRT) has been widely used as a treatment method (Lobo RA & Speroff L FertilSteril 61: 592-595, 1994), which was conceived that the cause of menopausal symptoms was due to decreased estrogen secretion. It is a method of artificially administering synthetic estrogen or a mixture of synthetic estrogen and synthetic progesterone. Clinically, it has been applied to the treatment of amenorrhea or menstrual abnormalities, menstrual movement, menopausal disorders, hormone therapy for prostate cancer or breast cancer, and osteoporosis. In addition, oral contraceptives consist of progesterone and estrogen. In addition, ovulation inducing agents, chromophene, and tamoxifen used in the treatment of breast cancer are non-steroidal antiestrogens. The administration of such synthetic estrogen hormone preparations is applied in the form of a tablet form orally administered or a patch form adhered to the skin, or a cream form for topical application.

However, long-term administration of synthetic estrogen has a serious problem that it can cause breast and uterine cancer, and many studies have reported (e.g., Hunt K etal, Br J Obstet Gynaecol 94:620-635, 1987; Ravnikar VA Women's Health Issues 2:75-82, 1992) In recent years, controversy has heated up not only among scholars but also in society as a whole, with many reliable claims that synthetic estrogen replacement therapy over a long period of time is likely to cause breast cancer. Although not yet clearly proven, it is believed that the risk of developing breast cancer in women receiving synthetic estrogen replacement therapy is related to estrogen exposure doses over time.

Therefore, studies to find phytoestrogen, a plant estrogen, as a safer substitute for synthetic estrogens used in the above-described hormone replacement therapy have been actively conducted in recent years.

The aforementioned phytoestrogen refers to a nonsteroidal compound present in plants that exerts an estrogenic effect in the body of an animal, and most of grains, fruits, and vegetables known to have anticancer action and effect on heart disease contain trace amounts of phytoestrogens. In particular, it contains relatively large amounts of processed soybean foods including soybeans (soy milk, tofu, miso, etc.), various cereals, fruits, alfalfa, and clover. It is known that isoflavones are mainly contained in legumes, and lignans are mainly contained in cereals.

The active effect of the above-described plant estrogen is only 1/100 to 1/1000 compared to the endogenous estrogen produced in the body, but the content is much larger, so that a sufficient estrogen effect can be obtained as a result. Representative activities of phytoestrogens are known to be effective in preventing bone loss and increasing bone density, reducing FSH and LH when administered to menopausal women, and alleviating menopausal symptoms, and genistein, one of the isoflavones, It has been reported that it has an anti-cancer effect of inhibiting the growth of cancer cells by inhibiting the protein tyrosine kinase of cells.

Therefore, it can be used for the prevention and treatment of osteoporosis in postmenopausal women, as well as lowering LDL (low density lipid) and increasing HDL (high density lipid), and acting as an estrogen agonist or antagonist showing anticancer effects in the endometrial and breast. In addition, request for effective phytoestrogens that can be used as a health functional food or therapeutic agent for the prevention and treatment of menopausal symptoms, which can be used effectively and safely for skin improvement, cataract and colon cancer prevention, and autoimmune disease prevention. This is rapidly increasing.

In response to this request, the current food and pharmaceutical industry has been widely seeking a safe natural hormone replacement plant that does not cause breast cancer or uterine cancer, but also does not act as an endocrine disorder substance while having the physiological activity of estrogen among natural resources. I keep trying.

Accordingly, an object of the present invention is to provide an effective method for producing a health functional food containing pomegranate extract that can effectively prevent and improve the symptoms of menopause or menopause in menopausal or menopausal women.

As described above, the health functional food containing the pomegranate extract according to the manufacturing method of the present invention as an active ingredient contains a large amount of safe phytoestrogens with little side effects while exhibiting excellent estrogen properties, so in menopausal or menopausal women It is expected to be able to effectively prevent and improve all symptoms that may be caused by estrogen deficiency.

In particular, the health functional food according to the manufacturing method of the present invention exhibits excellent effects on facial flushing, osteoporosis, and atherosclerotic heart disease, and has a very low body fat increase phenomenon as a side effect.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing a health functional food comprising a pomegranate extract as a natural estrogen source for menopausal or menopausal women.

Pomegranate is used as a main raw material in the production method of the present invention, and pomegranate is a plant native to southwestern Asia and northwest India, centered on Iran, and is now widely spread in subtropical and tropical regions. Since ancient times, pomegranate, especially black pomegranate, has been known as a tonic, and is known to have a good effect in preventing hypertension and arteriosclerosis. In addition, it contains a large amount of water-soluble sugars of 38 to 47%, and contains various vitamins and minerals.

Although there is no particular limitation on the kind of pomegranate in the manufacturing method of the present invention, black pomegranate is preferable, and a specific example of the pomegranate is black pomegranate from Iran.

According to the findings of the present inventors, the pomegranate concentrate has slight differences depending on the pomegranate origin and harvest time, but in particular, the black pomegranate concentrate contains a significant amount of phytoestrogens, specifically 10-20 ppm of catechin, It contains 20~30ppm of idzein, 01~05ppm of genistein, 80~140ppm of quercetin, 005~03ppm of 17β-estradiol and 001~010ppm of 2,3-di-MeO-estradiol.

The pomegranate concentrate prepared according to the manufacturing method of the present invention contains 35 to 42% by weight of moisture, 03 to 06% by weight of lipid, 07 to 12% by weight of protein, 12 to 16% by weight of ash, and 38 to 47% by weight of carbohydrates. Include. In the present invention, the pomegranate concentrate is used in the sense of including a concentrate to a pulp, a concentrate of pulp and seeds, and a concentrate from a raw material containing the skin.

Amino acids include 8 essential amino acids of threonine, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan, and lysine, as well as asphaltic acid, serine, glutamic acid, proline, glycine, alanine, cysteine, tyrosine, histidine, and arginine.

In addition, water-soluble vitamins include thiamine (vitamin B1), riboflavin (vitamin B2), ascorbic acid (vitamin C), niacin, and vitamin B6, and fatty acids include myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid. Etc., and as a weak acid component, glycolic acid and acetic acid are slightly contained.

The health functional food containing phytoestrogen according to the manufacturing method of the present invention contains 01 to 99% by weight of the pomegranate concentrated fruit juice, preferably 3 to 70% by weight, more preferably 5 to 50% by weight, based on the total weight of the composition. Include %. Most preferably, in the case of a tablet or granule form, it is 35 to 45% by weight, and in the case of a liquid form, it is 5 to 15% by weight.

In addition, optionally, the health functional food containing phytoestrogen according to the manufacturing method of the present invention may contain 01 to 50% by weight, respectively, of arrowroot, soybean, or isoflavone, which is a phytoestrogen derived from arrowroot and soybeans, based on the total weight of the composition. .

The health functional food containing phytoestrogen according to the manufacturing method of the present invention is not strictly limited, but may contain 01 to 80% by weight of chitooligosaccharide or cartilage-derived glucoseamine, which is helpful in improving skin and activating bone and cartilage tissue. have.

In addition, 001 to 4% by weight of vitamins such as vitamin E, vitamin C, nicotinic acid amide, riboflavin, and the like may be included if necessary.

When the health functional food containing phytoestrogen according to the manufacturing method of the present invention is in the form of a solid preparation such as tablets, granules, or capsules, collagen and/or mucopolysaccharide may be included, and in this case, the content is the total weight of the composition. Based on the range of 25 to 50% by weight and 50 to 100% by weight, respectively.

The health functional food containing phytoestrogen according to the manufacturing method of the present invention is formulated in the form of granules or conventional tablets by a compacting method using a roller compactor or a bang method by a wet or 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 if classified in more detail, they can be classified into excipients, binders, disintegrants, lubricants, and the like.

The excipient is selected in consideration of experience, cost, and compatibility with the active ingredient, and is usually used for the purpose of giving a certain volume to the tablet and improving tabletability. 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 include lactose, the content of which is all compositions It is in the range of 10 to 25% by weight based on weight.

In addition to improving the adhesion between the granules, the binder functions 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 and the properties of other ingredients, and if the amount is too small, the binding capacity is insufficient, and if the amount is too large, the tablet disintegration and dissolution rate are slowed. Regarding 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. In the present invention, a preferred binder in the present invention is collagen or mucopolysaccharide having an effect of improving skin and strengthening cartilage, and the ratio is as described above.

The lubricant has effects such as improving flow from the hopper to the die of the tablet press, preventing sticking to a punch or die, reducing resistance when taking out the tablet from the tablet press, and giving the tablet gloss. Examples of preferred lubricants include talc, magnesium stearate, stearic acid, calcium stearate, and fuming silicon dioxide. Particularly preferred lubricants include sun active iron and sodium alginate, and if the amount of the lubricant is too large, disintegration and dissolution of the tablet is delayed. Therefore, in the manufacturing method of the present invention, at least one lubricant is used in total. It is recommended to use it in a ratio of 01 to 05% by weight based on the tablet content.

The disintegrant expands upon contact with moisture to bring about the effect of disintegrating the tablet 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 the components, and those added for the purpose of binding agents may act as disintegrants rather than by interactions with other excipients, and vice versa. In some cases, the strict distinction between them is not absolute, but rather relative.

In the case of solid formulations of the above-described tablets, granules, or capsules, 500mg, 750mg, and 1000mg formulations can be prepared based on the weight of the pomegranate concentrate, and in the case of 750mg formulations, 6-12 tablets per day are taken orally. .

On the other hand, when the health functional food containing phytoestrogen according to the manufacturing method of the present invention is in a liquid form, it is preferable to add 3 to 10% by weight of polydextrose based on the total weight of the composition to form a stabilized colloid solution. .

It goes without saying that glucoseamine and the aforementioned vitamins may be included in the same amount as described above.

Most of the remaining balance is purified water.

In the case of the above-described liquid form, a vacuum-packed pack of 50 ㎖ or 100 ㎖ can be taken orally in 1 to 2 packets per day.

Hereinafter, a method of manufacturing a health functional food containing phytoestrogen 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 of refluxing for 05 to 2 hours under temperature conditions between 60°C and boiling point

(C) upper layer separation step

(D) solvent removal step

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

It is preferable to repeat the reflux step and the upper layer separation step 2 to 3 times.

The concentrate through the above-described solvent removal step is about 30% concentrate, and the total phytoestrogen content is 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 of refluxing for 05 to 2 hours

(C) upper layer separation step

(D) HCl addition step

(E) stirring step

(F) Glass filter filtration step

(G) After adding HCl, settling in a hot water bath at 60 to 100°C for 1 to 3 hours

(H) cooling stage

(I) Acetonitrile addition step

(J) solvent removal step

It is preferable to repeat the reflux step and the upper layer separation step 2 to 3 times. HCl is mixed with sulfuric acid or glucuronic acid by using a concentration of 01~10M, stirring thoroughly to make it homogeneous, removing fine pulverized particles of the skin, flesh and/or seeds using a glass filter and leaving it in a hot water bath. It hydrolyzes the estrogen that exists as a non-conjugated form of estrogen. Subsequently, acetonitrile is added to extract the intracellular components, and the solvent is removed using a vacuum distillation method or the like.

The concentrate passed through the above-described solvent removal step is about 40% concentrate, and the total phytoestrogen content is about 1420 to 1830 pg/ml.

Following the above-described acetonitrile addition step, (K) enzymatic hydrolysis at 37° C., (L) ethyl acetate addition and extraction step (preferably repeated 2-3 times), and finally (M) It is also possible to perform a solvent removal step.

Since estrogen is present in a conjugated form with sulfuric acid or glucuronic acid in many cases, in addition to hydrolysis with HCl described above, enzymatic hydrolysis steps can be performed independently or in parallel to make estrogen in a non-conjugated form. The hydrolysis step is performed by adding an acetic acid buffer solution adjusted to pH 52 with 02M acetic acid and sodium acetate, and then adding glucuronidase/arylsulfatase derived from Helix pomatia (manufactured by Boehringer Mannheim, Germany).

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

Next, a description will be given of the manufacturing method by the solid phase extraction method, which consists 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 of refluxing for 05 to 2 hours

(C) upper layer 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 steps of (G) 2 to 3 times. Separately collect and store the liquid passed through the column in step (D), and the eluate using a solution of acetone:methanol (1:9 concentration gradient) after performing steps (D) to (G), the column in step (D)

Combined with the pass-through, the solvent removal step of (H) is performed.

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

Hereinafter, the present invention will be described in more detail through examples, but this is only intended to illustrate the present invention and is not intended to limit the present invention.

Claims (5)

A method of manufacturing a health functional food containing phytoestrogen for improving the prevention of osteoporosis or hot flashes in menopausal or menopausal women consisting of the following steps:
(A) a solvent mixing step of adding at least one solvent selected from the group consisting of water, methanol, ethanol, n-hexane and ethyl acetate to the dried and pulverized pomegranate;
(B) reflux step of refluxing for 05 to 2 hours under a temperature condition between 60° C. and boiling point;
(C) After separation of the upper layer, adding and stirring HCl hydrolyzed by adding 01-10M HCl;
(D) a filter filtration step of removing fine pulverized particles using a glass filter;
(E) adding HCl again, and then adding HCl and standing in a hot water tank at 60-100° C. for 1 to 3 hours;
(F) after cooling, acetonitrile addition extraction step of adding acetonitrile to extract intracellular components;
(G) a solvent removal step of preparing a concentrated solution having a total phytoestrogen content of 1420 to 1830 pg/ml by distillation under reduced pressure; And
(D) Formulation step comprising 01 to 99% by weight of the concentrate. A method of manufacturing a health functional food containing phytoestrogen for improving the prevention of osteoporosis or hot flashes in menopausal or menopausal women consisting of the following steps:
(A) a solvent mixing step of adding at least one solvent selected from the group consisting of water, methanol, ethanol, n-hexane and ethyl acetate to the dried and pulverized pomegranate;
(B) reflux step of refluxing for 05 to 2 hours under a temperature condition between 60° C. and boiling point;
(C) passing through an ion exchange resin for purification by passing an ion exchange resin after separation of the upper layer;
(D) a solvent removal step of preparing a concentrated solution having a total phytoestrogen content of 1420 to 1830 pg/ml by distillation under reduced pressure;
(E) Enzymatic hydrolysis at 37° C. of adding glucuronidase/arylsulfatase derived from Helix pomatia in an acetic acid buffer solution;
(F) extraction step by adding ethyl acetate;
(G) a solvent removal step of preparing a concentrated solution having a total phytoestrogen content of 1560-1960 pg/ml by distillation under reduced pressure; And
(H) Formulation step comprising 01 to 99% by weight of the concentrate. The enzyme according to claim 1, wherein (F) glucuronidase/arylsulfatase derived from Helix pomatia is added in (F1) acetic acid buffer solution following the extraction step with addition of acetonitrile. After performing the hydrolysis step, (F2) addition of ethyl acetate and the extraction step, (G) a solvent removal step of preparing a concentrate having a total phytoestrogen content of 1540-1980 pg/ml by distillation under reduced pressure, and (H) the aforementioned concentrate A method of manufacturing a health functional food containing phytoestrogen for improving the prevention of osteoporosis or hot flashes in menopausal or postmenopausal women performing a formulation step comprising 01 to 99% by weight. The method of claim 2 or 3, wherein the acetic acid buffer solution in the enzymatic hydrolysis step is a 02M solution of pH 52, which is a phytoestrogen-containing health functional food for improving the prevention of osteoporosis or hot flashes in menopausal or menopausal women. The method of claim 1 or 2, wherein the concentrate is 10 to 20 ppm of catechin, 20 to 30 ppm of daidzein, 01 to 05 ppm of genistein, 80 to 140 ppm of quercetin, 005 to 03 ppm of 17β-estradiol and 2,3- Manufacturing method of health functional food containing phytoestrogen for improving the prevention of osteoporosis or hot flashes in menopausal or postmenopausal women containing di-MeO-estradiol 001~010ppm

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