TW200812604A - Pharmaceutical composition for prevention and/or treatment of bone loss - Google Patents

Abstract

A pharmaceutical composition for preventing and/or treating bone loss. The pharmaceutical composition includes an effective amount of a licorice, black bean, Cnidi Fructus and buckhorn.

Description

200812604 - Nine, invention description:

BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a pharmaceutical composition, and more particularly to a pharmaceutical composition for preventing and/or treating bone loss. [Prior Art] Osteoporosis can be divided into two categories: First, postmenopausal osteoporosis caused by a lack of female hormones. This condition occurs in women after menopause; the other reason is Due to aging, we call it "aged osteoporosis", which can occur regardless of whether men and women live old enough. From clinical studies, it can be found that when menopausal women have insufficient female hormone secretion, the rate of bone loss can be as high as three to five percent a year. When the bone is accelerated and lost, it will cause osteoporosis. When the bone structure becomes bad, it may cause the fracture to occur under the action of external force. After menopause, osteoporosis has some specific 10 sites. The bones of the human body can be roughly divided into two parts. One is the harder and denser part of the outer layer. We call it the hard bone. The softer part of the bone is also the soft part. Commonly known as the bone marrow, we call it the loose bone. The part of osteoporosis that occurs after menopause is the so-called loose bone. In terms of the human body, the proportion of loose bones and hard bones in different parts of the bone is different, and menopausal osteoporosis occurs in the body where the loose bone is the main component of the body, the most important of which is the spine, so it is easy Causes the compression fracture of the lumbar spine; the other is the terminal end of the long bone, that is, the terminal part of the limb bone, which is also prone to fracture of the wrist. Women with osteoporosis after menopause, easy

Client’s Docket No.: TT’s Docket No: 0973-A40927-TW/fmal/Kai/9/6/2006 5 200812604 w ’, the type of fracture caused is the two. Post-menopausal loosening (p0stmen〇pausai 〇ste〇p〇r〇sis) is a metabolic bone disease commonly seen in older women. In clinical studies, it can be found that § the rate of bone loss can be as high as 3% to 5% per year when the female hormones are discharged from women during menopause. As the average life expectancy increases, osteoporosis after menopause The incidence rate is on the rise. After menopause, the incidence of osteoporosis in women is mainly due to the loss of bone mass caused by the lack of female hormones caused by menopause (about 3 to 6% or more per year). This is because the normal olfactory hormone stimulates the "osteoblast" to produce the cell medium, which inhibits the activity of the osteoclast, and stimulates the "osteoclast" to maintain the balance of the bone. In the absence of female hormones, bone mass is lost, and the ions are released from the bones, which suppresses the concentration of parathyroid hormone and reduces the synthesis of active vitamin D, thus reducing the absorption of maternal ions in the gastrointestinal tract, resulting in the negative balance of calcium ions. To accelerate the occurrence of osteoporosis. _ Taiwan has gradually entered an aging society, and as a result, more people will develop osteoporosis. Postmenopausal osteoporosis is a metabolic bone disease commonly seen in older women. With the extension of the average life expectancy of Chinese people, the incidence of osteoporosis after menopause is on the rise. Modern medicine for the treatment of postmenopausal osteoporosis is mainly estrogen. Alternative therapy 'but long-term use has certain side effects (Colditz GA, Hankinson SE, Hunter DJ. The use of estrogen and progestins and the risk of breast cancer in postmenopausal women. N Eng J Med, 1995, 332·1589-1593; Grady D, Gebretsadik T,

Client's Docket No.: TT^ Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 6 200812604 ' Kelikowske Κ· Hormone replacement therapy and endometrial cancer risk: a meta-analysis. Obstet Gynecol, 1995,85 :304-313) Therefore, there is currently a need for a pharmaceutical composition that can be used to prevent and/or treat bone loss. SUMMARY OF THE INVENTION It is an object of the present invention to provide a pharmaceutical composition for preventing and treating bone loss in elderly or menopausal women. In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and/or treating bone loss, comprising an effective amount of licorice, black beans, cnidium and antlers, wherein the weight ratio of each component is licorice: black beans: Cnidium: antlers =1_10 : 2-10 : M0 : 1_1 〇〇 In order to achieve the above object, the present invention further provides a pharmaceutical composition for preventing and/or treating bone loss, comprising an effective amount of imperatorin, and a pharmacy An acceptable carrier or excipient. To achieve the above object, the present invention further provides a pharmaceutical composition for preventing and/or treating bone loss, comprising an effective amount of bergapten, and a pharmaceutically acceptable carrier or excipient. The above and other objects, features, and advantages of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; And/or the medical group for treating bone loss

Client’s Docket No.: TT’s Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 7 200812604 • Compound. In a consistent embodiment, the pharmaceutical composition of the present invention comprises licorice, tired bean bed, antler, etc. The weight ratio of each component is licorice: black bean: Cnidium: antler = 1-10: 2-10: 1-10 : 1-10, preferably 2_4 : 4-6 : 1-3 : Μ. Also, the pharmaceutical composition of the present invention also contains osthole (〇Sth〇1), imPeratorin, and bergapten. The preparation method of the pharmaceutical composition of the invention is a special processing process. First, the licorice is heated and extracted by an extract, wherein the extract used may be water ethanol. The black beans and the cnidium are then soaked in ethanol for about 2-40 hours, preferably about 10-20 hours, and heated for extraction. After that, the black bean, Cnidium extract and licorice extract are combined, and the volume ratio of each is licorice: black bean + Cnidium = 1: 2, concentrated under reduced pressure to obtain condensate ΗΜ 00 liter, 1 〇 _4 〇 a The antlers of the jin are known to be added to the "fine liquid" and dried, and the dried product is pulverized, granulated and prepared into a dosage form, which is the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention can be used for the prevention and/or treatment of bone loss, wherein bone loss includes osteoporosis or osteoporotic fracture. The 10 pharmaceutical composition can increase bone density (BMD), bone content (BMC) and bone cell alkaline phosphate (ALP) activity of one body. In general, the pharmaceutical composition of the present invention can increase bone mineral density of 5.15%, bone content of 3.77%, and bone cell alkaline phosphate (ALP) activity of 30.0%, and in a preferred embodiment, bone mineral density of 10.30% can be increased. , ιι·32% bone content and 68.0% bone cell alkaline strontium phosphate (ALP) activity. Moreover, the pharmaceutical composition can also increase the maximum stress, ultimate loading, young's modulus, and ultimate stress of a body bone tissue. In general, the pharmaceutical composition of the present invention can increase the maximum stress of 3.14%,

Client's Docket No·: TT^s Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 8 200812604 - 5.23% fracture strength, 7.49% Young's coefficient and 15.65% fracture strength In the preferred embodiment, the maximum stress of 10.24%, the breaking strength of 13.15%, the Young's modulus of 10.07%, and the breaking strength of 37.39% can be increased. The pharmaceutical composition of the present invention can be prepared into various forms such as tablets, capsules, granules, powders, liquids, or pills, etc., as needed. In another embodiment, the pharmaceutical composition of the present invention comprises an effective amount of imperatorin, and a pharmaceutically acceptable carrier or formulation, which may be in the form of an oral or injectable form, injectable form. It can be injected intramuscularly or subcutaneously. The pharmaceutical composition can increase the bone density, bone content and bone cell alkaline strontium phosphate activity of a body. Generally, the pharmaceutical composition of the invention can increase the bone density of 12.22%, the bone content of 28.91% and 6.0%. Osteoblastic acid sputum activity, in a preferred embodiment, can increase the osteoclast activity of bone cells by 90%. In another embodiment, the pharmaceutical composition of the present invention comprises an effective amount of bergapten, and a pharmaceutically acceptable carrier or excipient, which may be in the form of an oral or injectable form, and the injectable form may be a muscle. Injection or subcutaneous injection. The pharmaceutical composition can increase the bone density, bone content and bone cell amygdala (ALP) activity of a body. Generally, the pharmaceutical composition of the invention can increase the bone density of 12.22% and the bone content of 33.73%. And 5% of the bone cell alkaline phosphate activity, in a preferred embodiment can increase the bone fineness of 75%. Cellular acid filling (ALP) activity. The individual referred to in the present invention is an animal, including a human or non-human animal such as a dog, a seedling, a mouse, a rat, a cow, a sheep, a pig, a goat, or

Client’s Docket No.: TT,s Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 9 200812604 Non-human primate class. In one embodiment, the mammal is a human, and in another embodiment, the mammal is a squid, such as a mouse or a rat. The characteristics and advantages of the present invention are further illustrated by the following examples. 1. Preparation of the pharmaceutical composition of the present invention The pharmaceutical composition of the present invention comprises 30 kg of licorice, 60 kg of black beans, 10 kg of Cnidium, and antlers. 8 kg composition. First, the licorice was heated to 100 ° C, extracted with water, and 150 liter of the extract was taken. The black beans and the cnidium were soaked in 30% ethanol for 20 hours, then heated to 80 ° C for extraction, and combined with the licorice extract, the volume ratio of each was licorice: black bean + Cnidium = 1: 2, concentrated under reduced pressure. The concentrate was 30 liters, and the antler powder was added to the concentrate, followed by drying at 60T: for 48 hours. The dried product is pulverized, granulated and prepared into a dosage form. 2. Steps in animal experiments Using the National Experimental Animal Breeding and Research Center of the National Science Council, Sprague-Dawley breed female rats of 8 weeks old, after 4 weeks of feeding, underwent ovariectomy. They were randomly divided into (1) sham operation group, (2) ovariectomy control group, and (3) three groups of ovariectomy-administered pharmaceutical compositions, and Sprague-Dawley rats were anesthetized with 400 mg/kg trichloroacetaldehyde. Aseptic operation, the lumbar paravertebral incision was introduced into the dorsal side of the rat's abdomen. The bilateral ovaries of the ovariectomized control group and the ovariectomy + femoral supplement group were completely resected. The sham operation group did not cut the ovaries and sutured the blood. Postoperative feeding and drinking water as before. The medical treatment group started oral medicine after the next day.

Client's Docket No.: TFs Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 10 200812604 - Pharmaceutical composition, 1.2 grams per kilogram of body weight per day, once a day, 'sham operation group and ovariectomized group Carboxymethyl cellulose (CMC) was administered. The dose was adjusted once a week by body weight, fed and administered for 6 weeks, and all rats were sacrificed after 6 weeks. 3. Effect of pharmaceutical composition on bone length and weight After sacrifice of the above rats, the femur and tibia were taken out and stored in a refrigerator at -80 °C, and the muscles outside the femur and tibia were connected with the connective muscles. Group • • Weaving and shaving, using a microbalance to measure the weight of the bone. The length of the bone is measured with a micrometer (±0.05 mm), bone mass (BMD) and bone density (BMC) with a dual-energy X-ray absorptiometer ( DEXA, XR-26; Norland, Fort Atkinson, WI). After the femur was fixed in 4% formalin for two days, it was placed in EDTA (10%) at 4 °C for two weeks. After dehydration with alcohol, the femur was embedded in paraffin for 5 mm sectioning. Using Mayer, s hematoxylin_eosin solvent staining, and using Image_pr 春 spring plus (3·0 ed) software to determine the bone volume of the primary spongiosa. Referring to Fig. 1, the weight of the ovariectomized group was heavier than that of the pseudo-surgical group, and administration of the pharmaceutical composition of the present invention did not inhibit the increase in rat weight. Referring to Fig. 2, it can be seen from the bone tissue section that the pharmaceutical composition of the present invention has an effect of preventing bone loss. Other data are shown in Table 2. Table 1. Effect of pharmaceutical composition on bone tissue

Client’s Docket No.: TT5s Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 11 200812604

Bone volume, (%) 17.8±1·6 9.3 soil 1·5* 15·7 soil 1.4 § 14·9±1·9§ 13.4 soil 2·5§ (n=26) (n=26 Group + medicine group (1.2g/kg) (η=14) group + medicine group (〇.8g/kg) (n=13) group + medicine group (0.4g/kg) (η=13) bone length, Mm 胫骨4.03±0.02 4Ό2±〇.〇2 4.04士0.03 3.99±0.02 4.03 士0·01 Femur 3.62±0.04 3.65士0.05 3.67土0.05 3.62±〇.〇4 3.62±0.03 Bone weight, mg 胫骨758士10.5 657士12.7* 722±1〇.6§ 716.8士7.4§ 693.3士17.1§ Femur 958.1士10.3 862H3.9* 911.5士9.6§ 923土25·4§ 895士15·5§ Bone mass (BMD), (g /cm3) Tibia 0.109±0.009 0·097±0·011* 0.107 soil 0.010§ 0.102±0.006 0.105±0·006 Femur 0·131±0·007 0Λ22±0.008* 0·127±0·011§ 0.128±〇 〇28§ 0.127±0·016§ Bone mineral density (BMC), (g) tibia 0·29 (4).012 0·265±0·014* 0.295±0.024§ 0.290±0·002§ 0.275±0·003 Femur 0.411±0Ό14 0.357±〇·〇13* 0.402±0.022§ 0.415 Soil 0.025§ 0.40 (4)·014§ 4. Analysis of bone biomechanics using material testing system (MTS-858, MTS Sys Tem Inc., Minneapolis, MN) Three-point bending test to measure bone tissue biomechanics. The distance between the two ends of the bone is 20 mm, the speed of the pressure drop is 1 mm per minute, and the obtained speed and pressure are used as the regression line and belt.

Client's Docket No.: TT's Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 12 200812604 After entering the formula below, the Young's modulus and breaking strength can be obtained. Table 1. Pharmaceutical composition for tja woven biomechanics The effect of ovarian resection control group 襄 excision control group + oophorectomy control group + sham operation group nest resection control medicine group medical group medicine group (n=26) ”n=26) (1.2g/kg) (n=14) (0.8 o/ka\Γη—l Vi Maximum load, N 102.7 ± 2.9 85.9 ± 3.0 * 947 soil 2 · 8 § 91 · 7 ± 2.9 § Ultimate load, N 81 士 46 7〇 ·7±3·8* 80.〇h2.4§ 76.1 Soil 2.3§ Young's modulus, GPa 20Lftt6.2 170.8±6.6* 188.0±6.8§ 187·5 Soil 5·9§ Breaking strength (Ultimate Stress), Mpa 18.1 ± 0.9 11 · 5 ± 1. 5 * - 15.5 soil 1 · 0 § 15 · 8 ± 1 · 2 § 88. &amp; 1.5 74.4 ± 1.8 183.6 ± 4.2 § 5. Primary osteoblast culture Taken from the parietal bone of the Spraque-Dawley mouse embryo on the 18th day of pregnancy. After the sacrifice of the mother, the entire uterus is removed and placed in a sterile Petri dish. The fetus is removed from the uterus and taken care of. (calvarium) is separated, cut off the parietal bone and avoid suture, remove the periosteal layer on both sides of the bone, and then cut the bone into pieces, and slice the bone with 0.1% collagen (collagenase) Perform serial digestion for 5 times every 20 minutes to release the cells from the bone fragments. Collect the cells that have been digested for the last three times, and mix the three digested cells to form an osteoblast suspension. The cells are cultured in α- ΜΕΜ, which contains 1% penicillin-streptomycin

Client's Docket No·: TT^ Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 13 200812604 (penicillin-streptomycin) and 10% FBS, placed in a 37 ° C incubator with 5% CO 2 In the middle, when the cells were full, subculture was carried out, and only the first to fifth generations were used as experimental materials for all experiments. 6. Analyze the effect of various components in the pharmaceutical composition on cell proliferation. The above osteoblasts were cultured in 96 well cultured jujube, and 5, 15, 50, 100 /xg/ml (1) licorice, (7) black bean + Cnidium, (3) Concentration of Cnidium, (4) Black Bean and (5) Pharmaceutical Composition. After 48 hours of action, the cell culture medium was carefully aspirated, and cell proliferation was measured according to the procedure of Cell Proliferation ELISA, BrdU kit (Roche Applied Science). effect. 0.1% DMSO was added as a control group, and the cell proliferation rate in the control group was determined to be 100%. As a result, as shown in Figures 3A-3B, all components increased the proliferation of bone cells. ～ 佥 析 析 医 医 医 医 医 医 医 医 医 医 医 医 医 医 医 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨(2) After treatment of black bean + Cnidium, (3) Cnidium, (4) black bean and (5) pharmaceutical composition, 0.2% NP-40 was added to dissolve the cells, and the solution was shaken and centrifuged 1500 xg. Five minutes, the activity of ALP in the supernatant was measured using human 1^]^1. 〇.1% DMSO was added as a control group, and the activity of ALP in the A? group was 1%. As a result, as shown in Fig. 4, in addition to black beans, other ingredients increased the activity of bone cell ALp.

Client^ Docket No.: TT's Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 14 200812604 # IAnalyzing the effects of various components in the pharmaceutical composition on bone mineralization Differentiation of osteoblasts Culture medium (containing 50 pg/ml of vitamin C and 10 mM β-glycerophosphate g (p_giyCer〇ph〇Sphate)) and 5, 15, 50, 100/xg/ml of (1) licorice, (2) Black bean + Cnidium, (3) Cnidium, (4) Black Bean and (5) Concentrate of pharmaceutical composition, the culture medium is changed every three days, and after 14 days, the cells are fixed in 75% ethanol for 30 minutes, and then 4 mM is added. Alizarin red_S is allowed to act at room temperature for one hour to remove the dye that is not bonded to calcium. Finally, 10% of cetylpyridinium chloride is dissolved, using 550nm. The absorbance value measures the amount of bone nodules. 0.1% DMSO was added as a control group, and the degree of mineralization in the control group was determined to be 100%. As shown in Figures 5A-5B, in addition to black beans, the other four components can increase mineralization. L analysis of the effects of various components in the pharmaceutical composition on osteoclast differentiation. The bones of Spraque-Dawley mice were extracted by syringes for 6-8 weeks. After one day of sedimentation, 1×106 cells of hematopoietic cells were counted. In the dish, separate differentiation agents (50 ng/ml RANKL and .20 ng/ml] VUCSF) and 150 /xg/ml (1) licorice, (2) black beans + Cnidium, and (3) Cnidium (4) Black Bean and (5) Concentrate of the pharmaceutical composition, the differentiation agent was changed every three days, and after 8 days of culture, it was stained with tartrate-resistant acid phosphatase (TRAP) under a microscope. • Calculate cells that have stained TRAP and have more than three nuclei. 〇.1% DMSO was added as a control group. Referring to Figure 68, only the black bean component will slightly inhibit the differentiation of osteoclasts.

Client's Docket No·: TT^ Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 15 200812604 _Analysis of the effects of various components of jtL on osteoclasts塁' The five-day-old New Zealand rabbit was sacrificed. The long bones were cut with scissors and the mature osteoclasts on the bone wall were isolated. The cells were collected and planted in the AAS plate and given 150 Mg/ml (1). Concentrates of licorice, (7) black bean + Cnidium, (3) Cnidium, (4) black bean and (5) pharmaceutical composition, after 3 days of culture, add 1N sodium hydroxide (NaOH) to dissolve the cells and wash 3 Times, _ Image was taken under a microscope using a digital camera, and the area of the area absorbed by osteoclasts was calculated using image analysis software (Image-Pro Plus 3.0). The addition of 0.1% DMSO was used as a control group, and the osteoclast resorption rate of the control group was determined to be 100%. Referring to Figure 6B, none of the above five fractions affects the resorption of osteoclasts. 11. Analysis of the effects of imperatorin and bergapten on the survival of bone cells. The bone cells were plated in 96 well cultures with 0.3, 1, 3, 10 μΜ of imperatorin. After 48 hours of action with bergamot, carefully remove the cell culture medium, add ΜΤΤ (0.5 mg/ml) solution at 37 ° C for 30 minutes, then aspirate the solution to 100 μM of diterpene. (DMSO) to dissolve the cells, and finally absorb the absorbance at a wavelength of 550 nm using Bio-Kinetic Elisa Reader (ΒΙΟ-ΤΕΚ). 〇·1% DMSO was added as a control group, and the number of cells in the control group was determined to be 100%. Referring to Figure 7A, both praerupin and bergamot did not affect the survival of bone cells.

Client's Docket No.: TT^ Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 16 200812604 li·Analysis of the effects of imperatorin and bergamot on bone cell proliferation The above osteoblasts are seeded After treatment with 0.3, 1, 3, 10 μΜ of imperatorin and citron for 48 hours in 96 well culture dishes, carefully remove the cell culture medium according to Cell Proliferation ELISA, BrdU kit (Roche Applied Science) The process of measuring cell proliferation. DMSO was added as a control group, and the cell proliferation of the control group was determined to be 100%. Referring to Figure 7B, imperatorin and bergamot did not affect the proliferation of bone cells. 1β·Analysis of the effects of imperatorin and bergamot on the ALP activity of bone cells. The above osteoblasts were treated with imperatorin and bergamot at 〇·3, 1, 3, 10 μΜ, respectively, and 0.2% ΝΡ was added. 40 The cells were lysed, and the lysate was shaken and centrifuged at 1500 x g for five minutes, and the activity of ALP in the supernatant was measured using an ALP kit. DMSO containing 〇·〇〇/〇 was added as a control group, and the activity of ALP in the control group was set to 1.0%. As shown in Fig. 8A, both the imperatorin and the bergamot can increase the activity of ALP, and the effect of the use of imperatorin and bergamot on the collagen production of the bone cells; The 4-hydroxyproline content represents the amount of collagen synthesis in osteoblasts. After the osteoblasts were treated with prasin 3, i, 3, 10 μΜ of imperatorin and bergamot, 6NHC1 was lysed after cell differentiation, and the solution was placed in a glass tube at 116.

Client’s Docket No·: TT^s Docket No:0973-A40927-TW/fmayKai/9/6/2006 17 200812604 , Solution w hours. The hydrolyzate was vacuum dried, dissolved in an equal amount of water, and the content of 4-hydroxyproline was measured at a wavelength of 550 nm. DMSO containing 〇·!% was used as a control group, and the amount of collagen protein in the control group was determined to be 1% by weight. As a result, as shown in Fig. 8B, imperatorin and bergamot did not affect the ability of bone cells to secrete collagen. 15·H-Huhu and bergamot on bone mineralization | Osteoblasts were given differentiation medium (containing 50 pg/ml of vitamin C and 10 mM of β-glycerol _ vinegar (p_giyCer〇ph〇sphate)) The culture solution was changed every three days. At the same time, it was treated with 3, 1, 3, 10 μΜ of imperatorin and bergamot. After 14 days, it was fixed with 75% ethanol for 30 minutes, and then added with 40 mM. Alizarin red-S was allowed to act at room temperature for one hour to remove the dye that was not bonded to calcium. Finally, 10% of cetylpyridinium chloride was dissolved and dissolved by 550 nm absorbance. Bone summary content. The DMS of 〇.1% was added as a control group, and the mineralization of the bone nodules of the control group was set at 100. /. . As a result, as shown in the figure, both imperatorin and bergamot can increase mineralization. Jj. Analysis of ouqianhu complex 4 bergamot body shape peak i white (b〇ne mo卬hogenetic prot: ein, BMP-2) a solid performance of bone cells cultured in 6 well culture, respectively After adding 3, 6, and 12 hours of treatment with 〇3, 卜3, 10 shore 1 eugenin and bergamot, the cells were dissolved by adding TRIzol reagent at room temperature for 5 minutes, and the cell lysate was collected in a centrifuge tube. Add 5 ml of chloroform (cMoroform) to a uniform milky white solution and let stand at room temperature for 5 minutes.

Client’s Docket No.: TT^ Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 18 200812604 After the solution is layered, at 4. (: Centrifuge at 14000 xg for 15 minutes, then carefully extract the supernatant/monthly solution, add 0.25 ml of isopropanol (iSOpr〇pan〇i) and invert the tube to mix, then centrifuge at 14000 X g at 4QC. After a minute, the precipitated RNA can be obtained. After the supernatant is decanted, the sediment is washed with 75% alcohol, and then centrifuged at 14000 X g for 10 minutes. Finally, the RNA is air-dried and an appropriate amount of DEPC (diethyl pyrocarbonate) is added. Water dissolution 'quantified by OD26_0. The process of RNA conversion to CDNA is utilized

SupperscriptTM III reverse transcriptase, while the quantitative pcR is ABI Prism 7900' and the reagents for reaction are from Applied Biosystems. The glycerol preparation nitrate: acid dehydrogenase (GAPDH) was used as a control group, and the expression level of BMP-2 mRNA in the control group was determined to be 100%. Referring to Fig. 9A, the expression of BMP-2 mRNA increased with the increase of imperatorin and fragrant, as shown in Fig. 9B, at the concentration of fixed imperatorin and bergamot, bmP-2 The expression of mRNA also increases with time. _Analysis of the effects of imperatorin, fragrant sputum and BMP anti-agent (noggin) on the ALP activity of oblique bone cells. The above osteoblasts were (1) 10 μΜ of imperatorin and (2) 10 μΜ of bergamot , (3) 10 μΜ of imperatorin + noggin, (4) 10 μΜ bergamot + noggin and (5) ljttg / ml noggin treatment for 48 hours, adding 0.2% NP-40 to dissolve the cells, the solution oscillated The cells were further centrifuged at 1500 x g for five minutes, and the activity of ALP in the supernatant was measured using an ALP kit. 0.1% DMSO was added as a control group, and the ALP activity of the control group was determined.

Client’s Docket No.: TT’s Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 19 200812604 for /. . As a result, as shown in Fig. 9C, n〇ggin inhibited the ALP activity promoted by imperatorin and bergamot, and it can be seen from the results of Example 17_18 that imperatorin and bergamot may increase βΜρ_2. The amount of bone cells promotes differentiation. H is the effect of arsenic and fragrant house on the phosphorylation of SMADs, p3R and EPK proteins

The cells were cultured in a 6-well culture dish and treated with 1 〇/χΜ of eugenol and bergamot for 1, 3, 6, 12, and 24 hours, respectively, and the medium was aspirated. 'Add appropriate lysis buffer (lySis) Buffer) (containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 4 mM EDTA, 10 mM Na4P207, 100 mM NaF, 2 mM Na3V04, 1% (v/v) Triton X-100, 〇·25% (w/v) oxycholic acid Sodiimrdeoxycholate, 50 mM 4-(2,aminoethyl) benzene sulfonylfluoride, 50 pg/ml leupeptin, and 20 pg/ml aprotinin The cells were scraped off with a scraper, and after complete extraction, they were aspirated into a centrifuge tube and centrifuged at 13,000 rpm for 15 minutes. Add 30 pg of the same amount of protein to 5 times Laemmlibuffer, cook at 95 ° C for 5 minutes, separate the protein by 8% SDS-PAGE, transfer to PVDF membrane, and then add 4% BSA at room temperature for 1 hour. Wash three times with PBS containing 0.1% Tween-20 (PBST), add primary antibody (p-SMAD, p-ERK, P-38, respectively) for 1 hour at room temperature, wash three times with PBST, add labeled wasabi Oxidase goat anti-mouse or goat anti-rabbit secondary antibody 'reacted at room temperature for 1 hour, and finally washed with PBST 3 times, then detected by ECL reagent

Clienfs Docket No.: TT’s Docket No:0973-A40927-TW/final/Kai/9/6/2006 20 200812604 ’ Cold light analysis. G-tubuline was added as a control group. Referring to Figure 1, both Euphorbia and Fragrance can promote phosphorylation of SMADs, p38 and EPK proteins. 19·Analysis of the effects of p3_H preparation iSB203580) and ERK interesting preparation iPD98059) on phosphorylation of p38 and EPK I

All procedures were the same as in Example ,, and were treated with 1 μμ of p38 inhibitor (SB203580) and ERK inhibitor (PD98059) for 3 minutes only before treatment with imperatorin and bergamot. Add the undissolved p38 or ERK as the pair and the group. As shown in Figures 11A-11B, both the p3g inhibitor (SB203580) and the ERK inhibitor (PD98059) inhibited the scalification promoted by imperatorin and bergamot. - Analysis of the effects of P3_S inhibitor (SB203580) and ERK inhibitor fPD98059 on the ALP activity of bone cells. The above osteoblasts were (1) 1 μμΜ of SB203580, (2) ΙΟμΜ of PD98059, (3) 1〇μΜ· Imperatorin and (4) 1〇μΜ of bergamot, (5) ΙΟμΜ of praeruptorin + SB203580, (6) 1〇μΜ of bergamot + SB203580, (7) ΙΟμΜ of bergamot + PD98059 and 8) After treatment with 1 〇μΜ PD98059 for 48 hours, cells were dissolved by adding 〇·2% ΝΡ-40, and the lysate was shaken and centrifuged at 1500 xg for five minutes. The activity of ALP in the supernatant was measured using an ALP kit. The 〇.i%dMSO was added as a control group, and the ALP activity of the control group was determined to be 1%. Results As shown in Figure 11D, both the p38 inhibitor (SB203580) and the ERK inhibitor (PD98059) were inhibited.

Client's Docket No.: TT’s Docket No: 0973-A40927-TW/fmal/Kai/9/6/2006 200812604 ^ Bone cell ALP activity promoted by imperatorin and citron. 21·Divided n38 辔妷 辔妷 [DN-p38) and ERK 辔抶 (DN_ERK) slant bone cell ALP activity

All the steps were the same as in Example 19, except that the cell material of some experimental groups was changed to p38 mutant strain (DN-p38) and ERK mutant strain (DN-ERK) as follows: (1) DN-p38 cell line, (2) DN-ERK cell line, (3) DN-p38 cell line + 10 μΜ of imperatorin, (4) DN-p38 cell line + 10 μΜ of fragrant _ limonene, (5) DN_ERK cell line + 10 μΜ of Europe Formosan (6) DN-ERK cell line + 10 μΜ bergamot (7) normal bone cells + 10 μΜ of imperatorin (8) normal bone cells + 1 μμΜ of bergamotene. The 〇.1% DMSO was used as a control group, and the ALP activity of the control group was set at 1%. As shown in Fig. 11D, the p38 mutant strain (DN-p38) and the ERK mutant strain (DN-ERK) inhibited bone cell ALP activity promoted by imperatorin and bergamotene, and can be carried out by Examples 18-20. As a result, it was found that imperatorin and bergamot increased the role of bone cells via SMADs, p38 and EPK proteins. 22. Animals were tested with imperatorin and bergamot for three-week-old male rats (Sprague-Dawley) weighing 78 to 90 g to 400 mg/ml trichloroacetaldehyde (trichl〇roacetaidehyde) Monohydrate), remove the tip of the 22G needle, sterilize the needle and embed it in the tibia close to the knee. On the next day, the outer end of the needle will be covered by the outer skin. The injection is made with a hose made of 30G needle. The amount is 10 μΐ, respectively, given physiological saline, 3 μμΜ of imperatorin or fragrant lemon

Client's Docket No.: TT^ Docket No:0973-A40927-TW/fmaVKai/9/6/2006 22 200812604 • Citriene, after one week of continuous application, rest for one week, remove the mouse and remove the muscle and connective tissue. 'Measure the BMD and BMC with DEXA. After completing the above procedure, the tibia was stained with paraffin-embedded sections. It can be seen from Table 3 that both imperatorin and bergamot can increase BMD and BMC of the tibia, and the results of section 12 staining show that imperatorin and bergamot can promote bone synthesis. Table 2: Effect of BMD and BMC on Imperatorin and BMC and BMC In the control group, the amount of B. sinensis (BMD) (g/cm3) 0.09±0.001 0.101=t〇.〇02* 0.101±0.002* Bone mineral density ( BMC) (g) 0.083 ± 0.002 0.107 ± 0.003 * 0.111 ± 0.003 * BV / TV (%) 8.63 ± 0.2 ------ - = 18.1 ± 0.4 *

The pharmaceutical composition of the present invention increases the weight and bone density of the femur and tibia, and has been confirmed in other biochemical tests to increase the activity of ALp and the content of collagen to prevent and/or treat bone loss. Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of the invention is defined by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effect of the pharmaceutical composition of the present invention on the body weight of rats.

Client's Docket No.: TT^ Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 23 200812604 Piece Figure 2 shows the 3A-3B diagram of the bone tissue cut of the rats taking the pharmaceutical composition of the present invention. The effect of each component of the pharmaceutical composition of the invention on fine colonization. .曰 Figure 4 shows the effect of various components in the pharmaceutical composition on the activity of bone cell test (ALP). Effect of the influence of Figures 5A-5B shows that the components of the pharmaceutical composition are mineralized to bone cells. Figure 6A shows that Figure 6B of the composition of the pharmaceutical composition for osteoclast differentiation shows that the components of the pharmaceutical composition are broken. The effect of thin bones. Blowing the 7th AA shows the survival of bone cells by imperatorin and bergamot. Figure 7B shows that the formation of eucalyptus and bergamot on bone cell proliferation shows that imperatorin and bergamot are fine. m 〇&quot;匕 Effect of ALP activity shadowing Figure 8B shows the effect of imperatorin and fragrant lemon on the collagen secretion of bone cells. &quot;... Collagen 8C-8D shows that Euphorbia humilis and fragrant lemons are affected by bone mineralization. Figure 9A-9B shows that imperatorin and bergamot are related to bone ^ &amp; white (bone morphogenetic The protein, BMP-2) gene of the heart of the egg" clothing.

Client's Docket No.: TT's Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 200812604 Figure 9C shows Ouqian (nog (four) on bone cells ALP, = 素, bergamot and BMP antagonist brother 1 〇A shows the effect of eucalyptus phosphorylation. The effect of uranium on SMADs, P38 and EPK protein in Figure 10B shows the effect of citric acid acidification. %~ on SMADs, P38 and EPK protein phosphorus t丨A UB is not P38 The effect of inhibitor (SB2G358G) and ERK inhibitor __) on the acidification of P38 and protein. Figure 11C shows the effect of 38 inhibitors (SB2〇358〇) and ERK inhibitory state (4) on bone cell ALp activity. ^ 11D shows the effect of 7^ P38 mutant (DN-P38) and ERK mutant (DN-ERK) on bone cell ALp activity. Figure 12 shows the animal experiment of imperatorin and bergamot. [Main component symbol description]

Client’s Docket No.: TT^ Docket No:0973-A40927-TW/fmal/Kai/9/7/2006 25

Claims (1)

200812604 Stupid. #10, the scope of application for patents: &quot; 1. A pharmaceutical composition for preventing and / or treating bone loss, including an effective amount of licorice, black beans, cnidium and antlers, wherein the weight ratio of each component is licorice: black beans : Cnidium: Antlers = 1-10 : 2-10: 1-10: 1-10. 2. A pharmaceutical composition for preventing and/or treating bone loss as described in claim 1, wherein the weight ratio of the ingredients is licorice: black beans: Cnidium antler = 2-4: 4_6: 1-3: 1-3. 3. A pharmaceutical composition for preventing and/or treating loss of bone loss as described in claim 1 wherein the osthol comprises osthol. 4. The pharmaceutical composition for preventing and/or treating bone loss according to claim 1, wherein the ostrich comprises imperatorin 〇 5. The prevention described in claim 1 And/or a pharmaceutical composition for treating bone loss, wherein the cnidium comprises bergapten 〇6. The pharmaceutical composition for preventing and/or treating bone loss and loss as described in claim 1 wherein the bone mass Loss includes osteoporosis or osteoporotic fractures. 7. The pharmaceutical composition for preventing and/or treating bone loss according to claim 1, wherein the pharmaceutical composition increases bone mineral density (BMD) by more than 10.3%. 8. The pharmaceutical composition for preventing and/or treating bone loss according to claim 1, wherein the pharmaceutical composition increases bone content (BMC) by 11.3% or more. Client^ Docket No.: TT's Docket No:0973-A40927-TW/final/Kai/9/6/2006 26 200812604 9. Pharmaceutical composition for preventing and/or treating bone loss as described in claim 1 Wherein the pharmaceutical composition is increased by more than 90% of the osteoclast assay (ALP) activity. 10. The pharmaceutical composition for preventing and/or treating bone loss according to claim 1, wherein the pharmaceutical composition increases the maximum load of 10.2% or more of the bone tissue. 11. The pharmaceutical composition for preventing and/or treating bone loss according to claim 1, wherein the pharmaceutical composition increases the ultimate loading of more than 13.2% of the bone tissue. 12. The pharmaceutical composition for preventing and/or treating bone loss according to claim 1, wherein the pharmaceutical composition increases the young's modulus of the bone tissue by 10.1% or more. The pharmaceutical composition for preventing and/or treating bone loss according to the above item 1, wherein the pharmaceutical composition increases the ultimate stress of 34.8% or more of the bone tissue. 14. The pharmaceutical composition for preventing and/or treating bone loss according to claim 1, wherein the pharmaceutical composition is selected from the group consisting of: a tablet, a capsule, a granule, a powder, a liquid, or a pill. . 15. A pharmaceutical composition for preventing and/or treating bone loss, comprising: 'an effective amount of imperatorin; and a pharmaceutically acceptable carrier or excipient. 16. The pharmaceutical composition for preventing and/or treating bone loss according to claim 15, wherein the bone loss comprises osteoporosis or Client's Docket No.: TT5s Docket No:0973-A40927-TW/fmal/ Kai/9/6/2006 200812604 m • Osteoporotic fracture. The pharmaceutical composition for preventing and/or treating bone loss according to claim 15, wherein the pharmaceutical composition is increased by more than 95.0% of bone cell alkaline phosphate (ALP) activity. The pharmaceutical composition for preventing and/or treating bone loss according to the invention of claim 15, wherein the pharmaceutical composition increases the collagen content of bone cells by more than 102.0%. 19. A pharmaceutical composition for preventing and/or treating bone loss as described in claim 15 wherein the pharmaceutical composition increases mineralization capacity of more than 110.0% of bone cells. 20. The pharmaceutical composition for preventing and/or treating bone loss according to claim 15, wherein the pharmaceutical composition is increased by a bone mineral density (BMD) of 12.2% or more. 21. The pharmaceutical composition for preventing and/or treating bone loss according to claim 15, wherein the pharmaceutical composition is increased by more than 28.9% of bone content (BMC). The pharmaceutical composition for preventing and/or treating bone loss according to claim 15, wherein the pharmaceutical composition comprises an oral or injectable dosage form. 23. A pharmaceutical composition for preventing and/or treating bone loss, comprising: an effective amount of bergapten; and a pharmaceutically acceptable carrier or excipient. 24. Prevention and/or treatment of bones as described in Section 23 of the patent application Client's Docket No.: TTs Docket No:0973-A40927_TW/fmal/Kai/9/6/2006 28 200812604 • Pharmaceutical composition with loss of quality, The bone loss includes osteoporosis or ~ osteoporotic fracture. 25. The pharmaceutical composition for preventing and/or treating bone loss according to claim 23, wherein the pharmaceutical composition increases bone alkaline strontium phosphate (ALP) activity by more than 80.0%. The pharmaceutical composition for preventing and/or treating bone loss according to Item 23, wherein the pharmaceutical composition increases the collagen content of bone cells by more than 100.0%. 27. A pharmaceutical composition for preventing and/or treating bone loss as described in claim 23, wherein the pharmaceutical composition increases mineralization capacity of more than 105.0% of bone cells. 28. The pharmaceutical composition for preventing and/or treating bone loss according to claim 23, wherein the pharmaceutical composition is increased by a bone mineral density (BMD) of 12.2% or more. 29. The pharmaceutical composition for preventing and/or treating bone loss according to claim 23, wherein the pharmaceutical composition is increased by 33.7% to the bone content (BMC). 30. A pharmaceutical composition for preventing and/or treating bone loss as described in claim 23, wherein the pharmaceutical composition comprises an oral or injectable dosage form. Client’s Docket No.: TT,s Docket No:0973-A40927-TW/fmal/Kai/9/6/2006 29