Composition for remedying female climacteric syndrome symptoms

Abstract

The present disclosure relates to a composition for preventing, treating or remedying female climacteric syndrome symptoms, which contains an extract of Pueraria thomsonii flower or bud. The composition according to the present disclosure shows quick effects for preventing, remedying and/or treating female climacteric syndrome symptoms, particularly facial flushing and/or osteoporosis, and thus can be utilized for the hormone replacement therapy (HRT) used for preventing or remedying climacteric syndrome symptoms.

Claims

1. A method for treating or remedying female climacteric syndrome symptoms by administering to a subject in need thereof a therapeutically effective amount of a composition comprising an extract of buds of Pueraria thomsonii.

2. The method according to claim 1, wherein the Pueraria thomsonii bud extract is an ethanol extract.

3. The method according to claim 2, wherein the ethanol extract extract is a 60-90% ethanol extract.

4. The method according to claim 1, wherein the female climacteric syndrome symptom is facial flushing.

5. The method according to claim 1, wherein the female climacteric syndrome symptom is osteoporosis.

6. The method according to claim 1, wherein the female climacteric syndrome symptom is sweating.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a graph showing the activity of a P. thomsonii extract and a P. lobata extract according to an exemplary embodiment of the present disclosure for estrogen receptors.

(2) FIG. 2 is a graph showing the effect of remedying a vasomotor symptom of a P. thomsonii extract and a P. lobata extract according to an exemplary embodiment of the present disclosure.

(3) FIG. 3 is a graph showing the effect of remedying bone resorption of a P. thomsonii extract and a P. lobata extract according to an exemplary embodiment of the present disclosure.

(4) FIG. 4 is a graph showing the effect of remedying a vasomotor symptom of extracts of different parts of P. thomsonii according to an exemplary embodiment of the present disclosure.

(5) FIG. 5 is a graph showing the effect of remedying bone resorption of extracts of different parts of P. thomsonii according to an exemplary embodiment of the present disclosure.

BEST MODE

(6) Hereinafter, the present disclosure is described in detail through examples and test examples. However, the examples and test examples according to the present disclosure may be changed into various other forms, and it should not be construed that the scope of the present disclosure is limited to the examples and test examples. The examples and test examples of the present disclosure are provided to describe the present disclosure more completely to those of ordinary skill in the art.

Comparative Analysis of Plants in Genus Pueraria

Example 1. Preparation of Pueraria thomsonii and Pueraria lobata Extracts

(7) Pueraria thomsonii and Pueraria lobata were used in the following examples. The plants in the genus Pueraria were extracted at 70° C. for 4 hours with 85% ethanol as a solvent and then concentrated under reduced pressure to prepare each extract.

Example 2. Preparation of Tablet

(8) 300-mg tablets described in Table 1 were prepared using the P. thomsonii extract and the P. lobata extract prepared in Example 1 and an excipient (dextrin) as the balance.

(9) TABLE-US-00001 TABLE 1 Composition (%) P. thomsonii extract P. lobata extract Dextrin Preparation Example 1 — — 100 Preparation Example 2 30 — Balance Preparation Example 3 — 30 Balance

Example 3. Evaluation of Activity for Estrogen Receptors

(10) ERE (estrogen response element) reporter assay was performed to investigate the activity of the P. thomsonii extract and the P. lobata extract for estrogen receptors.

(11) After culturing 293T cells using a DMEM medium supplemented with 10% FBS in a 24-well plate for 24 hours, the medium was replaced with 500 μL of a 5% charcoal-stripped FBS containing phenol red-free medium per well and then transfection was carried out. DNA transfection was carried out using the Lipofectamine® reagent (Thermo Fisher Scientific) (0.1 μg of ERa, 0.1 μg of ERE and 10 ng of pRL-Tk) per well. After conducting the transfection for 4 hours, 10 ppm (DMSO 0.5 μL) of the P. thomsonii extract or the P. lobata extract was added. For a positive control group, 1 ppb (DMSO 0.5 μL) of 17β-estradiol (E2) was treated. For a negative control group (control, ctrl), DMSO of the same volume (0.5 μL) was treated. 24 hours after the treatment, luminescence was measured using the Dual-Luciferase® reporter assay system (Promega), and the experimental result is shown in FIG. 1 relative to the measurement value for the negative control group (1.0). The measurement value for firefly luciferase was corrected with the value for renilla luciferase.

(12) As shown in FIG. 1, the P. thomsonii extract showed better activity for estrogen receptors than the P. lobata extract.

Example 4. Activity of P. thomsonii Extract Depending on Solvent (Ethanol:Water) Composition

(13) After extracting dried Pueraria thomsonii at 70° C. for 4 hours using the solvents (ethanol:water) described in Table 2, an extract was prepared by concentrating under reduced pressure. ERE (estrogen response element) reporter assay was conducted to investigate the activity for estrogen receptors. The experiment was conducted in the same manner as in Example 3, and each extract was added at 10 ppm. The experimental result is shown in FIG. 2 relative to the measurement value for the negative control group (1.0).

(14) As shown in Table 2, the activity for estrogen receptors was superior for 35-95% ethanol solvents, and the most superior activity was achieved for 60-90% ethanol solvents.

(15) TABLE-US-00002 TABLE 2 Ethanol:water ratio Fold induction 95:5  27.4 85:15 35.7 75:25 31.1 65:35 33.4 55:45 28.7 45:55 27.1 35:65 28.6 25:75 18.9 10:90 14.2 100:0  12.9

Example 5. Effect of Relieving Climacteric Syndrome Symptoms of P. thomsonii Extract

(16) 60 women in their late 40s to 50s suffering from climacteric syndrome symptoms were divided randomly and were asked to take in the tablets prepared in Preparation Examples 1-3, 3 tablets a day. The degree of remedying climacteric syndrome symptoms was evaluated 2 weeks and 4 weeks after the intake.

(17) 2 weeks and 4 weeks after the intake, the degree of remedying climacteric syndrome symptoms was measured with a Kupperman index. The decrease (%) of the Kupperman index as compared to before the intake is shown in Table 3.

(18) The Kupperman index (KI) used in the present disclosure is a menopause index which rates the 11 symptoms occurring in postmenstrual women (facial flushing, sweating, insomnia, nervousness, depression, dizziness, fatigue, arthralgia, muscular pain, headache, palpitation, vaginal dryness). The menopausal score is calculated by multiplying each score by a weight and then summing them.

(19) TABLE-US-00003 TABLE 3 Preparation Preparation Preparation Example 1 Example 2 Example 3 2 4 2 4 2 4 weeks weeks weeks weeks weeks weeks Decrease of 2.3 5.3 17.6 32.0 7.5 16 Kupperman index (%)

(20) As shown in Table 3, the composition of Preparation Example 2 in which the P. thomsonii extract was mixed showed excellent effect of relieving climacteric syndrome symptoms as compared to the placebo group of Preparation Example 1 or the composition of Preparation Example 3 in which the P. lobata extract was mixed.

(21) In particular, the decrease in ‘facial flushing’ among the climacteric syndrome symptoms was investigated. As shown in Table 4, Preparation Example 2 showed the largest increase.

(22) From the result of Table 4, it can be seen that the P. thomsonii extract of the present disclosure has superior effect of decreasing and treating facial flushing.

(23) TABLE-US-00004 TABLE 4 Preparation Preparation Preparation Example 1 Example 2 Example 3 2 4 2 4 2 4 weeks weeks weeks weeks weeks weeks Decrease of 5 7 15 30 7 12 facial flushing (%)

Example 6. Effect of Remedying Vasomotor Symptoms

(24) It is known that 75% of postmenstrual women experience vasomotor symptoms. Facial flushing (hot flush) is the representative symptom where the skin on face, neck and chest is reddened abruptly, accompanied by unpleasant flushing and sweating. The change in the vasomotor symptoms can be evaluated by measuring the change in skin temperature. In an animal experiment, it can be measured by the skin temperature of the rat tail (Guidelines for evaluation of health functional food, ‘Helpful for health of postmenstrual women’, National Institute of Food and Drug Safety Evaluation).

(25) 11-12 week-old female Sprague-Dawley rats were subjected to sham operation (group 1, n=10) or ovariectomy (OVX) (groups 2-5, n=10 per each group). After a week from the operation, 0.01 mL of a sample per body weight (g) of each rat was administered orally every day for 4 weeks. 17β-Estradiol (E2) was administered to the positive control group and the P. thomsonii extract or the P. lobata extract was administered to the test groups. 4 weeks after the administration, the skin temperature at 2 cm where the tail starts was measured using an infrared thermometer. The measurement result is shown in FIG. 2.

(26) TABLE-US-00005 TABLE 5 Group Operation Sample Group 1 Sham Drinking water Group 2 OVX Drinking water Group 3 OVX 17β-Estradiol (E2), 0.5 mg/kg/day Group 4 OVX P. thomsonii extract, 100 mg/kg/day Group 5 OVX P. lobata extract, 100 mg/kg/day

(27) As seen from FIG. 2, the group to which the P. thomsonii extract was administered showed suppressed increase in the tail temperature caused by estrogen deficiency.

Example 7. Effect of Remedying Bone Resorption

(28) In order to measure CTX (C-terminal telopeptide of type I collagen), which is a marker related with bone resorption, the same rats as in Example 6 were orally administered with a sample for 8 weeks and then serum was collected. The CTX level in the serum was measured using the rat C-telopeptide of type I collagen ELISA kit (MyBioSource) according to the manufacturer's instructions. The result is shown in FIG. 3.

(29) As seen from FIG. 3, the group to which the P. thomsonii extract was administered showed significantly decreased CTX level as compared to the OVX group, suggesting that bone resorption was decreased.

(30) It was confirmed that the activity for estrogen receptors of the P. thomsonii extract was excellent as compared to the activity for estrogen receptors of the P. lobata extract (Example 3).

(31) It was confirmed that the P. thomsonii extract also had superior effect of relieving female climacteric syndrome symptoms (Example 5, Example 6 and Example 7).

(32) Accordingly, the Pueraria thomsonii extract exhibiting superior activity for estrogen receptors can be used effectively for preventing, treating or remedying female climacteric syndrome symptoms, particularly facial flushing or osteoporosis.

Comparative Analysis of Different Parts of Pueraria thomsonii

Example 8. Preparation of Extracts of Different Parts of Pueraria thomsonii

(33) After harvesting, drying and pulverizing the bud, root and leaf of Pueraria thomsonii, extracts were prepared by extracting 70° C. for 4 hours with 85% ethanol as a solvent and then concentrating under reduced pressure.

Example 9. Preparation of Tablet

(34) 300-mg tablets described in Table 6 were prepared using the extracts of the different parts of P. thomsonii and an excipient (dextrin) as the balance.

(35) TABLE-US-00006 TABLE 6 Composition (%) Bud Root Leaf Dextrin Preparation Example 4 100 Preparation Example 5 30 70 Preparation Example 6 30 70 Preparation Example 7 30 70

Example 10. Effect of Relieving Climacteric Syndrome Symptoms of Extracts of Different Parts of P. thomsonii

(36) 60 women in their late 40s to 50s suffering from climacteric syndrome symptoms were divided randomly and were asked to take in the tablets prepared in Preparation Examples 4-7, 3 tablets a day. The degree of remedying climacteric syndrome symptoms was evaluated 2 weeks and 4 weeks after the intake.

(37) 2 weeks and 4 weeks after the intake, the degree of remedying climacteric syndrome symptoms was measured with a Kupperman index. The decrease (%) of the Kupperman index as compared to before the intake is shown in Table 7.

(38) The Kupperman index (KI) used in the present disclosure is a menopause index which rates the 11 symptoms occurring in postmenstrual women (facial flushing, sweating, insomnia, nervousness, depression, dizziness, fatigue, arthralgia, muscular pain, headache, palpitation, vaginal dryness). The menopausal score is calculated by multiplying each score by a weight and then summing them.

(39) TABLE-US-00007 TABLE 7 Preparation Preparation Preparation Preparation Example 4 Example 5 Example 6 Example 7 2 4 2 4 2 4 2 4 weeks weeks weeks weeks weeks weeks weeks weeks Decrease of 3.5 7.8 18.5 33.9 9.4 20.0 5.4 10.7 Kupperman index (%)

(40) As shown in Table 7, the composition of Preparation Example 5 in which the bud extract was mixed showed excellent effect of relieving climacteric syndrome symptoms as compared to Preparation Example 4 (placebo group) or Preparation Example 6 and Preparation Example 7 in which the root or leaf extract was mixed.

(41) In particular, the decrease in ‘facial flushing’ among the climacteric syndrome symptoms was investigated. As shown in Table 8, Preparation Example 5 showed the largest increase.

(42) From the result of Table 8, it can be seen that the P. thomsonii bud extract of the present disclosure has superior effect of decreasing and treating facial flushing.

(43) TABLE-US-00008 TABLE 8 Preparation Preparation Preparation Preparation Example 4 Example 5 Example 6 Example 7 2 4 2 4 2 4 2 4 weeks weeks weeks weeks weeks weeks weeks weeks Decrease of facial 3.8 8.8 18.0 32.8 9.7 14.6 8.7 12.8 flushing (%)

Example 11. Effect of Remedying Vasomotor Symptoms

(44) It is known that 75% of postmenstrual women experience vasomotor symptoms. Facial flushing (hot flush) is the representative symptom where the skin on face, neck and chest is reddened abruptly, accompanied by unpleasant flushing and sweating. The change in the vasomotor symptoms can be evaluated by measuring the change in skin temperature. In an animal experiment, it can be measure by the skin temperature of the rat tail (Guidelines for evaluation of health functional food, ‘Helpful for health of postmenstrual women’, National Institute of Food and Drug Safety Evaluation).

(45) 11-12 week-old female Sprague-Dawley rats were subjected to sham operation (group 1, n=10) or ovariectomy (OVX) (groups 2-6, n=10 per each group). After a week from the operation, 0.01 mL of a sample per body weight (g) of each rat was administered orally every day for 4 weeks. 17β-Estradiol (E2) was administered to the positive control group and the extracts of different parts of P. thomsonii were administered to the test groups. 4 weeks after the administration, the skin temperature at 2 cm where the tail starts was measured using an infrared thermometer. The measurement result is shown in FIG. 4.

(46) TABLE-US-00009 TABLE 9 Group Operation Sample Group 1 Sham Drinking water Group 2 OVX Drinking water Group 3 OVX 17β-Estradiol (E2), 0.5 mg/kg/day Group 4 OVX P. thomsonii bud extract, 100 mg/kg/day Group 5 OVX P. thomsonii root extract, 100 mg/kg/day Group 6 OVX P. thomsonii leaf extract, 100 mg/kg/day

(47) As seen from FIG. 4, the group to which the P. thomsonii bud extract was administered showed suppressed increase in the tail temperature caused by estrogen deficiency.

Example 12. Effect of Remedying Bone Resorption

(48) In order to measure CTX (C-terminal telopeptide of type I collagen), which is a marker related with bone resorption, the same rats as in Example 8 were orally administered with a sample for 8 weeks and then serum was collected. The CTX level in the serum was measured using the rat C-telopeptide of type I collagen ELISA kit (MyBioSource) according to the manufacturer's instructions. The result is shown in FIG. 5.

(49) As seen from FIG. 5, the group to which the P. thomsonii bud extract was administered showed significantly decreased CTX level as compared to the OVX group, suggesting that bone resorption was decreased.

INDUSTRIAL APPLICABILITY

(50) The present disclosure provides a pharmaceutical composition and/or a food composition which is effective for preventing or remedying female climacteric syndrome symptoms and/or sweating, facial flushing or osteoporosis.

(51) It can be utilized for the hormone replacement therapy (HRT) used for preventing or remedying climacteric syndrome symptoms.

(52) In addition, because the composition according to the present disclosure has no cytotoxicity unlike existing therapeutic agents for female climacteric syndrome symptoms, particularly sweating, facial flushing or osteoporosis, and is safe with few side effects as much as it can be used as food, it can be utilized as a therapeutic agent for female climacteric syndrome symptoms, particularly sweating, facial flushing or osteoporosis.