Preparation method of liposome pomegranate extract containing phytoestrogen

Abstract

The present invention relates to a method for preparing a liposomal pomegranate extract containing phytoestrogen, and more specifically, when a pomegranate extract is prepared using a two-stage fermentation alcohol with a specific concentration and a nano-emulsion technique is used to perform nano-sized liposomal treatment, active ingredients such as phytoestrogen in the pomegranate extract are significantly increased, and thus, the amount of the active ingredients such as the phytoestrogen in the pomegranate extract is significantly increased, and the amount of the active ingredients such as the phytoestrogen in the pomegranate extract is significantly reduced. In addition, the stability of the active ingredients is maintained for a long period of time, so that the pomegranate extract (ESTROBAL) prepared by the preparation method of the present invention can be effectively used as a component of a health-care functional food or a common food which contributes to improving female climacteric symptoms and symptoms caused by estrogen imbalance.

Description

Technical Field

[0001] The invention relates to a method for preparing a liposome pomegranate extract containing high-concentration pomegranate effective components by using fermented alcohol, and a health functional food containing the liposome pomegranate extract as an effective component. Background Art

[0002] Generally speaking, the endocrine system is an organ system that produces and secretes various hormones involved in the maintenance of homeostasis, reproduction, production, behavior, etc. of an organism. It is composed of glands, hormones, and target cells. The hormones produced in the endocrine system circulate in the body through the blood, thereby participating in growth and development, male and female differentiation, etc. by transmitting information and instructions to each cell and tissue that becomes the target.

[0003] Estrogen, one of such endocrine hormones, is synthesized in the body from cholesterol, and its secretion sources are mainly the ovarian follicles and corpus luteum, and it is also secreted in the fetal placental system, adrenal glands, and testes during pregnancy. The process of estrogen secretion in the ovaries is as follows: gonadotropin secreted by the anterior pituitary gland transmits signals to the reproductive organs, thereby causing the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH), which are hormones that regulate the menstrual cycle and achieve pregnancy, and it acts on the ovaries again to cause the secretion of 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 implant, and in the second half, progesterone is secreted to put the endometrium in a better state for implantation. That is, estrogen is a representative female hormone that participates in endometrial hyperplasia, uterine muscle development, expression of secondary sexual characteristics, mediates the establishment of the menstrual cycle, causes changes in the mother during pregnancy, promotes the proliferation and secretion of mammary ducts, etc., but it also causes a wide range of effects on other parts of the body.

[0004] The above-mentioned estrogens are a general term for estradiol, estrone, estriol, etc., among which estradiol is known to be the most powerful. Estrogens act through receptors, and are mainly metabolized in the liver and become conjugated estrogens, which are then excreted from the body through urine.

[0005] In addition, in addition to the female urogenital system, estrogen also has excellent protective effects on various tissue functions such as bone tissue, heart, blood vessels, central nervous system, skin tissue, etc. Therefore, as the most effective prevention and treatment method for menopausal symptoms, hormone replacement therapy (hormone replacement therapy; HRT) (Lobo RA & Speroff L. FertilSteril 61; 592-595, 1994) is widely used. This is a method of artificially administering synthetic estrogen or a mixture of synthetic estrogen and synthetic progesterone considering that menopausal symptoms originate from reduced estrogen secretion. It is clinically used for the treatment of amenorrhea or irregular menstruation, artificial menstruation, menopausal disorders, hormone therapy for prostate cancer or breast cancer, osteoporosis, etc. In addition, oral contraceptives are composed of progesterone and estrogen. In addition, clomiphene (clomiphene) as an ovulation induction agent or tamoxifen (tamoxifen) for the treatment of breast cancer are non-steroidal anti-estrogens. These synthetic estrogens are taken orally in tablet form, or in the form of a patch that adheres to the skin or a cream that is applied topically.

[0006] However, many research reports point out that long-term use of synthetic estrogens can cause serious problems, namely, the possibility of causing breast cancer and uterine cancer (e.g., 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, most reliable arguments have argued that long-term use of synthetic estrogen replacement therapy is more likely to cause breast cancer, which has caused controversy among scholars and even the entire society. Although it has not yet been confirmed, it is currently believed that the risk of breast cancer in women receiving synthetic estrogen replacement therapy is related to the duration of exposure to estrogen doses.

[0007] Therefore, in recent years, research on exploring phytoestrogen as a safer alternative to synthetic estrogens used in the above-mentioned hormone replacement therapy has been very active.

[0008] The above-mentioned phytoestrogens refer to non-steroidal compounds existing in plants that exert estrogen effects in animals. Most of the grains, fruits and vegetables known to have anti-cancer effects and are effective against heart disease contain trace amounts of phytoestrogens, especially relatively more distributed in soybean processed foods including soybeans (soy milk, tofu, soybean paste, etc.) and various grains or fruits, alfalfa, clover, etc. It is known that isoflavones are mainly contained in legumes, and lignans are mainly contained in grains.

[0009] The activity of the above-mentioned phytoestrogens is only 1 / 100 to 1 / 1000 of the endogenous estrogen produced in the body, but its content is much larger, so it is finally possible to obtain sufficient estrogen effects. It is known that the representative activities of phytoestrogens are the effects of preventing bone loss and increasing bone density, and effectively reducing FSH and LH and relieving menopausal symptoms when applied to menopausal women. Genistein is an isoflavone that has been reported to inhibit cell protein tyrosine kinase, thereby having an anti-cancer effect of inhibiting the growth of cancer cells.

[0010] Therefore, there is a rapidly increasing demand for effective phytoestrogens that can be used as hormone replacement drugs for preventing and treating menopausal symptoms, which can not only be used to prevent and treat osteoporosis in menopausal women, lower LDL (low density lipid) and increase HDL (high density lipid), but also have the effect of estrogen agonists or antagonists that show anti-cancer effects on the endometrium and breast, and are effective and safe for improving the skin, preventing cataracts and colon cancer, and preventing autoimmune diseases.

[0011] Therefore, the inventors have devoted themselves to developing safe natural hormone replacement plants and extracts from natural resources, which have estrogen physiological activity and do not induce breast cancer or uterine cancer, etc., and do not have the effect of endocrine system disruptors. The inventors have confirmed that when the pomegranate extract is prepared by fermentation alcohol of a specific concentration in two stages and nanoemulsion technology is applied for nano-sized liposomes, the effective ingredients of phytoestrogens, etc. in the pomegranate extract are significantly increased, and the stability of the effective ingredients is maintained for a long time. Therefore, it is confirmed that the pomegranate extract prepared by the preparation method of the present invention can be used as an ingredient of health functional food or ordinary food, thereby completing the present invention, and the ingredient of the health functional food or ordinary food helps to improve women's menopausal symptoms and symptoms caused by estrogen imbalance.

[0012] Prior art literature

[0013] Patent Literature

[0014] (Patent Document 1) Korean Patent No. 10-0505746

[0015] (Patent Document 2) Korean Patent No. 10-0542478 Summary of the invention

[0016] Technical issues

[0017] The invention aims to provide a method for preparing a pomegranate extract containing high-concentration plant liposomes.

[0018] In addition, the present invention aims to provide a health functional food containing the liposomal pomegranate extract as an effective ingredient.

[0019] Technical Solution

[0020] To achieve the above-mentioned purpose, the present invention provides a method for preparing a pomegranate extract containing high concentration of phytoestrogens, comprising:

[0021] 1) preparing a pressed material by squeezing and filtering pomegranates;

[0022] 2) extracting and concentrating the pomegranate squeezed material obtained in the above step 1) using 65% (V / V) to 75% (V / V) fermentation alcohol to prepare a primary concentrate; and

[0023] 3) The primary concentrate obtained in the above step 2) is extracted and concentrated using 92% (V / V) to 97% (V / V) fermentation alcohol to prepare a secondary concentrate.

[0024] In addition, the present invention provides a health functional food containing liposome pomegranate extract as an effective ingredient, wherein the health functional food is used to prevent or improve menopausal symptoms of menopausal or postmenopausal women, such as osteoporosis, facial flushing, sweating, sleep disorders, depression, and obesity.

[0025] Effects of the Invention

[0026] In the present invention, when pomegranate extract is prepared by using two-stage fermentation alcohol with a specific concentration and nanoemulsion technology is applied to perform nano-sized liposome formation, it is confirmed that the effective ingredients such as phytoestrogens in the pomegranate extract are significantly increased, and the stability of the effective ingredients is maintained for a long time. Therefore, the pomegranate extract (ESTROBAL) prepared by the preparation method of the present invention can be effectively used as an ingredient of health functional foods or ordinary foods, and the ingredients of the health functional foods or ordinary foods help to improve women's menopausal symptoms and symptoms caused by estrogen imbalance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : is a figure which shows the preparation method of the liposomal pomegranate extract in this invention.

[0028] Figure 2 This is a graph confirming the average particle size of liposomes using nanoemulsion technology.

[0029] Figure 3 This is a graph confirming the average particle size of the liposomal pomegranate extract of the present invention prepared at an optimal mixing ratio.

[0030] Figure 4A and Figure 4BIt is an analysis chart of the effective ingredient (phytoestrogen) of the pomegranate extract of the present invention.

[0031] Figure 5 This is a graph confirming the stability of polyphenols which are active ingredients of the liposomal pomegranate extract of the present invention.

[0032] Figure 6 This is a graph confirming the stability of flavonoids as an effective ingredient of the liposomal pomegranate extract of the present invention. DETAILED DESCRIPTION

[0033] Hereinafter, the present invention is described in detail.

[0034] The present invention provides a method for preparing a pomegranate extract containing high concentration of phytoestrogens, comprising: 1) preparing a squeezed material by squeezing and filtering pomegranates;

[0035] 2) extracting and concentrating the pomegranate squeezed material obtained in the above step 1) using 65% (V / V) to 75% (V / V) fermentation alcohol to prepare a primary concentrate; and

[0036] 3) The primary concentrate obtained in the above step 2) is extracted and concentrated using 92% (V / V) to 97% (V / V) fermentation alcohol to prepare a secondary concentrate.

[0037] The pomegranate used in step 1) is not particularly limited, and cultivated or commercially available pomegranates may be used, but red pomegranates produced in Uzbekistan are preferably used.

[0038] The pomegranate of step 1) is peeled, and the squeezing of step 1) is preferably performed by a squeezing machine, and the squeezing is performed at an air pressure of 20 to 60, preferably 40 to 50. In addition, the filtration of step 1) can be preferably performed using a filter with a pore size of 5 to 15 meshes, more preferably using a filter with a pore size of 8 to 12 meshes, and most preferably using a filter with a pore size of 10 meshes.

[0039] In addition, in the above step 2), the pomegranate squeezed material of the above step 1) is preferably extracted 3 or more times by fermentation alcohol, preferably the extraction is repeated 3 to 4 times, and most preferably the extraction is performed by the following preparation method.

[0040] i) extracting the pomegranate squeezed material once using 65% (V / V) to 75% (V / V) fermented alcohol;

[0041] ii) extracting a second extract from the residue remaining after the first extraction in the above i) using 65% (V / V) to 75% (V / V) fermentation alcohol;

[0042] iii) extracting the residue remaining after the second extraction in the above ii) for a third time using 65% (V / V) to 75% (V / V) fermentation alcohol; and

[0043] iv) Mix the extracts from step i) to step iii) and filter them.

[0044] The fermented alcohol in steps i) to iii) above refers to fermenting starch or sugar and distilling it into alcohol. Specifically, it may refer to alcohol obtained by distillation, more specifically, ethanol with a purity of 60% to 100% obtained by continuous distillation, and most preferably refers to 70% (V / V) fermented alcohol.

[0045] Preferably, the fermentation alcohol of steps i) to iii) is added to the pomegranate squeezed material or the 1st to 3rd extract in an amount of 1 to 10 times, more preferably 6 to 8 times, for extraction. The extraction temperature is preferably 20° C. to 100° C., more preferably 15° C. to 25° C., and most preferably room temperature, but not limited thereto. In addition, the extraction time is preferably 1 hour to 24 hours, more preferably 3 hours to 5 hours, and most preferably 4 hours, but not limited thereto.

[0046] The residues in steps ii) to iii) refer to the residues remaining after filtering the extract once or twice using a filter with a pore size of 50 to 80 meshes, preferably a filter with a pore size of 60 meshes.

[0047] The concentration in step 2) is preferably carried out using a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the concentration of the first concentrate in step 2) is preferably 60 Brix to 70 Brix, more preferably 65 Brix.

[0048] In addition, in the above step 3), the concentrate of the above step 2) is preferably extracted three or more times by fermentation alcohol, and the extraction is preferably repeated three to four times, and most preferably extracted by the following preparation method.

[0049] v) extracting a primary extract from the primary concentrate using 92% (V / V) to 97% (V / V) fermentation alcohol;

[0050] vi) extracting the primary extract of v) above using 92% (V / V) to 97% (V / V) fermentation alcohol to obtain a secondary extract; and

[0051] vi) The secondary extract of vi) above is extracted a third time using 92% (V / V) to 97% (V / V) fermentation alcohol.

[0052] The fermented alcohol in steps v) to vi) above refers to fermenting starch or sugar and distilling it into alcohol. Specifically, it may refer to alcohol obtained by distillation, more specifically, ethanol with a purity of 60% to 100% obtained by continuous distillation, and most preferably refers to 95% (V / V) fermented alcohol.

[0053] It is preferred that 1 to 10 times, more preferably 6 to 8 times, of the fermentation alcohol in steps v) to vi) be added to the first concentrate or the first to second extract for extraction. The extraction temperature is preferably 20° C. to 100° C., more preferably 15° C. to 25° C., and most preferably room temperature, but not limited thereto. In addition, the extraction time is preferably 1 to 24 hours, more preferably 3 to 5 hours, and most preferably 4 hours, but not limited thereto.

[0054] The concentration in step 3) is preferably carried out using a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the secondary concentrate in step 3) is preferably 60 Brix to 70 Brix, more preferably 65 Brix.

[0055] In addition, it is preferred that the secondary concentrate of the present invention is further liposome-formed, but the present invention is not limited thereto.

[0056] The liposome is preferably prepared by mixing 35 to 50% by weight of the secondary concentrate, 20 to 40% by weight of caprylic / capric glyceramide, 5 to 10% by weight of ethanol, 1 to 10% by weight of an emulsifier, 20 to 40% by weight of glycerol, 15 to 25% by weight of lecithin, 1 to 5 parts by weight of glycolipid, 1 to 5% by weight of phosphatidylcholine, and 40 to 60% by weight of water and then performing high pressure emulsification. On the other hand, the emulsifier is preferably a glycerol fatty acid ester, but is not limited thereto. Furthermore, the liposomal preparation is preferably prepared by mixing 40 to 50% by weight of the secondary concentrate, 25 to 35% by weight of caprylic / capric glyceramide, 5 to 10% by weight of ethanol, 5 to 10% by weight of an emulsifier, 25 to 35% by weight of glycerol, 15 to 25% by weight of lecithin, 1 to 5 parts by weight of glycolipid, 1 to 5% by weight of phosphatidylcholine, and 45 to 55% by weight of water, followed by high-pressure emulsification.

[0057] The liposomal pomegranate extract prepared by liposomizing the secondary concentrate preferably has a particle size of 25 nm to 40 nm.

[0058] On the other hand, "emulsion" of the present invention refers to one or more liquid-liquid dispersion systems of liquid phase dispersion that are not mixed in a liquid phase, which usually have a particle size of several μm to tens of μm. Emulsions with this particle size are thermodynamically unstable and are eventually separated by various approaches, such as flocculation, precipitation, curdling, Ostwald ripening and aggregation. In addition, emulsions with an average diameter of dispersed phase particles of 20nm to 500nm are usually referred to as "nanoemulsions" (Flockhart, IR, etc., J.Pharm.Pharmacol., 50 (Supplement) 1998, 141). The particle size of nanoemulsions is small, so it is less affected by gravity, so that Brownian motion is carried out between particles, and therefore, compared to general emulsions, it is more stable to sedimentation and curdling. It is an emulsion, so it is not stable from a thermodynamic point of view, but the kinetic stability is improved than ordinary emulsions due to the influence of the above-mentioned Brownian motion. Furthermore, unlike microemulsions or normal emulsions, nanoemulsions can be diluted with an aqueous phase without a change in particle size (Colloid Surface A 2004; 251: 53-8).

[0059] The most representative surfactant that can produce such nanoemulsions is lecithin, an intercellular phospholipid. When lecithin is mixed with sufficient aqueous medium, it self-emulsifies, thereby forming double or multi-layer sheets and forming nano-sized particles, which are particles. When high-pressure emulsification is used in the self-emulsification process of lecithin, very fine nanoemulsions with a size of several nanometers to tens of nanometers can be produced.

[0060] In addition, the phytoestrogen of the present invention is preferably estradiol, and the liposomal pomegranate extract of the present invention has increased polyphenol and flavonoid contents compared to the extract extracted using water, C1 to C2 lower alcohols, fermented alcohol with a concentration different from that of the present invention, or a mixture thereof or the primary extract.

[0061] In a specific embodiment of the present invention, the inventors prepared pomegranate extract by fermentation alcohol of specific concentration in two stages, and applied nanoemulsion technology to perform nano-sized liposomes to prepare liposome pomegranate extract (refer to Figures 1 to 3 , Table 1).

[0062] In addition, the inventors conducted quantitative analysis on the polyphenols and flavonoids of the pomegranate extract according to the extraction conditions, and the results confirmed that the pomegranate extract prepared by the two-stage fermentation alcohol with a specific concentration in the present invention has a significantly higher content of polyphenols and flavonoids than the pomegranate extract prepared by other extraction conditions (see Table 2).

[0063] Furthermore, the present inventors analyzed the phytoestrogen content of the pomegranate extract of the present invention and confirmed that the pomegranate extract of the present invention contains a high concentration of phytoestrogen (β-Estradiol (E2)) (refer to Figure 4A and Figure 4B ).

[0064] In addition, the stability of the liposome pomegranate extract of the present invention was analyzed, and the results showed that the liposomes applied to the nanoemulsion in the liposome pomegranate extract of the present invention were not cleaved and stable under various temperature and pH conditions (see Table 4 and Table 5). Regarding long-term stability, it was confirmed that the liposomes applied to the nanoemulsion in the liposome pomegranate extract of the present invention were not cleaved and stable under neutral acidity and various temperature conditions for up to 230 days (see Table 6).

[0065] Furthermore, the present inventors confirmed the stability of the active ingredients in the liposomal pomegranate extract and found that the content of polyphenols and flavonoids as the active ingredients in the liposomal pomegranate extract of the present invention did not change within 10 months and maintained stability (see Figure 5 , Figure 6 ).

[0066] Therefore, in the present invention, the pomegranate extract is extracted by using two-stage fermentation alcohol with a specific concentration and is lipidated in nanometer size by applying nanoemulsion technology. The effective ingredients such as phytoestrogens in the pomegranate extract are significantly increased and the stability of the effective ingredients is maintained for a long time. Therefore, the pomegranate extract prepared by the preparation method of the present invention can be effectively used as an ingredient of health functional food or ordinary food, and the ingredient of the health functional food or ordinary food helps to improve women's menopausal symptoms and symptoms caused by estrogen imbalance.

[0067] In addition, the present invention provides a health functional food containing liposomal pomegranate extract as an effective ingredient, wherein the health functional food is used to prevent or improve menopausal symptoms of menopausal or postmenopausal women, such as osteoporosis, facial flushing, sweating, sleep disorders, depression, and obesity.

[0068] When the liposome pomegranate extract of the present invention is used as a health functional food, the extract can be directly added or used together with other foods or food ingredients, and can be used appropriately in a conventional manner. The liposome pomegranate extract of the present invention can be administered in a dry powder dosage form prepared by the preparation method of the present invention, or can be used in a liquid phase step dosage form before drying in the preparation method of the present invention, but is not limited thereto. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use (prevention, health care or therapeutic treatment). Generally, when manufacturing food or beverages, the composition of the present invention can be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw materials. However, when long-term intake is for health and hygiene purposes or for the purpose of regulating health, the amount can be less than or equal to the above range, and the active ingredient can be used in an amount greater than or equal to the above range because there are no safety issues.

[0069] The liposomal pomegranate extract prepared according to the present invention is formulated into granules or conventional tablets by wet granulation or dry granulation by compacting or slugging using a roller compactor or the like.

[0070] Generally, when manufacturing tablets, all additives except the main ingredients are collectively referred to as additives or excipients, which can be more specifically divided into excipients, binders, disintegrants, lubricants, etc.

[0071] Excipients are selected for tablet experience, cost, compatibility with the main ingredients, etc., and are generally used to give the tablet a certain volume and improve tableting performance. Examples of suitable excipients include lactose, lanolin, mannitol, starch, powdered sucrose, calcium phosphate, microcrystalline cellulose, colloidal silicon dioxide, etc., and lactose is a particularly preferred excipient in the present invention, and its content is in the range of 10% to 25% by weight based on the weight of the composition.

[0072] In addition to improving the adhesion between particles, the binder can also maintain the form of the tablet after compaction. The amount of the binder depends on the experience of the worker, the properties of other ingredients, etc. When the amount is too little, the binding capacity is insufficient, and when it is too much, it is over-hardened, thus reducing the disintegration and dissolution rate of the tablet. Preferred binder types and addition amounts can exemplify corn starch suspension, glucose solution, maltose, natural gum, cellulose derivatives (methylcellulose, carboxymethylcellulose, microcrystalline cellulose), gelatin, polyvidone, collagen, mucopolysaccharide, etc., but are not limited thereto.

[0073] Lubricants have the effects of improving the flow from the hopper to the die of the tablet press, preventing substances from adhering to the punch or die, reducing the resistance when taking out the tablet from the tablet press, and giving the tablet a gloss. Examples of preferred lubricants include talc, magnesium stearate, stearic acid, calcium stearate, and fumed silica, etc. Particularly preferred lubricants include SunActive Fe and sodium alginate. If the amount of lubricant is excessive, the disintegration and dissolution of the tablet are delayed, so it is excellent to use at least one of the lubricants in a ratio of 0.1% to 0.5% by weight based on the total tablet content in the present invention.

[0074] The disintegrant has the effect of swelling when wet to thereby disintegrate the tablet in the gastrointestinal tract, and preferred examples of the disintegrant include those mentioned above as the binder and the excipient.

[0075] Excipients, binders and disintegrants are usually appropriately combined in consideration of the relationships between the ingredients, and there are substances added for the purpose of binders that may act as disintegrants due to interaction with other excipients, and vice versa. Therefore, the strict distinction between them is not absolute but relative.

[0076] The solid preparation form of the above-mentioned tablets, granules or capsules can be prepared into 100 mg, 300 mg, 500 mg, 750 mg and 1000 mg dosage forms based on the weight of the liposomal pomegranate extract. Taking the 100 mg dosage form as an example, 1 to 10 tablets can be orally administered per day.

[0077] In addition, there is no particular limitation on the types of food. Examples of foods to which the liposomal pomegranate extract of the present invention can be added include meat, ham, bread, chocolate, candies, snacks, biscuits, pizza, instant noodles, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, teas, energy drinks, alcoholic beverages, and multivitamins, etc., which include all conventional health foods.

[0078] The health drink composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, just like conventional beverages. Natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners may use natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame. Based on 100 ml of the liposomal pomegranate extract of the present invention, the proportion of the natural carbohydrates is generally about 0.01 g to 0.04 g, preferably about 0.02 g to 0.03 g.

[0079] In addition, the liposome pomegranate extract of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, alcohol, and carbonating agents for carbonated beverages, etc. In addition, the liposome pomegranate extract of the present invention may contain pulp for making natural fruit juices, fruit juice drinks, and vegetable drinks. Such ingredients may be used alone or in combination.

[0080] Hereinafter, the present invention will be described in detail through examples, comparative examples, experimental examples and preparation examples.

[0081] However, the following examples, comparative examples, experimental examples and preparation examples are only used to exemplify the present invention, and the contents of the present invention are not limited to the following examples, comparative examples, experimental examples and preparation examples.

[0082] <Example 1> Preparation of pomegranate extract using fermented alcohol

[0083] Red pomegranates from Uzbekistan were washed in water three times, and then the pomegranate skin was removed. Then, they were squeezed using a squeezer at 40 to 50 atmospheres, and filtered to remove the residue. Then, 300 kg of pomegranate squeezed material from which seeds and residues were removed using a 10-mesh filter was loaded into an extractor equipped with a 2500-liter water jacket.

[0084] Next, 2100 l of 70% (V / V) fermentation alcohol (ethanol) was mixed and stirred at room temperature for 4 hours for primary extraction, and then the primary extract was passed through a filter with a pore size of 60 and transferred to a storage tank, and the first-stage primary extract was prepared by standing at a temperature of less than or equal to 5° C. for 24 to 48 hours.

[0085] The residue after extracting the first-stage 1 extract is mixed with 1200 l of 70% (V / V) fermented alcohol, and the mixture is extracted by stirring at room temperature for 4 hours, and then the extract is passed through a filter with a pore size of 60 and transferred to a storage tank, and allowed to stand at a temperature of less than or equal to 5° C. for 24 to 48 hours to prepare the first-stage 2 extract.

[0086] The residue after extracting the first stage 2 extract is mixed with 900 liters of 70% (V / V) fermentation alcohol and stirred at room temperature for 4 hours. The extract is passed through a filter with a pore size of 60 and transferred to a storage tank. The first stage 3 extract is prepared by standing at a temperature of less than or equal to 5° C. for 24 to 48 hours.

[0087] Then, the first stage 1, 2 and 3 extracts were combined and passed through a filter cloth, and then transferred to a vacuum concentrator and concentrated at a temperature of 60° C. to 70° C., thereby preparing a 1st concentrate of 65 Brix.

[0088] In addition, 2100 l of 95% (V / V) fermentation alcohol was mixed with the first concentrate concentrated with the 70% (V / V) fermentation alcohol, and extracted at room temperature for 4 hours, and then the extract was passed through a filter with a pore size of 60 and transferred to a storage tank, and allowed to stand at a temperature of less than or equal to 5° C. for 24 to 48 hours to prepare a second-stage first extract.

[0089] 1200 liters of 95% (V / V) fermented alcohol are mixed with the second-stage 1 extract, and the mixture is extracted by stirring at room temperature for 4 hours. The extract is then passed through a filter with a pore size of 60 and transferred to a storage tank. The extract is allowed to stand at a temperature of less than or equal to 5° C. for 24 to 48 hours to prepare a second-stage 2 extract.

[0090] The second stage 2 extract is mixed with 900 liters of 95% (V / V) fermented alcohol and stirred at room temperature for 4 hours, and then the extract is passed through a filter with a pore size of 60 and transferred to a storage tank, and allowed to stand at a temperature of less than or equal to 5° C. for 24 to 48 hours to prepare a second stage 3 extract.

[0091] Then, the second extreme tertiary extract was passed through a filter cloth and transferred to a vacuum concentrator, and concentrated at a temperature of 60° C. to 70° C., thereby preparing a secondary concentrate of 65 Brix, namely, the pomegranate extract of the present invention.

[0092] On the other hand, the preparation method of the present invention is shown in Figure 1 .

[0093] <Example 2> Liposomalization of pomegranate extract

[0094] The secondary concentrate prepared in the above-mentioned <Example 1>, that is, the pomegranate extract of the present invention, was lipidated.

[0095] Specifically, the process of lipidating the pomegranate extract using nanoemulsion technology is as follows: lecithin, caprylic / capric glyceramide, and auxiliary emulsifiers (phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and hydrogenated products thereof) as natural emulsifiers are added to the pomegranate extract of the present invention to prepare stable liposomes of nanometer size, thereby preparing liposomal pomegranate extract.

[0096] Specifically, in order to produce the pomegranate extract of the present invention prepared by <Example 1> as nanoemulsion liposomes, the ingredients required for preparing nanoemulsion liposomes were divided into the composition of [Table 1] below and placed in an oil phase tank and a water phase tank, and then passed through a high-pressure microfluidizer (Microfluidizer) at 1000 bar for 5 times to produce nano-sized liposomes. Then, the bubbles of the liposomes were removed, gradually cooled at room temperature, adsorbed on dextrose and dried with a fluidized bed dryer to form a powder.

[0097] On the other hand, in order to measure the particle size of the produced liposomes, 1 g of the lipidated secondary concentrate was dissolved in 100 ml (to prepare a 1% solution), and the particle size was measured using a Photal (OTSUKA Electronics) electrophoretic light scattering spectrophotometer apparatus.

[0098] [Table 1]

[0099]

[0100]

[0101] As a result, it was confirmed that Figure 2 As shown, the average size of the liposomes prepared using the comparative composition showed various particle sizes between 59.2 nm and 114 nm, but as Figure 3 As shown, the average size of the liposomes prepared by the experimental composition of the present invention (liposome pomegranate extract of the present invention) is 33 nm, with a very uniform particle size ( Figure 2 and Figure 3 ).

[0102] <Comparative Example 1> Comparative group of pomegranate extract prepared by fermentation alcohol

[0103] <1-1> Preparation of pomegranate extract using 70% (V / V) fermentation alcohol

[0104] Pomegranate extract (Comparative Group 1) was prepared using 70% (V / V) fermentation alcohol by the same method as in Example 1 above, except that 70% (V / V) fermentation alcohol was used to prepare both the 1st and 2nd concentrates after the pomegranate squeezed material was prepared by the same method as in Example 1 above.

[0105] <1-2> Preparation of pomegranate extract using 95% (V / V) fermentation alcohol

[0106] Pomegranate extract (Comparative Group 2) was prepared using 95% (V / V) fermentation alcohol by the same method as in Example 1 above, except that 95% (V / V) fermentation alcohol was used to prepare both the 1st and 2nd concentrates after the pomegranate squeezed material was prepared by the same method as in Example 1 above.

[0107] <1-3> Preparation of pomegranate extract using 95% (V / V) and 70% (V / V) fermentation alcohol Pomegranate extract was prepared using 95% (V / V) and 70% (V / V) fermentation alcohol by the same method as in <Example 1> above, except that 95% (V / V) fermentation alcohol was used to prepare a primary concentrate, and 70% (V / V) fermentation alcohol was used to prepare a secondary concentrate. (Comparative Group 3)

[0108] <1-4> Preparation of pomegranate extract using 60% (V / V) and 80% (V / V) fermentation alcohol Pomegranate extract was prepared using 60% (V / V) and 80% (V / V) fermentation alcohol by the same method as in <Example 1> above, except that 60% (V / V) fermentation alcohol was used to prepare a primary concentrate, and 80% (V / V) fermentation alcohol was used to prepare a secondary concentrate. (Comparative Group 4)

[0109] <Experimental Example 1> Quantitative Analysis of Polyphenols and Flavonoids

[0110] <1-1> Quantitative analysis of polyphenols and flavonoids according to extraction conditions

[0111] The contents of polyphenols and flavonoids in the pomegranate extracts prepared under different extraction conditions in <Example 1> and <Comparative Example 1> were quantified.

[0112] Specifically, the polyphenol content in the sample was measured by the Folin-Dens method, which uses the principle that phenolic substances react with phosphomolybdic acid to show a blue color. The method is to take 50 ml of the sample extraction solution, vacuum dry it at 40°C, and then make it constant with 5 ml of 80% ethanol solution.

[0113] 3 ml of the sample solution and 3 ml of the Fort-Danser reagent were mixed and left at room temperature for 3 minutes, and then 3 ml of the Na2CO3 solution was added and mixed, and the absorbance at 760 nm was measured after standing at room temperature for 1 hour. At this time, gallic acid was used as a standard substance for analysis.

[0114] In addition, for the total flavonoid content, 1 ml of the sample solution and 10 ml of diethylene glycol were mixed, 1 ml of 1N-NaOH solution was added thereto and mixed evenly, and the sample solution was reacted at 37°C for 1 hour and then the absorbance at 420 nm was measured, wherein the sample solution was obtained by taking 10 ml of the sample extraction solution and drying it in vacuum at 40°C and then fixing the volume with 5 ml of 50% methanol solution. At this time, quercetin was used as a standard substance for analysis.

[0115] The results are shown in the following [Table 2], and it was confirmed that the pomegranate extract of the present invention prepared in <Example 1> had significantly higher polyphenol and flavonoid contents than the pomegranate extract prepared under other conditions (Table 2).

[0116] [Table 2]

[0117]

[0118] <1-2> Further analysis of polyphenols and flavonoids content in pomegranate extract

[0119] In order to further verify the polyphenol and flavonoid contents of the pomegranate extract of the present invention (prepared in <Example 1>) with significantly excellent polyphenol and flavonoid contents in the above-mentioned Experimental Example <1-1>, the Korea Quality Inspection Center (Suwon, South Korea) was commissioned and the result report was received on October 26, 2023. On the other hand, as in the above-mentioned Experimental Example <1-1>, gallic acid and quercetin were used as standard substances.

[0120] As shown in the test report commissioned by Korea Quality Testing Center (Suwon, South Korea) to confirm the polyphenol and flavonoid contents of the pomegranate extract of the present invention, it was confirmed that the polyphenol (33.8 mg / g) and flavonoid (2.57 mg / g) contents of the pomegranate extract of the present invention prepared in <Example 1> showed high contents similar to those in <1-1> above (refer to other supporting documents).

[0121] <Experimental Example 2> Analysis of the Active Ingredients (Phytoestrogens) of Pomegranate Extract

[0122] In order to analyze the phytoestrogen (β-Estradiol (E2)) of the pomegranate extract of the present invention prepared in the above-mentioned <Example 1>, liquid chromatography-mass spectrometry analysis (LC / MS / MS) was commissioned to B&G Research Institute (Chuncheon City, Gangwon Province), and the results were received on April 16, 2022.

[0123] The LC / MS used in the present invention used an Agilent 1200 series HPLC of Agilent (Palo Alto, CA, USA) equipped with an autosampler (Agilent 1200 series G1313A, Autosampler), and in order to confirm the molecular weight of each separated substance, an Agilent 6410 Triple-quadrupole tandem LC / MS / MS (Agilent Technologies, Palo Alto, CA, USA) was used. The Extend C18 column was used for analysis in the LC / MS / MS ESI negative mode, and the analysis conditions are shown in [Table 3] below.

[0124] [Table 3]

[0125]

[0126]

[0127] The results are as follows Figure 4A and Figure 4B As shown, it was confirmed that 4.82 ppb of E2 (Sample No. 1) was detected in the pomegranate extract (Sample 1) of <Example 1>, and 19.8 ppb of E2 (Sample No. 2) was detected in the liposomal pomegranate extract of the present invention (Sample No. 2) of <Example 2>. Figure 4A and Figure 4B ).

[0128] That is, it was confirmed that in the liposomal pomegranate extract prepared by the composition of the present invention in <Example 2>, the E2 activity was maintained without loss by the nanoemulsion liposome.

[0129] <Experimental Example 3> Confirmation of the stability of the liposomal pomegranate extract of the present invention

[0130] In order to confirm the stability of the liposomal pomegranate extract of the present invention prepared in the above <Example 2>, the temperature and acidity stability were analyzed under the following four conditions by B&G Research Institute (Chuncheon City, Gangwon Province), and the results were received on April 16, 2022:

[0131] High temperature short time pasteurization (HTST): 130°C, 5 seconds;

[0132] High temperature pasteurization (HT): 130°C, 3 minutes;

[0133] Low temperature pasteurization (LT): 80°C, 30 minutes; and

[0134] Combined pasteurization (CT): 130℃, 3 minutes, then 80℃, 30 minutes.

[0135] In addition, we commissioned B&G Research Institute (Chuncheon City, Gangwon Province) to analyze long-term stability under the following three conditions and received the results on April 16, 2022:

[0136] High temperature (HT): 50℃;

[0137] Room temperature (RT): 25°C;

[0138] Low temperature (CT): 10℃.

[0139] In order to evaluate the temperature and acidity stability, as well as the long-term stability, 100 ml (10%) of water was mixed with 10 g of the liposomal pomegranate extract of the present invention prepared in <Example 2>, and then the mixture was sterilized at various temperatures and subjected to stability tests at various pH values. Under these conditions, the stability was determined based on the degree of oiling and suspension of the upper layer of the solution. In addition, the liposomal pomegranate extract prepared using the comparative composition of <Example 2> was evaluated in the same manner as above.

[0140] On the other hand, if the stability of the liposomes cannot be maintained under various sterilization conditions and acidity (liposome rupture), the oily components (oil) of the liposomes floating in the upper layer of the solution will form oils (stratification, segregation), and the samples that have oiled will be marked as stratified, and the samples that have not oiled will be marked as non-stratified.

[0141] Furthermore, the samples preserved in the long-term stability test were visually observed and evaluated as good if no layer separation occurred, and evaluated as clean if no suspension occurred.

[0142] As shown in [Table 4], it was confirmed that the liposomes applied to the nanoemulsion in the liposome pomegranate extract of the present invention were not cleaved and were stable under all conditions. However, as shown in [Table 5], it was confirmed that the liposome pomegranate extract prepared using the comparative composition of <Example 2> was oiled under pH of some acidic and alkaline conditions and sterilization conditions.

[0143] As shown in [Table 6], regarding long-term stability, it was confirmed that, in the liposome pomegranate extract of the present invention, the liposomes to which the nanoemulsion was applied were not cleaved and were stable up to 230 days under neutral acidity and various temperature conditions.

[0144] [Table 4]

[0145] pH HTST HT LT CT 3.0 Unstratified Unstratified Unstratified Unstratified 6.4 Unstratified Unstratified Unstratified Unstratified 9.0 Unstratified Unstratified Unstratified Unstratified

[0146] [Table 5]

[0147] pH HTST HT LT CT 3.0 Layering Layering Layering Layering 6.4 Unstratified Unstratified Layering Layering 9.0 Unstratified Layering Layering Layering

[0148] [Table 6]

[0149] pH Day 0 182 days 203 days 230 days 6.4 (HT conditions) Good & Clean Good & Clean Good & Clean Good & Clean 6.4 (RRT conditions) Good & Clean Good & Clean Good & Clean Good & Clean 6.4 (CCT conditions) Good & Clean Good & Clean Good & Clean Good & Clean

[0150] <Experimental Example 3> Confirmation of the stability of the active ingredients of the liposomal pomegranate extract of the present invention

[0151] In order to confirm the stability of the active ingredients in the liposomal pomegranate extract of the present invention prepared in the above <Example 2>, the stability of polyphenols and flavonoids was confirmed for 10 months at a relative humidity of 75% and a storage temperature of 40°C at B&G Research Institute (Chuncheon City, Gangwon Province), and the results were received on October 7, 2023. In addition, the stability of polyphenols and flavonoids was confirmed by measuring the content of polyphenols and flavonoids in the same manner as in the above <Experimental Example 1>. On the other hand, for comparative experiments, the first concentrate of <Example 1> was lipidated in the same manner as in the present invention and evaluated together (first concentrated liposome).

[0152] The results are as follows Figure 5 and Figure 6 As shown in FIG. 1 , it was confirmed that polyphenols and flavonoids as active ingredients in the liposomal pomegranate extract (secondary concentrated liposome) of the present invention did not change in content and maintained stability over 10 months (refer to Figure 5 , Figure 6 ).

[0153] <Preparation Example 1> Preparation of liposomal pomegranate extract powder

[0154] Using the liposomal pomegranate extract of <Example 2>, a liposomal pomegranate extract powder was prepared as described above and named In addition, The raw material inspection report is shown in other supporting documents.

Claims

1. A method for preparing a pomegranate extract containing high concentrations of phytoestrogens, characterized in that: The following steps are involved: 1) Preparing a pressed material by pressing and filtering pomegranate (Punica granatum L.); 2) extracting and concentrating the pomegranate squeezed material obtained in step 1) using 65% (V / V) to 75% (V / V) fermentation alcohol to prepare a primary concentrate; as well as 3) extracting and concentrating the primary concentrate obtained in step 2) using 92% (V / V) to 97% (V / V) fermentation alcohol to prepare a secondary concentrate.

2. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 1, characterized in that: The pomegranate in step 1) is red pomegranate produced in Uzbekistan.

3. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 1, characterized in that: The pomegranates in step 1) are peeled.

4. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 1, characterized in that: In step 2), the pomegranate squeezed material is repeatedly extracted three or more times using fermentation alcohol.

5. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 4, characterized in that: The step of repeating the extraction 3 times or more comprises the following steps: i) extracting the pomegranate squeezed material once using 65% (V / V) to 75% (V / V) fermented alcohol; ii) extracting a second extract from the residue remaining after the first extraction in step i) using 65% (V / V) to 75% (V / V) fermentation alcohol; iii) extracting the residue remaining after extracting the extract twice in step ii) for a third time using 65% (V / V) to 75% (V / V) fermentation alcohol; and iv) mixing the extracts from step i) to step iii) and filtering them.

6. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 5, characterized in that: The fermented alcohol in steps i) to iii) is 70% (V / V) fermented alcohol.

7. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 1, characterized in that: The primary concentrate in step 2) has a concentration of 60 Brix to 70 Brix.

8. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 1, characterized in that: In the step 3), the concentrate is repeatedly extracted three or more times using fermentation alcohol.

9. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 8, characterized in that: The step of repeating the extraction 3 times or more comprises the following steps: i) extracting a primary concentrate using 92% (V / V) to 97% (V / V) fermentation alcohol; ii) extracting the first extract of step i) using 92% (V / V) to 97% (V / V) fermentation alcohol to extract the second extract; and iii) extracting the second extracts from step ii) for a third time using 92% (V / V) to 97% (V / V) fermentation alcohol.

10. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 9, characterized in that: The fermented alcohol in steps i) to iii) is 95% (V / V) fermented alcohol.

11. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 1, characterized in that: The secondary concentrate in step 3) has a concentration of 60 Brix to 70 Brix.

12. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 1, characterized in that: The phytoestrogen is estradiol.

13. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 1, characterized in that: Further including: The secondary concentrate is subjected to a liposomal step.

14. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 13, characterized in that: The liposome is prepared by mixing 35 to 50 wt % of the secondary concentrate, 20 to 40 wt % of caprylic / capric glyceramide, 5 to 10 wt % of ethanol, 1 to 10 wt % of an emulsifier, 20 to 40 wt % of glycerol, 15 to 25 wt % of lecithin, 1 to 5 wt % of glycolipid, 1 to 5 wt % of phosphatidylcholine, and 40 to 60 wt % of water, followed by high pressure emulsification.

15. The method for preparing a pomegranate extract containing high concentration of phytoestrogens according to claim 13, characterized in that: The liposomal pomegranate extract prepared by liposomizing the secondary concentrate has a particle size of 25 nm to 40 nm.

16. A health functional food for improving symptoms of menopausal or postmenopausal women, The invention comprises liposomal pomegranate extract, and the symptom is any one selected from the group consisting of osteoporosis, facial flushing, sweating, sleep disorder, depression, and obesity.

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