WO2017200224A1 - Composition comprising the combined extract for preventing or treating the abnormal regulation of female hormone and the use thereof - Google Patents

Composition comprising the combined extract for preventing or treating the abnormal regulation of female hormone and the use thereof Download PDF

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Publication number
WO2017200224A1
WO2017200224A1 PCT/KR2017/004529 KR2017004529W WO2017200224A1 WO 2017200224 A1 WO2017200224 A1 WO 2017200224A1 KR 2017004529 W KR2017004529 W KR 2017004529W WO 2017200224 A1 WO2017200224 A1 WO 2017200224A1
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WO
WIPO (PCT)
Prior art keywords
extract
mulberry fruit
lycii fructus
asparagi radix
abnormal regulation
Prior art date
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PCT/KR2017/004529
Other languages
French (fr)
Inventor
Soon Il Kim
Jong Hoon Ryu
Original Assignee
Olmanfood Co., Ltd.
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Publication date
Priority claimed from KR1020170039996A external-priority patent/KR101801130B1/en
Application filed by Olmanfood Co., Ltd. filed Critical Olmanfood Co., Ltd.
Publication of WO2017200224A1 publication Critical patent/WO2017200224A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
    • A61K36/605—Morus (mulberry)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • A61K36/815—Lycium (desert-thorn)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8965—Asparagus, e.g. garden asparagus or asparagus fern

Definitions

  • the present invention relates to a composition and health functional food comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus for treating and preventing an abnormal regulation of female hormone and the use thereof.
  • Abnormal regulation of female hormone incudes various diseases caused by the abnormal regulation on female hormone such as estrogen or progesterone, for example, female climacteric syndrome, sexual desire disorder, hypo-estrogenemia, sex hormone hyper-secretion, precocious puberty, and the like as well as the resulting syndrome caused thereby, for example, over-sensitiveness, anxiety disorder, depression, vertigo, facial flushing response, hidrosis, sleep disturbance, hypotrophy, memory deficiency, business ability degradation, stamina degradation, decline in exercise capacity, decrease in body hair, skin aging, osteopenia, increase in visceral fat, and the like.
  • female climacteric syndrome sexual desire disorder, hypo-estrogenemia, sex hormone hyper-secretion, precocious puberty, and the like as well as the resulting syndrome caused thereby, for example, over-sensitiveness, anxiety disorder, depression, vertigo, facial flushing response, hidrosis, sleep disturbance, hypotrophy, memory deficiency, business ability
  • the climacteric syndrome of women is used to refer to a series of symptoms and conditions associated with menopause caused by hypofuction of ovary before and after natural menopause. Menopause may give rises to various syndrome caused by hormonal changes, such as decline of estrogen synthesis, the increased level of FSH (Follicular stimulating hormone) and LH (Luteinizing hormone) etc.
  • FSH Follicular stimulating hormone
  • LH Luteinizing hormone
  • the said symptoms and conditions include malfunctions of endocrine system, decreases in estrogen level, disorder of metabolism, menstrual disturbance caused by disturbances of cardiovascular and vegetative nervous system, hidrorrhea, nausea, dizziness, and tinnitus, palpitation and insomnia, restlessness and irascibility, soreness of loin and knees, benumbedness and itch of skin, hypomnesis, and even emotional abnormality and the like. (US2005/0123629 A1).
  • HRT Hemmone-replacement Therapy
  • the estrogenic extract of Asparagus conchinchinensis (LOUR) MERR of Liliaceae Family can be used to treat one or more disease states associated with the estrogen receptor and climacteric symptoms such as hot-flashes (PCT/WO2009/033025A1).
  • Mulberry fruit a fruit of Mori alba L. belonged to Moraceae, has been reported to contain various saccharide, tannic acid, vitamins, linoleic acid etc and used to be edible fruit for a long time in Korea (B. S. Cheong et al., Dohaehyangyak Daesajeon, Youngrim press, pp545-548, 1998).
  • Asparagi radix a radix of Asparagus conchinchinensis (LOUR) MERR distributed over southern area and Ulleung island in South Korea and belonged to Liliaceae, has been reported to contain asparagine, sitosterol, steroid saponin etc and known to show anti-bacterial and anti-tumor activities (B. S. Cheong et al., Dohaehyangyak Daesajeon, Youngrim press, pp164-1165, 1998).
  • LOUR Asparagus conchinchinensis
  • lycii fructus a fruit of Lycium chinesis or Lycium barbarum L. belonged to Solanaceae, has been reported to contain carotene, nicotic acid etc and to show lowering effect on blood sugar, blood cholesterol and blood pressure as an edible fruit for a long time in Korea (B. S. Cheong et al., Dohaehyangyak Daesajeon, Youngrim press, pp826-828, 1998).
  • the inventors of the present invention have performed various animal model test to determine the reducing effect on tail skin temperature and anti-fatigue activity using by treadmill as well as brief clinical test on volunteers suffering with climacteric syndrome, and finally completed present invention by confirming that the novel combination showed stronger synergistic treating and improving effect on the abnormal regulation of female hormone than sole plant extract.
  • the inventors of the present invention have performed various animal model test to determine the reducing effect on tail skin temperature and anti-fatigue activity using by treadmill as well as brief clinical test on volunteers suffering with climacteric syndrome, and finally completed present invention by confirming that the novel combination showed stronger synergistic treating and improving effect on the abnormal regulation of female hormone than sole plant extract.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus for treating and preventing an abnormal regulation of female hormone.
  • mulberry fruit disclosed herein comprises fruit of Mori alba L.
  • Asparagi radix includes a radix of Asparagus conchinchinensis (LOUR) MERR, specifically, that distributed over southern area and Ulleung island in South Korea.
  • LOUR Asparagus conchinchinensis
  • lycii fructus disclosed herein comprises a fruit of Lycium chinesis or Lycium barbarum L.
  • extract is soluble in water, spirit, C 1 -C 4 lower alcohol, or the mixtures thereof, preferably water or 10-90% ethanol or spirit in water, more preferably, water.
  • the "an extract of at least two combined herbs” disclosed herein comprise the an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus, preferably, (A) combinations of mulberry fruit and Asparagi radix; (B) combinations of Asparagi radix and lycii fructus ; or (C) combinations of mulberry fruit, Asparagi radix and lycii fructus; more preferably, the combination(s) with the weight mixed ratio of 0.01-100: 0.01-100 weight part (w/w), more and more preferably, 0.1-10: 0.1-10 weight part (w/w), most preferably, 0.5-5: 0.5 - 5 weight part (w/w) in case of (A) combinations of mulberry fruit and Asparagi radix; (B) combinations of Asparagi radix and lycii fructus ; or the combination with the weight mixed ratio of 0.01-100: 0.01-100: 0.01-100 weight part (w/
  • abnormal regulation of female hormone incudes various diseases caused by the abnormal regulation on female hormone such as estrogen or progesterone, for example, female climacteric syndrome, sexual desire disorder, hypo-estrogenemia, sex hormone hyper-secretion, precocious puberty and the like, specifically, female climacteric syndrome and the resulting syndrome caused thereby, for example, hidrorrhea, nausea, dizziness, tinnitus, palpitation, insomnia, restlessness, irascibility, soreness of loin and knees, benumbedness and itch of skin, hypomnesis, arthralgia, myalgia, colpoxerosis, headache, over-sensitiveness, anxiety disorder, depression, vertigo, facial flushing response, sleep disturbance, hypotrophy, memory deficiency, business ability degradation, stamina degradation, decline in exercise capacity, decrease in body hair, skin aging, osteopenia, increase in visceral fat, and the like, more specifically
  • a pharmaceutical composition comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus as an active ingredient in an amount effective to prevent and treat an abnormal regulation of female hormone, together with a pharmaceutically acceptable carrier or diluents.
  • the herbs which can be used in the present invention, include the same genus plants which would be apparent to those skilled in the art and have been used for identical or similar purpose and can be substituted for the prevention and treatment of the diseases.
  • the dried each plant materials i.e., mulberry fruit, Asparagi radix and lycii fructus was mixed together with specific mixed ratio; the plant materials were mixed with 1 to 50-fold, preferably, 5 to 15-fold weight (w/v) of water, spirit, C 1 -C 4 lower alcohol, such as methanol, ethanol, butanol or the mixtures thereof, preferably water or 10-90% ethanol in water, more preferably, water and extracted by reflux extraction, cold water extraction, ultra-sonication or other conventional extraction, preferably by reflux extraction with the temperature ranging from 10 to 150°C, preferably, 50 to 100°C for the period ranging from 0.5 to 20 hours, preferably, 2 to 16 hours; the residue was filtered and the filtrate was dried at the temperature ranging from 40 to 80°C, preferably from 50 to 70°C to obtain inventive combined extract of the present invention.
  • the plant materials were mixed with 1 to 50-fold, preferably, 5 to 15-fold weight (w/v) of water, spirit, C
  • the present invention also provides a method for preparing an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus comprising the steps of; drying the mulberry fruit, Asparagi radix and lycii fructus and mixed together with specific mixed ratio at 1 st step; mixing the plant materials with 1 to 50-fold weight (w/v) of water, spirit, C 1 -C 4 lower alcohol or the mixture thereof and extracting the solution by reflux extraction, cold water extraction, ultra-sonication or other conventional extraction at the temperature ranging from 10 to 150°C for the period ranging from 0.5 to 20 hours at 2 nd step; filtering the residue and drying the filtrate or the supernatant at the temperature ranging from 40 to 80°C to obtain inventive combined extract of the present invention.
  • the combined inventive extract prepared by the above-described method showed stronger synergistic treating and improving effect on the abnormal regulation of female hormone than sole plant extract, therefore the inventive extract can be useful in treating or preventing Abnormal regulation of female hormone.
  • the pharmaceutical composition comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus prepared by the above-described method , as an active ingredient in an amount effective to prevent and treat Abnormal regulation of female hormone, together with a pharmaceutically acceptable carrier or diluents.
  • an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus prepared by the above-described method for manufacture of medicines employed for treating or preventing Abnormal regulation of female hormone in mammals including human as an active ingredient in amount effective to treat or prevent Abnormal regulation of female hormone.
  • a method of treating or preventing Abnormal regulation of female hormone in a mammal comprising administering to said mammal an effective amount of an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus prepared by the above-described method, together with a pharmaceutically acceptable carrier thereof into the mammals including human suffering from said disease.
  • composition of the present invention has no toxicity and adverse effect therefore can be used with safe.
  • prevent means the inhibition of such those diseases in a mammal which is prone to be caught by those disease and the term “treat” used herein means (a) the inhibition of the development of disease or illness; (b) the alleviation of disease or illness; or (c) the elimination of disease or illness.
  • pharmaceutically acceptable carriers or excipients comprises “pharmaceutical additives, the inactive ingredients used to make up a medication. They include dyes, flavors, binders, emollients, fillers, lubricants, preservatives, and many more classifications. Common excipients include cornstarch, lactose, talc, magnesium stearate, sucrose, gelatin, calcium stearate, silicon dioxide, shellac and glaze, which has been well-known in the art ( See , Home-page of Food and Drug Administration or drug information online) or previous literature, for example, Rowe, Raymond C et al., Handbook of Pharmaceutical Excipients, Pharmaceutical Press, 7th Edition, 2012)
  • the inventive extract preferably should be present between 0.01 to 99 %, and more preferably between 0.02 to 50 %.
  • the above composition does not limit the invention in no way.
  • the present invention may be prepared by mixing the inventive extract with the pharmaceutically acceptable carriers, adjuvants or diluents as pharmaceutical composition for prevention and treatment of purposed disease or disorders.
  • composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrlidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
  • the formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like.
  • the desirable dose of the inventive extract varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging from 0.0001 to 100 mg/kg, preferably 0.001 to 100 mg/kg by weight/day of the inventive compounds of the present invention.
  • the dose may be administered in single or divided into several times per day.
  • composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intrathecal, epidural or intracerebroventricular injection.
  • inventive composition of the present invention also can be used as a main component or additive and aiding agent in the preparation of various functional health food and health care food.
  • the present invention provides a health functional food comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus as an active ingredient for alleviating or preventing Abnormal regulation of female hormone.
  • the present invention also provides a health functional food comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus, and a sitologically acceptable additive for alleviating or preventing Abnormal regulation of female hormone.
  • a health functional food defined herein the functional food having enhanced functionality such as physical functionality or physiological functionality by adding the composition of the present invention to conventional food to prevent or improve the purposed diseases in human or mammal.
  • a health care food defined herein "the food containing the combination of the present invention showing no specific intended effect but general intended effect in a small amount of quantity as a form of additive or in a whole amount of quantity as a form of powder, granule, capsule, pill, tablet etc.
  • a sitologically acceptable additive comprises "any substance the intended use which results or may reasonably be expected to result-directly or indirectly-in its becoming a component or otherwise affecting the characteristics of any food", and can be classified into three groups according to its origin, i.e., (1) chemically synthetic additive such as ketones, glycine, potassium citrate, nicotinic acid, etc; (2) natural additive such as persimmon dye, licorice extract, crystalline cellulose, gua dum etc; (3) the mixed additive therewith such as sodium L-glutamate, preservatives, tar dye etc, or various categories according to its function in the food, for example, thickening agent, maturing agent, bleaching agent, sequestrant, humectant, anti-caking agent, clarifying agents, curing agent, emulsifier, stabilizer, thickener, bases and acid, foaming agents, nutrients, coloring agent, flavoring agent, sweetener, preservative agent, anti-oxidant, etc, which has been well-known
  • direct additive a substance that becomes part of the food in trace amounts due to its packaging, storage or other handling.
  • health care foods or health functional foods can be contained in food, health beverage, dietary supplement etc, and may be formulated into a form of pharmaceutically dosing form such as a powder, granule, tablet, suspension, emulsion, syrup, chewing tablet, capsule, beverage etc; or the food form, for example, bread, rice cake, dry fruit, candy, chocolate, chewing gum, ice cream, milk such as low-fat milk, lactose-hydrolyzed milk, goat-milk, processed milk, milk product such as fermented milk, butter, concentrated milk, milk cream, butter oil, natural cheese, processed cheese, dry milk, milk serum etc, processed meat product such as hamburger, ham, sausage, bacon etc, processed egg product, fish meat product such as fish cake etc, noodle products such as instant noodles, dried noodles, wet noodles, fried noodles, non-fried noodles, gelatinized dry noodles, cooked noodles, frozen noodles, Pasta etc, tea product such as tea bag, leached tea etc, health drinks such as fruit drinks, vegetable drinks,
  • above described combination can be added to food or beverage for prevention and improvement of purposed disorder.
  • the amount of above described combination in food or beverage as a functional health food or health care food may generally range from about 0.01 to 100 w/w % of total weight of food for functional health food composition.
  • the preferable amount of the combination of the present invention in the functional health food, health care food or special nutrient food may be varied in accordance to the intended purpose of each food, it is preferably used in general to use as an additive in the amount of the combination of the present invention ranging from about 0.01 to 5% in food such as noodles and the like, from 40 to 100% in health care food on the ratio of 100% of the food composition.
  • the health beverage composition of present invention contains above combination as an essential component in the indicated ratio
  • the other component can be various deodorant or natural carbohydrate etc such as conventional beverage.
  • natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc.
  • natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartame et al.
  • the amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 ml of present beverage composition.
  • the other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese, chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al.
  • the other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination.
  • the ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition.
  • Examples of addable food comprising aforementioned extract or compound therein are various food, beverage, gum, vitamin complex, health improving food and the like.
  • Inventive combination of the present invention has no toxicity and adverse effect therefore; they can be used with safe.
  • the inventive composition comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus a prepared by the above-described method showed stronger synergistic treating and improving effect on the abnormal regulation of female hormone than sole plant extract. Accordingly, the inventive combination may be useful to alleviate, treat or prevent an abnormal regulation of female hormone in the form of a pharmaceutical composition, health functional food, food care food etc.
  • the extract was formulated into capsule according to the composition and method disclosed in Table 1 to use as a test sample in the brief clinical tests.
  • the extract was formulated into capsule according to the composition and method disclosed in Table 1 to use as a test sample in the brief clinical tests by substituting the extract with MLC1.
  • the extract was formulated into capsule according to the composition and method disclosed in Table 1 to use as a test sample in the brief clinical tests by substituting the extract with MLC2.
  • mice prepared in the above step underwent a bilateral ovariectomy under sodium pentobarbital anesthesia (50mg/kg, i.p.) according to the methods disclosed in previous literature (Park et al., J. Korean Neurosurg . Soc ., 2010, 48 :p309).
  • mice The back of mice was excised with scissors and the bilateral ovaries were excised and sutured tightly. The excised back was sutured, and disinfected with administrating antibiotics. The body temperature of the mice was maintained by heating with heating pad and the mice were used in following experiments as experimental models of climacteric syndrome.
  • test samples were orally administrated (800mg/kg) as test sample groups and equivalent amounts of physiological saline solution were orally administrated into the mice as control group(Sham) and negative-control group (OVX-saline) respectively.
  • mice 2 weeks after the administration of the test samples, the body weight of the mice was determined and the mice were restrained in a holder in a conscious state for 5 mins.
  • the tail skin temperature was measured at the dorsal surface of the tail about 1 cm from its base with a thermo tracer ( FLIR T600, USA) as a basal temperature (T B ).
  • the mice were forced to run (15m/min) on a motor driven treadmill (Exer-3/6, Columbus instruments, USA) for 10 mins. After termination of the forced exercise, tail skin temperature was measured at every 1 min, three times, using by thermo tracer as Forced exercise Temperature (T E ).
  • T E1 tail skin temperature at 1 min after the forced exercise.
  • T E2 tail skin temperature at 2 mins after the forced exercise.
  • T E3 tail skin temperature at 3 mins after the forced exercise.
  • TST in negative control group (OVX + saline) showed significantly increased, which means the successfully performed surgery and the inventive combinations of the present invention showed synergistically decreasing effect on the tail skin temperature than the comparative test sample treated with only the extract of Asparagi radix (AR) as can be seen in Table 2.
  • mice 2 weeks after the administration of the test samples, the ovariectomized mice was forced to run with increasing the percentage of slope to 10 °.
  • the speed of treadmill was started at the speed of 15 m/mins, and increased, step by step, at the speed of 5 m/mins for every 3 mins intervals, resulting in 30 m/mins finally.
  • mice 2 weeks after the administration of the test samples, the ovariectomized mice was performed to inhalation anesthesia with isofluran ® and the blood was delivered from the heart of mice.
  • the blood was kept in vacutainer tube for 30 mins at room temperature, and centrifuged to obtain a serum to store in a refrigerator.
  • the blood level of ALT, AST, ALP, total cholesterol, HDL, LDL, calcium and inorganic-phosphorous was analyzed at an analysis institute (Green Cross Labcell Inc. Yongin-city, Korea).
  • the blood level of ALT, AST, ALP, total cholesterol, HDL, LDL, calcium and inorganic-phosphorous in test sample groups was similar to those in the control group and negative control group.
  • the inventive combination of the present invention showed synergistically reducing effect on the hot flush syndrome and increasing effect on the locomotion capacity in the ovariectomized mice than that in the comparative test sample treated with only the extract of Asparagi radix (AR).
  • each climacteric symptom was classified into four categories according to the severity of climacteric symptom, i.e., no symptoms (i), mere symptom (ii), moderate symptom (iii) and severe symptom (iv).
  • the inventive combination of the present invention showed synergistically treating effect on various for climacteric symptoms in the brief clinical test than that in the comparative test sample treated with only the extract of Asparagi radix (AR).
  • Acute toxicity test was performed by using four-weeks-old ICR mouse (Orientbio, Japan) according to method disclosed in the literature (Haschek WM and Rousseaux CG, Handbook of toxicologic pathology, Published by Academic press, Inc,. New York, pp293-295, 1991).
  • the inventive combined extract of the present invention dissolved in water was administrated orally to each group consisting of 5 mice in a dose of 2g/kg/10ml. After administration, the mortality of the mice and clinical symptom, body weight change was observed and hematologic test and hematological biochemistry test was performed. Whether abdominal organ and thoracic organ is abnormal was observed with the naked eye by an autopsy.
  • Powder preparation was prepared by mixing above components and filling sealed package.
  • Tablet preparation was prepared by mixing above components and entabletting.
  • Capsule preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.
  • Injection preparation was prepared by dissolving active component and then filling all the components in 2 ml ample and sterilizing by conventional injection preparation method.
  • Liquid medicine was prepared by dissolving the components to distilled water with a proper dose of lemon scent, mixing, adjusting to 100 ml with distilled water in brown bottle and sterilizing by conventional liquid medicine preparation method.
  • Vitamin mixture optimum amount
  • Vitamin A acetate 70 ⁇ g
  • Vitamin E 1.0 mg
  • Vitamin B 1 0.13 mg
  • Vitamin B 2 0.15 mg
  • Vitamin B 6 0.5 mg
  • Vitamin B 12 0.2 ⁇ g
  • Vitamin C 10 mg
  • Citric acid 1000 mg
  • Health beverage preparation was prepared by dissolving active component, mixing, stirring at 85°C for 1 hour, filtering and then filling all the components in 2 lcontainer and sterilizing by conventional health beverage preparation method.
  • the inventive composition comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus a prepared by the above-described method showed stronger synergistic treating and improving effect on the abnormal regulation of female hormone than sole plant extract. Accordingly, the inventive combination may be useful to alleviate, treat or prevent an abnormal regulation of female hormone in the form of a pharmaceutical composition, health functional food, food care food etc.

Abstract

The present invention relates to compositions comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus for treating and preventing an abnormal regulation of female hormone. As described in the present invention, the inventive composition showed stronger synergistic treating and improving effect on the abnormal regulation of female hormone than sole plant extract. Accordingly, the inventive combination may be useful to alleviate, treat or prevent an abnormal regulation of female hormone in the form of a pharmaceutical composition, health functional food, food care food etc.

Description

COMPOSITION COMPRISING THE COMBINED EXTRACT FOR PREVENTING OR TREATING THE ABNORMAL REGULATION OF FEMALE HORMONE AND THE USE THEREOF
The present invention relates to a composition and health functional food comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus for treating and preventing an abnormal regulation of female hormone and the use thereof.
Abnormal regulation of female hormone incudes various diseases caused by the abnormal regulation on female hormone such as estrogen or progesterone, for example, female climacteric syndrome, sexual desire disorder, hypo-estrogenemia, sex hormone hyper-secretion, precocious puberty, and the like as well as the resulting syndrome caused thereby, for example, over-sensitiveness, anxiety disorder, depression, vertigo, facial flushing response, hidrosis, sleep disturbance, hypotrophy, memory deficiency, business ability degradation, stamina degradation, decline in exercise capacity, decrease in body hair, skin aging, osteopenia, increase in visceral fat, and the like.
The climacteric syndrome of women is used to refer to a series of symptoms and conditions associated with menopause caused by hypofuction of ovary before and after natural menopause. Menopause may give rises to various syndrome caused by hormonal changes, such as decline of estrogen synthesis, the increased level of FSH (Follicular stimulating hormone) and LH (Luteinizing hormone) etc. The said symptoms and conditions include malfunctions of endocrine system, decreases in estrogen level, disorder of metabolism, menstrual disturbance caused by disturbances of cardiovascular and vegetative nervous system, hidrorrhea, nausea, dizziness, and tinnitus, palpitation and insomnia, restlessness and irascibility, soreness of loin and knees, benumbedness and itch of skin, hypomnesis, and even emotional abnormality and the like. (US2005/0123629 A1).
The insufficient release of estrogen level after menopause also gives rise to various syndromes, for example, hot flush, urogenital symptoms, osteoporosis, depression, sleep disturbance, skin aging etc (Na-Ra HAN, et al., Cysteine Prevents menopausal syndromes in Ovariectomized Mouse, Reproductive Sciences, 2016, Vol. 23(5), pp670-679).
HRT (Hormone-replacement Therapy) has used successfully to treat the abnormal regulation of female hormone including female climacteric syndrome. However the therapy give rises to various adverse responses such as breast cancer, stroke, heart attack, venous thrombosis, cardiovascular disease, etc.
Accordingly, there has been still needed to develop more effective drug to treat treat the abnormal regulation of female hormone including female climacteric syndrome with little adverse response in order to overcome the problem of the conventionally used synthetic hormonal therapy.
One of such attempts, the estrogenic extract of Asparagus conchinchinensis (LOUR) MERR of Liliaceae Family can be used to treat one or more disease states associated with the estrogen receptor and climacteric symptoms such as hot-flashes (PCT/WO2009/033025A1).
Mulberry fruit, a fruit of Mori alba L. belonged to Moraceae, has been reported to contain various saccharide, tannic acid, vitamins, linoleic acid etc and used to be edible fruit for a long time in Korea (B. S. Cheong et al., Dohaehyangyak Daesajeon, Youngrim press, pp545-548, 1998).
Asparagi radix, a radix of Asparagus conchinchinensis (LOUR) MERR distributed over southern area and Ulleung island in South Korea and belonged to Liliaceae, has been reported to contain asparagine, sitosterol, steroid saponin etc and known to show anti-bacterial and anti-tumor activities (B. S. Cheong et al., Dohaehyangyak Daesajeon, Youngrim press, pp164-1165, 1998).
lycii fructus, a fruit of Lycium chinesis or Lycium barbarum L. belonged to Solanaceae, has been reported to contain carotene, nicotic acid etc and to show lowering effect on blood sugar, blood cholesterol and blood pressure as an edible fruit for a long time in Korea (B. S. Cheong et al., Dohaehyangyak Daesajeon, Youngrim press, pp826-828, 1998).
However, there has been not reported or disclosed about the synergistic effect of the extract of combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus for treating and preventing an abnormal regulation of female hormone in any of the above cited literatures, the disclosures of which are incorporated herein by reference.
To investigate the synergistic effect on the abnormal regulation of female hormone disease of novel combination of mulberry fruit, Asparagi radix and lycii fructus, the inventors of the present invention have performed various animal model test to determine the reducing effect on tail skin temperature and anti-fatigue activity using by treadmill as well as brief clinical test on volunteers suffering with climacteric syndrome, and finally completed present invention by confirming that the novel combination showed stronger synergistic treating and improving effect on the abnormal regulation of female hormone than sole plant extract.
However, there has been not reported or disclosed about the synergistic effect of the extract of combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus for treating and preventing an abnormal regulation of female hormone in any of the above cited literatures, the disclosures of which are incorporated herein by reference.
To investigate the synergistic effect on the abnormal regulation of female hormone disease of novel combination of mulberry fruit, Asparagi radix and lycii fructus, the inventors of the present invention have performed various animal model test to determine the reducing effect on tail skin temperature and anti-fatigue activity using by treadmill as well as brief clinical test on volunteers suffering with climacteric syndrome, and finally completed present invention by confirming that the novel combination showed stronger synergistic treating and improving effect on the abnormal regulation of female hormone than sole plant extract.
Accordingly, the present invention provides a pharmaceutical composition comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus for treating and preventing an abnormal regulation of female hormone.
The term "mulberry fruit" disclosed herein comprises fruit of Mori alba L.
The term "Asparagi radix" disclosed herein includes a radix of Asparagus conchinchinensis (LOUR) MERR, specifically, that distributed over southern area and Ulleung island in South Korea.
The term "lycii fructus" disclosed herein comprises a fruit of Lycium chinesis or Lycium barbarum L.
The term "extract" disclosed herein is soluble in water, spirit, C1-C4 lower alcohol, or the mixtures thereof, preferably water or 10-90% ethanol or spirit in water, more preferably, water.
The "an extract of at least two combined herbs" disclosed herein comprise the an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus, preferably, (A) combinations of mulberry fruit and Asparagi radix; (B) combinations of Asparagi radix and lycii fructus ; or (C) combinations of mulberry fruit, Asparagi radix and lycii fructus; more preferably, the combination(s) with the weight mixed ratio of 0.01-100: 0.01-100 weight part (w/w), more and more preferably, 0.1-10: 0.1-10 weight part (w/w), most preferably, 0.5-5: 0.5 - 5 weight part (w/w) in case of (A) combinations of mulberry fruit and Asparagi radix; (B) combinations of Asparagi radix and lycii fructus ; or the combination with the weight mixed ratio of 0.01-100: 0.01-100: 0.01-100 weight part (w/w), more and more preferably, 0.1-10: 0.1-10: 0.1-10 weight part (w/w), most preferably, 0.5-5: 0.5-5: 0.5 - 5 weight part (w/w) in case of (C) (C) combinations of mulberry fruit, Asparagi radix and lycii fructus in the present invention.
The term "Abnormal regulation of female hormone" disclosed herein incudes various diseases caused by the abnormal regulation on female hormone such as estrogen or progesterone, for example, female climacteric syndrome, sexual desire disorder, hypo-estrogenemia, sex hormone hyper-secretion, precocious puberty and the like, specifically, female climacteric syndrome and the resulting syndrome caused thereby, for example, hidrorrhea, nausea, dizziness, tinnitus, palpitation, insomnia, restlessness, irascibility, soreness of loin and knees, benumbedness and itch of skin, hypomnesis, arthralgia, myalgia, colpoxerosis, headache, over-sensitiveness, anxiety disorder, depression, vertigo, facial flushing response, sleep disturbance, hypotrophy, memory deficiency, business ability degradation, stamina degradation, decline in exercise capacity, decrease in body hair, skin aging, osteopenia, increase in visceral fat, and the like, more specifically, hidrorrhea, dizziness, palpitation, insomnia, restlessness, irascibility, over-sensitiveness, anxiety disorder, depression, vertigo, facial flushing response, arthralgia, myalgia, colpoxerosis, or headache etc.
Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus as an active ingredient in an amount effective to prevent and treat an abnormal regulation of female hormone, together with a pharmaceutically acceptable carrier or diluents.
It is another object of the present invention to provide a use of an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus for manufacture of medicament employed for treating or preventing an abnormal regulation of female hormone in human or mammal.
It is the other object of the present invention to provide a method for treating an abnormal regulation of female hormone in a mammal or animal suffering from said disease comprising administering an effective amount of an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus, together with a pharmaceutically acceptable carrier to said mammal or animal in need thereof
The herbs, which can be used in the present invention, include the same genus plants which would be apparent to those skilled in the art and have been used for identical or similar purpose and can be substituted for the prevention and treatment of the diseases.
Hereinafter, the present invention is described in detail.
For the preparation of ginseng of the present invention, for example, the dried each plant materials, i.e., mulberry fruit, Asparagi radix and lycii fructus was mixed together with specific mixed ratio; the plant materials were mixed with 1 to 50-fold, preferably, 5 to 15-fold weight (w/v) of water, spirit, C1-C4 lower alcohol, such as methanol, ethanol, butanol or the mixtures thereof, preferably water or 10-90% ethanol in water, more preferably, water and extracted by reflux extraction, cold water extraction, ultra-sonication or other conventional extraction, preferably by reflux extraction with the temperature ranging from 10 to 150℃, preferably, 50 to 100℃ for the period ranging from 0.5 to 20 hours, preferably, 2 to 16 hours; the residue was filtered and the filtrate was dried at the temperature ranging from 40 to 80℃, preferably from 50 to 70℃ to obtain inventive combined extract of the present invention.
Accordingly, the present invention also provides a method for preparing an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus comprising the steps of; drying the mulberry fruit, Asparagi radix and lycii fructus and mixed together with specific mixed ratio at 1st step; mixing the plant materials with 1 to 50-fold weight (w/v) of water, spirit, C1-C4 lower alcohol or the mixture thereof and extracting the solution by reflux extraction, cold water extraction, ultra-sonication or other conventional extraction at the temperature ranging from 10 to 150℃ for the period ranging from 0.5 to 20 hours at 2nd step; filtering the residue and drying the filtrate or the supernatant at the temperature ranging from 40 to 80℃ to obtain inventive combined extract of the present invention.
The combined inventive extract prepared by the above-described method showed stronger synergistic treating and improving effect on the abnormal regulation of female hormone than sole plant extract, therefore the inventive extract can be useful in treating or preventing Abnormal regulation of female hormone.
Accordingly, it is another object of the present invention to provide the pharmaceutical composition comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus prepared by the above-described method, as an active ingredient in an amount effective to prevent and treat Abnormal regulation of female hormone, together with a pharmaceutically acceptable carrier or diluents.
In accordance with the other aspect of the present invention, there is also provided a use of an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus prepared by the above-described method for manufacture of medicines employed for treating or preventing Abnormal regulation of female hormone in mammals including human as an active ingredient in amount effective to treat or prevent Abnormal regulation of female hormone.
In accordance with the other aspect of the present invention, there is also provided a method of treating or preventing Abnormal regulation of female hormone in a mammal comprising administering to said mammal an effective amount of an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus prepared by the above-described method, together with a pharmaceutically acceptable carrier thereof into the mammals including human suffering from said disease.
Inventive composition of the present invention has no toxicity and adverse effect therefore can be used with safe.
The term "prevent" defined herein means the inhibition of such those diseases in a mammal which is prone to be caught by those disease and the term "treat" used herein means (a) the inhibition of the development of disease or illness; (b) the alleviation of disease or illness; or (c) the elimination of disease or illness.
The term "pharmaceutically acceptable carriers or excipients" defined herein comprises "pharmaceutical additives, the inactive ingredients used to make up a medication. They include dyes, flavors, binders, emollients, fillers, lubricants, preservatives, and many more classifications. Common excipients include cornstarch, lactose, talc, magnesium stearate, sucrose, gelatin, calcium stearate, silicon dioxide, shellac and glaze, which has been well-known in the art ( See , Home-page of Food and Drug Administration or drug information online) or previous literature, for example, Rowe, Raymond C et al., Handbook of Pharmaceutical Excipients, Pharmaceutical Press, 7th Edition, 2012)
In terms of pharmaceutical composition of the present invention, the inventive extract preferably should be present between 0.01 to 99 %, and more preferably between 0.02 to 50 %. The above composition does not limit the invention in no way.
Therefore, the present invention may be prepared by mixing the inventive extract with the pharmaceutically acceptable carriers, adjuvants or diluents as pharmaceutical composition for prevention and treatment of purposed disease or disorders.
The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrlidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like.
The desirable dose of the inventive extract varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging from 0.0001 to 100 mg/kg, preferably 0.001 to 100 mg/kg by weight/day of the inventive compounds of the present invention. The dose may be administered in single or divided into several times per day.
The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intrathecal, epidural or intracerebroventricular injection.
The inventive composition of the present invention also can be used as a main component or additive and aiding agent in the preparation of various functional health food and health care food.
The present invention provides a health functional food comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus as an active ingredient for alleviating or preventing Abnormal regulation of female hormone.
The present invention also provides a health functional food comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus, and a sitologically acceptable additive for alleviating or preventing Abnormal regulation of female hormone.
The term "a health functional food" defined herein" the functional food having enhanced functionality such as physical functionality or physiological functionality by adding the composition of the present invention to conventional food to prevent or improve the purposed diseases in human or mammal.
It is the other object of the present invention to provide a health care food comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus, together with a sitologically acceptable additive for the prevention or alleviation of Abnormal regulation of female hormone.
The term "a health care food" defined herein "the food containing the combination of the present invention showing no specific intended effect but general intended effect in a small amount of quantity as a form of additive or in a whole amount of quantity as a form of powder, granule, capsule, pill, tablet etc.
The term "a sitologically acceptable additive" defined herein comprises "any substance the intended use which results or may reasonably be expected to result-directly or indirectly-in its becoming a component or otherwise affecting the characteristics of any food", and can be classified into three groups according to its origin, i.e., (1) chemically synthetic additive such as ketones, glycine, potassium citrate, nicotinic acid, etc; (2) natural additive such as persimmon dye, licorice extract, crystalline cellulose, gua dum etc; (3) the mixed additive therewith such as sodium L-glutamate, preservatives, tar dye etc, or various categories according to its function in the food, for example, thickening agent, maturing agent, bleaching agent, sequestrant, humectant, anti-caking agent, clarifying agents, curing agent, emulsifier, stabilizer, thickener, bases and acid, foaming agents, nutrients, coloring agent, flavoring agent, sweetener, preservative agent, anti-oxidant, etc, which has been well-known in the art or previous literature ( See , "Codex General Standard for Food Additives" (GSFA, Codex STAN 192-1995) in Home-page of GSFA Online).
If a substance is added to a food for a specific purpose in that food, it is referred to as a direct additive and indirect food additives are those that become part of the food in trace amounts due to its packaging, storage or other handling.
The term "health care foods or health functional foods" disclosed herein can be contained in food, health beverage, dietary supplement etc, and may be formulated into a form of pharmaceutically dosing form such as a powder, granule, tablet, suspension, emulsion, syrup, chewing tablet, capsule, beverage etc; or the food form, for example, bread, rice cake, dry fruit, candy, chocolate, chewing gum, ice cream, milk such as low-fat milk, lactose-hydrolyzed milk, goat-milk, processed milk, milk product such as fermented milk, butter, concentrated milk, milk cream, butter oil, natural cheese, processed cheese, dry milk, milk serum etc, processed meat product such as hamburger, ham, sausage, bacon etc, processed egg product, fish meat product such as fish cake etc, noodle products such as instant noodles, dried noodles, wet noodles, fried noodles, non-fried noodles, gelatinized dry noodles, cooked noodles, frozen noodles, Pasta etc, tea product such as tea bag, leached tea etc, health drinks such as fruit drinks, vegetable drinks, carbonated soft drinks, soymilk drinks, lactic beverage mixed beverage, etc, seasoning food such as soy sauce, soybean paste, red pepper paste, chunjang (a kind of fermented soybean product colored by caramel), cheonggukjang (natural fermented soybean by B. subtillis), mixed paste, vinegar, sauce, ketchup, curry, dressing etc, margarine, shortening, pizza etc, but not intended herein to limit thereto, for preventing or improving of purposed disease.
Also, above described combination can be added to food or beverage for prevention and improvement of purposed disorder. The amount of above described combination in food or beverage as a functional health food or health care food may generally range from about 0.01 to 100 w/w % of total weight of food for functional health food composition. In particular, although the preferable amount of the combination of the present invention in the functional health food, health care food or special nutrient food may be varied in accordance to the intended purpose of each food, it is preferably used in general to use as an additive in the amount of the combination of the present invention ranging from about 0.01 to 5% in food such as noodles and the like, from 40 to 100% in health care food on the ratio of 100% of the food composition.
Providing that the health beverage composition of present invention contains above combination as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartame et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 ㎖ of present beverage composition.
The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese, chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising aforementioned extract or compound therein are various food, beverage, gum, vitamin complex, health improving food and the like.
Inventive combination of the present invention has no toxicity and adverse effect therefore; they can be used with safe.
As described in the present invention, the inventive composition comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus a prepared by the above-described method showed stronger synergistic treating and improving effect on the abnormal regulation of female hormone than sole plant extract. Accordingly, the inventive combination may be useful to alleviate, treat or prevent an abnormal regulation of female hormone in the form of a pharmaceutical composition, health functional food, food care food etc.
It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.
The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.
Comparative Example 1. Preparation of the extract of Asparagi radix
1kg of Asparagi radix was mixed with 10 L of distilled water and refluxed for 5 hrs at the above 90℃ in water bath repeatedly. The solution was filtered with filter paper (Whatman laboratory Filter GE), concentrated under vaccuo with evaporator (Eyela, Rotary Evaporator N-1000) and dried with freeze dryer (Eyela, Freeze Dryer FDU-540) to afford 250g of the extract of Asparagi radix (designated as "AR" hereinafter).
Example 1. Preparation of the extract of combination ( MLC0 )
500g of each dried fruit of mulberry fruit and Asparagi radix procured from Kyungdong market located in Seoul (KOREA) was mixed together (mixed ratio, 1:1 w/w) and 10 fold volume of water was poured thereto. The mixture was refluxed at the above 90℃ for 5 hrs in water bath repeatedly. The solution was filtered with filter paper (Whatman laboratory Filter GE), concentrated under vaccuo with evaporator (Eyela, Rotary Evaporator N-1000) and dried with freeze dryer (Eyela, Freeze Dryer FDU-540) to afford 25g of the extract of the combination of (1) mulberry fruit and (2) Asparagi radix (designated as "MLC0" hereinafter).
The extract was formulated into capsule according to the composition and method disclosed in Table 1 to use as a test sample in the brief clinical tests.
Ingredient Amount
MLC0 10 mg
Crystalline cellulose 3 mg
Lactose 14.8 mg
Magnesium stearate 0.2 mg
Capsule preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.
Example 2. Preparation of the extract of combination ( MLC1 )
500g of each dried fruit of Asparagi radix and lycii fructus procured from Kyungdong market located in Seoul (KOREA) was mixed together (mixed ratio, 1:1 w/w) and 10 fold volume of water was poured thereto. The mixture was refluxed at the above 90℃ for 5 hrs in water bath repeatedly. The solution was filtered with filter paper (Whatman laboratory Filter GE), concentrated under vaccuo with evaporator (Eyela, Rotary Evaporator N-1000) and dried with freeze dryer (Eyela, Freeze Dryer FDU-540) to afford 20g of the extract of the combination of (1) Asparagi radix and (2) lycii fructus (designated as "MLC1" hereinafter).
The extract was formulated into capsule according to the composition and method disclosed in Table 1 to use as a test sample in the brief clinical tests by substituting the extract with MLC1.
Example 3. Preparation of the extract of combination ( MLC2 )
1,500g of each dried fruit of mulberry fruit, Asparagi radix and lycii fructus procured from Kyungdong market located in Seoul (KOREA) was mixed together (mixed ratio, 1:1 w/w) and 10 fold volume of water was poured thereto. The mixture was refluxed at the above 90℃ for 5 hrs in water bath repeatedly. The solution was filtered with filter paper (Whatman laboratory Filter GE), concentrated under vaccuo with evaporator (Eyela, Rotary Evaporator N-1000) and dried with freeze dryer (Eyela, Freeze Dryer FDU-540) to afford 95g of the extract of the combination of (1) mulberry fruit, (2) Asparagi radix and (3) lycii fructus (designated as "MLC2" hereinafter).
The extract was formulated into capsule according to the composition and method disclosed in Table 1 to use as a test sample in the brief clinical tests by substituting the extract with MLC2.
Experimental Example 1. The effect on the climacteric symptoms in ovariectomized mice (animal model)
1.1. Reagent and drugs.
All the reagent and drugs were procured from Sigma-Aldrich Chemistry Co. and the other reagents were commercially available in the market or company.
1.2. experimental animals
7 weeks old female ICR mice weighing 26-30g (Orient Bio Co. Ltd, Korea) were bred and were used for experiment after five days adaptation. The room was controlled by automatic light system from 7:00 A.M. to 7:00 P.M, for 12 hours, and the temperature was adjusted to 23±2℃, humidity was adjusted to 50±10℃%. The animal feed and tap water were fed freely. All the animal experiments were performed with the approval of IACUC (Institutional Animal Care and Use Committee of Kyung Hee University, KHUASP (SE)-16-125).
1.3. experimental procedure
1-3-1. preparation of ovariectomized mice
The mice prepared in the above step underwent a bilateral ovariectomy under sodium pentobarbital anesthesia (50mg/kg, i.p.) according to the methods disclosed in previous literature (Park et al., J. Korean Neurosurg . Soc., 2010, 48:p309).
The back of mice was excised with scissors and the bilateral ovaries were excised and sutured tightly. The excised back was sutured, and disinfected with administrating antibiotics. The body temperature of the mice was maintained by heating with heating pad and the mice were used in following experiments as experimental models of climacteric syndrome.
1-3-2. treatment of samples
2 weeks after the surgery, the test samples were orally administrated (800mg/kg) as test sample groups and equivalent amounts of physiological saline solution were orally administrated into the mice as control group(Sham) and negative-control group (OVX-saline) respectively.
1-3-3. determination of tail skin temperature
In order to investigate the effect of the test samples prepared in Comparative Examples and examples on tail skin temperature, following animal model test using by ovariectomized mice was performed according to the modified method disclosed in the literature (Hideki Shuto et al., Forced exercise-induced flushing of tail skin in ovariectomized mice as a new experimental model of menopausal hot flushes, J. Pharmacol . Sci., 2005, 98; pp323).
2 weeks after the administration of the test samples, the body weight of the mice was determined and the mice were restrained in a holder in a conscious state for 5 mins. The tail skin temperature was measured at the dorsal surface of the tail about 1 cm from its base with a thermo tracer ( FLIR T600, USA) as a basal temperature (TB). The mice were forced to run (15m/min) on a motor driven treadmill (Exer-3/6, Columbus instruments, USA) for 10 mins. After termination of the forced exercise, tail skin temperature was measured at every 1 min, three times, using by thermo tracer as Forced exercise Temperature (TE).
The tail skin temperature before forced exercise were assessed three times to obtain the average of three determined values) and the changes in tail temperature were assessed using Total TST (ΔTST) using by following empirical formulae 1
[Empirical Formulae 1]
Total ΔTST = (TE1-TB) + (TE2-TB) + (TE3-TB)
*TB : average basal tail skin temperature before forced exercise
TE1: tail skin temperature at 1 min after the forced exercise.
TE2: tail skin temperature at 2 mins after the forced exercise.
TE3: tail skin temperature at 3 mins after the forced exercise.
At the result, TST in negative control group (OVX + saline) showed significantly increased, which means the successfully performed surgery and the inventive combinations of the present invention showed synergistically decreasing effect on the tail skin temperature than the comparative test sample treated with only the extract of Asparagi radix (AR) as can be seen in Table 2.
synergistic effect on climacteric hot flush
Mean±SEM*Group Basal tem.(℃) Exercise Tem. ΔTST n
Sham 27.0 ± 0.4 28.5 ± 0.5 4.4 ± 0.7 9
Negative control(OVX+saline) 29.9 ± 0.8 34.8 ± 0.6* 14.7 ± 2.4* 9
OVX + AR, 800mg/kg 28.4 ± 0.5 33.2 ± 0.7 14.3 ± 1.9 10
OVX + MLC0, 800mg/kg 27.3 ± 0.1 29.1 ± 0.5 4.9 ± 1.0** 10
OVX + MLC1, 800mg/kg 27.6 ± 0.2 29.5 ± 0.6 4.8 ± 2.1 8
OVX + MLC2, 800mg/kg 27.1 ± 0.5 28.2 ± 0.7** 0.9 ± 0.2 10
OVX + oyster calcium, 800mg/kg 28.2 ± 0.3 32.9 ± 0.5 4.7 ± 0.2 10
*; significant comparing with the control group, P<0.05,**; significant comparing with the negative control group, P<0.05,
1-3-4. Anti-fatigue activity using by treadmill
In order to investigate the effect of the test samples prepared in Comparative Examples and examples on the anti-fatigue activity, following animal model test using by ovariectomized mice was performed according to the modified method disclosed in the literature (Minghua et al., Mechanisms of Aging and Development, 2003, 124:771).
2 weeks after the administration of the test samples, the ovariectomized mice was forced to run with increasing the percentage of slope to 10 °. The speed of treadmill was started at the speed of 15 m/mins, and increased, step by step, at the speed of 5 m/mins for every 3 mins intervals, resulting in 30 m/mins finally. The maximum running time of each mice which the mice could not run anymore, was determined.
As shown in Table 3, the maximum running time in control group and negative control group showed 26.2 ± 1.4 mins and 13.7 ± 1.0 mins, respectively, while the maximum running time in test sample groups treated with the inventive combinations of the present invention was synergistically increased than that in the comparative test sample treated with only the extract of Asparagi radix (AR).
synergistic effect on anti-fatigue activity
Mean±SEM*Group Weight (g) Maximum rung time (mins) n
Sham 31.2 ± 0.4 26.2± 1.4 10
Negative control (OVX+saline) 31.1 ± 0.5 13.7± 1.0* 10
OVX + AR, 800mg/kg 31.2 ± 0.2 20.1 ± 1.2 10
OVX + MLC0, 800mg/kg 31.5 ± 0.7 31.1 ± 5.7** 9
OVX + MLC1, 800mg/kg 31.3 ± 0.6 35.1 ± 1.9** 8
OVX + MLC2, 800mg/kg 30.6 ± 0.6 55.9± 1.9** 10
OVX + oyster calcium, 800mg/kg 30.4 ± 0.7 13.1 ± 1.3 10
*; significant comparing with the control group, P<0.05,**; significant comparing with the negative control group, P<0.05,
1-3-5. Effect on Blood Biochemical parameter
In order to investigate the effect of the test samples prepared in Comparative Examples and examples on the blood biochemical parameters, following animal model test using by ovariectomized mice was performed as follows.
2 weeks after the administration of the test samples, the ovariectomized mice was performed to inhalation anesthesia with isofluran ® and the blood was delivered from the heart of mice. The blood was kept in vacutainer tube for 30 mins at room temperature, and centrifuged to obtain a serum to store in a refrigerator. The blood level of ALT, AST, ALP, total cholesterol, HDL, LDL, calcium and inorganic-phosphorous was analyzed at an analysis institute (Green Cross Labcell Inc. Yongin-city, Korea).
As shown in Table 4, the blood level of ALT, AST, ALP, total cholesterol, HDL, LDL, calcium and inorganic-phosphorous in test sample groups was similar to those in the control group and negative control group.
Analysis on blood biochemical parameters
GroupMean±SEM* Sham Negative control (OVX+saline) OVX + MLC0, 800mg/kg OVX + MLC1, 800mg/kg OVX + MLC2, 800mg/kg OVX + oyster calcium, 800mg/kg
ALT (U/L) 25.7 ± 2.9 21.4 ± 0.9 28.8 ± 2.8 29.0 ± 4.4 30.5 ± 4.0 25.6 ± 7.4
AST(U/L) 51.6± 2.5 59.1± 3.8 57.4± 2.4 53.9± 1.4 64.0± 9.5 58.0± 2.0
ALP (U/L) 73.1± 3.8 74.0± 4.6 69.1± 4.3 65.9± 6.2 68.7± 5.6 65.3± 4.0
TC*(mg/dL) 112.9± 8.3 120.9± 9.5 119.0± 7.5 107.9± 4.7 123.1± 13.2 118.4± 8.6
HDL(mg/dL) 99.8± 5.6 106.4± 4.8 106.6 ± 3.9 102.4 ± 4.2 103.3± 7.0 107.5 ± 5.8
LDL(mg/dL) 14.9± 1.7 16.8± 2.2 16.6± 1.6 12.7± 0.9 19.4± 2.9 17.1± 2.2
Calcium(mg/dL) 9.5± 0.1 9.6± 0.1 9.5± 0.1 9.3± 0.1 9.4± 0.1 9.3± 0.1
IP*(mg/dL) 14.9± 1.7 6.3± 0.1* 6.0± 0.2* 6.0± 0.2* 5.9± 0.3* 6.0± 0.2*
n 9 8 8 7 10 8
*: TC: Total Cholesterol, IP: Inorganic Phosphorous**; significant comparing with the control group, P<0.05
1.1.Static Analysis
The statistical significance of all results was tested by ANOVA (one way analysis of variance) test and the significance was verified with Student-Newman-Keuls -test (p<0.05).
In summary, the inventive combination of the present invention showed synergistically reducing effect on the hot flush syndrome and increasing effect on the locomotion capacity in the ovariectomized mice than that in the comparative test sample treated with only the extract of Asparagi radix (AR).
Example 2. Brief clinical test
In order to verify the treating activity of the inventive combinations on climacteric syndrome, following brief clinical test was performed according to the method disclosed in the literature (Kupperman S et al., "Comparative clinical evaluation of estrogenic preparation by menopausal and amenorrheal indices, Journal of Clinical Endocrinology, 1953; 13:pp688-703).
50 female volunteers aged about 50 years lived in Seoul who had suffered with various climacteric syndromes listed in Table 5 were randomly divided into four groups and the three kinds of capsules, i.e., (a) comparative test sample capsules prepared in Comparative Example 1, (b) test sample capsules prepared in Example 1 and (c) placebo capsules which does not contain the test sample, were orally administrated into the volunteers, 2 capsules per once, twice a day for 4 weeks. The degree of each climacteric symptom was classified into four categories according to the severity of climacteric symptom, i.e., no symptoms (i), mere symptom (ii), moderate symptom (iii) and severe symptom (iv).
As can be seen in Table 6, the control group which is not treated with test samples did not showed any significant changes on the improvement of climacteric symptoms whereas the test sample groups treated with the inventive combinations of the present invention showed synergistically potent treating effect on climacteric syndromes than the comparative test sample treated with only the extract of Asparagi radix (AR).
Kupperman score for climacteric symptoms
No Symptoms (i) (ii) (iii) (iv)
1 Hot flush 0 4 8 12
2 hidrorrhea 0 2 4 6
3 insomnia 0 2 4 6
4 over-sensitiveness 0 1 2 3
5 depression 0 1 2 3
6 vertigo 0 1 2 3
7 Fatigue 0 1 2 3
8 arthralgia/myalgia 0 1 2 3
9 Headache 0 1 2 3
10 Palpitation 0 1 2 3
11 colpoxerosis 0 1 2 3
*severity: no symptoms (i), mere symptom (ii), moderate symptom (iii) and severe symptom (iv).
the change on Kupperman score for climacteric symptoms
Sample Score (Before the test) Score (After the test) Change
Control 30.8 26.9 -3.9
AR 31.9 25.1 -6.8
MLC0 29.8 14.1 -15.7
MLC1 31.5 15.6 -15.9
MLC2 30.2 8.7 -21.5
In summary, the inventive combination of the present invention showed synergistically treating effect on various for climacteric symptoms in the brief clinical test than that in the comparative test sample treated with only the extract of Asparagi radix (AR).
Experimental Example 3. Acute toxicity test of oral administration
Acute toxicity test was performed by using four-weeks-old ICR mouse (Orientbio, Japan) according to method disclosed in the literature (Haschek WM and Rousseaux CG, Handbook of toxicologic pathology, Published by Academic press, Inc,. New York, pp293-295, 1991).
The inventive combined extract of the present invention dissolved in water was administrated orally to each group consisting of 5 mice in a dose of 2g/kg/10ml. After administration, the mortality of the mice and clinical symptom, body weight change was observed and hematologic test and hematological biochemistry test was performed. Whether abdominal organ and thoracic organ is abnormal was observed with the naked eye by an autopsy.
As the result, there was no toxicity effect on clinical symptom, mortality, body weight change and gross finding in all the animals. Accordingly these results suggested that the mixed herbal extract of the present invention were potent and safe substance with over 5000 mg/kg of the minimum lethal dose of oral administration.
Hereinafter, the formulating methods and kinds of excipients of pharmaceutical compositions or health functional food will be described, but the present invention is not limited to them.
Preparation of powder
MLC0 : 500 mg
Lactose : 100 mg
Talc : 10 mg
Powder preparation was prepared by mixing above components and filling sealed package.
Preparation of tablet
MLC1 : 500 mg
Corn Starch : 100 mg
Lactose : 100 mg
Magnesium Stearate : 2 mg
Tablet preparation was prepared by mixing above components and entabletting.
Preparation of capsule
MLC2 : 500 mg
Crystallized cellulose : 3 mg
Lactose : 14.8 mg
Magnesium Stearate : 0.2 mg
Capsule preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.
Preparation of injection
MLC0 : 100 mg
Mannitol : 180 mg
Distilled water for injection : 2974 mg
Na2HPO412H2O : 26 mg
Injection preparation was prepared by dissolving active component and then filling all the components in 2 ml ample and sterilizing by conventional injection preparation method.
Preparation of liquid
MLC1 : 1000 mg
Isomerized sugar : 10 g
Mannitol : 5 g
Distilled water : optimum amount
Liquid medicine was prepared by dissolving the components to distilled water with a proper dose of lemon scent, mixing, adjusting to 100 ml with distilled water in brown bottle and sterilizing by conventional liquid medicine preparation method.
Preparation of health functional food
MLC2 : 500 mg
Vitamin mixture : optimum amount
Vitamin A acetate : 70 ㎍
Vitamin E : 1.0 mg
Vitamin B1 : 0.13 mg
Vitamin B2 : 0.15 mg
Vitamin B6 : 0.5 mg
Vitamin B12 : 0.2 ㎍
Vitamin C : 10 mg
Biotin : 10 ㎍
Amide nicotinic acid : 1.7 mg
Folic acid : 50 ㎍
Calcium pantothenic acid : 0.5 mg
Mineral mixture : optimum amount
Ferrous sulfate : 1.75 mg
Zinc oxide : 0.82 mg
Magnesium carbonate : 25.3 mg
Monopotassium phosphate : 15 mg
Dicalcium phosphate : 55 mg
Potassium citrate : 100 mg
Magnesium chloride : 24.8 mg
The above-mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.
Preparation of health beverage
MLC1 : 1000 mg
Citric acid : 1000 mg
Oligosaccharide : 100 g
Apricot concentration : 2 g
Taurine : 1 g
Distilled water : 900 ㎖
Health beverage preparation was prepared by dissolving active component, mixing, stirring at 85℃ for 1 hour, filtering and then filling all the components in 2 ℓcontainer and sterilizing by conventional health beverage preparation method.
The invention being thus described, may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
As described in the present invention, the inventive composition comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus a prepared by the above-described method showed stronger synergistic treating and improving effect on the abnormal regulation of female hormone than sole plant extract. Accordingly, the inventive combination may be useful to alleviate, treat or prevent an abnormal regulation of female hormone in the form of a pharmaceutical composition, health functional food, food care food etc.

Claims (15)

  1. A pharmaceutical composition comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus for treating and preventing an abnormal regulation of female hormone.
  2. The pharmaceutical composition according to claim 1, said lycii fructus is a fruit of Lycium chinesis or Lycium barbarum L.
  3. The pharmaceutical composition according to claim 1, said at least two combined herbs is (A) combinations of mulberry fruit and Asparagi radix; or (B) combinations of Asparagi radix and lycii fructus.
  4. The pharmaceutical composition according to claim 3, said at least two combined herbs is (A) combinations of mulberry fruit and Asparagi radix; or (B) combinations of Asparagi radix and lycii fructus the combination(s) with the weight mixed ratio of 0.01-100: 0.01-100 weight part (w/w) in case of (A) combinations of mulberry fruit and Asparagi radix; (B) combinations of Asparagi radix and lycii fructus.
  5. The pharmaceutical composition according to claim 1, said at least two combined herbs is (C) combinations of mulberry fruit, Asparagi radix and lycii fructus.
  6. The pharmaceutical composition according to claim 5, said at least two combined herbs is (C) combinations of mulberry fruit, Asparagi radix and lycii fructus with the weight mixed ratio of 0.01-100: 0.01-100: 0.01-100 weight part (w/w).
  7. The pharmaceutical composition according to claim 1, said extract is soluble in water, spirit, C1-C4 lower alcohol, or the mixtures thereof.
  8. The pharmaceutical composition according to claim 1, said Abnormal regulation of female hormone is selected from the group consisting of female climacteric syndrome, sexual desire disorder, hypo-estrogenemia, sex hormone hyper-secretion, and precocious puberty.
  9. The pharmaceutical composition according to claim 8, said female climacteric syndrome is selected from the group consisting of hidrorrhea, nausea, dizziness, tinnitus, palpitation, insomnia, restlessness, irascibility, soreness of loin and knees, benumbedness and itch of skin, hypomnesis, arthralgia, myalgia, colpoxerosis, headache, over-sensitiveness, anxiety disorder, depression, vertigo, facial flushing response, sleep disturbance, hypotrophy, memory deficiency, business ability degradation, stamina degradation, decline in exercise capacity, decrease in body hair, skin aging, osteopenia, and increase in visceral fat.
  10. A use of an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus for manufacture of medicament employed for treating or preventing an abnormal regulation of female hormone in human or mammal.
  11. A method for treating an abnormal regulation of female hormone in a mammal or animal suffering from said disease comprising administering an effective amount of an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus, together with a pharmaceutically acceptable carrier to said mammal or animal in need thereof.
  12. A health functional food comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus as an active ingredient for alleviating or preventing an abnormal regulation of female hormone.
  13. The health functional food according to claim 12, said health functional food is provided as powder, granule, tablet, capsule or beverage type.
  14. A health care food comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus for alleviating or preventing an abnormal regulation of female hormone, together with a sitologically acceptable additive.
  15. A food additive comprising an extract of at least two combined herbs consisting of mulberry fruit, Asparagi radix and lycii fructus as an active ingredient for alleviating or preventing an abnormal regulation of female hormone.
PCT/KR2017/004529 2016-05-18 2017-04-28 Composition comprising the combined extract for preventing or treating the abnormal regulation of female hormone and the use thereof WO2017200224A1 (en)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
KR20070112546A (en) * 2006-05-22 2007-11-27 동신대학교산학협력단 Food composition comprising extracts from lycium chinenses mill
JP2010538090A (en) * 2007-09-07 2010-12-09 バイオノボ・インコーポレーテッド Estrogenic extracts of Lilyaceae families and their use
KR101451755B1 (en) * 2013-08-05 2014-10-16 동우당제약(주) A composition for preventing bone metabolism-related diseases and increasing bone function comprising mix extracts of Mori Fructus and Aralia Continentalis
KR20160013227A (en) * 2016-01-14 2016-02-03 김득수 Production method of young antler tea

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Publication number Priority date Publication date Assignee Title
KR20070112546A (en) * 2006-05-22 2007-11-27 동신대학교산학협력단 Food composition comprising extracts from lycium chinenses mill
JP2010538090A (en) * 2007-09-07 2010-12-09 バイオノボ・インコーポレーテッド Estrogenic extracts of Lilyaceae families and their use
KR101451755B1 (en) * 2013-08-05 2014-10-16 동우당제약(주) A composition for preventing bone metabolism-related diseases and increasing bone function comprising mix extracts of Mori Fructus and Aralia Continentalis
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