Functional composition for improvement and prevention of estrogen deficiency in menopausal women

Abstract

The present invention relates to a food composition for improvement and prevention of estrogen deficiency, the composition containing a thistle extract or a thistle and thyme complex extract as an active ingredient. The present invention has advantages of alleviating estrogen deficiency in climacteric women and improving symptoms caused by estrogen deficiency, such as facial flushing, sweating, fatigue, anxiety, depression, memory impairment, insomnia, increased blood cholesterol, weight gain, and reduced bone density.

Claims

1. A composition which is effective for improving the blood lipid content in a human, improving hyperlipidemia in a human caused by the blood lipid content in a human, and improving estrogen deficiency in a human consisting essentially of dextrin, thistle extract, thyme extract and a solvent selected from the group consisting of acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane and petroleum ether.

Description

DESCRIPTION OF DRAWINGS

(1) FIGS. 1 to 8 are graphs illustrating results of measuring the concentration of blood estradiol in test animals.

(2) FIG. 9 is a graph illustrating weight gain in test animals.

(3) FIG. 10 is a graph illustrating a change in weight of test animals.

(4) FIG. 11 is a graph illustrating an increment in blood alkaline phosphatase of test animals.

(5) FIG. 12 is a graph illustrating an increment in blood osteocalcin of test animals.

(6) FIG. 13 is a graph illustrating the improvement ratios of climacteric symptoms of test women, measured by a KI assay.

(7) FIG. 14 is a graph illustrating MRS scores of test women, measured by an MRS assay.

BEST MODE

(8) Before the present invention is described, the meaning of the terms used in the present invention will be described.

(9) In the present specification, the term extract has a meaning commonly used as a crude extract in the art, but also includes a meaning of a fractionated product obtained by additionally fractionating the extract in a wide sense. That is, a thistle extract includes not only those obtained by using the above-described extraction solvent, but also those obtained by additionally applying a purification process. For example, a fraction obtained by allowing the extract to pass through an ultrafiltration membrane having a predetermined molecular weight cut-off value, and a fraction obtained through various purification methods such as separation by means of various chromatography methods (those manufactured for separation according to the size, charge, and hydrophobicity or hydrophilicity) additionally carried out are included in the thistle complex extract of the present invention.

(10) In the present specification, containing as an active ingredient means including an amount of a thistle extract sufficient to achieve the efficacy or activity. The present invention is a composition extracted from thistle which is a natural plant material, and has no side effects on the human body even though the composition is administered in excess, and accordingly, the upper limit of the amount of thistle extract included in the composition of the present invention may be selected within an appropriate range and carried out by a person skilled in the art.

(11) Hereinafter, the composition of the present invention will be described in detail.

(12) The thistle extract according to the present invention is extracted by using an organic solvent, and those which may be used as an extraction solvent are as follows.

(13) First, examples of those suitable as a polar solvent include (i) water, (ii) alcohol having 1 to 6 carbon atoms (preferably, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO), and (v) dimethyl sulfoxide (DMSO), and the like.

(14) Moreover, examples of those suitable as a non-polar solvent include acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, methylene chloride, petroleum ether, THF, and the like.

(15) It is preferred that the present invention uses (a) water, (b) an anhydrous or lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, and the like), (c) a mixed solvent of the lower alcohol and water, (d) acetone, (e) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butyelene glycol, (i) hexane, (j) diethyl ether, and the like as the extraction solvent, and it is more preferred that for facilitated extraction, the extraction is carried out by treating thistle with water, ethanol, or a mixture of water and ethanol.

(16) Further, the thistle extract used in the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and lyophilization or spray drying.

(17) Therefore, the present invention may prepare an extract by using the aforementioned extraction solvent, and a method for preparing a thistle extract will be described below.

(18) 1) Preparation of Thistle Extract

(19) {circle around (1)} Thistle is sorted and ground, and {circle around (2)} then an extraction is carried out repeatedly by using a solvent selected from water including purified water with a volume amount (v/w<) which is about 1 to 25 times, preferably 7 to 15 times the weight of the ground substance, a lower alcohol having 1 to 4 carbon atoms, such as methanol, ethanol, and butanol, or a mixed solvent thereof, more preferably water, ethanol, or a mixed solvent thereof, {circle around (3)} at an extraction temperature of 0 to 120 C., preferably 50 to 100 C., {circle around (4)} for about 1 hour to 10 days, preferably about 3 hours to 6 hours {circle around (5)} by means of an extraction method such as cold brew extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, or heated extraction, preferably hot water extraction or reflux cooling extraction, and {circle around (6)} about one time to ten times, preferably two times to eight times. Moreover, a thistle extract is obtained by {circle around (7)} filtering the extract by a filter cloth, {circle around (8)} concentrating the filtrate under vacuum, and then {circle around (9)}-{circle around (1)} lyophilizing the filtrate or {circle around (9)}-{circle around (2)} mixing the concentrate with dextrin according to the blending ratio and spray drying the resulting mixture.

(20) Further, the thistle extract may be prepared through a subcritical extraction method which extracts the extract by using a subcritical fluid, or a supercritical extraction method which extracts the extract by using a supercritical fluid.

(21) As a thistle extract used in a test to be described below, an extract prepared by using a hot water extraction and an organic solvent extraction (ethanol, methanol) is used, and each extraction condition is as follows.

(22) Thistle Extract 1 is prepared by carrying out a primary extraction at 121 C. for 5 hours using purified water with a volume amount which is 7 times the weight of the ground substance, carrying out a secondary extraction by adding purified water with a volume amount which is 5 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(23) Thistle Extract 2 is prepared by carrying out a primary extraction at 100 C. for 5 hours using purified water with a volume amount which is 7 times the weight of the ground substance, carrying out a secondary extraction by adding purified water with a volume amount which is 5 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(24) Thistle Extract 3 is prepared by carrying out a primary extraction at 70 C. for 5 hours using purified water with a volume amount which is 7 times the weight of the ground substance, carrying out a secondary extraction by adding purified water with a volume amount which is 5 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(25) Thistle Extract 4 is prepared by carrying out a primary extraction at 70 C. for 5 hours using 40% ethanol with a volume amount which is 7 times the weight of the ground substance, carrying out a secondary extraction by adding 40% ethanol with a volume amount which is 5 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(26) Thistle Extract 5 is prepared by carrying out a primary extraction at 50 C. for 5 hours using 90% ethanol with a volume amount which is 7 times the weight of the ground substance, carrying out a secondary extraction by adding 90% ethanol with a volume amount which is 5 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(27) Thistle Extract 6 is prepared by carrying out a primary extraction at 70 C. for 5 hours using 40% methanol with a volume amount which is 7 times the weight of the ground substance, carrying out a secondary extraction by adding 40% methanol with a volume amount which is 5 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(28) Thistle Extract 7 is prepared by carrying out a primary extraction at 50 C. for 5 hours using 90% methanol with a volume amount which is 7 times the weight of the ground substance, carrying out a secondary extraction by adding 90% methanol with a volume amount which is 5 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(29) 2) Preparation of Thyme Extract

(30) A thyme extract is also prepared by the same preparation method as the thistle extract.

(31) As a thyme extract used in a test to be described below, an extract prepared by using a hot water extraction and an organic solvent extraction (ethanol, methanol) is used, and each extraction condition is as follows.

(32) Thyme Extract 1 is prepared by carrying out a primary extraction at 121 C. for 6 hours using purified water with a volume amount which is 6 times the weight of the ground substance, carrying out a secondary extraction by adding purified water with a volume amount which is 6 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(33) Thyme Extract 2 is prepared by carrying out a primary extraction at 100 C. for 6 hours using purified water with a volume amount which is 6 times the weight of the ground substance, carrying out a secondary extraction by adding purified water with a volume amount which is 6 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(34) Thyme Extract 3 is prepared by carrying out a primary extraction at 70 C. for 6 hours using purified water with a volume amount which is 6 times the weight of the ground substance, carrying out a secondary extraction by adding purified water with a volume amount which is 6 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(35) Thistle Extract 4 is prepared by carrying out a primary extraction at 70 C. for 6 hours using 40% ethanol with a volume amount which is 6 times the weight of the ground substance, carrying out a secondary extraction by adding 40% ethanol with a volume amount which is 6 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(36) Thistle Extract 5 is prepared by carrying out a primary extraction at 50 C. for 6 hours using 90% ethanol with a volume amount which is 6 times the weight of the ground substance, carrying out a secondary extraction by adding 90% ethanol with a volume amount which is 6 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(37) Thistle Extract 6 is prepared by carrying out a primary extraction at 70 C. for 6 hours using 40% methanol with a volume amount which is 6 times the weight of the ground substance, carrying out a secondary extraction by adding 40% methanol with a volume amount which is 6 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(38) Thistle Extract 7 is prepared by carrying out a primary extraction at 50 C. for 6 hours using 90% methanol with a volume amount which is 6 times the weight of the ground substance, carrying out a secondary extraction by adding 90% methanol with a volume amount which is 6 times the weight of the ground substance thereto, concentrating the resulting extract under vacuum, and then mixing the extract and dextrin at 1:1, and spray drying the resulting mixture.

(39) 3) Preparation of Thistle and Thyme Complex Extract

(40) A thistle extract and a thyme extract which correspond to each condition are mixed at a ratio of 6:4. For example, Thistle and Thyme Complex Extract 1 is prepared by mixing Thistle Extract 1 and Thyme Extract 1, Thistle and Thyme Complex Extract 2 is prepared by mixing Thistle Extract 2 and Thyme Extract 2, and in this manner, Thistle and Thyme Complex Extracts 3 to 7 are prepared. And extracts, in which Thistle Extract 1 and Thyme Extract 1 are mixed at a ratio of 2:8, 4:6, and 8:2, are additionally prepared.

(41) The thistle extract according to the present invention is prepared as a food composition. That is, the thistle extract according to the present invention may be prepared as a food composition for prevention and improvement of estrogen deficiency, the composition containing a thistle extract as an active ingredient.

(42) When the thistle extract according to the present invention is prepared as a food composition, the thistle extract contains an ingredient typically added when a food is prepared, and for example, protein, carbohydrate, fat, a nutrient, and a seasoning agent correspond to the ingredient. Furthermore, when the thistle extract according to the present invention is prepared as a drink preparation, the thistle extract may contain a flavoring agent, natural carbohydrate, and the like as an additional ingredient in addition to the thistle extract as an active ingredient. For example, the natural carbohydrate includes monosaccharides (glucose, fructose, and the like), disaccharides (maltose, sucrose, and the like), oligosaccharides, polysaccharides (dextrin, cyclodextrin, and the like), and sugar alcohols (xylitol, sorbitol, erythritol, and the like). As the flavoring agent, it is possible to use natural flavoring agents (for example, thaumatin, stevia extract, and the like) and synthetic flavoring agents (for example, saccharin, aspartame, and the like).

(43) Meanwhile, even when a thistle and thyme complex extract is prepared as a food composition, the preparation method thereof is the same as those described above. The effects of the thistle extract according to the present invention will be described in detail below by way of Test Examples.

TEST EXAMPLE 1

Animal Test

(44) <1-1> Test Subject

(45) After 7-week-old female rats having a body weight of 200 to 250 g were acclimated in a laboratory for 1 week, a menopausal model was induced by performing an ovariectomy on some of the female rats, and a non-menopausal model was induced by performing a sham-operation that cuts open the abdomen and then sutures the abdomen on the other female rats. Furthermore, after each model was fed with a solid feed for 1 week and recovered from the wound, five female rats were assigned to a control in which the ovary was not excised (hereinafter, referred to as a normal control) and five female rats were assigned to a control in which the ovary was excised (hereinafter, referred to as a negative control), 30 female rats were assigned to a first experimental group (hereinafter, referred to as a first experimental group) in which the ovary was excised, and 21 female rats were assigned to a second experimental group (hereinafter, referred to as a second experimental group) in which the ovary was excised.

(46) The first experimental group was divided into 7 groups of 3 female rats per group, and Thistle Extracts 1 to 7 dissolved at a concentration of 50 mg/kg in physiological saline were orally administered to each group. After the second experimental group was divided into 10 groups of 3 female rats per group, Thistle and Thyme Complex Extracts 1 to 7 dissolved at a concentration of 50 mg/kg in physiological saline were orally administered to 7 groups, thistle and thyme complex extracts in which Thistle Extract 1 and Thyme Extract 1 were mixed at a ratio of 2:8, 4:6, and 8:2 were orally administered to the other 3 groups, and the same amount of physiological saline was orally administered to the normal control and the negative control. For reference, the administration period was 8 weeks, the breeding conditions were a temperature of 242 C. and a humidity of 50 to 55%, and water and diet were provided without restriction.

(47) <1-2> Measurement of Estrogen in Body

(48) A reduction of estrogen is a representative cause of the occurrence of women climacteric symptoms. Accordingly, in the present invention, the concentration of blood estradiol-17 after the administration of a drug was measured in order to confirm effects of the thistle extract on estrogen. For reference, estradiol is a representative estrogen, and is used as an estrogen measurement subject due to the very high activity compared to estron or estriol.

(49) Specifically, blood was collected from each group in which the rats in <1-1> Test Subject were completely administered, was centrifuged (1,000 g, 15 min, 4 C.), and stored at 80 C. in a deep freezer prior to an analysis. Furthermore, blood estradiol was measured by using Rat estradiol (E2) ELISAKit (Cayman, Ann Arbor, Mich., USA). The significance was verified at a level of p<0.05 by a statistical treatment of the test result through a statistical program SPSS (version 12.0), and the result of measuring blood estradiol for each extract is illustrated in FIGS. 1 to 8. That is, the results according to Thistle Extract 1 and Thistle and Thyme Complex Extract 1 are illustrated in FIG. 1, the results according to Thistle Extract 2 and Thistle and Thyme Complex Extract 2 are illustrated in FIG. 2, the results according to Thistle Extract 3 and Thistle and Thyme Complex Extract 3 are illustrated in FIG. 3, the results according to Thistle Extract 4 and Thistle and Thyme Complex Extract 4 are illustrated in FIG. 4, the results according to Thistle Extract 5 and Thistle and Thyme Complex Extract 5 are illustrated in FIG. 5, the results according to Thistle Extract 6 and Thistle and Thyme Complex Extract 6 are illustrated in FIG. 6, the results according to Thistle Extract 7 and Thistle and Thyme Complex Extract 7 are illustrated in FIG. 7, and the results according to the mixture ratio of Thistle Extract 1 and Thyme Extract 1 are illustrated in FIG. 8.

(50) For reference, the results of the normal control (Sham) and the negative control (OVX-Con) are each included in FIGS. 1 to 7 for a smooth comparison.

(51) Judging from the fact that the concentration of blood estradiol in the negative control (OVX-Con) is significantly lower than that in the normal control (Sham) as illustrated in FIG. 1, it can be seen that the activity of estrogen is reduced due to the menopause. In addition, judging from the fact that the concentration of blood estradiol in the first experimental group (OVX-Cirs) was shown to be higher than that in the negative control (OVX-Con), it can be seen that the thistle extract is effective for alleviating a sharp drop in concentration of estrogen after menopause. Furthermore, judging from the fact that the concentration of estradiol in the second experimental group (OVX-CT) was measured to be higher than that in the first experimental group (OVX-Cirs), it can be seen that the thistle and thyme complex extract is more effective for an increase in concentration of blood estrogen.

(52) Judging from the fact that FIGS. 2 to 7 also had a slight difference from FIG. 1, and exhibited the results in a similar pattern as FIG. 1, it can be seen that the efficacies of the thistle extracts and the thistle and thyme complex extracts are not significantly affected by the extraction solvent or the temperature.

(53) Furthermore, FIG. 8 is a result of measuring the concentration of estradiol according to the mixture ratio of Thistle Extract 1 and Thyme Extract 1, and it can be seen that when a mixture, in which Thistle Extract 1 and Thyme Extract 1 were mixed at a ratio of 8:2, was administered to the test subjects, the highest concentration of estradiol was measured.

(54) <1-3> Measurement of Body Weight

(55) Weight gain is one of the representative symptoms in women during the climacteric period. Accordingly, in the present invention, a change in body weight according to the administration period was measured in order to confirm effects of the thistle extract on the body weight.

(56) Specifically, the body weights of each group {in the case of the first experimental group, a group to which Thistle Extract 1 was administered and in the case of the second experimental group, a group to which Thistle and Thyme Complex Extract 1 (at a ratio of 6:4) was administered} were measured for a total of 9 weeks from the period when the drug was administered for 8 weeks prior to the administration of the drug until the period of 1 week after the administration of the drug. The significance was verified at a level of p<0.05 compared to the normal control by a statistical treatment of the test result through a statistical program SPSS (version 12.0), and the result thereof is illustrated in FIGS. 9 and 10.

(57) Judging from the fact that the body weight in the negative control (OVX-Con) is significantly increased compared to that in the normal control (Sham) as illustrated in FIG. 9, it can be seen that the menopause affects an increase in body weight to some degree. Furthermore, it can be seen that an increase in body weight in the first experimental group (OVX-Cirs) was relatively less than that in the negative control (OVX-Con). Further, as illustrated in FIG. 10, it can be seen that after 9 weeks, the first experimental group (OVX-Cirs) made no big difference in the increase in body weight as compared to the normal control (Sham). Accordingly, it can be seen that the thistle extract is effective for alleviating an increase in body weight after the menopause.

(58) Meanwhile, judging from the fact that the body weight in the second experimental group (OVX-CT) was increased at a lower ratio than that in the first experimental group (OVX-Cirs), it can be seen that the thistle and thyme complex extract more effectively acts in an increment in body weight in the climacteric period.

(59) <1-4> Measurement of Cholesterol in Body

(60) Healthy young women have lower blood total cholesterol, LDL-cholesterol, and triglyceride levels and a higher HDL-cholesterol level than those of men at the same age. However, when the menopausal period comes, it is known that the triglyceride and HDL-cholesterol levels are not usually changed, but the total cholesterol and LDL-cholesterol levels are increased, and these changes increase the incidence rate of vascular diseases after the women's menopausal period. Accordingly, in the present invention, the concentration of blood cholesterol after administration of a drug was measured in order to confirm effects of the thistle extract and the thistle and thyme complex extract on blood cholesterol.

(61) Specifically, blood was collected from each group in which the administration was completed in <1-1> Test Subject { in the case of the first experimental group, a group to which Thistle Extract 1 was administered and in the case of the second experimental group, a group to which Thistle and Thyme Complex Extract 1 (at a ratio of 6:4) was administered}. Furthermore, sera were separated, and the concentrations of triglyceride, total cholesterol, and HDL-cholesterol in the serum were measured at a wavelength of 400 to 600 nm by Dry Chemistry Analyzer 3500i (Fuji, Japan) using a slide for automatic measurement (Fuji Film, Japan). In the case of HDL-cholesterol, the HDL-cholesterol level in the supernatant was measured by precipitating chylomicron, low-density lipoprotein and very low density lipoprotein with a precipitating agent. The LDL-cholesterol level was calculated by the following Friedewald equation.
LDL Cholesterol Level={Total Cholesterol Level(HDL-Cholesterol LevelTriglyceride Level/5)}

(62) The study results were expressed as meanstandard deviation, the significance was verified at a level of p<0.05 by a statistical treatment of the study result through a statistical program SPSS (version 12.0), and the results are shown in Table 1.

(63) TABLE-US-00001 TABLE 1 Total cholesterol Triglyceride HDL-cholesterol LDL-cholesterol Classification level level level level Normal control 84.86 5.67* 63.92 4.59* 50.80 5.64* 46.84 5.75* Negative control 117.36 8.25 89.80 8.93 40.60 4.18 94.72 13.28 First experimental 94.80 4.44* 71.24 4.15* 48.40 4.18* 60.65 6.75* group Second experimental 89.92 11.80* 67.23 3.61* 49.60 2.06* 53.77 11.39* group

(64) (Unit: mg/dl)

(65) As a result of the experiments, as in Table 1, blood total cholesterol, triglyceride, and LDL-cholesterol levels were significantly increased and the HDL-cholesterol level was decreased in the negative control as compared to those in the normal control. Conversely, in the first experimental group and the second experimental group, the blood total cholesterol, triglyceride, and LDL-cholesterol levels were significantly lower and the HDL-cholesterol level was higher than those in the negative control (OVX-Con).

(66) Accordingly, through the result of the present test example, it can be seen that the thistle extract and the thistle and thyme complex extract, which are the compositions of the present invention, are effective for improving the blood lipid content, hyperlipidemia caused by the blood lipid content, and the like in a menopausal situation.

(67) <1-5> Measurement of Bone Index

(68) The reduced bone density is one of the symptoms occurring in climacteric women, and is responsible for causing diseases such as osteoporosis. Accordingly, in the present invention, the concentration of blood alkaline phosphatase (hereinafter, referred to as ALP) and the concentration of osteocalcin (hereinafter, referred to as OC) after administration of a drug were measured in order to confirm effects of the thistle extract and the thistle and thyme complex extract on bone density.

(69) Specifically, blood was collected from each group in which the administration was completed in <1-1> Test Subject {in the case of the first experimental group, a group to which Thistle Extract 1 was administered and in the case of the second experimental group, a group to which Thistle and Thyme Complex Extract 1 (at a ratio of 6:4) was administered}. The concentration of blood ALP was analyzed by using an automatic analyzer (ADVIA 1650, Bayer, Tokyo, Japan), and the concentration of blood OC was analyzed by using ELISA kit (Metra OC, Quidel Corporation, San Diego, Calif., USA).

(70) The significance was verified at a level of p<0.05 compared to the normal control by a statistical treatment of the test result through a statistical program SPSS (version 12.0), and the result thereof is illustrated in FIGS. 11 and 12. As a result of the experiments, as illustrated in FIGS. 11 and 12, the concentrations of blood ALP and OC in the negative control (OVXCon) were increased as compared to those in the normal control (Sham). The result described above can be considered as a procedure in which the bone reformation procedure is increased by reduced bone density caused by ovariectomy, and bone replacement indices are enhanced during the bone reformation procedure. Accordingly, during the procedure, the concentration of blood ALP, which is a representative bone replacement index, is increased, and the concentration of OC, which is a bone reabsorption and bone formation index, is increased (Hertrampf T., Schleipen B., Offermanns C., Velders M., Laudenbach U., Diel P., Comparison of The Bone Protective Effects of Anisoflavone-Rich Diet With Dietary and Subcutaneous Administrations of Genistein in Ovariectomized Rats. Toxicol. Lett. 2009, 184, 198-203.). In contrast, the concentrations of blood ALP and OC in the first experimental group (OVX-Cirs) were reduced as compared to those in the negative control (OVX-Con). Further, in the second experimental group (OVX-CT), the concentrations of blood ALP and OC were reduced than those in the first experimental group, so that a numerical value closest to the normal control (Sham) was produced.

(71) Accordingly, judging from the fact that the concentrations of blood ALP and OC were relatively less increased, it can be seen that the thistle extract and the thistle and thyme complex extract are effective for alleviating a decrease in bone density caused by the menopause.

TEST EXAMPLE 2

Human Body Test

(72) <2-1> Test Subject

(73) Among 60 middle-aged women at the age of 45 years or older, who agreed to the present assay, 20 women were arbitrarily classified into a control (Placebo) in which a placebo was taken, a first experimental group (Cirs) in which a thistle extract was taken, and a second experimental group in which a thistle and thyme complex extract (CT) was taken per group, and were allowed to take 400 mg of a placebo or an experimental drug containing a thistle extract or a thistle and thyme complex extract daily for 12 weeks.

(74) <2-2> Korean Version KI (Kupperman's Index)

(75) This is an assay published by setting 11 representative symptoms in the climacteric period as items as in Table 2 based on clinical treatment experiences of climacteric disorders by Kupperman in 1953 and grasping the degree and characteristics of climacteric disorders, and is a self-questionnaire assay method which is most commonly used as a measure of evaluating the symptom among studies related to women climacteric symptoms up until now. The degree and characteristics of each symptom in the climacteric period are grasped, the products obtained by multiplying a score for each item were added up, and 20 points or less, 20 to 40 points, 40 to 60 points, and 60 points or more are determined as a mild symptom, an intermediate symptom, and a serious state, respectively. A result in which the improvement rate was calculated after the intake is illustrated in FIG. 13, and the significance was verified at a level of p<0.05 by a statistical treatment of the result through a statistical program SPSS (version 12.0).

(76) TABLE-US-00002 TABLE 2 Not experienced Sometimes Frequently Always Symptom at all experienced experienced experienced 1. Facial flushing 2. Sweating 3. Insomnia 4. Nervousness 5. Depression 6. Vertigo 7. Fatigue 8. Arthralgia, Myalgia 9. Headache 10. Palpitation 11. Colpoxerosis, Reduced secretions

(77) As illustrated in FIG. 13, the control (Placebo) in which the placebo was taken exhibited a symptom improvement rate after the intake for 3 months by 4.2%, whereas the first experimental group (Cirsium) reduced women climacteric symptoms by 30%, which were evaluated by KI, and thus significantly improved the climacteric symptoms as compared to the control (Placebo). Further, in the second experimental group (C+Thyme), it can be seen that the women climacteric symptoms were reduced by 45% or more.

(78) Accordingly, through the present result, it was confirmed that the thistle extract and the thistle and thyme complex extract could effectively improve complicated women climacteric symptoms, such as facial flushing, sweating, insomnia, nervousness, depression, vertigo, fatigue, arthralgia and myalgia, palpitation, colpoxerosis, and reduced secretions.

(79) <2-3> Korean Version Menopause Rating Scale (MRS)

(80) The MRS assay is an assay method produced in 1996 by newly complementing a MENopause-specific Quality of Life Questionnaire (MENQOL) assay produced by complementing the Kuppermanindex by Hildich et al., in 1992. While the MRS has been cited globally in numerous studies, the reliability and validity thereof have been recognized. The MRS is composed of 11 items in total, such as physical symptoms, mental symptoms, and urogenital symptoms, and has advantages in that the number of items is small, and the procedure is simple and convenient. The MRS as described above evaluates symptoms from the criteria as in the following Table 3.

(81) The total score is calculated, and from the total score, 0 to 4 points are in a good climacteric state, meaning a happy climacteric period, and 5 to 8 points are in a mild climacteric state, showing that care is required. 9 to 15 points are in a severe climacteric state, and diagnosis and treatment by experts are required because a patient with these points has a climacteric disorder. 16 points or higher are in a serious climacteric state, and are a numerical value showing that long-term plans and treatments are considerably required.

(82) The result is illustrated in FIG. 14, and the significance was verified at a level of p<0.05 by statistically analyzing points before and after the intake by each experimental group through a statistical program SPSS (version 12.0).

(83) TABLE-US-00003 TABLE 3 State None Slight Fair Severe Very severe Symptom (0) (1) (2) (3) (4) Facial flushing and sweating (an experience in which the face feels flushed or feverish and an experience in which sweat suddenly appears) Heart discomfort (suddenly palpitates and the pulse becomes fast or skips) Sleep problem (difficult to sleep or readily awake from sleep and difficult to continue sleep) Depressed mood (feeling despondent, sadness, readily shed tears, and no motivation) Hypersensitivity (feeling in which nerves become sharp, aggressive feeling, and tension) Anxiety (inner irritation and feeling of panic) Physical and mental fatigue (fatigue, lethargy, memory loss, reduced concentration, and forgetfulness) Sexual problems (sexual desire changes, changes in sexual behavior, and changes in sexual gratification) Urination problems (difficult to urinate, frequently urinate, and incontinence) Vaginal dryness (dryness or burning of vaginal areas, discomfort during sexual intercourse) Joint and muscle discomfort (joint pain, symptoms similar to rheumatism) Total score

(84) As a result of the test, the control (Placebo) did not exhibit a statistically significant difference in the MRS score after the intake for 3 months, as illustrated in FIG. 14. In contrast, in the first experimental group (Cirsium), it was confirmed that after the intake for 3 months, the MRS score was decreased by approximately 30%, and thus, the women climacteric symptoms were significantly reduced. Further, judging from the fact that in the second experimental group (C+Thyme), the results after the intake for 3 months exhibited a greater difference than those in the first experimental group, and the symptoms were reduced, it can be seen that it is possible to more effectively improve climacteric symptoms.

INDUSTRIAL APPLICABILITY

(85) The thistle extract and the thistle and thyme complex extract according to the present invention may alleviate estrogen deficiency which is a symptom in which estrogen in climacteric women is reduced, and thus may be provided as an associated functional food or medicine, and the like.