Title of Invention

"COMPOSITION FOR PREVENTING AND TREATING CLIMACTERIC SYMPTOMS COMPRISING THE EXTRACT OF SOPHORAE FRUCTUS"

Abstract A food composition characterized in that the composition comprises 30 ~ 50 weight % of hot water extract of Sophorae Fructus, 30-50 weight % of seaweed calcium powder, 1-10 weight % of crystalline cellulose, 0.1-2 weight % of hydrolyzed milk protein, 0.1-2 weight % of green tea ex-powder, 0.1-2 weight % of shark cartilage extract powder, 0.1-2 weight % of chito-oligosaccharide, 0.1-2 weight % of vitamin C, 0.1 - 2 weight % of collagen peptide, 0.1-2 weight % of grape seeds extract powder, 0.1 -2 weight % of enzyme mixture comprising amylase, protease, cellulase, lipase and lactase, 0.1 - 0.3 weight % of vitamin D3 powder, and 0.1-2 weight % of magnesium stearate as an effective ingredient for preventing or improving metabolic bone disease.
Full Text COMPOSITION FOR PREVENTING AND TREATING CLIMACTERIC SYMPTOMS COMPRISING THE EXTRACT OF SOPHORAE FRUCTUS
The application claims the priority of Korean Patent Application No. 10-2003-0084329 filed on November 26,2003.
FIELD OF THE INVENTION
The present invention relates to a composition and method for preventing and treating climacteric symptoms including osteoporosis.
BACKGROUND OF THE INVENTION
Climacteric symptoms are caused by the decrease of secretion of male or female hormones. Especially, in the case of women, estrogen is less secreted as an ovary gets old, resulting in climactic symptoms for about 2 — 10 years before and after menopause. High fever, sweat, insomnia, depression, urinary incontinence, pain, osteoporosis, myocardial infraction, cerebral apoplexy and hypertension are the representative climacteric symptoms.
Among those symptoms, osteoporosis is the most typical one, which is caused by the decrease of total bone mass induced as osteoclasts exceed osteoblasts in their activities. Once osteoporosis is developed, the width of cortical bone becomes narrower, the cavity of bone manow is expanded and bone column of reticular tissue becomes lower, resulting in porosity in bones. As

osteoporosis gets serious, physical strength of bones is further declined, causing lumbago, arthralgia and bone breaking even by a slight impact.
Until now, the methods to prevent and treat climacteric symptoms such as hormone replacement therapy, non-steroid medicines and medicinal therapy for osteoporosis, etc. have been developed. The most effective method of them seems to be hormone replacement therapy. However, long-term administration of a hormone carries side effects such as headache, ganing weight, possibility of tumorigenesis, etc. Therefore, a safer and more effective treatment agent or method is required.
Recently, studies have been actively conducted to develop a novel substance having art excellent pharmaceutical effect with fewer side effects during long-term administration, so as to substitute estrogen. One of the attractive candidates for substituting estrogen is phytoestrogen, which is included in soybeans, etc. The phytoestrogen has a similar structure to human estrogen, so that it has influence on diseases involved in hormone or anti-hormone activities in vivo. So, the possibility of using phytocstrogeu us a food supplementary agent to take the place of hormone replacement therapy has been examined. The representative phytoestrogens, known so far, are isoflavone compounds such as daidzein, genistein, formononetin, biochanin A, etc., coumestan compounds like coumestrol, etc., lignan compounds such as enterolactone, etc., and phenol compounds like enterodiol, etc.
Korea patent No. 348148 discloses the extract ofPueraria root having huge

amount of phytoestrogen and preventing and treating effects for osteoporosis. In addition, the extract of Pueraria root has also been reported to have enough amount of daidzein, a kind of phytoestrogen, to have influence on the prevention and treatment of osteoporosis (Kim C. S. et al,, Korean J. Food Scl Technol., 34(4), 710-718,2002). Soybean powder has been reported to have an effect of improving osteoporosis, too (Yang S. B. et al., Korean Journal of Bone Metabolism, 6(1), 11-17,1999).
Sophorae Fntctus is a fruit of a Sophora japonica Linne. The Sophora japonica Linne, a deciduous arbor, belongs to a pea family (Leguminosae), and largely inhabits Korea, Japan and China. The contents are varied from the parts of the tree and have different medical actions content by content.
Sophorae Flos, a flower of a Sophora japonica Linne, is known to have such medicinal actions as anti-inflammation, anti-ulcer, declining of blood pressure, and preventing and treating effects of arteriosclerosis (Kim C. M. et al., Dictionary of Traditional Chinese Medicine, Vol. 1, Jungdam Publishing, 496-509,1998).
Sophorae resina, a resin of it Sophora japonica Linne, has been used for treatment of tetanus. All the leaves, branches, bark and root bark of the Sophora japonica Linne have an antimicrobial activity (Yook C. S. et al., K. H. Pharma. Sci., 17,75 -87,1989).
Sophorae Fructus, a fruit of Sophora japonica Linne, has a blood sugar increasing activity and an antimicrobial activity, and thus has been used for treatment of hemorrhoids, uterine hemorrhage, hematuria, hematemesis, hemoptysis and anal prolapse (Kim C. M. et al., Dictionary of Traditional Chinese

Medicine, Vol. I, Jungdam Publishing, 496-509,1998). SUMMARY OF THE INVENTION
The present inventors have endeavored to find a novel substance available for prevention and treatment of climacteric symptoms without side effects, and have completed the invention by confirming that an extract ofSohporae Fructus, a fruit of Sophora japonica Linne, has an excellent activity of preventing and treating climacteric symptoms.
Thus, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating climacteric symptoms comprising the extract of Sophorae Fructus as an effective ingredient.
It is another object of the present invention to provide a food composition for preventing or improving climacteric symptoms comprising the extract of Sophorae Fructus as an effective ingredient.
lit is another object of the present invention to provide a method for preventing or treating climacteric symptoms, which comprises administering a pharmaceutical composition comprising the extract of Sophorae Fructus to a subject.
lit is another object of the present invention to provide a method for preventing weight gaining, which comprises administering a pharmaceutical
composition comprising the extract of Sophorae Fructus to a subject.
It is another object of the present invention to provide a use of the extract of Sophorae Fructus for the preparation of a medicament for preventing or the

treating climacteric symptoms.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG 1 shows the effect of: an extract of Sophorae Fructus of the present Invention on the osteoblst proliferation, confirmed by MTT method (R-G: a group treated with an extract of Sophorae Fructus of Example 1, R-A: a group treated with an enzyme extract of Sophorae Fructus of Example 2, R-P: a group treated with a food composition comprising an extract of Sophorae Fructus of Example 3, S-S: a group treated with soybean ex-powder, E: a group treated with 17-beta estradiol, control: a group treated with cell culture medium, LPS: a group treated with I'ipopolysaccharide).
FIG 2A shows the IL-1 beta secretion inhibitoiy effect of an extract of Sophorae Fructus of the present invention, confirmed by ELISA (R-G: a group treated with an extract of Sophorae Fructus of Example 1, R-A: a group treated with aji enzyme extract of Sophorae Fructus of Example 2, R-P: a group treated with a food composition comprising an extract of Sophorae Fructus of Example 3, S-S: a group treated with soybean ex-powder, E: a group treated with 17-beta estradiol, control: a group treated with cell culture medium).
FIG 2B shows the IL-6 secretion inhibitory effect of an extract of Sophorae Fructus of the present invention, confirmed by ELISA (R-G: a group treated with an extract of Sophorae Fructus of Example 1, R-A: a group treated with an enzyme extract of Sophorae Fructus of Example 2, R-P: a group treated with a

food composition comprising an extract of Sophorae Fnictus of Example 3, S-S: a group treated with soybean ex-powder, E: a group treated with 17-beta estradiol, control: a group treated with cell culture medium).
FIG 3 shows the expressions of IL-1 beta and IL-6 inhibited by an extract of Sophorae Fructus of the present invention, confirmed by RT-PCR (R-G: a group treated with an extract of Sophorae Fnictus of Example 1, R-A: a group treated with an enzyme extract of Sophorae Fnictus of Example 2, R-P: a group treated with a food composition comprising an extract of Sophorae Fructus of Example 3, S-S: a group treated with soybean ex-powder, E: a group treated with 17-beta estradiol).
FIG 4 A presents the result of ELISA showing the IGF-1 secretion promoting effect of an extract of Sophorae Fructus of the present invention (R-G: 'a group treated with an extract of Sophorae Fructus of Example 1, R-A: a group treated with an enzyme extract of Sophorae Fructus of Example 2, R-P: a group treated with a food composition comprising an extract of Sophorae Fructus of Example 3, S-S: a group treated with soybean ex-powder, E: a group treated with 17-beta estradiol, control: a group treated with cell culture medium).
FIG 4B presents the result of ELISA showing the TGF'ß secretion promoting effect of an extract of Sophorae Fructus of the present invention (R-G: a group treated with an extract of Sophorae Fnictus of Example 1, R-A: a group treated with an enzyme extract of Sophorae Fructus of Example 2, R-P: a group treated with a food composition comprising an extract of Sophorae Fructus of

Example 3, S-S: a group treated with soybean ex-powder, E: a group treated with 17-beta estradiot, control: a group treated with cell culture medium).
FIG 5 presents the result of RT-PCR reflecting the expression of IGF-1 and TGF-J3 induced by an extract ofSophorae Fructus of the present invention (R-G: a group treated with an extract of Sophorae Fructus of Example 1, R-A: a group treated with an enzyme extract of Sophorae Fructus of Example 2, R-P: a group treated with a food composition comprising an extract of Sophorae Fructus of Example 3, S-S: a group treated with soybean ex-powder, E: a group treated with 17-beta estradiol).
FIG 6 presents the result of ELISA showing the nitric oxide (NO) generation promoting effect of an extract of Sophorae Fructus of the present invention (R-G: a group treated with an extract ofSophorae Fructus of Example 1, R-A: a group treated with an enzyme extract of Sophorae Fructus of Example 2, R-P: a group treated with a food composition comprising an extract of Sophorae Fructus of Example 3, S-S: a group treated with soybean ex-powder, E: a group treated with 17-beta estradiol, control: a group treated with cell culture medium).
FIG 7 presents the result of RT-PCR showing the level of expression of endothelial nitric oxide synthase (ecNOS) induced by an extract of Sophorae Fructus of the present invention. GABDH was used for loading control (R-G: a group treated with an extract ofSophorae Fructus of Example 1, R-A: a group treated with an enzyme extract of Sophorae Fructus of Example 2, R-P: a group treated with a food composition comprising an extract of Sophorae Fructus of

Example 3, S-S: a group treated with soybean ex-powder, E: a group treated with 17-beta estradiol, *:R-P, E vs S-S, statistically significant when p FIG 8 presents the number of positive osteoclasts observed under an optical microscope after staining with TRAP to measure a osteoclast differentiation inhibitory activity of an extract of Sophorae Fructus of the present invention (R-G: a group treated with an extract of Sophorae Fructus of Example 1, R-A: a group treated with an enzyme extract of Sophorae Fructus of Example 2, R-P: a group treated with a food composition comprising an extract of Sophorae Fructus of Example 3, E: a group treated with 17-beta estradiol, S-S: a group treated with soybean ex-powder).
FIG 9 presents a osteoclast differentiation inhibitory activity of an extract of Sophorae Fructus of the present invention, which was analyzed by measuring optical density after staining with TRAP (Control: a group treated with cell culture medium, R-G: a group treated with an extract of Sophorae Fructus of Example 1, R-A: a group treated with an enzyme extract of Sophorae Fructus of Example 2, R-P: a. group treated with a food composition comprising an extract of Sophorae Fructus of Example 3, S-S: a group treated with soybean ex-powder, E: a group treated with 17-beta estradiol).
FIG. 10 is a graph showing the,weight changes in ovari-ectomized rats administered with an extract of Sophorae Fructus of trie present invention (E: a group administered with 17-beta estradiol, R-G: a group administered with an extract of Sophorae Fructus of Example 1, R-A: a group administered with an

enzyme extract of Sophorae Fructus of Example 2, S-S: a group administered with soybean ex-powder).
FIG 11 is a calibration curve showing the relations between Dpd (Deoxypyridinoline) concentration and optical density, by which Dpd concentration in a blood plasma of ovari-ectomized rats administered with an extract of Sophorae Fructus of the present invention, could be measured (Y--O.H28X+l.6102,R=0.9902).
FIG 12 is a graph showing the changes of Dpd concentration in a blood plasma of ovari-ectomized rats administered with an extract of Sophorae Fructus of the present invention (E: a group administered with 17-beta estradiol, R-A: a group administered with an enzyme extract of Sophorae Fructus of Example 2, R-G: a group administered with an extract of Sophorae Fructus of Example 1, R-P: a group administered with a food composition comprising an extract of Sophorae Fructiis of Example 3, S-S: a group treated with soybean ex-powder).
FIG 13 is a graph showing the difference between before and after experiments in Dpd concentration in a blood plasma of ovari-ectomized rats administered with an extract of Sophorae Fructus of the present invention (Control: a group administered with water, E: a group administered with 17-beta estradiol, R-A: a group administered with an enzyme extract of Sophorae Fructus of Example 2, R-G: a group administered with an extract of Sophorae Fructus of Example 1, R-P: a group administered with a food composition comprising an extract of Sophorae Fructus of Example 3, S-S: a group administered with

soybean ex-powder).
FIG 14 is a graph showing the comparison of Dpd inhibitory activity of an extract of Sophorae Fructus of the present invention (E: a group administered with 17-beta estradiol, R-G: a group administered with an extract of Sophorae Fructus of Example I, S-S: a group administered with soybean ex-powder, R-P: a group administered with a food composition comprising an extract of Sophorae Fructus of Example 3, R-A: a group administered with an enzyme extract of Sophorae Fructus of Example 2, *: not statistically significant when p FIG 15 is a graph showing the changes of calcium concentration in a blood plasma of ovari-ectomized rats administered with an extract of Sophorae Fructus of the present invention (E: a group administered with estradiol, R-A: a group administered with an enzyme extract of Sophorae Fructus of Example 2, R-G: a group administered with an extract of Sophorae Fructus of Example 1, R-P: a group administered with a food composition comprising an extract of Sophorae Fructus of Example 3, S-S: a group iidministered with soybean ex-powder).
FIG 16A is a microphotograph showing the tibia of ovari-ectomized rats administered with an extract of Sophorae Fructus of the present invention (Magnification: X16, A: a normal group.,(non-ovari-ectomized group), B: control 1 (sham-operated group), C: control 2 (ovari-ectomized group), D: a,group administered with 17-p.estradiol, E: a group administered with an extract of Sophorae Fructus of Example 1, F: a group administered with an enzyme extract

of Sophorae Fructus of Example 2, G: a group administered with a food composition comprising an extract of Sophorae Fructus of Example 3, H: a group administered with soybean ex-powder).
FIG I6B presents the area of trabecular bone of the tibia of ovari-ectomized rats administered with an extract of Sophorae Fructus of the present invention (E: a group administered with 17-beta estradiol, R-A: a group administered with an enzyme extract of Sophorae Fructus of Example 2, R-G: a group administered with an extract of Sophorae Fructus of Example I, R-P: a group administered with a food composition comprising an extract of Sophorue Fructus of Example 3, S-S: a group administered with soybean ex-powder, *: statistically significant when p FIG 17A is a microphotograph showing the lumbar of ovari-ectomized rats administered with an extract of Sophorae Fructus of the present invention (Magnification: XI6, A: a normal group (non-ovari-ectornized group), B: control 1 (sham-operated group), C: control 2 (ovari-ectomlzed group), D: a group administered with 17- P estradiol, F.: a group administered with un extract of Sophorae Fructus of Example 1a group administered with an enzyme extract of Sophorae Fructus of Example 2, G: a group administered with a food composition comprising an extract of Sophorae Fructus of Example 3, H: a group administered with soybean ex-powder).
FIG 17B presents the area of trabecular bone of the lumbar of ovari-ectomized rats administered with an extract of Sophorae Fructus of the present

invention (E: a group administered with 17-beta estradiol, R-A: a group administered with an enzyme extract of Sophorae Fructus of Example 2, R-G: a group administered with an extract of Sophorae Fructus of Example 1, R-P: a group administered with a food composition comprising an extract of Sophorae Fructus of Example 3, S-S: a group administered with soybean ex-powder, *: statistically significant when p DETAILED DESCRIPTION OF THE INVENTION
In order to achieve the above objects of the invention, the present invention provides a pharmaceutical composition for preventing or treating climacteric symptoms comprising the extract of Sophorae Fructus as an effective ingredient.
The present invention also provides a food composition for preventing or improving climacteric symptoms comprising the extract of Sophorae Fructus as an effective ingredient
The present invention also provides a method of preventing or treating climacteric symptoms, which comprises administering a pharmaceutical composition comprising the extract of Sophorae Fructus to a subject.
The present invention also provides a method of preventing weight gaining, which comprises administering a pharmaceutical composition comprising the extract of Sophorae Fructus to a subject.
The present invention further provides a use oJ; the extract of Sophorae Fructus for the preparation of a medicament for preventing or treating climacteric symptoms.

The present invention will be described in detail,
"Sophorae Fructus" of the present invention refers to u fruit of Sophora japonica inLne, a deciduous arbor belonging to a pea family (Leguminosae). More particularly, it means a mature fruit of Sophora japonica Linne.
It is preferable for the present invention that Sophorae Fructus, as a mature fruit of Sohpora japonica Linne, ought to have its unique color and flavor without other taste and smell. The peel of the fruit has to be khaki brown or brown, and a seed had better be black or black brown.
It is preferable to prepare an extract of Sophorate Fructus of the present invention by hydrothermal extraction, but not always limited thereto. The ratio of Sophorae Fructits to water for hydrothermal extraction is not specially limited, but for Ig of Sophorae Frvctus, water can be used by 3 to 20 times (based on weight) preferably, 5 to 10 times.
The temperature for extraction is preferably room temperature under atmospheric pressure. The extraction time varies depending on extraction temperature, but preferably ranges from 1 to 6 hours, more preferably 2 to 4 hours. Also, extraction efficiency may further enhanced by stirring with a shaker during extraction.
Sophorae Fructus can be used either right after being rinsed after cropping or after being dried. Sophorae Fructus can be dried either in the sun, in the shade, by hot air or naturally. In addition, Sophorae Fructus or its dried body can be crushed into powder to enhance the efficiency of the extraction.
Preferably, dried Sophorae Fructus can be pulverized in 20—40 mesh size, and drinking water is added to the Sophorae Fructus powder, wherein the ratio of

Sophorae Fructus powder to water is 1 to 3 ~20, preferably 1 to 5 -10. Then, hydrothermal extraction is carried out for 1 to 3 hours at 100- I30t:, preferably 120 —125 "C. The Sophorae Frucfus extract can be prepared by centrifuging the hydrothermal extract and removing the precipitation to obtain supernatant.
An enzyme extract of Sophorae Fructus \$ also obtained by treating the hydrothermal extract of Sophorae Fructus of the invention with an enzyme. Precisely, the hydrothermal extract prepared by the above method is treated with an enzyme by 0.01 ~1 %(v/v), followed by a reaction for 4—24 hours. After concentration, the reaction solution is freeze-dried, resulting in an enzyme extract. At this time, one of a -amylase, 0 -amylase and pectinase can be used as an enzyme.
The extract of Sophorae Fructus of the present invention has an effect of preventing and treating climacteric symptoms. The "climacteric symptoms" as used herein refers to diseases that can be caused by the lack of a hormone, and especially for women, they are caused by the deficiency in estrogen, which results from the blockage of functions of ovary. The representative climacteric symptoms are classified into metabolic bone diseases such as osteoporosis, lumbago, rheumatoid arthritis, degenerative arthritis, rickets, osteomalacia and Paget's disease of bone, cardiovascular diseases such as angina pectoris and arteriosclerosis, and degenerative neurological diseases such as Parkinson's disease. Particularly, the metabolic bone disease is developed by the break of balance between osteoclasts and osteoblasts, and osteoporosis is the most representative one. The extract of Sophorae Frucfus of the present invention has

an excellent effect of preventing and treating osteoporosis.
The effect of preventing and treating climacteric symptoms of the extract of Sophorae Fructus of the present invention has been confirmed by in vitro and in vivo experiments.
Through in vitro experiments, the extract of Sophorae Fructus of the present invention was proved to promote osteoblast proliferation (see FIG. 1) but inhibit the secretion of bone-absorptive cytokines, IL-1 beta and IL-6 (see FIG 2A, 2B and 3). Besides, the extract of Sophorae Fructus of the present invention promoted the expression of IGF-1 and TGF-ßwhich are growth factors involved in bone-regeneration (see FIG. 4A, 4B and 5), accelerated the generation of nitric oxide (see FIG. 6 and 7), and effectively inhibited osteoclast differentiation (see FIG 8 and 9). Such activities were observed well even under the low concentration of the extract of Sophorae Fructus of the present invention.
For in vivo experiments with the extract of Sophorae Fructus, ovari-ectomized rats were used. The extract of Sophorae Fructus of the present invention was administered to rats that could not secret estrogen because its ovary was removed. Then, weight changes, the level of Dpd, an index for bone replacement rate in a serum, which increases as bone matrixes are decomposed by osteoclasts, and calcium concentration varying with the activation of osteoblasts are investigated. As a result, the extract of Sophorae Fructus of the present invention worked as a substitute for estrogen and so prevented weight gaining (see FIG 10), inhibited the increase of Dpd (see FIG. 12 - FIG 14) and increased calcium concentration in blood (see FIG 15). In addition, the extract of

Sophorae Fructus of the present invention was confirmed to inhibit the restriction of trabecular bone of the tibia and the lumbar, which can be used as an index for bone density in ovari-ectomized rats (see FIG 16 and 17).
Thus, the present invention provides a pharmaceutical composition for preventing or treating of climacteric symptoms comprising the extract of Sophorae Fructus as an effective ingredient. The climacteric symptoms include all diseases induced by the lack of hormones, especially estrogen. For example, metabolic bone diseases such as osteoporosis, lumbago, rheumatoid arthritis, degenerative arthritis, rickets, osteomalacia arid Paget's disease of bone, cardiovascular diseases such as angina pectoris- and arteriosclerosis, and degenerative neurological diseases such as Parkinson's disease, etc., are included in the symptoms. And osteoporosis is the most representative one.
The pharmaceutical composition of the present invention can include a pharmaceutically effective amount of the extract of Sophorae Fructus of the invention singly or additionally include one or more phElrmaceutically acceptable carriers, binders or diluents. The term of 'pharmaceutically effective amount" as used herein means the amount of an extract enough to prevent or treat the symptoms.
The pharmaceutically effective amount of the extract of Sophorae Fructus of the present invention was determined to be 1 ~ 600 mg/day/weight kg in this invention, and more preferably 1 ~ 100 mg/day/weight kg. Though, the effective amount can vary depending on seriousness of a disease, age, weight, body condition and sex of a patient, administration methods, and duration of treatment,

The term of "pharmaceutically acceptable" as used herein means a composition that can be physiologically acceptable for humans, and does not cause side effects such as stomach trouble, allergic reactions like dizziness, etc., as being administered to humans. The carriers, binders and diluents are exemplified by lactose, dextrose, sucrose, sorbitol, manitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrolidon, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil,
.The mentioned pharmaceutical composition c;in further include fillers, anticoagulants, lubricants, wetting agents, perfumes, emulsifying agents and antiseptics. The pharmaceutical composition of the present invention can also be formulated by the known methods in the pertinent art, to give satisfactory results after administration, for example, immediate absorption, sustaining or delayed release of an active ingredient The composition caa be formulated into the forms of powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterilized ampoule and sterilized powder.
The pharmaceutical composition of the present invention can be administered via several roue's including oral, intracutaneous, subcutaneous, intravenous or intramuscular. The effective dosage can be determined by considering administration method, age, sex, weight arid seriousness of a disease of a patient, etc.
The pharmaceutical composition of the prese