Female menopause alleviation use of composition containing composite extract of red clover and pomegranate as active ingredient
10709750 ยท 2020-07-14
Assignee
Inventors
Cpc classification
A23L33/105
HUMAN NECESSITIES
A61K36/48
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
A61K31/352
HUMAN NECESSITIES
A61K2236/00
HUMAN NECESSITIES
A61K31/352
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K36/48
HUMAN NECESSITIES
International classification
A61K47/46
HUMAN NECESSITIES
A61K31/352
HUMAN NECESSITIES
A23L33/105
HUMAN NECESSITIES
A61K36/48
HUMAN NECESSITIES
Abstract
The present invention relates to: a natural composition containing a composite extract of pomegranates and red clover, exhibiting synergistic effects with respect to a menopausal symptom alleviation effect, an estrogen-like effect, an anti-obesity effect, a hyperlipidemia inhibitory effect, a fatty liver protective effect, an osteoporosis inhibitory effect and the like without any side effects; and various uses thereof, in particular, pharmaceutical, bromatological and cosmetic uses.
Claims
1. A method for reducing a menopausal disorder, comprising: administering a composition comprising a composite of pomegranate extract and red clover extract as an active ingredient to a subject in need thereof; wherein the weight ratio of the pomegranate extract to the red clover extract is 1:1-4 wherein the red clover extract comprises 50-150 mg/g of isoflavone in relation to total red clover extract, and wherein the composite of pomegranate extract and red clover extract comprises 30-100 mg/g of isoflavone and 0.15-0.4 mg/g of ellagic acid.
2. The method for reducing a menopausal disorder according to claim 1, wherein the menopausal disorder shows at least one menopausal symptom selected from the group consisting of facial flushing, sweating, insomnia, nervousness, depression, dizziness, poor concentration, arthralgia, headache, tachycardia, vaginal dryness, fatigue, excitement, sleeplessness, memory loss, anxiety and atherosclerosis.
3. The method for reducing a menopausal disorder according to claim 1, wherein the menopausal disorder reduction shows at least one effect selected from the group consisting of menopausal symptom relief, anti-obesity, hyperlipidemia inhibition, fatty liver inhibition, osteoporosis inhibition, liver protection and uterus protection.
4. The method according to claim 1, wherein the weight ratio of the pomegranate extract to the red clover extract is 1:2.
5. The method according to claim 1, wherein the composite of pomegranate extract and red clover extract is present in 50-150 mg per 100 ml of the composition.
6. The method according to claim 1, wherein the pomegranate extract comprises 0.5-1 mg/g of ellagic acid in relation to total pomegranate extract.
7. The method according to claim 1, wherein the red clover extract is obtained by extraction using water, lower alcohol with 1-4 carbons, or mixtures thereof as a solvent.
8. The method according to claim 1, wherein the red clover extract is a red clover extract powder.
9. The method according to claim 1, wherein the pomegranate extract is a pomegranate extract powder.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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(7) A to G are arranged, starting from upper left; A shows sham control, B shows OVX control, C shows red clover extract 40 mg/kg administered group, D shows pomegranate extract 20 mg/kg administered group, E shows red clover:pomegranate extract 2:1 mixture (g/g) 120 mg/kg administered group, F shows red clover:pomegranate extract 2:1 mixture (g/g) 60 mg/kg administered group.
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BEST MODE FOR CARRYING OUT THE INVENTION
(35) Hereinafter, the present disclosure will be explained in detail with reference to preparation examples and working examples to help the understanding of the present disclosure. However, the preparation examples and working examples according to the present disclosure may be modified in a variety of different forms and should not be construed as limiting the scope of the present disclosure. The preparation examples and working examples of the present disclosure are provided to explain the present disclosure more fully to those having ordinary knowledge in the art to which the present disclosure pertains.
0. Method for Preparing a Mixture Including a Red Clover Extract and a Pomegranate Extract
(36) (1) Preparation of a Red Clover Extract
(37) Red clover was cleaned first. Aerial parts were only separated from red clover and 80-120 kg red clover aerial parts in total weight were prepared. 70% ethanol was added in weight about 3 times larger than the weight of red clover, and extraction was performed at 75 C.-80 C. and 0.05 MPa for 2.5 hrs 23 times, yielding 300 kg red clover extract. 100 mesh filtration followed separation through a solid/liquid separation process. The filtered red clover extract was heated at 70 C. and 0.06 Mpa for 6 hours to bring it to a concentrate, yielding 25 kg red clover concentrate extract. The concentrate was spray dried with the addition of 0-1 kg maltodextrin and passed through 8 mesh, yielding 10 kg red clover extract powder containing 10% isoflavone.
(38) (2) Preparation of a Pomegranate Extract
(39) After removing stains from 1000 kg pomegranates, spoiled fruits were sorted out, followed by cleaning. The pomegranate fruits left after sorting were cut to remove pericarps and pressed to deseed, yielding 450 kg pomegranate pulp. After filtration, sterilization at 100-105 C. for 60 seconds, and cooling at 48-55 C. was performed. Amylolysis was performed at 48-55 C. for 30 minutes with the addition of 70100 ml pectinase per 1000 L pomegranate juice. Subsequently, for good eating viscosity with maintaining turbidity and color, 900 g bentonite per 10000 L pomegranate juice was fed, and stirred at 48-55 C. for 10 minutes. Subsequently, 1.5 mm and 1 mm vacuum filtration and concentration by heating (at 80 C. under 475 mbar to 12 brix, at 87 C. under 626 mbar to 17 brix, at 95 C. under 847 mbar to 31 Brix, at 70 C. under 312 mbar to 43 Brix and at 49 C. under 118 mbar to 65 Brix in a sequential order) was performed, followed by 0.15 mm filtration, yielding a liquid pomegranate concentrate containing 1.83.0 mg/g ellagic acid. Subsequently, the liquid pomegranate concentrate was mixed with dextrin and gone through a spray drying process to produce pomegranate concentrate powder containing 0.51.0 mg/g ellagic acid.
(40) (3) Preparation of a Composite Extract of Red Clover and Pomegranate
(41) 40 kg the prepared red clover extract powder (containing 10% isoflavone) and 20 kg the prepared pomegranate extract powder (containing 0.8 mg/g ellagic acid) were mixed and stirred, yielding 60 kg a final composite extract of red clover and pomegranate (containing 66.7 mg/g isoflavone and 0.26 mg/g ellagic acid).
1. Effect of a Combination of a Red Clover Extract and a Pomegranate Extract
(42) An experiment was designed with expectation of a remarkable synergistic effect of diversity of isoflavonoid content at a particular mixture ratio of a red clover extract and a pomegranate extract.
(43) In the study, with the addition of a pomegranate extract, an effect of magnified biological activity of a red clover extract on menopausal disorders was evaluated using ovariectomy (OVX) rat model. That is, from 28 days after OVX, red clover extract:pomegranate extract 2:1 (g/g) mixture 120 mg/kg and 60 mg/kg were dissolved in or diluted with sterile distilled water and orally administered at a dose of 5 ml/kg once daily for 84 days (12 weeks; 3 months), and evaluation was conducted based on 5 pharmacological effects including an estrogen-like effect, an anti-obesity effect, a hyperlipidemia inhibitory effect, a protective effect against fatty liver and an osteoporosis inhibitory effect.
(44) To evaluate the estrogen-like effect and the anti-obesity effect, changes in body weight and body weight gain, water and food consumption, urine volume and fecal excretion, serum estradiol level, and weight of abdominal fat pad and uterus, and changes in abdominal fat pad thickness and mean adipocyte diameter, thickness of uterus total, epithelium and mucosa, and percentage of uterine glands in the mucosa were each evaluated. Furthermore, the liver protective effect against fatty liver was evaluated by measuring changes in liver weight, serum Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) levels, mean hepatocyte diameter and region with hepatic degeneration showing fatty change, and the hyperlipidemia reduction effect was evaluated using changes in serum total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride (TG) levels, and to evaluate the osteoporosis reduction effect, namely, the bone protective effect, histological changes in wet, dry and ash weights of femur, tibia and lumbar vertebrae, bone mineral density (BMD) (
Example 1-1. Experimental Animal and Experimental Method
(45) (1) Experimental Animal
(46) Female virgin Sprague-Dawley Rats (6 weeks old, OrientBio, Seungnam, Korea) were used, and in the experiment, all experimental animals were handled according to the guidelines for animal ethics approved by the Animal Care and Ethics Committee of Daegu Haany University, and the experiment was performed with prior approval [Approval No. DHU2013-042].
(47) (2) Experimental Method
(48) The experiment was performed after dividing the experimental animals into groups (total 7 groups; 8 mice per group) (Table 1,
(49) That is, the experiment was performed after dividing into sham control, OVX control, red clover dry extract single formula 40 mg/kg administered group after OVX surgery, pomegranate extract single formula 20 mg/kg administered group after OVX surgery, red clover:pomegranate extract 2:1 (g/g) mixed formula 120 mg/kg administered group after OVX surgery, and red clover:pomegranate extract 2:1 (g/g) mixed formula 60 mg/kg administered group after OVX surgery.
(50) Optimal amounts of red clover extract, pomegranate extract or red clover:pomegranate extract 2:1 (g/g) mixture were suspended or dissolved in sterile distilled water, and orally administered at a dose of 5 ml/kg once daily for 84 days from 28 days after OVX. That is, red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg were each orally administered for 84 days, and in sham control and OVX control, only sterile distilled water as a vehicle was orally administered at the same dose for the same duration, instead of the test material (Table 1).
(51) TABLE-US-00001 TABLE 1 Group Operation Dose (mg/kg/day) Animal No. Anti-climacterium Effect of RC:PCP 2:1 Mixtures (g/g) Control Sham operate Vehicle, distilled water R01~R08 5 ml/kg Control OVX operate Vehicle, distilled water R09~R16 5 ml/kg Reference OVX operate RC 40 mg/kg R17~R24 Reference OVX operate PCP 20 mg/kg R25~R32 Active OVX operate RC:PCP 2:1 mixtures (g/g) R33~R40 120 mg/kg Active OVX operate RC:PCP 2:1 mixtures (g/g) R41~R48 60 mg/kg OVX = Bilateral ovariectomy PCP = Pomegranate Concentrate Powder RC = Red clover dry extracts
(52) For induction of menopausal disorders by OVX, an experiment was performed as follows.
(53) That is, all experimental animals except sham control were randomly selected and underwent bilateral ovariectomy to induce osteoporosis. On the other hand, in sham operated group, both ovaries were detected by the same method but incisions were not made, and abdominal cavities were closed again. At 28 days after surgery, based on body weights (OVX rats: 323.814.77 g, 304-354 g; sham operated rats: 253.259.08 g, 240-265 g), 8 mice per group were selected from all the experimental animals including shame operated group and used for experiments.
(54) The effect on menopausal disorders was evaluated based on 5 pharmacological effects including an estrogen-like effect, an anti-obesity effect, a hyperlipidemia inhibitory effect, a protective effect against fatty live and an osteoporosis inhibitory effect.
(55) More specifically, the anti-obesity and estrogen-like effects were tested as follows. The body weights on the date of OVX surgery, 1 day before administration (27 days after OVX), the date of start of administration, and at least once a week from 1 day after start of administration to the date of final sacrifice were each recorded, and change amounts of body weight, i.e., body weight gains for the recovery period after OVX (menopausal disorder induction period) and 12 weeks after start of administration were measured. The water and food consumption, urine volume and fecal excretion (28, 49 and 83 days after start of administration), serum estradiol level, weight of abdominal fat pad and uterus, and histopathological changes in uterus and abdominal fat pad (changes in abdominal fat pad thickness and mean adipocyte diameter, thickness of uterus total, epithelium and mucosa, and percentage of uterine glands in the mucosa) were observed.
(56) Furthermore, to ascertain the protective effect against fatty liver, an experiment was performed as follows. Liver weight, serum AST and ALT levels, and histological changes of liver (changes in mean hepatocyte diameter and region with hepatic degeneration showing fatty change) were observed.
(57) The hyperlipidemia inhibitory effect was ascertained through changes in serum TC, LDL, HDL and TG levels.
(58) The osteoporosis reduction effect was ascertained through histological changes in wet, dry and ash weights of femur, tibia and lumbar vertebrae, BMD (
Example 1-2. Change in Body Weight and Body Weight Gain
(59) At 28 days after OVX surgery, as compared to sham control, only experimental animals showing remarkable increases in body weight were selected and used (OVX rats: 323.8114.77 g, 304-354 g; sham operated rats: 253.259.08 g, 240-265 g), and significant (p<0.01) increases in body weight and body weight gain for a 4 week-induction period after OVX were found in all OVX operated groups as compared to sham control, but red clover:pomegranate extract 2:1 (g/g) mixture 120 mg/kg administered group started to show significant (p<0.01 or p<0.05) decreases in body weight 49 days after start of administration as compared to OVX control, and red clover:pomegranate extract 2:1 (g/g) mixture 60 mg/kg administered group started to show significant (p<0.01 or p<0.05) decreases in body weight 56, 63 and 70 days after start of administration respectively as compared to OVX control. Furthermore, all test substance administered groups also showed significant (p<0.01) decreases in body weight gain for the duration of administration of 84 days as compared to OVX control. Particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 mg/kg administered group showed significant (p<0.01 or p<0.05) decreases in body weight 77 days after start of administration as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered group, and red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered group also showed significant (p<0.01) decreases in body weight gain for the duration of administration of 84 days as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered group (Table 2).
(60) TABLE-US-00002 TABLE 2 Body weights (g) Body weight gains (g) At OVX At 27 days after At first treatment At sacrifice OVX recovery Treatment Groups [A]* OVX [B] [C]* [D]* [B A] [D C] Controls Sham 157.88 4.73 253.25 9.08 234.88 8.41 314.50 23.63 95.83 9.04 79.63 20.98 OVX 159.13 8.01 323.88 17.13.sup.a 298.75 13.33.sup.a 455.25 20.95.sup.a 164.75 15.68.sup.a 156.50 16.27.sup.e RC 40 mg/kg 158.38 7.80 320.75 8.88.sup.a 296.00 11.31.sup.a 415.50 13.56.sup.ab 162.38 7.07.sup.a 119.50 6.50.sup.ef PCP 20 mg/kg 156.38 4.41 324.13 15.99.sup.a 298.38 13.26.sup.a 418.88 15.12.sup.ab 167.75 14.61.sup.a 120.50 8.47.sup.ef RC:PCP 2:1 mixture (g/g) 120 mg/kg 157.88 6.83 323.50 15.69.sup.a 299.38 14.68.sup.a 395.00 9.07.sup.abcd 165.63 12.33.sup.a 95.63 9.27.sup.efgh 60 mg/kg 157.25 7.30 327.63 17.94.sup.a 301.00 16.78.sup.a 407.63 18.05.sup.ab 170.38 14.32.sup.a 106.63 8.75.sup.efgh *All animals were fasted overnight. .sup.ap < 0.01: comparison with sham control according to LSD test .sup.bp < 0.01: comparison with OVX control according to LSD test .sup.cp < 0.05: comparison with red clover extract 40 mg/kg administered group according to LSD test .sup.dp < 0.01: comparison with pomegranate extract 20 mg/kg administered group according to LSD test .sup.ep < 0.01: comparison with sham control according to MW test .sup.fp < 0.01: comparison with OVX control according to MW test .sup.gp < 0.01: comparison with red clover extract 40 mg/kg administered group according to MW test .sup.hp < 0.01: comparison with pomegranate extract 20 mg/kg administered group according to MW test
(61) In the case of OVX control, the body weight gain for the duration of administration changed by 96.55% as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60/kg administered groups had changes by 23.64, 23.00, 38.90, and 31.87% respectively as compared to OVX control.
Example 1-3. Change in Food Consumption
(62) Significant (p<0.01) increases in food consumption were found in OVX control respectively 28, 49 and 83 days after start of administration as compared to sham control, changes in food consumption related to administration of red clover extract, pomegranate extract and red clover:pomegranate extract 2:1 (g/g) mixture compared to OVX control were not found at each of 28, 49 and 83 days when measured, and significant changes in food consumption were not found in all groups administered with all doses of red clover:pomegranate extract 2:1 (g/g) mixture as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Table 3).
(63) In the case of OVX control, the food consumption at 28, 49 and 83 days after start of administration changed by 30.53, 32.50 and 37.35% as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups changed in food consumption by 5.60, 6.48, 8.86, 3.17% respectively 28 days after start of administration, by 0.68, 7.11, 8.95, 5.94% respectively 49 days after start of administration, and by 1.40, 0.98, 1.77, 4.57% respectively 83 days after start of administration, as compared to OVX control.
(64) TABLE-US-00003 TABLE 3 Food consumption (g/24 hrs/rat): Water consumption (ml/24 hrs/rat): Days after initial treatment Days after initial treatment Groups 28 49 83 28 49 83 Controls Sham 15.55 1.42 14.19 1.70 12.47 1.56 26.75 3.49 37.00 3.55 32.25 4.77 OVX 20.29 2.61.sup.a 18.80 2.61.sup.a 17.13 1.62.sup.a 33.38 5.13.sup.b 48.88 5.25.sup.d 45.25 5.97.sup.a RC 40 mg/kg 19.16 3.54.sup.b 18.93 4.06.sup.a 16.89 2.82.sup.a 34.75 7.25.sup.b 46.13 4.73.sup.d 44.75 12.34.sup.b PCP 20 mg/kg 18.98 3.06.sup.b 17.47 2.07.sup.b 16.96 2.40.sup.a 36.13 7.70.sup.a 47.25 10.43.sup.e 52.00 13.91.sup.a RC:PCP 2:1 mixture (g/g) 120 mg/kg 18.49 1.94.sup.b 17.12 2.97.sup.b 16.83 2.30.sup.a 35.38 4.78.sup.a 46.00 4.54.sup.d 41.38 5.95.sup.c 60 mg/kg 19.65 3.50.sup.a 19.92 1.74.sup.a 17.92 4.40.sup.a 38.38 8.60.sup.a 51.13 8.82.sup.d 50.50 9.30.sup.a .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to LSD test .sup.dp < 0.01 and .sup.ep < 0.05: comparison with sham control according to MW test
Example 1-4. Change in Water Consumption
(65) Significant (p<0.01 or p<0.05) increases in water consumption were found in OVX control respectively 28, 49 and 83 days after start of administration as compared to sham control, changes in water consumption related to administration of red clover extract, pomegranate extract and red clover:pomegranate extract 2:1 (g/g) mixture compared to OVX control were not found at each of 28, 49 and 83 days when measured, and significant (p<0.05) decreases in water consumption were found in red clover:pomegranate extract 2:1 (g/g) mixture 120 mg/kg administered group merely 83 days after start of administration as compared to pomegranate extract 20 mg/kg administered group, and significant changes in water consumption were not found in all groups administered with all doses of red clover:pomegranate extract 2:1 (g/g) mixture as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Table 3).
(66) In the case of OVX control, the water consumption at 28, 49 and 83 days after start of administration changed by 24.77, 32.09 and 40.31% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups changed in water consumption by 4.12, 8.24, 5.99, 14.98% respectively 28 days after start of administration, by 5.63, 3.32, 5.88, 4.60% respectively 49 days after start of administration, and by 1.10, 14.92, 8.56, 11.60% respectively 83 days after start of administration, as compared to OVX control.
Example 1-5. Change in Urine Volume
(67) When respectively measured 28, 49 and 83 days after start of administration, significant changes in urine volume were not found in OVX control as compared to sham control, while red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered group showed significant (p<0.01) increases in urine volume as compared to OVX control when respectively measured 28, 49 and 83 days after start of administration, and particularly, significant (p<0.01 or p<0.05) increases in urine volume were found at all the measurement dates as compared to red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered group (Table 4).
(68) In the case of OVX control, the urine volume at 28, 49 and 83 days after start of administration changed by 1.61, 4.35 and 11.99% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in urine volume by 5.82, 1.59, 80.95, 65.08%, respectively, 28 days after start of administration, by 53.33, 33.33, 115.83, 82.08%, respectively, 49 days after start of administration, and by 36.70, 34.25, 163.00, 115.29%, respectively, 83 days after start of administration, as compared to OVX control.
(69) TABLE-US-00004 TABLE 4 Urine volume (ml/24 hrs/rat): Fecal excretion (g/24 hrs/rat): Days after initial treatment Days after initial treatment Groups 28 49 83 28 49 83 Controls Sham 2.33 0.81 2.88 0.50 3.65 1.00 8.80 2.53 8.23 2.12 9.61 3.33 OVX 2.36 0.75 3.00 0.50 4.09 0.77 9.97 3.13 10.44 4.14 12.09 2.47 RC 40 mg/kg 2.50 0.90 4.60 1.06.sup.ab 5.59 0.77.sup.ac 12.81 4.31.sup.i 14.91 3.07.sup.ab 16.43 1.73.sup.hj PCP 20 mg/kg 2.33 0.71 4.00 0.98.sup.ac 5.49 0.81.sup.ac 10.76 4.50 14.58 2.76.sup.ab 16.02 1.03.sup.hk RC:PCP 2:1 mixture (g/g) 120 mg/kg 4.28 0.93.sup.abdf 6.48 1.10.sup.abdf 10.75 1.88.sup.abdf 12.48 4.38 19.86 3.31.sup.abdf 19.41 1.33.sup.hjlm 60 mg/kg 3.90 1.02.sup.abdf 5.46 0.60.sup.abef 8.80 1.69.sup.abdf 13.33 2.04.sup.hk 18.54 2.05.sup.abeg 18.98 1.64.sup.hjlm .sup.ap < 0.01: comparison with sham control according to LSD test .sup.bp < 0.01 and .sup.cp < 0.05: comparison with OVX control according to LSD test .sup.dp < 0.01 and .sup.ep < 0.05: comparison with red clover extract 40 mg/kg administered group according to LSD test .sup.fp < 0.01 and .sup.gp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to LSD test .sup.mp < 0.01: comparison with pomegranate extract 20 mg/kg administered group according to MW test .sup.hp < 0.01 and .sup.ip < 0.05: comparison with sham control according to MW test .sup.jp < 0.01 and .sup.kp < 0.05: comparison with OVX control according to MW test .sup.lp < 0.01: comparison with red clover extract 40 mg/kg administered group according to MW test
Example 1-6. Change in Fecal Excretion
(70) OVX control showed insignificantly slight increases in in fecal excretion respectively 28, 49 and 83 days after start of administration when measured as compared to sham control, while red clover:pomegranate extract 2:1 (g/g) mixture 120 mg/kg administered group showed significant (p<0.01 or p<0.05) increases in fecal excretion respectively 49 and 83 days after start of administration as compared to OVX control, and red clover:pomegranate extract 2:1 (g/g) mixture 60 mg/kg administered group showed significant (p<0.01 or p<0.05) increases in fecal excretion respectively 28, 49 and 83 days after start of administration when measured as compared to OVX control, and particularly, significant (p<0.01 or p<0.05) increases in fecal excretion were found in red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered group respectively 49 and 83 days after start of administration as compared to red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered group. In the case of OVX control, the fecal excretion at 28, 49 and 83 days after start of administration changed by 13.29, 26.96 and 25.76% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups changed in fecal excretion by 28.46, 7.87, 25.15, 33.63% respectively 28 days after start of administration, by 42.80, 39.56, 90.16, 77.55% respectively 49 days after start of administration 49, and by 35.89, 32.54, 60.58, 57.03% respectively 83 days after start of administration, as compared to OVX control. (Table 4)
Example 1-7. Change in Abdominal Fat Pad Weight
(71) Significant (p<0.01) increases in absolute weight of fat deposited in left abdominal wall and relative weight to body weight were each found in OVX control as compared to sham control, while significant (p<0.01) decreases in abdominal fat pad weight were found in each of red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups as compared to OVX control. Particularly, significant (p<0.01) decreases in abdominal fat pad weight were found in red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Table 5,
(72) In the case of OVX control, the absolute weight of fat deposited in left abdominal wall and relative weight to body weight changed by 307.47 and 183.68% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in absolute weight by 40.94, 36.38, 64.82, 55.77% respectively, and changes in abdominal fat pad relative weight by 35.49, 30.82, 59.59, 50.86% respectively, as compared to OVX control.
(73) TABLE-US-00005 TABLE 5 Absolute wet-weight (g) Relative wet-weight (% of body weight) Groups Abodminal fat pad Uterus Liver Abodminal fat pad Uterus Liver Controls Sham 3.608 1.146 0.648 0.148 7.374 0.837 1.140 0.351 0.209 0.055 2.349 0.245 OVX 14.703 2.682.sup.f 0.082 0.007.sup.f 7.372 0.351 3.235 0.604.sup.f 0.018 0.002.sup.f 1.623 0.118.sup.f RC 40 mg/kg 8.683 1.261.sup.fh 0.104 0.014.sup.fh 7.680 0.606 2.087 0.270.sup.fh 0.025 0.004.sup.fh 1.849 0.147.sup.fi PCP 20 mg/kg 9.355 1.279.sup.fh 0.101 0.014.sup.fh 7.663 0.351 2.238 0.329.sup.fh 0.024 0.004.sup.fh 1.830 0.066.sup.fh RC:PCP 2:1 mixture (g/g) 120 mg/kg 5.173 1.220.sup.ghjl 0.136 0.023.sup.fhjl 8.477 0.444.sup.abde 1.307 0.296.sup.hjl 0.034 0.006.sup.fhjl 2.146 0.096.sup.hjl 60 mg/kg 6.504 0.979.sup.fhjl 0.125 0.011.sup.fhjl 8.514 0.736.sup.abce 1.590 0.181.sup.fhjl 0.031 0.003.sup.fhjl 2.091 0.188.sup.ghkl .sup.ap < 0.01: comparison with sham control according to LSD test .sup.bp < 0.01: comparison with OVX control according to LSD test .sup.cp < 0.01 and .sup.dp < 0.05: comparison with red clover extract 40 mg/kg administered group according to LSD test .sup.ep < 0.01: comparison with pomegranate extract 20 mg/kg administered group according to LSD test .sup.fp < 0.01 and .sup.gp < 0.05: comparison with sham control according to MW test .sup.hp < 0.01 and .sup.ip < 0.05: comparison with OVX control according to MW test .sup.jp < 0.01 and .sup.kp < 0.05: comparison with red clover extract 40 mg/kg administered group according to MW test .sup.lp < 0.01: comparison with pomegranate extract 20 mg/kg administered group according to MW test
Example 1-8. Change in Uterus Weight
(74) Significant (p<0.01) decreases in absolute uterus weight and relative uterus weight to body weight were each found in OVX control as compared to sham control, while significant (p<0.01) increases in uterus weight were each found in all test substance administered groups including red clover:pomegranate extract 2:1 (g/g) mixture administered groups as compared to OVX control. Particularly, significant (p<0.01) increases in uterus weight were found in each of red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Table 5,
(75) In the case of OVX control, the absolute uterus weight and relative uterus weight to body weight changed by 87.37 and 91.35% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in absolute weight by 26.76, 23.39, 66.36, 52.75% respectively, and changes in relative uterus weight by 38.78, 33.80, 91.02, 70.17% respectively, as compared to OVX control.
Example 1-9. Change in Liver Weight
(76) Significant (p<0.01) decreases in relative liver weight to body weight were found in OVX control as compared to sham control, while significant (p<0.01 or p<0.05) increases in relative liver weight were each found in all candidate substance administered groups including red clover:pomegranate extract 2:1 (g/g) mixture administered groups as compared to OVX control. Particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups showed significant (p<0.01) increases in absolute liver weight as compared to OVX control, and significant (p<0.01 or p<0.05) increases in absolute and relative liver weights were each found as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Table 5).
(77) In the case of OVX control, absolute liver weight and relative liver weight to body weight changed by 0.03 and 30.91% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in absolute weight by 4.18, 3.96, 14.99, 15.49% respectively, and changes in relative liver weight by 13.95, 12.75, 32.21, 28.83% respectively, as compared to OVX control.
Example 1-10. Change in Bone Weight
(78) Significant (p<0.01) decreases in wet/dry relative weights of femur, tibia and lumbar vertebrae (L5) to body weight as well as significant decreases in ash absolute and relative weights of femur, tibia and L5 were found in OVX control as compared to sham control, while remarkable increases in bone wet/dry relative weights were found in all test substance administered groups including pomegranate extract 20 mg/kg administered groups as compared to OVX control, and significant (p<0.01 or p<0.05) increases in relative and absolute bone ash weights were each found. Particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered group showed remarkable increases in bone wet weight as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups, and significant (p<0.01 or p<0.05) increases in bone dry/ash absolute and relative weights were each found (Tables 6-8).
(79) In the case of OVX control, the relative weight of wet, dry and ash bone of femur to body weight changed by 29.34, 31.02 and 42.92% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in wet relative weight by 7.81, 12.49, 17.16, 14.78% respectively, changes in dry bone relative weight by 12.82, 11.11, 29.59, 24.58% respectively, and changes in ash femur relative weight by 25.36, 29.70, 53.23, 42.77% respectively, as compared to OVX control.
(80) In the case of OVX control, the relative weights of wet, dry and ash bone of tibia to body weight changed by 27.38, 32.82 and 44.13% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in wet relative weight by 7.22, 9.78, 22.73, 18.15% respectively, changes in dry bone relative weight by 13.83, 19.44, 36.30, 29.55% respectively, and changes in ash tibia relative weight by 30.03, 32.77, 62.18, 50.77% respectively, as compared to OVX control.
(81) In the case of OVX control, the relative weights of wet, dry and ash bone of L5 to body weight changed by 29.62, 34.86 and 44.49% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in wet relative weight by 6.91, 10.84, 34.79, 27.46% respectively, changes in dry bone relative weight by 15.82, 15.44, 42.68, 31.84% respectively, and changes in ash L5 relative weight by 26.19, 18.74, 48.36, 39.84% respectively, as compared to OVX control.
(82) TABLE-US-00006 TABLE 6 Absolute weight (g) Relative weight (% of body weight) Groups Wet Dry Ash Wet Dry Ash Controls Sham 0.996 0.106 0.688 0.073 0.432 0.050 0.317 0.026 0.219 0.013 0.137 0.009 OVX 1.020 0.082 0.685 0.043 0.356 0.040.sup.a 0.224 0.012.sup.a 0.151 0.012.sup.a 0.078 0.009.sup.a RC 40 mg/kg 1.003 0.107 0.706 0.061 0.407 0.034.sup.c 0.241 0.027.sup.a 0.170 0.016.sup.ac 0.098 0.009.sup.ac PCP 20 mg/kg 1.055 0.058 0.702 0.082 0.425 0.021.sup.c 0.252 0.014.sup.ac 0.168 0.017.sup.ad 0.102 0.004.sup.ac RC:PCP 2:1 mixture (g/g) 120 mg/kg 1.036 0.083 0.772 0.033.sup.aceg 0.474 0.029.sup.bceg 0.262 0.014.sup.ace 0.195 0.008.sup.aceg 0.120 0.007.sup.aceg 60 mg/kg 1.048 0.096 0.765 0.039.sup.aceg 0.455 0.030.sup.ce 0.257 0.021.sup.ac 0.188 0.012.sup.aceg 0.112 0.009.sup.aceh .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01 and .sup.dp < 0.05: comparison with OVX control according to LSD test .sup.ep < 0.01 and .sup.fp < 0.05: comparison with red clover extract 40 mg/kg administered group according to LSD test .sup.gp < 0.01 and .sup.hp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to LSD test
(83) TABLE-US-00007 TABLE 7 Absolute weight (g) Relative weight (% of body weight) Groups Wet Dry Ash Wet Dry Ash Controls Sham 0.717 0.065 0.520 0.056 0.343 0.037 0.228 0.012 0.165 0.009 0.110 0.016 OVX 0.754 0.047 0.504 0.016 0.278 0.037.sup.a 0.166 0.007.sup.a 0.111 0.005.sup.a 0.061 0.010.sup.a RC 40 mg/kg 0.737 0.058 0.524 0.039 0.331 0.025.sup.c 0.178 0.015.sup.ad 0.126 0.011.sup.ac 0.080 0.006.sup.ac PCP 20 mg/kg 0.760 0.037 0.554 0.029.sup.d 0.341 0.025.sup.c 0.182 0.013.sup.ac 0.132 0.009.sup.ac 0.081 0.006.sup.ac RC:PCP 2:1 mixture (g/g) 120 mg/kg 0.803 0.041.sup.ade 0.597 0.032.sup.aceg 0.393 0.030.sup.aceg 0.203 0.010.sup.aceg 0.151 0.007.sup.aceg 0.099 0.008.sup.bceg 60 mg/kg 0.796 0.019.sup.adf 0.585 0.042.sup.ace 0.376 0.031.sup.bceh 0.196 0.010.sup.aceh 0.144 0.009.sup.aceh 0.092 0.010.sup.acfh .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01 and .sup.dp < 0.05: comparison with OVX control according to LSD test .sup.ep < 0.01 and .sup.fp < 0.05: comparison with red clover extract 40 mg/kg administered group according to LSD test .sup.gp < 0.01 and .sup.hp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to LSD test
(84) TABLE-US-00008 TABLE 8 Absolute weight (g) Relative weight (% of body weight) Groups Wet Dry Ash Wet Dry Ash Controls Sham 0.388 0.046 0.249 0.039 0.144 0.013 0.123 0.014 0.079 0.010 0.046 0.002 OVX 0.396 0.051 0.234 0.027 0.115 0.009.sup.a 0.087 0.010.sup.a 0.051 0.005.sup.i 0.025 0.003.sup.a RC 40 mg/kg 0.386 0.073 0.248 0.022 0.133 0.009.sup.bc 0.093 0.016.sup.a 0.060 0.006.sup.il 0.032 0.003.sup.ac PCP 20 mg/kg 0.404 0.033 0.249 0.022 0.126 0.007.sup.ac 0.096 0.007.sup.a 0.059 0.006.sup.il 0.030 0.002.sup.ac RC:PCP 2:1 mixture (g/g) 120 mg/kg 0.463 0.043.sup.adeh 0.290 0.018.sup.aceg 0.149 0.009.sup.ceg 0.117 0.010.sup.ceg 0.073 0.004.sup.kmo 0.038 0.003.sup.aceg 60 mg/kg 0.451 0.051.sup.bdh 0.277 0.033.sup.bcfh 0.145 0.007.sup.cfg 0.111 0.013.sup.bceh 0.068 0.007.sup.jknp 0.036 0.002.sup.aceg .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01 and .sup.dp < 0.05: comparison with OVX control according to LSD test .sup.ep < 0.01 and .sup.fp < 0.05: comparison with red clover extract 40 mg/kg administered group according to LSD test .sup.gp < 0.01 and .sup.hp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to LSD test .sup.ip < 0.01 and .sup.jp < 0.05: comparison with sham control according to MW test .sup.kp < 0.01 and .sup.lp < 0.05: comparison with OVX control according to MW test .sup.mp < 0.01 and .sup.np < 0.05: comparison with red clover extract 40 mg/kg administered group according to MW test .sup.op < 0.01 and .sup.pp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to MW test
Example 1-11. Serum Biochemical Change (1): AST, ALT, TC, LDL, HDL and TG
(85) Significant (p<0.01) increases in serum Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total cholesterol (TC), Low density lipoprotein (LDL) and Triglyceride (TG) levels as well as significant (p<0.05) decreases in High density lipoprotein (HDL) level were found in OVX control as compared to sham control, while significant (p<0.01 or p<0.05) decreases in serum AST, ALT, TC, LDL and TG levels and significant increases in serum HDL level were each found in of all test substance administered groups as compared to OVX control. Particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups showed significant (p<0.01) increases in serum HDL level as compared to OVX control, and significant (p<0.01 or p<0.05) decreases in serum AST, ALT, TC, LDL and TG levels and significant increases in HDL level were each found as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Table 9).
(86) In the case of OVX control, the serum AST and ALT levels changed by 77.03 and 72.53% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in AST level by 14.50, 12.10, 31.63, 25.80% respectively, and changes in serum ALT level by 20.70, 16.77, 34.93, 29.30% respectively, as compared to OVX control.
(87) In the case of OVX control, the serum TC and LDL levels changed by 105.00 and 163.08% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in TC level by 24.00, 20.98, 40.39, 36.78% respectively, and changes in serum DL level by 22.37, 24.56, 44.30, 39.62% respectively, as compared to OVX control.
(88) In the case of OVX control, the serum HDL and TG levels changed by 18.62 and 121.81% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in HDL level by 5.88, 13.73, 52.92, 41.83% respectively, and changes in serum TG level by 27.51, 24.69, 42.68, 36.92% respectively, as compared to OVX control.
(89) TABLE-US-00009 TABLE 9 Serum biochemistrical values Groups AST (U/L) ALT (U/L) TC (mg/dl) LDL (mg/dl) HDL (mg/dl) TG (mg/dl) Controls Sham 96.86 12.54 68.25 14.62 62.50 13.48 32.50 10.94 23.50 4.81 53.88 13.15 OVX 171.50 21.79.sup.a 117.75 19.56.sup.h 128.13 19.70.sup.a 85.50 15.92.sup.a 19.13 3.23.sup.b 119.50 29.09.sup.h RC 40 mg/kg 146.63 6.80.sup.ac 93.38 7.93.sup.hj 97.38 10.01.sup.ac 66.38 11.61.sup.ac 20.25 2.19 86.63 10.84.sup.hj PCP 20 mg/kg 150.75 7.81.sup.ac 98.00 7.58.sup.hk 101.25 14.36.sup.ac 64.50 11.07.sup.ac 21.75 3.96 90.00 12.06.sup.hk RC:PCP 2:1 mixture (g/g) 120 mg/kg 117.25 13.33.sup.acdf 76.63 9.16.sup.jln 76.38 9.13.sup.cdf 47.63 9.83.sup.bcdf 29.13 4.09.sup.bcdf 68.50 10.69.sup.ijln 60 mg/kg 127.25 15.98.sup.acef 83.25 7.59.sup.ijmn 81.00 14.01.sup.bcef 51.63 7.67.sup.aceg 27.13 4.05.sup.cdg 75.38 7.61.sup.hjmo
Example 1.12. Serum Biochemical Change (2): Estradiol
(90) Significant (p<0.01) decreases in serum estradiol level were found in OVX control as compared to sham control, while significant (p<0.01 or p<0.05) increases in serum estradiol level were found in each of red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups as compared to OVX control. Particularly, significant (p<0.01 or p<0.05) increases in serum estradiol level were found in each of red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (
(91) In the case of OVX control, the serum estradiol level changed by 86.58% as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in serum estradiol level by 165.55, 141.18, 307.56, 247.90% respectively as compared to OVX control.
Example 1-13. Serum Biochemical Change (3): Osteocalcin and bALP
(92) Significant (p<0.01) increases in serum osteocalcin level as well as significant (p<0.01) decreases in bALP level were found in OVX control as compared to sham control, while significant (p<0.01 or p<0.05) decreases in serum osteocalcin level as well as significant increases in serum bALP level were each found in all candidate substance administered groups as compared to OVX control. Particularly, significant (p<0.01 or p<0.05) decreases in serum osteocalcin level and significant increases in bALP level were each found in red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (
(93) In the case of OVX control, the serum osteocalcin and bALP levels changed by 69.33 and 49.94% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in osteocalcin level by 17.75, 13.83, 31.27, 27.36% respectively, and changes in serum bALP level 26.37, 21.16, 64.38, 47.53% respectively, as compared to OVX control.
Example 1-14. Change in Bone Mineral Density
(94) OVX control significantly (p<0.01) decreased in bone mineral density of total, neck and mid-shaft region of femur and tibia as well as total bone mineral density of fourth lumbar vertebrae (L4) as compared to sham control, while significant (p<0.01 or p<0.05) increases in bone mineral density were each found in all test substance administered groups all over the measured regions as compared to OVX control. Particularly, remarkable increases in bone mineral density were found in each of red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups all over the measured regions as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Table 10).
(95) OVX control had changes in bone mineral density of femur total, neck and mid-shaft region by 18.42, 20.06 and 16.44% as compared to normal control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in total bone mineral density by 5.67, 5.46, 14.64, 11.13% respectively, changes in neck bone mineral density by 9.33, 6.65, 22.12, 16.77% respectively, and changes in mid-shaft bone mineral density by 7.94, 5.81, 16.47, 13.51% respectively, as compared to OVX control.
(96) OVX control had changes in bone mineral density of tibia total, neck and mid-shaft region by 22.65, 20.24 and 28.62% respectively as compared to normal control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in total bone mineral density by 8.92, 6.75, 19.28, 16.75% respectively, changes in neck bone mineral density by 8.39, 6.45, 18.71, 14.95% respectively, and changes in mid-shaft bone mineral density by 13.22, 11.01, 30.21, 19.38% respectively, as compared to OVX control.
(97) OVX control had changes in L4 total bone mineral density by 21.38% as compared to normal control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in L4 total bone mineral density by 9.60, 7.43, 18.74, 15.20% respectively as compared to OVX control.
(98) TABLE-US-00010 TABLE 10 Control References RC:PCP 2:1 mixture(g/g) Groups Sham OVX RC 40 mg/kg PCP 20 mg/kg 120 mg/kg 60 mg/kg Femur Total 0.149 0.010 0.121 0.006.sup.a 0.128 0.004.sup.ac 0.128 0.005.sup.ac 0.139 0.005.sup.aceg 0.135 0.004.sup.acfh Neck 0.158 0.012 0.126 0.003.sup.i 0.138 0.010.sup.ik 0.134 0.004.sup.ik 0.154 0.008.sup.kmo 0.147 0.004.sup.jko Mid-shaft 0.126 0.005 0.106 0.004.sup.a 0.114 0.003.sup.ac 0.112 0.004.sup.ac 0.123 0.003.sup.ceg 0.120 0.003.sup.aceg Tibia Total 0.134 0.010 0.104 0.003.sup.a 0.113 0.007.sup.ac 0.111 0.003.sup.ad 0.124 0.004.sup.aceg 0.121 0.005.sup.aceg Neck 0.146 0.007 0.116 0.006.sup.a 0.126 0.005.sup.ad 0.124 0.004.sup.ad 0.138 0.005.sup.bceg 0.134 0.006.sup.aceg Mid-shaft 0.119 0.012 0.085 0.007.sup.i 0.096 0.005.sup.ik 0.095 0.005.sup.il 0.111 0.008.sup.kmo 0.102 0.004.sup.iknp L4-Total 0.139 0.009 0.109 0.005.sup.a 0.120 0.005.sup.ac 0.118 0.006.sup.ad 0.130 0.008.sup.bceg 0.126 0.004.sup.ach .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01 and .sup.dp < 0.05: comparison with OVX control according to LSD test .sup.ep < 0.01 and .sup.fp < 0.05: comparison with red clover extract 40 mg/kg administered group according to LSD test .sup.gp < 0.01 and .sup.hp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to LSD test .sup.ip < 0.01 and .sup.jp < 0.05: comparison with sham control according to MW test .sup.kp < 0.01 and .sup.lp < 0.05: comparison with OVX control according to MW test .sup.mp < 0.01 and .sup.np < 0.05: comparison with red clover extract 40 mg/kg administered group according to MW test .sup.op < 0.01 and .sup.pp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to MW test
Example 1-15. Change in Bone Strength
(99) OVX control significantly (p<0.01) decreased in bone strength of mid-shaft region of femur and tibia as compared to sham control, while significant (p<0.01 or p<0.05) increases in bone strength of femur and tibia were each found in all candidate substance administered groups including three doses of red clover:pomegranate extract 2:1 (g/g) mixture administered groups as compared to OVX control. Particularly, significant (p<0.01 or p<0.05) increases in bone mineral density in mid-shaft regions of femur and tibia were found in each of red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (
(100) In the case of OVX control, the bone strength of femur and tibia mid-shaft regions changed by 51.06 and 53.22% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in femur mid-shaft region bone strength by 51.66, 48.93, 87.20, 78.09% respectively, and changes in bone strength of tibia mid-shaft region by 26.38, 24.75, 80.90, 50.49% respectively, as compared to OVX control.
Example 1-16. Histological Change (1): Abdominal Wall Fat Pad, Uterus and Liver
(101) (1) Abdominal Wall Fat Pad
(102) Significant (p<0.01) increases in abdominal wall fat pad thickness caused by remarkable increases in abdominal wall fat deposition and significant (p<0.01) increases in mean adipocyte diameter were each found in OVX control as compared to sham control, while significant (p<0.01) decreases in abdominal wall fat pad and adipocyte diameter were each found in all test substance administered groups as compared to OVX control. Particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups showed presumably significant (p<0.01 or p<0.05) inhibition of abdominal wall fat deposition and adipocyte hypertrophy as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Table 11,
(103) The abdominal wall fat pad thickness and mean adipocyte diameter changed by 122.59 and 92.42% respectively in OVX control as compared to normal control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in abdominal wall fat pad thickness by 21.81, 18.61, 43.66, 35.33% respectively, and changes in mean adipocyte diameter by 25.72, 19.19, 37.39, 35.67% respectively, as compared to OVX control.
(104) (2) Uterus
(105) Disuse atrophy contributing to remarkable decreases in uterine mucosa occurred in OVX control, and significant (p<0.01) decreases in total, epithelium and mucosa thickness of uterus and percentage of uterine glands in the mucosa were each found in OVX control as compared to sham control, while significant (p<0.01) increases in total, epithelium and mucosa thickness of uterus and percentage of uterine glands in the mucosa were each found in all candidate substance administered groups as compared to OVX control, and particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups showed presumably significant (p<0.01 or p<0.05) histological inhibition of disuse atrophy of uterus as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Table 11,
(106) The mean thickness of total and epithelium of uterus changed by 83.08 and 74.62% respectively in OVX control as compared to normal control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in mean total uterus thickness 62.99, 34.94, 167.13, 102.53% respectively, and changes in uterine epithelium mean thickness by 73.09, 96.22, 148.66, 133.62% respectively, as compared to OVX control.
(107) The mucosa mean thickness of uterus and percentage of uterine glands in the mucosa changed by 81.70 and 83.16% respectively in OVX control as compared to normal control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in uterine mucosa mean thickness by 56.88, 47.96, 174.02, 137.75% respectively, and changes in percentage of uterine glands in the mucosa by 91.41, 127.57, 314.70, 264.53% respectively, as compared to OVX control.
(108) (3) Liver
(109) Hepatic steatosis characterized by hypertrophy caused by remarkable fat deposition in hepatocytes occurred in OVX control, and significant (p<0.01) increases in region with hepatic fatty degeneration and mean hepatocyte diameter were each found in OVX control as compared to sham control, while significant (p<0.01 or p<0.05) decreases in hepatic steatosis were each found in all test substance administered groups including red clover extract 40 mg/kg administered groups as compared to OVX control, and particularly, significant (p<0.01) decreases in region with hepatic fatty degeneration and mean hepatocyte diameter were found in red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Table 11,
(110) The region with hepatic fatty degeneration and mean hepatocyte diameter changed by 1001.19 and 152.94% respectively in OVX control as compared to normal control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in region with hepatic fatty degeneration by 38.93, 29.44, 65.97, 59.16% respectively, and changes in mean hepatocyte diameter by 20.83, 19.11, 45.46, 35.65% respectively, as compared to OVX control.
(111) TABLE-US-00011 TABLE 11 Control References RC:PCP 2:1 mixture(g/g) Groups Sham OVX RC 40 mg/kg PCP 20 mg/kg 120 mg/kg 60 mg/kg Fat pads Total Th (mm) 4.43 0.92 9.85 1.60.sup.a 7.70 1.01.sup.ac 8.02 0.54.sup.ac 5.55 0.71.sup.bcdf 6.37 0.57.sup.acef Adipocyte 82.20 12.37 158.17 19.42.sup.a 117.50 14.07.sup.ac 127.82 15.14.sup.ac 99.03 10.07.sup.bcef 101.74 11.48.sup.acef DM (m) Uterus Total Th (mm) 3.21 0.65 0.54 0.11.sup.h 0.89 0.10.sup.hi 0.73 0.09.sup.hi 1.45 0.36.sup.hikl 1.10 0.10.sup.hikl Epl Th (m) 38.47 5.18 9.76 2.26.sup.a 16.90 1.51.sup.ac 19.16 1.72.sup.ac 24.28 2.31.sup.acdf 22.81 2.02.sup.acdg Mucosa Th (m) 967.28 220.32 176.98 21.58.sup.h 227.65 31.82.sup.hi 261.87 32.06.sup.hi 484.97 56.84.sup.hikl 420.77 94.19.sup.hikl UG percentage 28.78 4.83 4.85 2.17.sup.a 9.28 1.53.sup.ac 11.03 3.19.sup.ac 20.10 3.13.sup.acdf 17.67 2.71.sup.acdf (%) Liver FC region (%) 4.74 3.02 52.14 11.22.sup.a 31.84 4.83.sup.ac 39.79 7.25.sup.ac 17.74 4.16.sup.acdf 21.29 4.91.sup.acdf Hepatocyte 12.81 1.30 32.39 5.08.sup.h 25.65 2.24.sup.hi 26.20 2.16.sup.hj 17.67 3.20.sup.hikl 20.85 2.27.sup.hikl DM (m) .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01: comparison with OVX control according to LSD test .sup.dp < 0.01 and .sup.ep < 0.05: comparison with red clover extract 40 mg/kg administered group according to LSD test .sup.fp < 0.01 and .sup.gp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to LSD test .sup.hp < 0.01: comparison with sham control according to MW test .sup.ip < 0.01 and .sup.jp < 0.05: comparison with OVX control according to MW test .sup.kp < 0.01: comparison with red clover extract 40 mg/kg administered group according to MW test .sup.lp < 0.01: comparison with pomegranate extract 20 mg/kg administered group according to MW test
(112) Abbreviations in Table 11 are as follows:
(113) Th=thickness,
(114) DM=diameter,
(115) Epi=epithelium,
(116) UG=uterine gland,
(117) FC=fatty change.
Example 1-17. Histological Change (2): Femur, Tibia and Lumbar Vertebrae (L4)
(118) Trabecular bone and cortical bone developed relatively well were observed in femur, tibia and L4 of sham operated group, while remarkable histological decreases of trabecular bone and cortical bone caused by remarkable increases in number and activity of osteoclast cells were found in OVX control, but remarkable increases in bone mass and thickness of trabecular bone and cortical bone caused by osteoclast cell activity inhibition of femur, tibia and L4 were found in all test substance administered groups including pomegranate extract 20 mg/kg administered groups, and particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups had remarkable osteoclast cell activity inhibition and bone mass preservation effects as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Tables 12-14,
(119) (1) Bone Mass and Structure
(120) In the case of OVX control, each of the trabecular bone mass, mean trabecular bone length, thickness and number, and mid-shaft region conical bone thickness of femur, tibia and L4 significantly (p<0.01) decreased as compared to sham control, while significant (p<0.01 or p<0.05) increases in trabecular bone mass, mean trabecular bone length, thickness and number, and conical bone thickness of femur, tibia and L4 were found in all candidate substance administered groups including red clover:pomegranate extract 2:1 (g/g) mixture administered groups as compared to OVX control, and particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered group had a remarkable effect on inhibition of reduction in histological markers of bone mass and structure as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups.
(121) In the case of OVX control, the trabecular bone mass and mid-shaft cortical bone thickness of femur changed by 66.40% and 25.69% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in trabecular bone mass by 43.75, 20.91, 111.04, 82.21% respectively, and changes in cortical bone thickness by 17.00, 14.54, 27.73, 24.76% respectively, as compared to OVX control.
(122) In the case of OVX control, the trabecular bone mass and mid-shaft cortical bone thickness of tibia changed by 63.65 and 46.83% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in trabecular bone mass by 35.78, 23.25, 101.87, 72.78% respectively, and changes in cortical bone thickness by 16.05, 11.62, 58.61, 29.80% respectively as compared to OVX control.
(123) In the case of OVX control, the trabecular bone mass and mid-shaft cortical bone thickness of L4 changed by 44.84 and 38.59% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in trabecular bone mass by 30.88, 24.90, 56.05, 44.72% respectively, and changes in cortical bone thickness by 23.16, 17.86, 40.78, 39.53% respectively, as compared to OVX control.
(124) (2) Bone Resorption
(125) In the case of OVX control, each of the osteoclast cell number and ratio (OS/BS) of femur, tibia and L4 significantly (p<0.01) increased as compared to sham control, while remarkable decreases in osteoclast cell number and OS/BS of femur, tibia and L4 were found in all candidate substance administered groups as compared to OVX control, and particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups had a remarkable effect on inhibition of bone resorption increase as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups (Tables 12-14).
(126) In the case of OVX control, the osteoclast cell number and OS/BS of femur changed by 280.30 and 229.41% respectively as compared to case sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in osteoclast cell number by 37.05, 30.28, 60.16, 52.59% respectively, and changes in OS/BS by 21.94, 10.44, 44.78, 35.85% respectively, as compared to OVX control.
(127) In the case of OVX control, the osteoclast cell number and OS/BS of tibia changed by 145.69 and 351.02% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in osteoclast cell number by 29.82, 23.86, 44.21, 37.89% respectively, and changes in OS/BS 24.34, 17.04, 68.83, 51.81% respectively, as compared to OVX control.
(128) In the case of OVX control, the osteoclast cell number and OS/BS of L4 changed by 188.89 and 254.62% respectively as compared to sham control, and red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg administered groups had changes in osteoclast cell number by 18.68, 13.19, 52.75, 45.60% respectively, and changes in OS/BS by 21.41, 16.01, 62.69, 54.16% respectively, as compared to OVX control.
(129) TABLE-US-00012 TABLE 12 Control References RC:PCP 2:1 mixture (g/g) Groups Sham OVX RC 40 mg/kg PCP 20 mg/kg 120 mg/kg 60 mg/kg Bone mass and structure TBV, BV/TV 47.27 11.66 15.88 2.97.sup.h 22.83 2.96.sup.hj 19.20 1.70.sup.hj 33.52 6.51.sup.hjlm 28.94 2.85.sup.hjlm Tbn 23.00 3.07 11.38 1.92.sup.a 16.00 2.27.sup.ac 15.63 1.69.sup.ac 20.50 0.93.sup.bcdf 18.38 1.30.sup.acef Tbl 6.81 1.68 1.99 0.21.sup.h 3.42 0.49.sup.hj 3.08 0.90.sup.hj 5.78 0.42.sup.jlm 5.08 0.25.sup.hjlm Tbt 144.33 11.68 90.54 15.24.sup.a 112.08 11.01.sup.ac 104.83 7.23.sup.ac 133.80 8.50.sup.bcdf 124.82 6.89.sup.acef Cbt-shaft 848.67 91.46 630.65 103.16.sup.h 737.86 49.96.sup.hj 722.33 20.24.sup.hj 805.56 37.42.sup.jlm 786.77 22.00.sup.jlm Bone resorption Ocn 8.35 2.38 31.38 6.02.sup.h 19.75 2.31.sup.hj 21.88 3.00.sup.hj 12.50 2.62.sup.hjlm 14.88 2.23.sup.hjlm OS/BS 4.28 1.58 14.09 1.96.sup.a 11.00 0.95.sup.ac 12.62 1.69.sup.a 7.78 1.32.sup.acdf 9.04 1.35.sup.acef .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01: comparison with OVX control according to LSD test .sup.dp < 0.01 and .sup.ep < 0.05: comparison with red clover extract 40 mg/kg administered group according to LSD test .sup.fp < 0.01 and .sup.gp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to LSD test .sup.mp < 0.01: comparison with pomegranate extract 20 mg/kg administered group according to MW test .sup.hp < 0.01 and .sup.ip < 0.05: comparison with sham control according to MW test .sup.jp < 0.01 and .sup.kp < 0.05: comparison with OVX control according to MW test .sup.lp < 0.01: comparison with red clover extract 40 mg/kg administered group according to MW test
(130) TABLE-US-00013 TABLE 13 Control References RC:PCP 2:1 mixture (g/g) Groups Sham OVX RC 40 mg/kg PCP 20 mg/kg 120 mg/kg 60 mg/kg Bone mass and structure TBV, BV/TV 52.14 5.29 18.95 2.79.sup.g 25.73 2.74.sup.gi 23.36 2.47.sup.gi 38.26 8.22.sup.gikm 32.75 5.50.sup.gikm Tbn 36.13 6.47 11.13 1.36.sup.g 14.63 1.60.sup.gi 14.38 2.26.sup.gi 23.13 2.53.sup.gikm 22.00 2.00.sup.gikm Tbl 7.41 0.74 2.34 0.52.sup.a 3.28 0.49.sup.ac 2.98 0.19.sup.ad 5.23 0.76.sup.bcef 4.36 0.63.sup.acef Tbt 124.58 12.32 77.05 5.96.sup.a 94.69 13.44.sup.ac 91.32 14.36.sup.ad 115.76 14.42.sup.cef 112.71 12.97.sup.bcef Cbt-shaft 813.67 87.66 432.65 32.47.sup.g 502.08 53.06.sup.gi 482.92 31.92.sup.gi 686.23 66.91.sup.hikm 561.55 37.01.sup.gilm Bone resorption Ocn 14.50 2.20 35.63 5.68.sup.a 25.00 2.00.sup.ac 27.13 3.40.sup.a 19.88 3.64.sup.acef 22.13 2.53.sup.acf OS/BS 6.49 1.31 29.28 3.21.sup.g 22.16 1.62.sup.gi 24.29 3.70.sup.gj 9.31 1.34.sup.gikm 14.11 4.38.sup.gikm .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01 and .sup.dp < 0.05: comparison with OVX control according to LSD test .sup.ep < 0.01: comparison with red clover extract 40 mg/kg administered group according to LSD test .sup.fp < 0.01: comparison with pomegranate extract 20 mg/kg administered group according to LSD test .sup.gp < 0.01 and .sup.hp < 0.05: comparison with sham control according to MW test .sup.ip < 0.01 and .sup.jp < 0.05: comparison with OVX control according to MW test .sup.kp < 0.01 and .sup.lp < 0.05: comparison with red clover extract 40 mg/kg administered group according to MW test .sup.mp < 0.01: comparison with pomegranate extract 20 mg/kg administered group according to MW test
(131) TABLE-US-00014 TABLE 14 Control References RC:PCP 2:1 mixture (g/g) Groups Sham OVX RC 40 mg/kg PCP 20 mg/kg 120 mg/kg 60 mg/kg Bone mass and structure TBV, BV/TV 56.33 5.80 31.07 2.17.sup.a 40.67 2.21.sup.ac 38.81 3.14.sup.ad 48.49 5.58.sup.aceg 44.97 3.01.sup.acfg Tbn 24.50 4.00 10.88 2.10.sup.l 14.88 1.55.sup.ik 13.38 1.51.sup.il 19.88 2.17.sup.jkmo 17.25 1.49.sup.ikmo Tbl 4.45 0.47 2.32 0.33.sup.i 2.89 0.33.sup.ik 2.82 0.17.sup.il 3.89 0.23.sup.jkmo 3.66 0.44.sup.jkmo Tbt 148.78 14.67 109.21 12.86.sup.i 125.25 6.43.sup.il 124.18 6.91.sup.il 136.42 6.01.sup.ikmo 132.19 2.27.sup.ikp Cbt-shaft 233.01 32.31 143.10 19.90.sup.a 176.25 15.35.sup.ac 168.67 9.71.sup.ad 201.46 11.24.sup.acfg 199.67 15.11.sup.acfg Bone resorption Ocn 7.88 1.89 22.75 2.76.sup.a 18.50 3.07.sup.ac 19.75 1.49.sup.ad 10.75 1.67.sup.bceg 12.38 1.30.sup.aceg OS/BS 5.75 0.93 20.40 2.67.sup.a 16.03 2.06.sup.ac 17.13 1.55.sup.ac 7.61 1.54.sup.ceg 9.35 1.26.sup.aceg .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01 and .sup.dp < 0.05: comparison with OVX control according to LSD test .sup.ep < 0.01 and .sup.fp < 0.05: comparison with red clover extract 40 mg/kg administered group according to LSD test .sup.gp < 0.01 and .sup.hp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to LSD test .sup.ip < 0.01 and .sup.jp < 0.05: comparison with sham control according to MW test .sup.kp < 0.01 and .sup.lp < 0.05: comparison with OVX control according to MW test .sup.mp < 0.01 and .sup.np < 0.05: comparison with red clover extract 40 mg/kg administered group according to MW test .sup.op < 0.01 and .sup.pp < 0.05: comparison with pomegranate extract 20 mg/kg administered group according to MW test
(132) For reference, what is meant by abbreviations in Tables 12-14 is as follows:
(133) Cbt=Cortical bone thickness (Cross thickness; m),
(134) Tbl=Trabecular bone length (Longitudinal thickness; mm),
(135) Tbn=Trabecular bone number (N/epiphyseal),
(136) Tbt=Trabecular bone thickness (Cross thickness; m),
(137) TV/BV=Trabecular bone mass (%),
(138) OS/BS=Osteoclast cell surface/bone surface (%),
(139) Ocn=Osteoclast cell number (N/epiphyseal).
Summary of Example 1
(140) In the study, the effect of the mixture including the composite extract of pomegranate and red clover in enhancing bioactivity against menopausal disorders was evaluated using OVX Rat model that is known to be similar to menopausal disorders after human menopause [Tominaga et al., 2001, 2002; Iwamoto et al., 2005; Cheng and Tian, 2012].
(141) That is, from 28 days after OVX, red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, and red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg were dissolved in or diluted with sterile distilled water and orally administered at a dose of 5 ml/kg once daily for 84 days (12 weeks; 3 months), and evaluation was conducted based on 5 pharmacological effects including an estrogen-like effect, an anti-obesity effect, a hyperlipidemia inhibitory effect, a protective effect against fatty liver, and an osteoporosis inhibitory effect.
(142) To evaluate the estrogen-like effect and anti-obesity effect, changes in body weight and body weight gain, water and food consumption, urine volume and fecal excretion, serum estradiol level, and weights of abdominal fat pad and uterus, and changes in abdominal fat pad thickness and mean adipocyte diameter, thickness of uterus total, epithelium and mucosa, and percentage of uterine glands in the mucosa were each evaluated. Furthermore, the liver protective effect against fatty liver was evaluated by measuring changes in liver weight, serum AST and ALT levels, mean hepatocyte diameter and region with hepatic degeneration showing fatty change, the hyperlipidemia reduction effect was evaluated using changes in serum total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride (TG) levels, and to evaluate the osteoporosis reduction effect, namely, the bone protective effect, histological changes in wet, dry and ash weights, bone mineral density (BMD), bone strength, serum osteocalcin and bone specific alkaline phosphatase (bALP) levels, bone mass and structure and bone resorption of femur, tibia and lumbar vertebrae were each measured. In the experiment, evaluation was conducted by comparing the results of red clover:pomegranate extract 2:1 (g/g) mixture to the results in rats administered with each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg.
(143) As a result of the experiment, remarkable increases in body weight and body weight gain, food and water consumption, abdominal fat pad weight, and serum AST, ALT, TC, LDL, TG and osteocalcin levels as well as remarkable decreases in uterus, liver, femur, tibia and L5 weights, serum bALP, and estradiol levels were found in OVX control as compared to sham vehicle control, and indication of remarkable increases in abdominal wall fat pad thickness, hypertrophy of adipocytes, hepatic steatosis, disuse atrophy of uterus, and reductions in bone mass and structure of femur, tibia and L4 was each histologically and histomorphometrically detected together with remarkable increases of bone resorption marker (Ocn and OS/BS). That is, a variety of disorders such as obesity, hyperlipidemia, hepatic steatosis and osteoporosis were induced by OVX. On the other hand, signs of disorders induced by OVX were remarkably inhibited by continuous oral administration of red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg for 84 days. Particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups had a significant effect on reduction of disorders induced by OVX such as obesity, hyperlipidemia, hepatic steatosis and osteoporosis as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups. Accordingly, the addition of the pomegranate extract (red clover:pomegranate extract 2:1 (g/g) mixture) at an optimal ratio is expected to synergistically increase an effect of red clover extract on reduction of OVX induced menopausal disorders through the increased diversity of isoflavonoid contained, but the present disclosure is not limited thereto.
(144) In the study, food and water consumption remarkably increased without any significant change in urine and fecal excretion due to OVX, resulting in remarkable increases in body weight and deposition of abdominal wall fat and hypertrophy of adipocytes. On the other hand, signs of obesity induced by OVX were remarkably inhibited by continuous oral administration of each of red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg for 84 days, and particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups had presumably significant inhibition of increases in body fat deposition and body weight as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups. Furthermore, remarkable increases in urine volume and fecal excretion (28, 49 and 83 days after start of administration) when measured were each found in red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered rats as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered rats. These results serve as an evidence demonstrating that the addition of pomegranate extract at an optimal ratio (red clover:pomegranate extract 2:1 (g/g) mixture) synergistically increases an effect of red clover extract on reduction of obesity, and red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg is thought to have very effective activities on obesity through increased peristalsis of a digestive tract and a diuretic effect together with an estrogenic effect through regulation of CCK [Asarian and Geary, 2007; Thammacharoen et al., 2008] and glucagon [Geary and Asarian, 2001] generally known as digestive hormones related to satiety of isoflavonoid, but the present disclosure is not limited thereto. Generally, it is known that as peristalsis of the digestive tract increases, fecal excretion increases, leading to an anti-obesity effect such as body weight decrease [Hyland et al., 2010; Bertrand et al., 2011; Snedeker and Hay, 2012], and a diuretic effect also produces a similar body weight decrease effect [Dagnault et al., 1996; Verlander et al., 1998].
(145) In the experiment results, due to OVX, significant decreases in uterus weight occurred, and histopathologically remarkable decreases in not only uterus total, mucosa and epithelium thickness but also uterine glands in the mucosa occurred. On the other hand, indication of endometrial atrophy was remarkably inhibited by oral administration of each of red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg, and particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups showed presumably significant inhibition of endometrial atrophy as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups.
(146) In the study results, the serum TC, LDL and TG levels were remarkably decreased and the HDL level was remarkably increased by oral administration of red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg, and particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups had a significant anti-hyperlipidemia effect as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered group, and it is determined that the addition of pomegranate extract at a ratio according to the present disclosure synergistically increases an anti-hyperlipidemia effect of red clover extract at least under the experimental conditions, and the anti-hyperlipidemia effect will be transferred via HMG-CoA, the enzyme involved in cholesterol synthesis [Di Croce et al., 1996].
(147) In the study, remarkable hepatic steatosis in OVX control was each bio-chemically and histologically ascertained. On the other hand, signs of fatty liver induced by OVX were remarkably inhibited by oral administration of each of red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg, and particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups had a significant liver protective effect as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups. These results also serve as an evidence demonstrating that a liver protective effect of a mixture of pomegranate extract and red clover extract at an optimal ratio synergistically increases. As a result of the experiment, remarkable increases in serum osteocalcin level as well as decreases in femur, tibia and L5 weights and serum bALP level were found in OVX control as compared to sham vehicle control, and indication of decreases in bone mass and structure of femur, tibia and L4 as well as remarkable increases of bone resorption marker (Ocn and OS/BS) was each histologically and histomorphometrically ascertained, so it was observed that osteoporosis was induced by OVX. On the other hand, signs of osteoporosis were remarkably inhibited by continuous oral administration of each of red clover extract 40 mg/kg, pomegranate extract 20 mg/kg, red clover:pomegranate extract 2:1 (g/g) mixture 120, 60 mg/kg for 84 days, and particularly, red clover:pomegranate extract 2:1 (g/g) mixture 120 and 60 mg/kg administered groups had a significant osteoporosis reduction effect as compared to each of red clover extract 40 mg/kg and pomegranate extract 20 mg/kg administered groups. Accordingly, it was observed that a mixture of pomegranate extract and red clover extract at a ratio according to the present disclosure synergistically increased an osteoporosis reduction effect.
2. Study about Setting of a Mixture Ratio of a Pomegranate Extract and a Red Clover Extract
Example 2-1. Experimental Animal and Experimental Method
(148) (1) Experimental Animal
(149) Female virgin Sprague-Dawley Rats (6 weeks old) were purchased, all experimental animals except sham control were randomly selected and underwent bilateral ovariectomy to induce menopause. On the other hand, in sham group, both ovaries were detected by the same method but incisions were not made, and abdominal cavities were closed again. At 28 days after surgery, based on body weights (OVX rats: 312.2112.3 g, sham operated rats: 255.1212.11 g), 4 mice per group were selected from all experimental animals and used for experiments.
(150) (2) Experimental Method
(151) The effect on menopausal disorders was evaluated based on 3 pharmacological effects including an anti-obesity effect, a hyperlipidemia inhibitory effect and an osteoporosis inhibitory effect.
(152) The test material was suspended or dissolved in sterile distilled water, and orally administered at a dose of 5 ml/kg once daily for 56 days from the 28th day after OVX. Sham and OVX controls were orally administered with the vehicle, sterile distilled water alone instead of the test material at the same dose for the same period.
(153) Changes in body weight were measured and recorded on the date of OVX surgery, 12 days before administration (27 days after OVX), the start date of administration, and the date of final sacrifice. The hyperlipidemia inhibitory effect was ascertained by measuring serum TC in blood taken at the time of final sacrifice, and the osteoporosis reduction effect was ascertained through BMD measurements of femur.
Example 2-2. Estrogen Activity Result in Proportion to Setting of a Mixture Ratio of a Pomegranate Extract and a Red Clover Extract
(154) To ascertain a synergistic effect of a mixture of red clover extract and pomegranate extract on a menopausal symptom relief effect in menopause induced model, 40 mg/kg of red clover extract intake was mixed with a pomegranate extract as in Table 1, and a synergistic effect of the added pomegranate extract on menopausal symptom relief was measured through an anti-obesity effect, a hyperlipidemia inhibitory effect and an osteoporosis inhibitory effect in menopause induced animal model.
(155) As a result, the extent of menopausal symptom relief in menopause induced model through OVX was determined by oral administration of with a mixture of red clover extract (RC) 40 mg/kg and pomegranate extract (PCP) for 8 weeks.
(156) In menopause induced model through OVX, changes in body weight increased by about 146 g, and when compared to red clover alone treated group, improvements were detected in the case in which a mixture including 10 mg/kg or higher of pomegranate extract was prescribed, and significant improvements were detected in 20 mg/kg or higher of pomegranate extract, but dose dependent improvements were not detected in a mixture including 40 mg/kg or higher of pomegranate extract.
(157) Similar to changes in body weight, there was a significant improvement in BMD changes at 20 mg/kg or higher, but there was no dose dependent improvement.
(158) As a result of measuring serum TC, there was a significant improvement at 10 mg/kg, but there was no dose dependent change between 80 mg/kg and 160 mg/kg.
(159) It was seen from these results that significant improvements in menopause induced model were achieved by pomegranate extract (PCP) mixture administration than red clover extract (RC) alone administration, and dose dependent results were obtained at an optimal concentration of administration of 1080 mg/kg.
(160) Accordingly, in the case of preparing a mixture of 4080 mg/kg of red clover extract with 1080 mg/kg of pomegranate extract as formula for preparing a mixture of red clover extract (RC) and pomegranate extract (PCP), a synergistic effect was obtained.
3. Verification of a Synergistic Effect in Proportion to a Mixture Ratio of a Pomegranate Extract and a Red Clover Extract
Example 3-1. Experimental Animal and Experimental Method
(161) (1) Experimental Animal
(162) Female virgin ddY mice (6 weeks old, Kiwa Laboratory Animal, Wakayama, Japan) were used, and in the experiment, all experimental animals were handled according to the guidelines for animal ethics approved by the Animal Care and Ethics Committee of Daegu Haany University, and the experiment was done with prior approval. [Approval No. DHU2014-020]
(163) (2) Experimental Method
(164) The experiment was performed after dividing the experimental animals into groups (total 14 groups; 8 mice per group). (Table 15,
(165) That is, the experiment was performed after dividing into sterile distilled water 10 ml/kg administered vehicle control after sham surgery (hereinafter sham control), sterile distilled water 10 ml/kg administered vehicle control after OVX surgery (hereinafter OVX control), pomegranate extract single formula 120 mg/kg administered group (PCP) after OVX, and 9 types of red clover:pomegranate extract mixed formula (g/g) administered groups after OVX surgery including red clover:pomegranate extract 1:1 (g/g) mixed formula 120 mg/kg administered group after OVX surgery (1-1), red clover:pomegranate extract 1:2 (g/g) mixed formula 120 mg/kg administered group after OVX surgery (1-2), red clover:pomegranate extract 1:4 (g/g) mixed formula 120 mg/kg administered group after OVX surgery (1-4), red clover:pomegranate extract 1:6 (g/g) mixed formula 120 mg/kg administered group after OVX surgery (1-6), red clover:pomegranate extract 1:8 (g/g) mixed formula 120 mg/kg administered group after OVX surgery (1-8), red clover:pomegranate extract 2:1 (g/g) mixed formula 120 mg/kg administered group after OVX surgery (2-1), red clover:pomegranate extract 4:1 (g/g) mixed formula 120 mg/kg administered group after OVX surgery (4-1), red clover:pomegranate extract 6:1 (g/g) mixed formula 120 mg/kg administered group after OVX surgery (6-1), and red clover:pomegranate extract 8:1 (g/g) mixed formula 120 mg/kg administered group after OVX surgery (8-1).
(166) Optimal amounts of red clover extract or pomegranate extract were suspended or dissolved in sterile distilled water, and orally administered at a dose of 10 ml/kg once daily for 84 days from 28 days after OVX. That is, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixture 120 mg/kg were each orally administered for 84 days, and sham control and OVX control were orally administered with only sterile distilled water as a vehicle at the same dose for the same period instead of the test material, and according to earlier researchers' method [Chiba et al., 2003; Murakami et al., 2007; Tousen et al., 2011], 0.03 ug of 17-estradiol (Sigma-Aldrich, St. Louise, Mo., USA) was dissolved in 0.2 ml of sterile saline, and subcutaneously administered to back skin at a dose of 0.2 ml/mouse (0.03 g/head/day) once daily for 84 days from 28 days after OVX surgery (Table 15,
(167) TABLE-US-00015 TABLE 15 Group Operation Dose (mg/kg/day) Animal No. Selection of the Fittest Anti-climacterium Herbal Compositions on OVX Mice Control Sham operate Distilled water 10 ml/kg; once a day for 84 days M01~M08 Control OVX operate Distilled water 10 ml/kg; once a day for 84 days M09~M16 Reference OVX operate 17-estradiol (0.03 g/head); once a day for 84 days M17~M24 Reference OVX operate RC (120 mg/kg); once a day for 84 days M25~M32 Reference OVX operate PCP (120 mg/kg); once a day for 84 days M33~M40 Active OVX operate RC:PCP 1:1 (60:60 mg/kg); once a day for 84 days M41~M48 Active OVX operate RC:PCP 1:2 (40:80 mg/kg); once a day for 84 days M49~M56 Active OVX operate RC:PCP 1:4 (24:96 mg/kg); once a day for 84 days M57~M64 Active OVX operate RC:PCP 1:6 (17:103 mg/kg); once a day for 84 days M65~M72 Active OVX operate RC:PCP 1:8 (13:107 mg/kg); once a day for 84 days M73~M80 Active OVX operate RC:PCP 2:1 (80:40 mg/kg); once a day for 84 days M81~M88 Active OVX operate RC:PCP 4:1 (96:24 mg/kg); once a day for 84 days M89~M96 Active OVX operate RC:PCP 6:1 (103:17 mg/kg); once a day for 84 days M97~M104 Active OVX operate RC:PCP 8:1 (107:13 mg/kg); once a day for 84 days M105~M112 OVX = Bilateral ovariectomy PCP = Pomegranate Concentrate Powder RC = Red clover dry extracts RC:PCP = RC and PCP mixed formula (g/g)
(168) For induction of menopausal disorders by OVX, an experiment was performed as follows.
(169) That is, all experimental animals except sham control were randomly selected and underwent bilateral ovariectomy to induce estrogen deficient menopausal disorder. On the other hand, in sham operated group, both ovaries were detected by the same method but incisions were not made, and abdominal cavities were closed again. At 27 days after surgery, based on body weights (OVX mice: 30.711.65 g, 28.0-35.1 g; sham operated mice: 26.991.46 g, 24.4-28.7 g), 8 mice per group were selected from all experimental animals including sham operated group, and used for experiments.
(170) The effect on menopausal disorders was evaluated based on 5 pharmacological effects including an estrogen-like effect, an anti-obesity effect, a hyperlipidemia inhibitory effect, a protective effect against fatty liver and an osteoporosis inhibitory effect, and an optimal mixture ratio was determined by comparing to each of red clover extract and pomegranate extract single formula, and using mixed formula showing statistically significant (p<0.01 or p<0.05) increase in efficacy at the same time as red clover:pomegranate extract mixed formula showing a synergistic effect.
(171) More specifically, the anti-obesity and estrogen-like effects were tested as follows. The body weights on OVX surgery date, 1 day before administration (27 days after OVX), the start date of administration, and from the first day after start of administration up to the time of final sacrifice at least once a week were each recorded, and change amounts of body weights, i.e., body weight gains, for the recovery period after OVX (menopausal disorder induction period) and 12 weeks after start of administration were measured. Food consumption (1, 3, 7, 28, 56 and 83 days after start of administration), serum estradiol levels, amounts of body fat and abdominal fat, weights of abdominal fat pad and uterus, histological changes of uterus and abdominal fat pads (changes in abdominal fat pad thickness and mean adipocyte diameter, thickness of uterus total, epithelium and mucosa, and percentage of uterine glands in the mucosa (
(172) Furthermore, to ascertain the protective effect against fatty liver, testing was conducted as follows. Liver weight, serum AST and ALT levels, and histological changes of liver (changes in mean hepatocyte diameter and region with hepatic degeneration showing fatty change) were observed.
(173) The hyperlipidemia inhibitory effect was ascertained through changes in serum TC, LDL, HDL and TG levels.
(174) The osteoporosis reduction effect was ascertained through histological changes in wet, dry and ash weights of femurs, BMD (
Example 3-2. Change in Body Weight and Body Weight Gain
(175) At 28 days after OVX surgery, as compared to sham control, only experimental animals showing remarkable increases in body weight were selected and used (OVX mice: 30.711.65 g, 28.0-35.1 g; sham operated mice: 26.991.46 g, 24.4-28.7 g), so significant (p<0.01) increases in body weight and body weight gain for a 4 week-induction period after OVX were found in all OVX operated groups as compared to sham control, but estradiol, pomegranate extract and red clover extract single formula administered groups started to show significant (p<0.01 or p<0.05) decreases in body weight from 14, 21 and 28 days after start of administration as compared to OVX control, all 9 types of red clover:pomegranate extract mixture administered groups each started to show significant (p<0.01 or p<0.05) decreases in body weight 14, 21 or 28 days after start of administration as compared to OVX control, and all test substance administered groups also showed significant (p<0.01 or p<0.05) decreases in body weight gain for the duration of administration of 84 days as compared to OVX control. Particularly, red clover:pomegranate extract 1:1, 2:1 and 4:1 mixture administered groups started to show significant (p<0.01 or p<0.05) decreases in body weight from 28, 42 and 49 days after start of administration as compared to each of red clover extract and pomegranate extract single formula administered groups, and significant (p<0.01) decreases in body weight gain for the duration of administration were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 as compared to each of red clover extract and pomegranate extract single formula administered groups (Table 16,
(176) In the case of OVX control, the body weight gain for the duration of administration changed by 351.54% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 52.56, 44.46, 41.91, 67.72, 54.51, 51.28, 53.33, 52.30, 74.19, 64.40, 48.89 and 44.55% respectively as compared to OVX control.
(177) TABLE-US-00016 TABLE 16 Periods Body weights (g) Body weight gains (g) At OVX At initial At sacrifice OVX recovery Treatment Groups [A]* treatment [C]* [D]* [B-A] [D-C] Controls Sham 23.61 1.64 24.75 1.81 28.00 2.23.sup. 1.14 1.58 3.25 1.92.sup. OVX 24.21 1.47 30.78 2.12.sup.a 42.90 2.28.sup.a 4.01 1.23.sup.a 14.68 2.88.sup.f Estradiol 24.65 0.99 28.71 1.61.sup.a 35.68 2.19.sup.ab 4.06 1.18.sup.a 6.96 1.21.sup.fg RC 23.93 1.47 28.98 1.73.sup.a 36.44 1.52.sup.ab 4.36 0.95.sup.a 8.15 0.95.sup.fg PCP 24.60 1.19 28.78 2.02.sup.a 37.30 2.38.sup.ab 4.18 1.74.sup.a 8.53 1.31.sup.fg RC:PCP 1:1 24.41 0.97 28.53 1.65.sup.a .sup.33.26 1.85.sup.abde 4.11 1.50.sup.a .sup.4.74 1.55.sup.gij 1:2 24.69 1.33 28.56 1.78.sup.a 35.24 2.54.sup.ab 3.88 1.46.sup.a 6.68 2.55.sup.fg 1:4 24.40 0.97 28.44 1.52.sup.a 35.59 2.05.sup.ab 4.04 1.72.sup.a 7.15 1.86.sup.fg 1:6 24.65 1.02 28.75 1.72.sup.a 35.60 2.47.sup.ab 4.10 1.55.sup.a 6.85 2.47.sup.fg 1:8 24.66 1.11 28.69 1.31.sup.a 35.69 1.95.sup.ab 4.03 1.51.sup.a 7.00 2.58.sup.fg 2:1 24.58 1.08 28.50 1.54.sup.a .sup.32.29 2.53.sup.abce 3.93 1.31.sup.a .sup.3.79 2.28.sup.gij 4:1 24.31 1.86 28.41 0.88.sup.a .sup.33.64 1.45.sup.abde 4.10 1.63.sup.a .sup.5.23 1.80.sup.gij 6:1 23.86 1.77 28.03 1.35.sup.a 35.53 2.02.sup.ab 4.16 1.95.sup.a 7.50 1.11.sup.fg 8:1 24.61 1.77 28.73 1.12.sup.a 36.86 4.74.sup.ab 4.11 1.52.sup.a 8.14 4.99.sup.fh Values are expressed as mean standard deviation of 8 mice. *All animals were fasted overnight. .sup.ap < 0.01: comparison with sham control according to LSD test .sup.bp < 0.01: comparison with OVX control according to LSD test .sup.cp < 0.01 and .sup.dp < 0.05: comparison with red clover extract administered group according to LSD test .sup.ep < 0.01: comparison with pomegranate extract administered group according to LSD test .sup.fp < 0.01: comparison with sham control according to MW test .sup.gp < 0.01 and .sup.hp < 0.05: comparison with OVX control according to MW test .sup.ip < 0.01: comparison with red clover extract administered group according to MW test .sup.jp < 0.01: comparison with pomegranate extract administered group according to MW test RC:PCP = a mixture of red clover extract and pomegranate extract (g/g).
Example 3-3. Change in Food Consumption
(178) In OVX control, significant (p<0.01) increases in food consumption were found at all measurement dates from 1 day to 83 days after start of administration as compared to sham control, and when compared to OVX control, only estradiol administered group started to show significant (p<0.01 or p<0.05) decreases in food consumption from 7 days after start of administration, while changes in food consumption were not found in red clover extract and pomegranate extract single formula, and 9 types of red clover:pomegranate extract (g/g) mixed formula administered groups as compared to OVX control, and significant changes in food consumption were not found in the 9 types of red clover:pomegranate extract (g/g) mixed formula administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (Table 17).
(179) In the case of OVX control, the food consumption changed by 58.37, 75.12, 79.42, 72.71, 75.06 and 95.69% respectively 1, 3, 7, 28, 56 and 83 days after start of administration as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in food consumption by 0.87, 1.21, 4.52, 0.59, 2.78, 1.34, 0.93, 1.96, 2.36, 0.22, 0.70 and 0.46% respectively 1 day after start of administration as compared to OVX control, changes by 5.24, 0.92, 2.22, 1.19, 1.41, 2.02, 1.42, 0.48, 1.23, 1.25, 1.52 and 0.15% respectively 3 days after start of administration, changes by 13.04, 2.04, 2.05, 1.44, 0.37, 2.51, 0.66, 0.70, 2.27, 0.91, 0.22 and 0.68% respectively 7 days after start of administration, changes by 16.34, 2.35, 1.00, 0.79, 1.73, 4.05, 0.25, 0.48, 0.56, 0.34, 2.52 and 0.74% respectively 28 days after start of administration, changes by 20.01, 1.12, 0.59, 2.86, 1.89, 2.76, 1.35, 1.19, 0.75, 0.30, 2.73 and 0.46% respectively 56 days after start of administration, and changes by 27.82, 1.01, 0.57, 1.57, 0.24, 2.64, 4.01, 0.71, 1.08, 0.54, 0.85 and 0.95% respectively 83 days after start of administration.
(180) TABLE-US-00017 TABLE 17 Points Food consumption (g/24 hrs/rat): Days after initial treatment Groups 1 3 7 28 56 83 Controls Sham 7.20 0.72 6.35 0.67 6.29 0.72 6.59 0.77 7.31 0.90 7.54 0.78 OVX 11.40 1.44.sup.a 11.11 0.98.sup.a 11.29 1.03.sup.a 11.38 1.33.sup.a 12.80 1.41.sup.a 14.75 1.57.sup.d Estradiol 11.49 1.41.sup.a 10.53 0.93.sup.a 9.82 0.93.sup.ac 9.52 1.01.sup.ab 10.24 0.98.sup.ab 10.65 1.54.sup.de RC 11.26 1.18.sup.a 11.01 1.53.sup.a 11.06 1.06.sup.a 11.11 0.92.sup.a 12.66 1.62.sup.a 14.60 1.08.sup.d PCP 11.91 1.38.sup.a 11.36 1.67.sup.a 11.52 1.20.sup.a 11.26 1.31.sup.a 12.87 1.55.sup.a 14.67 1.34.sup.d RC:PCP 1:1 11.46 1.12.sup.a 10.98 1.34.sup.a 11.13 1.24.sup.a 11.29 1.31.sup.a 12.43 1.36.sup.a 14.52 1.40.sup.d 1:2 11.08 1.19.sup.a 10.96 1.25.sup.a 11.33 1.25.sup.a 11.57 1.19.sup.a 13.04 0.91.sup.a 14.79 1.35.sup.d 1:4 11.24 1.13.sup.a 11.34 1.11.sup.a 11.58 1.32.sup.a 11.84 1.44.sup.a 12.45 0.84.sup.a 14.38 1.68.sup.d 1:6 11.29 0.94.sup.a 11.27 1.00.sup.a 11.37 1.48.sup.a 11.40 1.62.sup.a 12.63 1.55.sup.a 14.16 2.87.sup.d 1:8 11.62 1.01.sup.a 11.17 1.40.sup.a 11.37 1.55.sup.a 11.43 1.35.sup.a 12.95 0.92.sup.a 14.65 2.04.sup.d 2:1 11.66 0.93.sup.a 11.25 0.95.sup.a 11.04 1.61.sup.a 11.31 1.14.sup.a 12.90 1.33.sup.a 14.59 1.62.sup.d 4:1 11.42 1.46.sup.a 11.25 0.99.sup.a 11.40 1.26.sup.a 11.34 1.26.sup.a 12.76 1.17.sup.a 14.83 1.23.sup.d 6:1 11.48 1.31.sup.a 10.94 0.91.sup.a 11.27 1.14.sup.a 11.66 1.16.sup.a 12.45 1.34.sup.a 14.63 1.61.sup.d 8:1 11.34 1.99.sup.a 11.13 1.13.sup.a 11.37 1.58.sup.a 11.46 1.25.sup.a 12.86 0.72.sup.a 14.89 1.17.sup.d Values are expressed as mean standard deviation of 8 mice. .sup.ap < 0.01: comparison with sham control according to LSD test .sup.bp < 0.01 and .sup.cp < 0.05: comparison with OVX control according to LSD test .sup.dp < 0.01: comparison with sham control according to MW test .sup.ep < 0.01: comparison with OVX control according to MW test
Example 3-4. Change in Abdominal Fat Pad Weight
(181) Significant (p<0.01) increases in absolute weight of abdominal fat pad and relative weight to body weight were each found in OVX control as compared to sham control, while significant (p<0.01) decreases in abdominal fat pad weight were found in all administered groups including estradiol as compared to OVX control, and particularly, significant (p<0.01 or p<0.05) decreases in abdominal fat pad weight were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (Table 18,
(182) In the case of OVX control, the absolute weight of abdominal fat pad changed by 2375.48% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 82.20, 50.38, 45.23, 73.29, 49.86, 54.48, 45.27, 48.56, 80.93, 70.89, 52.72 and 49.28% respectively as compared to OVX control.
(183) In the case of OVX control, the relative weight of abdominal fat pad to body weight changed by 1491.59% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 78.86, 41.76, 36.56, 65.51, 39.48, 45.64, 34.56, 38.46, 74.73, 62.83, 42.68 and 38.75% as compared to OVX control.
Example 3-5. Change in Uterus Weight
(184) Significant (p<0.01) decreases in absolute weight of uterus and relative weight to body weight were each found in OVX control as compared to sham control, while significant (p<0.01) increases in uterus weight were each found in all test substance administered groups including red clover extract and pomegranate extract single formula as compared to OVX control. Particularly, significant (p<0.01) increases in uterus weight were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (Table 18,
(185) In the case of OVX control, the absolute weight of uterus changed by 88.57% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 288.64, 47.73, 45.00, 90.45, 50.00, 42.73, 45.45, 44.55, 125.00, 90.00, 71.82 and 52.73% respectively as compared to OVX control.
(186) In the case of OVX control, the relative weight of uterus to body weight changed by 92.53% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 372.56, 74.57, 67.51, 145.55, 84.03, 72.76, 76.97, 73.48, 200.64, 142.46, 109.43 and 80.95% respectively as compared to OVX control.
Example 3-6. Change in Liver Weight
(187) Significant (p<0.01) decreases in relative weight of liver to body weight were found in OVX control as compared to sham control, while significant (p<0.01 or p<0.05) increases in relative liver weight were each found in all candidate substance administered groups including all 9 types of red clover:pomegranate extract mixed formula administered groups as compared to OVX control. Particularly, significant (p<0.01 or p<0.05) increases in relative liver weight were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups. On the other hand, when compared to sham control, significant changes in absolute liver weight were not found in all OVX control, and significant changes in absolute liver weight were not found in estradiol, red clover extract and pomegranate extract single formula, and 9 types of red clover:pomegranate extract mixed formula administered groups as compared to OVX control (Table 18).
(188) In the case of OVX control, the absolute liver weight changed by 4.28% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 5.37, 3.34, 4.95, 10.29, 1.80, 0.24, 1.10, 1.49, 12.28, 11.71, 1.66 and 0.54% respectively as compared to OVX control.
(189) In the case of OVX control, the relative weight of liver to body weight changed by 31.75% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 27.16, 21.96, 21.28, 42.26, 19.79, 21.18, 19.73, 22.57, 49.99, 42.54, 23.11 and 18.01% respectively as compared to OVX control.
(190) TABLE-US-00018 TABLE 18 Organs Absolute wet-weight (g) Relative wet-weight (% of body weight) Groups Abdominal fat pad Uterus Liver Abdominal fat pad Uterus Liver Controls Sham 0.136 0.107 0.241 0.082 1.205 0.281 0.492 0.390 0.858 0.286 4.291 0.819 OVX 3.370 0.467.sup.a 0.028 0.009.sup.a 1.257 0.174 7.835 0.814.sup.a 0.064 0.022.sup.a 2.929 0.369.sup.a Estradiol 0.600 0.536.sup.bc 0.107 0.040.sup.ac 1.324 0.073 1.656 1.432.sup.bc 0.303 0.124.sup.ac 3.724 0.307.sup.c RC 1.672 0.556.sup.ac 0.041 0.006.sup.ad 1.299 0.138 4.563 1.417.sup.ac 0.112 0.018.sup.ac 3.571 0.429.sup.c PCP 1.846 0.415.sup.ac 0.040 0.004.sup.ac 1.319 0.097 4.970 1.163.sup.ac 0.107 0.013.sup.ac 3.552 0.388.sup.c RC:PCP 1:1 0.900 0.289.sup.acfg 0.052 0.005.sup.aceg 1.386 0.095 2.702 0.855.sup.acfg 0.157 0.010.sup.aceg 4.166 0.165.sup.ceg 1:2 1.690 0.644.sup.ac 0.041 0.007.sup.ad 1.234 0.192 4.741 1.527.sup.ac 0.118 0.022.sup.ac 3.508 0.525.sup.bd 1:4 1.534 0.644.sup.ac 0.039 0.007.sup.ad 1.254 0.140 4.259 1.641.sup.ac 0.111 0.020.sup.ac 3.549 0.576.sup.d 1:6 1.844 0.561.sup.ac 0.040 0.005.sup.ad 1.234 0.121 5.127 1.277.sup.ac 0.113 0.021.sup.ac 3.506 0.420.sup.bd 1:8 1.734 0.515.sup.ac 0.040 0.006.sup.ad 1.275 0.131 4.822 1.219.sup.ac 0.111 0.014.sup.ac 3.590 0.487.sup.c 2:1 0.643 0.354.sup.aceg 0.062 0.010.sup.aceg 1.411 0.092.sup.f 1.980 1.067.sup.aceg 0.193 0.032.sup.aceg 4.392 0.428.sup.ceg 4:1 0.981 0.310.sup.aceg 0.052 0.010.sup.aceg 1.404 0.107.sup.f 2.912 0.890.sup.aceg 0.155 0.028.sup.aceg 4.174 0.286.sup.ceh 6:1 1.593 0.370.sup.ac 0.047 0.016.sup.ac 1.278 0.088 4.491 1.034.sup.ac 0.134 0.050.sup.ac 3.605 0.309.sup.bc 8:1 1.709 0.663.sup.ac 0.042 0.008.sup.ac 1.263 0.103 4.799 2.144.sup.ac 0.116 0.029.sup.ac 3.456 0.352.sup.bd Values are expressed as mean standard deviation of 8 mice. .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to MW test .sup.cp < 0.01 and .sup.dp < 0.05: comparison with OVX control according to MW test .sup.ep < 0.01 and .sup.fp < 0.05: comparison with red clover extract alone administered group according to MW test .sup.gp < 0.01 and .sup.hp < 0.05: comparison with pomegranate extract alone administered group according to MW test
Example 3-7. Change in Femur Weight
(191) Significant (p<0.01) decreases in relative wet weight of femur to body weight as well as decreases in absolute and relative weights of dry and ash bone were found in OVX control as compared to sham control, while remarkable increases in femur weight were each found in all test substance administered groups including estradiol as compared to OVX control. Particularly, significant (p<0.01 or p<0.05) increases in relative wet weight of femur and increases in absolute and relative weights of dry and ash bone were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (Table 19).
(192) In the case of OVX control, the femur absolute wet weight and relative wet weight to body weight changed 4.19 and 37.68% respectively as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in absolute wet weight by 5.79, 1.27, 2.68, 3.67, 0.85, 0.56, 2.82, 0.71, 4.94, 3.39, 0.56 and 0.58% respectively, and changes in relative wet weight by 27.75, 19.30, 18.35, 33.90, 23.11, 21.48, 23.97, 19.47, 39.91, 31.87, 21.50 and 18.54% respectively, as compared to OVX control.
(193) In the case of OVX control, the femur absolute dry weight and relative dry weight to body weight changed by 21.64 and 49.05% respectively as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in absolute dry weight by 14.93, 8.96, 8.46, 17.16, 10.70, 10.45, 12.69, 9.70, 20.65, 16.92, 10.70 and 9.95% respectively, and changes in relative dry weight by 38.22, 28.18, 24.70, 50.75, 35.13, 33.14, 35.43, 31.69, 60.65, 48.83, 33.29 and 29.00% respectively, as compared to OVX control.
(194) In the case of OVX control, the femur absolute ash weight and relative ash weight to body weight changed by 34.74 and 57.77% respectively as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in absolute ash weight by 30.35, 21.39, 19.40, 38.81, 23.88, 25.87, 23.88, 22.39, 51.24, 36.32, 24.38 and 21.39% respectively, and changes in relative ash weight by 57.03, 43.35, 37.53, 79.44, 51.03, 52.74, 49.80, 47.40, 102.33, 74.11, 50.56 and 43.57% respectively, as compared to OVX control.
(195) TABLE-US-00019 TABLE 19 Items Absolute weight (g) Relative weight (% of body weight) Groups Wet Dry Ash Wet Dry Ash Controls Sham 0.092 0.005 0.064 0.003 0.039 0.003 0.331 0.028 0.213 0.025 0.139 0.019 OVX 0.089 0.006 0.050 0.003.sup.g 0.025 0.003.sup.a 0.207 0.013.sup.a 0.118 0.010.sup.g 0.059 0.007.sup.a Estradiol 0.094 0.007 0.058 0.004.sup.gi 0.033 0.002.sup.ac 0.264 0.032.sup.ac 0.162 0.016.sup.gi 0.092 0.007.sup.ac RC 0.090 0.004 0.055 0.002.sup.gi 0.031 0.003.sup.ac 0.246 0.015.sup.ac 0.151 0.011.sup.gi 0.084 0.010.sup.ac PCP 0.091 0.005 0.055 0.002.sup.gi 0.030 0.003.sup.ac 0.244 0.019.sup.ac 0.147 0.010.sup.gi 0.081 0.008.sup.ac RC:PCP 1:1 0.092 0.003 0.059 0.003.sup.gikm 0.035 0.001.sup.bvdf 0.277 0.017.sup.acdf 0.177 0.006.sup.gikm 0.105 0.006.sup.acdf 1:2 0.089 0.005 0.056 0.004.sup.gi 0.031 0.004.sup.ac 0.254 0.021.sup.ac 0.159 0.018.sup.gi 0.088 0.009.sup.ac 1:4 0.089 0.005 0.056 0.003.sup.gi 0.032 0.004.sup.ac 0.251 0.022.sup.ac 0.156 0.014.sup.gi 0.089 0.016.sup.ac 1:6 0.091 0.006 0.057 0.004.sup.gi 0.031 0.004.sup.ac 0.256 0.015.sup.ac 0.159 0.008.sup.gin 0.088 0.012.sup.ac 1:8 0.088 0.007 0.055 0.005.sup.g 0.031 0.004.sup.ac 0.247 0.021.sup.ac 0.155 0.015.sup.gi 0.086 0.011.sup.ac 2:1 0.093 0.003 0.061 0.002.sup.hikm 0.038 0.002.sup.cdf 0.289 0.021.sup.acdf 0.189 0.016.sup.gikm 0.118 0.012.sup.acdf 4:1 0.092 0.005 0.059 0.003.sup.giln 0.034 0.003.sup.acef 0.272 0.016.sup.acef 0.175 0.012.sup.gikn 0.102 0.010.sup.acdf 6:1 0.089 0.004 0.056 0.005.sup.gi 0.031 0.003.sup.ac 0.251 0.012.sup.ac 0.157 0.011.sup.gi 0.088 0.009.sup.ac 8:1 0.089 0.007 0.055 0.005.sup.g 0.031 0.003.sup.ac 0.245 0.029.sup.ac 0.152 0.021.sup.gi 0.084 0.014.sup.ac Values are expressed as mean standard deviation of 8 mice. .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01: comparison with OVX control according to LSD test .sup.dp < 0.01 and .sup.ep < 0.05: comparison with red clover extract alone administered group according to LSD test .sup.fp < 0.01: comparison with pomegranate extract alone administered group according to LSD test .sup.gp < 0.01 and .sup.hp < 0.05: comparison with sham control according to MW test .sup.ip < 0.01 and .sup.jp < 0.05: comparison with OVX control according to MW test .sup.kp < 0.01 and .sup.lp < 0.05: comparison with red clover extract alone administered group according to MW test .sup.mp < 0.01 and .sup.np < 0.05: comparison with pomegranate extract alone administered group according to MW test
Example 3-8. Serum Biochemical Change (1): AST, ALT, TC, LDL, HDL and TG
(196) Significant (p<0.01) increases in serum Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total cholesterol (TC), Low density lipoprotein (LDL) and Triglyceride (TG) levels as well as significant (p<0.01) decreases in High density lipoprotein (HDL) level were found in OVX control as compared to sham control, while significant (p<0.01) decreases in serum AST, ALT, TC, LDL and TG levels and significant (p<0.01) increases in serum HDL level were each found in all test substance administered groups including red clover extract single formula as compared to OVX control. Particularly, significant (p<0.01 or p<0.05) decreases in serum AST, ALT, TC, LDL and TG levels and increases in serum HDL level were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (Table 20).
(197) In the case of OVX control, the serum AST and ALT levels changed by 92.30 and 100.66% respectively as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in serum AST level by 33.98, 13.94, 12.56, 26.73, 14.10, 13.71, 12.25, 12.17, 32.67, 25.96, 16.26 and 13.10% respectively, and changes in serum AST level by 31.58, 17.11, 16.28, 31.74, 16.78, 19.08, 18.82, 17.60, 37.17, 30.43, 19.74 and 18.26% respectively, as compared to OVX control.
(198) In the case of OVX control, the serum TC and LDL levels changed by 100.00 and 189.26% respectively as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in serum TC level by 25.94, 20.01, 17.85, 28.87, 20.64, 19.94, 18.27, 17.50, 36.33, 26.85, 21.27 and 19.46% respectively, and changes in serum LDL level by 26.87, 14.98, 11.96, 26.87, 14.57, 13.40, 12.92, 12.03, 36.49, 23.71, 15.40 and 15.05% respectively, as compared to OVX control.
(199) In the case of OVX control, the serum HDL and TG levels changed by 50.45 and 305.74% respectively as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in serum HDL level by 52.09, 37.17, 33.25, 60.99, 36.91, 32.98, 34.55, 34.03, 87.70, 59.42, 39.27 and 34.29% respectively, and changes in serum TG level by 34.80, 17.99, 17.07, 31.56, 19.48, 18.98, 18.48, 17.74, 43.55, 28.56, 18.65 and 18.23% respectively, as compared to OVX control.
(200) TABLE-US-00020 TABLE 20 Items Serum biochemistrical values Groups AST (U/L) ALT (U/L) TC (mg/dl) LDL (mg/dl) HDL (mg/dl) TG (mg/dl) Controls Sham 84.38 13.95 37.88 11.98 89.63 16.87 62.88 10.11 96.38 11.78 37.00 10.39 OVX 162.35 15.84.sup.a 76.00 10.54.sup.a 179.25 21.41.sup.a 181.88 17.94.sup.a 47.75 11.20.sup.a 150.13 20.90.sup.a Estradiol 107.13 12.43.sup.ac 52.00 10.03.sup.ac 132.75 21.06.sup.ac 133.00 10.54.sup.ac 72.63 11.04.sup.ac 97.88 18.16.sup.ac RC 139.63 8.86.sup.ac 63.00 5.66.sup.ac 143.38 10.80.sup.ac 154.63 13.05.sup.ac 65.50 5.10.sup.ac 123.13 9.11.sup.ac PCP 141.88 7.64.sup.ac 63.63 4.84.sup.ac 147.25 12.28.sup.ac 160.13 7.41.sup.ac 63.63 6.05.sup.ac 124.50 9.38.sup.ac RC:PCP 1:1 118.88 13.26.sup.acdf 51.88 7.26.sup.acef 127.50 10.85.sup.acef 133.00 13.60.sup.acdf 76.88 8.25.sup.acef 102.75 12.96.sup.acdf 1:2 139.38 10.72.sup.ac 63.25 7.46.sup.ac 142.25 10.35.sup.ac 155.38 16.23.sup.ac 65.38 9.55.sup.ac 120.88 14.80.sup.ac 1:4 140.00 10.00.sup.ac 61.50 11.33.sup.ac 143.50 12.36.sup.ac 157.50 11.63.sup.ac 63.50 6.59.sup.ac 121.63 11.59.sup.ac 1:6 142.38 10.49.sup.ac 62.00 6.39.sup.ac 146.50 8.25.sup.ac 158.38 12.44.sup.ac 64.25 10.58.sup.ac 122.38 14.04.sup.ac 1:8 142.50 9.44.sup.ac 62.63 6.72.sup.ac 147.88 13.88.sup.ac 160.00 9.96.sup.ac 64.00 9.70.sup.a 123.50 12.14.sup.ac 2:1 109.25 7.74.sup.acdf 47.75 11.31.sup.bcdf 114.13 12.93.sup.bcdf 115.50 11.15.sup.bcdf 89.63 7.13.sup.cdf 84.75 13.64.sup.cdf 4:1 120.13 15.07.sup.acdf 52.88 6.10.sup.aceg 131.13 8.24.sup.aceg 138.75 8.81.sup.acef 76.13 9.75.sup.acef 107.25 9.85.sup.acef 6:1 135.88 10.01.sup.ac 61.00 8.09.sup.ac 141.13 9.63.sup.ac 153.88 13.92.sup.ac 66.50 13.64.sup.ac 122.13 9.25.sup.ac 8:1 141.00 8.35.sup.ac 62.13 11.01.sup.ac 144.38 14.81.sup.ac 144.50 12.24.sup.ac 64.13 10.43.sup.ac 122.75 9.68.sup.ac Values are expressed as mean standard deviation of 8 mice. .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01: comparison with OVX control according to LSD test .sup.dp < 0.01 and .sup.ep < 0.05: comparison with red clover extract alone administered group according to LSD test fp < 0.01 and .sup.gp < 0.05: comparison with pomegranate extract alone administered group according to LSD test
Example 3-9. Serum Biochemical Change (2): Estradiol
(201) Significant (p<0.01) decreases in serum estradiol level were found in OVX control as compared to sham control, while significant (p<0.01) increases in serum estradiol level were each found in all test substance administered groups including pomegranate extract single formula as compared to OVX control. Particularly, significant (p<0.01) increases in serum estradiol level were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (
(202) In the case of OVX control, the serum estradiol level changed by 72.46% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 185.69, 37.14, 30.62, 84.78, 37.86, 36.23, 35.87, 31.34, 116.30, 83.70, 49.64 and 44.02% respectively as compared to OVX control.
Example 3-10. Serum Biochemical Change (3): Osteocalcin and bALP
(203) Significant (p<0.01) increases in serum osteocalcin level as well as significant (p<0.01) decreases in bALP level were found in OVX control as compared to sham control, while significant (p<0.01 or p<0.05) decreases in serum osteocalcin level and increases in serum bALP level were each found in all candidate substance administered groups including red clover extract and pomegranate extract single formula as compared to OVX control. Particularly, significant (p<0.01) decreases in serum osteocalcin level and increases in serum bALP level were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (
(204) In the case of OVX control, the serum osteocalcin and bALP levels changed by 77.11 and 42.35% respectively as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in serum osteocalcin level by 31.95, 18.80, 17.20, 31.43, 19.54, 19.35, 17.27, 16.83, 34.29, 30.05, 20.62 and 18.39% respectively, and change in serum bALP level by 39.72, 22.04, 17.04, 36.32, 22.87, 19.17, 18.92, 17.31, 49.67, 34.38, 24.84 and 22.84% respectively, as compared to OVX control.
Example 3-11. Chaste in Bone Mineral Density
(205) OVX control significantly (p<0.01) decreased in each of the mean total body bone mineral density and femur total bone mineral density as compared to sham control, while significant (p<0.01) increases in total body bone mineral density and total femur bone mineral density were found in all test substance administered groups including all 9 types of red clover:pomegranate extract mixed formulas as compared to OVX control. Particularly, significant (p<0.01) increases in mean total body bone mineral density and total femur bone mineral density were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (Table 21,
(206) In the case of OVX control, the mean total body bone mineral density changed by 12.75% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 9.86, 4.41, 3.48, 8.00, 5.10, 4.29, 4.00, 3.77, 10.84, 7.42, 5.57 and 5.16% respectively as compared to OVX control.
(207) In the case of OVX control, the total femur bone mineral density changed by 12.56% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 10.78, 5.23, 5.18, 9.20, 6.73, 5.93, 5.61, 5.39, 11.53, 8.97, 7.42 and 5.02% respectively as compared to OVX control.
Example 3-12. Change in Amounts of Body Fat and Abdominal Fat
(208) Significant (p<0.01) increases in amounts of body fat and abdominal fat were found in OVX control as compared to sham control, while significant (p<0.01) decreases in amounts of body fat and abdominal fat were each found in all test substance administered groups including estradiol as compared to OVX control. Particularly, significant (p<0.01 or p<0.05) decreases in amounts of body fat and abdominal fat were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (Table 21).
(209) In the case of OVX control, the amount of body fat changed by 213.34% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 33.02, 15.21, 13.20, 31.06, 20.16, 15.67, 14.24, 13.90, 46.56, 27.58, 18.22 and 15.32% respectively as compared to OVX control.
(210) In the case of OVX control, the amount of abdominal fat changed by 279.17% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 35.00, 17.21, 15.43, 35.30, 18.61, 17.54, 16.07, 15.42, 43.69, 32.08, 19.54 and 17.66% respectively as compared to OVX control.
(211) TABLE-US-00021 TABLE 21 Items Bone mineral density (g/cm.sup.2) Fat density (% of body mass) Groups Total body Right femur Total body Abdominal cavity Controls Sham 0.0247 0.0006.sup. 0.0268 0.0006.sup. 11.16 2.15.sup. 10.85 1.50.sup. OVX 0.0216 0.0003.sup.a 0.0234 0.0004.sup.a 34.97 3.52.sup.a 41.15 4.55.sup.a Estradiol 0.0237 0.0004.sup.ac 0.0259 0.0008.sup.bc 23.42 4.24.sup.ac 26.75 4.22.sup.ac RC 0.0225 0.0005.sup.ac 0.0246 0.0004.sup.ac 29.65 1.99.sup.ac 34.06 4.04.sup.ac PCP 0.0223 0.0004.sup.ac 0.0246 0.0003.sup.ac 30.35 2.21.sup.ac 34.80 2.81.sup.ac RC:PCP 1:1 .sup.0.0233 0.0005.sup.acdf .sup.0.0256 0.0007.sup.aceg .sup.24.11 4.25.sup.acdf .sup.26.62 4.66.sup.acdf 1:2 0.0227 0.0005.sup.ac 0.0250 0.0011.sup.ac 27.92 5.30.sup.ac 33.49 4.25.sup.ac 1:4 0.0225 0.0003.sup.ac 0.0248 0.0007.sup.ac 29.49 2.00.sup.ac 33.93 4.26.sup.ac 1:6 0.0224 0.0004.sup.ac 0.0247 0.0006.sup.ac 29.99 3.28.sup.ac 34.53 4.18.sup.ac 1:8 0.0224 0.0006.sup.ac 0.0247 0.0009.sup.ac 30.10 2.58.sup.ac 34.80 3.48.sup.ac 2:1 .sup.0.0239 0.0005.sup.acdf 0.0261 0.0009.sup.cdf .sup.18.68 3.99.sup.acdf .sup.23.17 3.39.sup.acdf 4:1 0.0232 0.0004.sup.acef .sup.0.0255 0.0007.sup.aceg 25.32 3.08.sup.acef .sup.27.94 3.74.sup.acdf 6:1 0.0228 0.0007.sup.ac 0.0252 0.0009.sup.ac 28.59 2.77.sup.ac 33.11 4.36.sup.ac 8:1 0.0227 0.0008.sup.ac 0.0246 0.0007.sup.ac 29.61 2.64.sup.ac 33.88 3.55.sup.ac Values are expressed as mean standard deviation of 8 mice. .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01: comparison with OVX control according to LSD test .sup.dp < 0.01 and .sup.ep < 0.05: comparison with red clover extract alone administered group according to LSD test .sup.fp < 0.01 and .sup.gp < 0.05: comparison with pomegranate extract alone administered group according to LSD test
Example 3-13. Change in Bone Strength
(212) OVX control significantly (p<0.01) decreased in bone strength of femur mid-shaft region as compared to sham control, while significant (p<0.01 or p<0.05) increases in femur strength were each found in all candidate substance administered groups including red clover extract single formula as compared to OVX control. Particularly, significant (p<0.01 or p<0.05) increases in femur strength were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (
(213) In the case of OVX control, the bone strength of femur mid-shaft region changed by 51.62% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 61.41, 35.17, 32.31, 59.11, 39.58, 44.06, 40.40, 40.85, 78.65, 56.44, 33.33 and 32.61% respectively as compared to OVX control.
Example 3-14. Histological Change (1): Abdominal Fat Pad, Uterus and Liver
(214) (1) Abdominal Fat Pad
(215) Due to remarkable abdominal fat deposition, significant (p<0.01) increases in abdominal fat pad thickness and mean adipocyte diameter were each found in OVX control as compared to sham control, while significant (p<0.01) decreases in abdominal fat pad thickness and mean adipocyte diameter were each found in all test substance administered groups including pomegranate extract single formula as compared to OVX control. Particularly, significant (p<0.01 or p<0.05) decreases in abdominal fat pad thickness and mean adipocyte diameter were each found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (
(216) In the case of OVX control, the abdominal fat pad thickness changed by 329.04% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 52.23, 25.90, 22.25, 40.72, 23.97, 24.59, 24.45, 22.71, 55.21, 38.83, 26.54 and 25.98% respectively as compared to OVX control.
(217) In the case of OVX control, the mean adipocyte diameter changed by 270.68% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 48.06, 28.54, 25.14, 57.23, 31.57, 31.50, 27.72, 26.29, 63.70, 51.62, 28.55 and 27.24% respectively as compared to OVX control.
(218) (2) Uterus
(219) Disuse atrophy contributing to remarkable decreases in uterine mucosa occurred in OVX control, and significant (p<0.01) decreases in total, epithelium and mucosa thickness of uterus and percentage of uterine glands in the mucosa were each found in OVX control as compared to sham control, while significant (p<0.01 or p<0.05) increases in total, epithelium and mucosa thickness of uterus and percentage of uterine glands in the mucosa were each found in all candidate substance administered groups including 9 types of red clover:pomegranate extract mixed formulas as compared to OVX control, and particularly, significant (p<0.01 or p<0.05) increases in total, epithelium and mucosa thickness of uterus and percentage of uterine glands in the mucosa were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (Table 22,
(220) The mean thickness of uterus total and epithelium changed by 73.99 and 77.79% respectively in OVX control as compared to normal control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in uterus total mean thickness by 181.27, 37.05, 30.46, 77.23, 39.43, 37.12, 33.70, 31.79, 98.58, 61.95, 40.74 and 40.64% respectively, and changes in uterine epithelium mean thickness by 170.17, 51.29, 44.04, 133.75, 71.36, 62.15, 61.74, 46.04, 205.44, 119.58, 83.78 and 57.71% respectively, as compared to OVX control.
(221) The mucosa mean thickness of uterus and percentage of uterine glands in the mucosa changed by 78.74 and 76.90% respectively in OVX control as compared to normal control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in uterine mucosa mean thickness by 196.04, 46.06, 39.48, 102.37, 55.72, 47.16, 44.15, 41.20, 125.29, 84.08, 53.63 and 53.00% respectively, and changes in percentage of uterine glands in the uterine mucosa by 180.37, 92.88, 80.37, 164.14, 109.49, 91.62, 84.40, 80.88, 186.05, 159.76, 112.69 and 98.34% respectively, as compared to OVX control.
(222) TABLE-US-00022 TABLE 22 Items Left uterine horn tissues Total thickness Epithelium Mucosa layer Uterine gland Groups (m) thickness (m) thickness (m) regions (%) Controls Sham 2177.82 573.19.sup. 34.58 6.17.sup. 951.22 242.86.sup. 53.94 12.02.sup. OVX .sup.566.47 128.78.sup.a .sup.7.68 1.26.sup.a 202.25 46.58.sup.a 12.46 3.07.sup.a Estradiol 1593.32 371.85.sup.bc 20.75 4.52.sup.ac 598.74 153.51.sup.ac 34.93 4.60.sup.ac RC 776.35 97.07.sup.ac 11.62 3.02.sup.ac 295.40 45.06.sup.ac 24.03 6.13.sup.ac PCP 739.04 84.66.sup.ad 11.06 2.72.sup.ac 282.10 37.26.sup.ac 22.47 4.28.sup.ac RC:PCP 1:1 .sup.1003.98 173.11.sup.aceg .sup.17.96 4.26.sup.aceg .sup.409.30 31.93.sup.aceg .sup.32.91 3.70.sup.aceg 1:2 789.85 116.09.sup.ad 13.16 4.17.sup.ac 314.95 79.42.sup.ac 26.10 6.70.sup.ac 1:4 776.76 98.02.sup.ac 12.46 3.50.sup.ac 297.63 45.24.sup.ac 23.88 3.85.sup.ac 1:6 757.35 66.86.sup.ac 12.42 4.31.sup.ad 291.55 34.89.sup.ac 22.98 5.17.sup.ac 1:8 746.54 64.96.sup.ac 11.22 2.41.sup.ac 285.57 40.39.sup.ac 22.54 4.56.sup.ac 2:1 .sup.1124.93 125.59.sup.aceg .sup.23.46 4.93.sup.aceg .sup.455.64 92.39.sup.aceg .sup.35.65 6.07.sup.aceg 4:1 .sup.917.42 54.64.sup.aceg .sup.16.87 4.11.sup.aceg .sup.372.29 44.26.sup.aceg .sup.32.37 4.67.sup.acfg 6:1 797.22 129.15.sup.ac 14.12 4.61.sup.ac 310.71 36.93.sup.ac 26.50 5.18.sup.ac 8:1 796.67 146.28.sup.ac 12.11 3.99.sup.ad 309.44 44.82.sup.ac 24.71 4.22.sup.ac .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to MW test .sup.cp < 0.01 and .sup.dp < 0.05: comparison with OVX control according to MW test .sup.ep < 0.01 and .sup.fp < 0.05: comparison with red clover extract alone administered group according to MW test .sup.gp < 0.01: comparison with pomegranate extract alone administered group according to MW test
(223) (3) Liver
(224) Hepatic steatosis characterized by hypertrophy caused by remarkable fat deposition in hepatocytes occurred in OVX control, and significant (p<0.01) increases in region with hepatic fatty degeneration and mean hepatocyte diameter were each found in OVX control as compared to sham control, while significant (p<0.01) decreases in hepatic steatosis were each found in all test substance administered groups including estradiol as compared to OVX control, and particularly, significant (p<0.01) decreases in region with hepatic fatty degeneration and mean hepatocyte diameter were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (Table 23,
(225) Region with hepatic fatty degeneration and mean hepatocyte diameter changed by 500.66 and 224.65% respectively in OVX control as compared to normal control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in region with hepatic fatty degeneration by 44.73, 24.38, 21.08, 43.20, 25.72, 23.91, 23.20, 22.21, 51.85, 41.47, 27.38 and 23.67% respectively, and changes in mean hepatocyte diameter by 42.21, 26.50, 25.88, 49.87, 30.22, 30.02, 29.80, 25.35, 55.39, 45.10, 27.18 and 26.68% respectively, as compared to OVX control.
(226) TABLE-US-00023 TABLE 23 Items Abdominal fat pads Hepatic tissues Total thickness Mean adipocyte Steatosis regions Mean hepatocyte Groups (m) diameters (m) (%) diameters (m) Controls Sham 1.41 0.45.sup. 35.05 10.25.sup. 13.08 3.16.sup. 9.83 3.34.sup. OVX 6.04 0.75.sup.a 129.93 24.06.sup.a 78.58 6.76.sup.a 31.93 4.92.sup.a Estradiol 2.89 0.27.sup.ac 67.49 14.77.sup.ac 43.43 6.53.sup.ac 18.45 3.66.sup.ac RC 4.48 0.51.sup.ac 92.85 12.70.sup.ac 59.42 7.08.sup.ac 23.47 3.83.sup.ac PCP 4.70 0.63.sup.ac 97.27 15.68.sup.ac 62.01 7.82.sup.ac 23.66 3.59.sup.ac RC:PCP 1:1 .sup.3.58 0.49.sup.acdf .sup.55.57 12.55.sup.acdf .sup.44.63 6.94.sup.acdf .sup.16.00 3.73.sup.acdf 1:2 4.59 0.45.sup.ac 88.91 15.64.sup.ac 58.37 7.54.sup.ac 22.28 4.15.sup.ac 1:4 4.55 0.65.sup.ac 89.01 13.89.sup.ac 59.80 6.51.sup.ac 22.34 2.75.sup.ac 1:6 4.56 0.59.sup.ac 93.91 15.69.sup.ac 60.35 8.89.sup.ac 22.41 3.35.sup.ac 1:8 4.67 0.61.sup.ac 95.77 18.09.sup.ac 61.13 10.91.sup.ac 23.83 2.67.sup.ac 2:1 .sup.2.71 0.93.sup.acdf 47.17 10.84.sup.cdf .sup.37.84 9.05.sup.acdf .sup.14.24 4.07.sup.bcdf 4:1 3.69 0.50.sup.acef .sup.62.87 14.30.sup.acdf .sup.45.99 8.44.sup.acdf .sup.17.53 3.43.sup.acdf 6:1 4.44 0.53.sup.ac 92.84 14.14.sup.ac 57.06 7.96.sup.ac 23.25 4.91.sup.ac 8:1 4.47 0.75.sup.ac 94.54 14.59.sup.ac 59.98 7.88.sup.ac 23.41 4.31.sup.ac Values are expressed as mean standard deviation of 8 mice. .sup.ap < 0.01 and .sup.bp < 0.05: comparison with sham control according to LSD test .sup.cp < 0.01: comparison with OVX control according to LSD test .sup.dp < 0.01 and .sup.ep < 0.05: comparison with red clover extract alone administered group according to LSD test .sup.fp < 0.01: comparison with pomegranate extract alone administered group according to LSD test
Example 3-15. Histological Change (2): Femur
(227) Trabecular bone and cortical bone developed relatively well were observed in femur of sham operated group, while decreases in trabecular bone and cortical bone mass caused by remarkable increases in number and activity of osteoclast cells were found in OVX control, but remarkable increases in bone mass and thickness of trabecular bone and cortical bone caused by osteoclast cell activity inhibition of femur were found in all test substance administered groups including red clover extract single formula, and particularly, remarkable osteoclast cell activity inhibition and bone mass preservation effects were provided in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups (Tables 24, 25 and
(228) (1) Bone Mass and Structure
(229) OVX control significantly (p<0.01) decreased in each of the trabecular bone mass, mean trabecular bone length, thickness and number, and cortical bone thickness of mid-shaft region of femur as compared to sham control, while significant (p<0.01 or p<0.05) increases in femur trabecular bone mass, mean trabecular bone length, thickness and number, cortical bone thickness were found in all candidate substance administered groups including pomegranate extract single formula as compared to OVX control, and particularly, significant (p<0.01) increases in femur trabecular bone mass, mean trabecular bone length, thickness and number, and cortical bone thickness were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each red clover extract and pomegranate extract single formula administered groups.
(230) The trabecular bone mass and mean trabecular bone number of femur changed by 53.66 and 59.41% respectively in OVX control as compared to normal control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in trabecular bone mass by 73.01, 32.47, 25.54, 59.92, 31.08, 30.51, 26.55, 26.42, 81.75, 58.54, 33.70 and 30.75% respectively, and changes in mean trabecular bone number by 92.68, 46.34, 36.59, 102.44, 46.34, 43.90, 36.59, 34.15, 139.02, 97.56, 53.66 and 46.34% respectively, as compared to OVX control.
(231) The mean length and thickness of femur trabecular bone changed by 53.47 and 55.82% respectively in OVX control as compared to normal control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in mean length of trabecular bone by 89.04, 23.62, 20.88, 79.43, 27.47, 26.57, 25.18, 21.16, 99.20, 72.68, 27.65 and 24.04% respectively, and changes in mean thickness of trabecular bone by 70.56, 35.69, 33.29, 104.28, 44.40, 43.67, 34.65, 34.98, 129.10, 101.26, 40.19 and 37.58% respectively, as compared to OVX control.
(232) In the case of OVX control, the femur cortical bone thickness changed by 27.80% as compared to sham control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes by 21.85, 16.11, 13.83, 25.23, 18.97, 16.70, 15.84, 15.01, 30.43, 24.33, 20.10 and 14.00% respectively as compared to OVX control.
(233) TABLE-US-00024 TABLE 24 Items Left femur tissues Groups TV/BV Tbn Tbl Tbt Controls Sham 37.28 5.04.sup. 12.63 1.69.sup. 1029.36 145.55.sup. 73.44 11.98 OVX 17.28 2.91.sup.a 5.13 0.64.sup.e 478.99 80.94.sup.e 32.45 7.07.sup.e Estradiol 29.89 5.24.sup.ab 9.88 1.36.sup.eg .sup.905.51 146.34.sup.g 55.34 8.99.sup.eg RC 22.89 2.05.sup.ab 7.50 0.93.sup.eg 592.13 35.08.sup.eg 44.03 3.97.sup.eg PCP 21.69 1.85.sup.ab 7.00 1.20.sup.eg 578.99 39.41.sup.eg 43.25 2.49.sup.eg RC:PCP 1:1 .sup.27.63 2.47.sup.abcd 10.38 1.30.sup.fgij .sup.859.44 173.38.sup.egij 66.28 14.40.sup.gij 1:2 22.65 2.58.sup.ab 7.50 0.76.sup.eg 610.56 60.98.sup.eg 46.85 8.26.sup.eg 1:4 22.55 2.72.sup.ab 7.38 0.92.sup.eg 606.29 36.21.sup.eg 46.62 8.71.sup.eh 1:6 21.86 2.30.sup.ab 7.00 + 1.07.sup.eg 599.59 55.83.sup.eg 43.69 3.85.sup.eg 1:8 21.84 1.57.sup.ab 6.88 0.83.sup.eg 580.37 66.93.sup.eh 43.80 5.38.sup.eg 2:1 .sup.31.40 4.14.sup.abcd 12.25 1.04.sup.egij 954.18 140.48.sup.gij 74.33 14.63.sup.gij 4:1 .sup.27.39 3.68.sup.abcd 10.13 1.89.sup.fgij .sup.827.11 142.28.sup.fgij 65.30 11.84.sup.gij 6:1 23.10 2.83.sup.ab 7.88 0.83.sup.eg 611.46 38.35.sup.eg 45.49 5.74.sup.eg 8:1 22.59 3.67.sup.ab 7.50 0.93.sup.eg 594.17 49.46.sup.eg 44.64 3.53.sup.eg Values are expressed as mean standard deviation of 8 mice. .sup.ap < 0.01: comparison with sham control according to LSD test .sup.bp < 0.01: comparison with OVX control according to LSD test .sup.cp < 0.01: comparison with red clover extract alone administered group according to LSD test .sup.dp < 0.01: comparison with pomegranate extract alone administered group according to LSD test .sup.ep < 0.01 and .sup.fp < 0.05: comparison with sham control according to MW test .sup.gp < 0.01 and .sup.hp < 0.05: comparison with OVX control according to MW test .sup.ip < 0.01: comparison with red clover extract alone administered group according to MW test .sup.jp < 0.01: comparison with pomegranate extract alone administered group according to MW test
(234) (2) Bone Resorption
(235) In the case of OVX control, the osteoclast cell number and ratio (OS/BS) of femur significantly (p<0.01) increased as compared to sham control, while significant (p<0.01 or p<0.05) decreases in femur osteoclast cell number and OS/BS were each found in all test substance administered groups including 9 types of red clover:pomegranate extract mixed formulas as compared to OVX control, and particularly, significant (p<0.01) decreases in femur osteoclast cell number and OS/BS were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups.
(236) In OVX control, the osteoclast cell number and OS/BS of femur changed by 209.09 and 146.31% respectively as compared to normal control, and estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula administered groups had changes in osteoclast cell number by 55.15, 19.12, 16.91, 46.32, 23.53, 20.59, 19.85, 18.38, 58.82, 44.85, 21.32 and 19.85% respectively, and changes in OS/BS by 37.50, 18.29, 15.47, 35.88, 20.81, 18.67, 15.92, 20.12, 45.43, 35.05, 17.87 and 15.10% respectively, as compared to OVX control.
(237) TABLE-US-00025 TABLE 25 Items Left femur tissues Groups Cbt Ocn OS/BS Controls Sham 209.33 18.14 5.50 0.93 8.52 1.57 OVX .sup.151.14 11.14.sup.e 17.00 2.00.sup.a 21.00 2.39.sup.e Estradiol 184.17 8.40.sup.eg 7.63 1.06.sup.ab 13.12 1.41.sup.eg RC 175.49 4.41.sup.eg 13.75 1.49.sup.ac 17.16 1.89.sup.eg PCP 172.05 8.17.sup.eg 14.13 1.64.sup.ac 17.75 1.46.sup.eg RC:PCP 1:1 189.27 7.10.sup.fgij .sup.9.13 1.13.sup.abcd 13.46 1.88.sup.egij 1:2 179.81 8.89.sup.eg 13.00 1.85.sup.ab 16.63 1.74.sup.eg 1:4 176.39 9.83.sup.eg 13.50 1.51.sup.ab 17.08 1.65.sup.eg 1:6 175.08 10.48.sup.eg 13.63 1.51.sup.ab 17.65 1.26.sup.eg 1:8 173.84 9.20.sup.eg 13.88 1.89.sup.ab 16.77 3.23.sup.eh 2:1 197.13 9.00.sup.gij .sup.7.00 0.76.sup.bcd 11.46 1.37.sup.egij 4:1 .sup.187.92 10.08.sup.fgij .sup.9.38 1.30.sup.abcd 13.64 1.99.sup.egij 6:1 181.52 12.63.sup.eg 13.38 1.69.sup.ab 17.24 1.48.sup.eg 8:1 172.30 6.68.sup.eg 13.63 2.33.sup.ab 17.82 0.98.sup.eg Values are expressed as mean standard deviation of 8 mice. .sup.ap < 0.01: comparison with sham control according to LSD test .sup.bp < 0.01: comparison with OVX control according to LSD test .sup.cp < 0.01: comparison with red clover extract alone administered group according to LSD test .sup.dp < 0.01 comparison with pomegranate extract alone administered group according to LSD test .sup.ep < 0.01 and .sup.fp < 0.05: comparison with sham control according to MW test .sup.gp < 0.01 and .sup.hp < 0.05: comparison with OVX control according to MW test .sup.ip < 0.01: comparison with red clover extract alone administered group according to MW test .sup.jp < 0.01: comparison with pomegranate extract alone administered group according to MW test
Summary of Example 3
(238) In the study, optimal mixture formula of red clover extract and pomegranate extract was evaluated using OVX ddY mouse model being currently used as experimental animal model for various menopausal disorders including osteoporosis [Yamaguchi et al., 1999; Ku and Lee, 2005; Han et al., 2007; Shin et al., 2007]. That is, from 28 days after OVX, red clover extract and pomegranate extract single formula, and 9 types of red clover:pomegranate extract mixed formulas (1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1, g/g) 120 mg/kg was dissolved in or diluted with sterile distilled water and orally administered at a dose of 10 ml/kg once daily for 84 days, and evaluation was based on 5 pharmacological effects including an estrogen-like effect, an anti-obesity effect, a hyperlipidemia inhibitory effect, a protective effect against fatty liver and an osteoporosis inhibitory effect. To evaluate the estrogen-like effect and anti-obesity effect, changes in body weight and body weight gain, water and food consumption, amounts of body fat and abdominal fat, serum estradiol level, and weights of abdominal fat pad and uterus were evaluated together with histological changes such as abdominal fat pad thickness and mean adipocyte diameter, thickness of uterus total, epithelium and mucosa, and percentage of uterine glands in the mucosa, and the protective effect against fatty liver was evaluated by measuring liver weight, serum AST and ALT levels and histological change (mean hepatocyte diameter and region with hepatic degeneration showing fatty change), the hyperlipidemia alleviation effect was evaluated using changes in serum TC, LDL, HDL and TG levels, and to evaluate the osteoporosis reduction effect, namely, the bone protective effect, histological changes in wet, dry and ash weights of femur, BMD, bone strength, serum osteocalcin and bALP levels, femur bone mass and structure, and bone resorption were each measured. In the experiment, mixed formula showing statistically significant (p<0.01 or p<0.05) increases in efficacy at the same time when compared to each of red clover extract and pomegranate extract single formula was determined as red clover:pomegranate extract mixed formula showing a synergistic effect, and evaluation was conducted by comparing to results in 17-estradiol 0.03 g/head/day subcutaneously administered mice [Chiba et al., 2003; Murakami et al., 2007; Tousen et al., 2011].
(239) The administration dose of mixed formula used in the experiment was set higher 12 times than a human clinical dose of 600 mg, i.e., 600 mg/60 kg)12=120 mg/kg, in consideration of a body surface area of mouse, and the dose of each of red clover extract and pomegranate extract single formula was also set as 120 mg/kg for direct comparative evaluation of efficacy, and according to earlier researchers' method [Chiba et al., 2003; Murakami et al., 2007; Tousen et al., 2011], 0.03 g of 17-estradiol was dissolved in 0.2 ml of sterile saline and subcutaneously administered to back skin at a dose of 0.2 ml/mouse (0.03 g/head/day) once daily for 84 days from 28 days after OVX surgery.
(240) As a result of the experiment, remarkable increases in body weight and body weight gain, food consumption, amounts of body fat and abdominal fat, abdominal fat pad weight, serum AST, ALT, TC, LDL, TG and osteocalcin levels, and decreases in uterus, liver and femur weights, serum bALP and estradiol levels, mean total body bone mineral density and femur bone mineral density were found in OVX control as compared to sham vehicle control, and indication of remarkable increases in abdominal fat pad thickness and hypertrophy of adipocytes, hepatic steatosis, disuse atrophy of uterus, and decreases in bone mass of femur was each histologically detected together with remarkable increases of bone resorption marker (Ocn and OS/BS), so typical estrogen deficient menopausal disorders (obesity, hyperlipidemia, hepatic steatosis and osteoporosis) were induced by OVX. On the other hand, signs of menopausal disorders related to estrogen deficient menopause induced by OVX were remarkably inhibited by continuous administration of estradiol, red clover extract and pomegranate extract single formula, and all 9 types of red clover:pomegranate extract mixed formulas for 84 days, and particularly, a significant effect on reduction of on estrogen deficient menopausal disorders induced by OVX such as obesity, hyperlipidemia, hepatic steatosis and osteoporosis was provided in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups. Accordingly, it was observed that red clover:pomegranate extract mixture formula at an optimal ratiored clover:pomegranate extract 2:1, 1:1 and 4:1 even synergistically increased an effect of red clover extract and pomegranate extract on reduction of OVX induced menopausal disorders through increased diversity of isoflavonoids contained, and among them, it was observed that red clover:pomegranate extract 2:1 mixed formula synergistically increased a best menopausal disorder reduction effect, but the present disclosure is not limited thereto.
(241) In the study, food consumption remarkably increased due to OVX, resulting in remarkable increases in body weight as well as fat deposition and hypertrophy of adipocytes. On the other hand, signs of estrogen deficient obesity induced by OVX were remarkably inhibited by continuous administration of each of red clover extract and pomegranate extract single formula, and all 9 types of red clover:pomegranate extract mixed formulas for 84 days, and particularly, red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered group in a sequential order showed presumably significant inhibition of body fat deposition, adipocyte hypertrophy and body weight increase as compared to each of red clover extract and pomegranate extract single formula administered groups. These results serve as an evidence demonstrating that red clover:pomegranate extract mixed formula at an optimal ratiored clover:pomegranate extract 2:1, 1:1 and 4:1 synergistically increases an effect of red clover extract and pomegranate extract on reduction of obesity related to menopausal disorders, and among them, it was observed that red clover:pomegranate extract 2:1 (g/g) mixed formula showed a best anti-obesity effect, but the present disclosure is not limited thereto.
(242) In the study, due to OVX, significant decreases in uterus weight and significant decreases in serum estradiol level occurred, and histologically remarkable decreases in uterus total, mucosa and epithelium thickness and decreases in uterine glands in the mucosa occurred. On the other hand, signs of estrogen deficient uterus atrophy were remarkably inhibited by administration of estradiol, red clover extract and pomegranate extract single formula, red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula, and particularly, red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered group in a sequential order showed significant increases in serum estradiol level and presumable inhibition of uterus atrophy as compared to each of red clover extract and pomegranate extract single formula administered groups, which serves as an evidence demonstrating that red clover:pomegranate extract mixed formula at an optimal ratiored clover:pomegranate extract 2:1, 1:1 and 4:1 synergistically increases an estrogenic effect of red clover extract and pomegranate extract through increased diversity of isoflavonoids contained, and among them, it was observed that red clover:pomegranate extract 2:1 (g/g) mixed formula showed a best estrogenic effect, but the present disclosure is not limited thereto.
(243) In the study, the serum TC, LDL and TG levels were remarkably decreased and the HDL level was increased by subcutaneous administration of estradiol and oral administration of all red clover extract and pomegranate extract single or mixed formula, and particularly, significant increases in anti-hyperlipidemia effect were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups, which serves as an evidence demonstrating that red clover:pomegranate extract mixed formula at an optimal ratiored clover:pomegranate extract 2:1, 1:1 and 4:1 synergistically increases an anti-hyperlipidemia effect of red clover extract and pomegranate extract at least under the experimental conditions via HMG-CoA, the enzyme involved in cholesterol synthesis [Di Croce et al., 1996], and among them, it was observed red clover:pomegranate extract 2:1 (g/g) mixed formula showed a best anti-hyperlipidemia effect, but the present disclosure is not limited thereto.
(244) In the study, remarkable hepatic steatosis in OVX control was bio-chemically and histologically ascertained. On the other hand, signs of fatty liver induced by OVX were remarkably inhibited by administration of estradiol, red clover extract and pomegranate extract single formula, and 9 types of red clover:pomegranate extract mixed formulas, and particularly, significant increases in liver protective effect were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups, which serves as an evidence demonstrating that red clover:pomegranate extract mixed formula at an optimal ratio synergistically increases a liver protective effect of red clover extract and pomegranate extract, and among them, it was observed that red clover:pomegranate extract 2:1 (g/g) mixed formula showed a best liver protective effect, but the present disclosure is not limited thereto.
(245) As a result of the experiment, remarkable increases in serum osteocalcin level and decreases in femur weight and serum bALP level were found in OVX control as compared to sham vehicle control, and indication of decreases in femur bone mass were histologically ascertained together with remarkable increases of bone resorption marker (Ocn and OS/BS), so it was observed that estrogen deficient osteoporosis was induced by OVX. On the other hand, signs of estrogen deficient osteoporosis were remarkably inhibited by administration of estradiol, red clover extract and pomegranate extract single formula, and red clover:pomegranate extract 1:1, 1:2, 1:4, 1:6, 1:8, 2:1, 4:1, 6:1 and 8:1 (g/g) mixed formula, and particularly, significant increases in effect on reduction of estrogen deficient osteoporosis were found in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups, which serves as an evidence demonstrating that red clover:pomegranate extract mixed formula at an optimal ratiored clover:pomegranate extract 2:1, 1:1 and 4:1 synergistically increases an effect of red clover extract and pomegranate extract on reduction of estrogen deficient osteoporosis through increased diversity of isoflavonoids contained, and among them, it was observed that red clover:pomegranate extract 2:1 (g/g) mixed formula showed a best reduction effect for estrogen deficient osteoporosis.
(246) As a result of the experiment, signs of menopausal disorders (obesity, hyperlipidemia, hepatic steatosis and osteoporosis) related to estrogen deficient menopause induced by OVX in ddY mice were remarkably inhibited by continuous administration of estradiol, red clover extract and pomegranate extract single formula, and all 9 types of red clover:pomegranate extract mixed formulas for 84 days, and particularly, a significant effect on reduction of menopausal disorders was provided in the order of red clover:pomegranate extract 2:1, 1:1 and 4:1 (g/g) mixture administered groups as compared to each of red clover extract and pomegranate extract single formula administered groups, so it was observed that red clover:pomegranate extract mixed formula red clover:pomegranate extract 2:1, 1:1 and 4:1 at an optimal ratio even synergistically increased a menopausal disorder reduction effect of red clover extract and pomegranate extract for OVX induced menopausal disorders through increased diversity of isoflavonoids, and among them, it was determined that red clover:pomegranate extract 2:1 mixed formula showed a best menopausal disorder alleviation effect. Accordingly, red clover:pomegranate extract 2:1 (g/g) mixture is expected to have a very high possibility of being developed as a new, more effective drug for treatment of menopausal disorders, and particularly, will be effective in reducing estrogen deficient obesity, hyperlipidemia, hepatic steatosis and osteoporosis.