CN102166204A - Pharmaceutical composition for preventing and/or treating bone loss - Google Patents
Pharmaceutical composition for preventing and/or treating bone loss Download PDFInfo
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- CN102166204A CN102166204A CN2011100666786A CN201110066678A CN102166204A CN 102166204 A CN102166204 A CN 102166204A CN 2011100666786 A CN2011100666786 A CN 2011100666786A CN 201110066678 A CN201110066678 A CN 201110066678A CN 102166204 A CN102166204 A CN 102166204A
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Abstract
The invention provides a pharmaceutical composition which comprises an effective dose of bergapten and pharmaceutically acceptable carriers or excipients, and the pharmaceutical composition can be used for preventing and/or treating bone loss.
Description
The application is a Chinese patent application (denomination of invention: the medical composition of prevention and/or treatment bone-loss; The applying date: on October 18th, 2006; Application number: dividing an application 200610135591.9).
Technical field
The invention relates to a kind of medicine group compositions, and especially in regard to a kind of medical composition that prevents and/or treat bone-loss.
Prior art
Osteoporosis can be divided into two big classes: the one, and because of estrogen lacks " osteoporosis after the menolipsis " caused, this disease is sent out well on one's body the women after menolipsis; The reason of another kind of generation then is to produce owing to aging, and we are referred to as " veteran form osteoporosis ", no matter as long as this disease men and women lives enough oldly, all may take place.Can find that from clinical studies report when the estrogen hyposecretion of climacteric women, the speed that bone runs off 1 year can be up to 3 percent to 5 percent.After bone quicken to run off, will cause osteoporosis, when bony structure become bad the time, just may under the effect of external force, cause the generation of fracture.Osteoporosis has some specific predilection sites after climacteric, but the bone rough segmentation of human body is two major parts, a part that is outer than hard and compact, and we are called os osseum; The middle softer part of bone just generally is commonly called as the position of bone marrow in addition, and we are referred to as loose bone, and the position that osteoporosis is sent out well after climacteric is exactly the part of so-called loose bone.With human body, the ratio of contained loose bone of different parts bone and os osseum is all inequality, and climacteric osteoporosis sends out well that loose bone is the position of main composition composition in human body, and wherein main is exactly the vertebra position, so cause the compression fracture of lumbar vertebra easily; Be exactly the terminal of long bone in addition, i.e. the terminal region of bones of limbs also causes the fracture of wrist portion easily.The women of osteoporosis after climacteric, the type that easily causes fracture is exactly these two kinds.
Osteoporosis after the menolipsis (postmenopausal osteoporosis) is the normal a kind of metabolic osteopathy of suffering from of elderly woman.Can find in the clinical studies report that when the estrogen hyposecretion of climacteric women, the speed that bone runs off 1 year can be up to 3 percent to 5 percent, along with the prolongation of human average life, osteoporotic sickness rate is in rising trend after the menolipsis.After the menolipsis due to the women's osteoporosis morbidity machine alternation of hosts essential factor menolipsis women He Ermeng lack technical staff's sclerotin of causing knowing this run off in a large number (descending 3～6% even higher every year approximately).Cause just often oestrogen can stimulate " osteoblast " (osteoblast) to produce cell medium, suppresses " osteoclast " activity (osteoclast) on the one hand, and order one side stimulation " osteoblast " is so that sclerotin is kept poised state.In case shortage estrogen, then sclerotin runs off in a large number, calcium ion disengages from bone in a large number, constrained parathyroid gland concentration, cause the synthetic minimizing of activated vitamin D, absorb so lowered the technical staff that calcium ion knows this at gastrointestinal tract, cause calcium ion " negative balance ", quicken the generation of osteoporosis.
Aging society is stepped in Taiwan gradually, and will there be more Crinis Carbonisatus bone growth promoting matter osteoporosis in association thus.Osteoporosis after the menolipsis (postmenopausal osteoporosis) is the normal a kind of metabolic osteopathy of suffering from of elderly woman.Along with the prolongation of compatriots' average life, osteoporotic sickness rate is in rising trend after the menolipsis.Modern medicine to menolipsis after osteoporotic Therapeutic Method mainly be controversies in hormone replacement in the elderly, but life-time service has certain side effect (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, Kelikowske K.Hormonereplacement therapy and endometrial cancer risk:a meta-analysis.ObstetGynecol, 1995,85:304-313), therefore, need a kind of medical composition that can be used to prevent and/or treat bone-loss at present badly.
Summary of the invention
Purpose of the present invention can be used to prevent and treat old man or menolipsis women's bone-loss for a kind of medical composition is provided.
For reaching above-mentioned purpose, the invention provides the medical composition of a kind of prevention and/or treatment bone-loss, the Radix Glycyrrhizae, Semen sojae atricolor, Fructus Cnidii and the Cornu Cervi that comprise effective dose, wherein the weight ratio of each composition is a Radix Glycyrrhizae: Semen sojae atricolor: Fructus Cnidii: Cornu Cervi=1-10: 2-10: 1-10: 1-10.
For reaching above-mentioned purpose, the present invention provides the medical composition of a kind of prevention and/or treatment bone-loss in addition, comprises the imperatorin (imperatorin) of effective dose, and pharmaceutically acceptable carrier or excipient.
For reaching above-mentioned purpose, the present invention provides the medical composition of a kind of prevention and/or treatment bone-loss in addition, comprises the bergaptene (bergapten) of effective dose, and pharmaceutically acceptable carrier or excipient.
Brief description of drawingsfig
The 1st figure shows the influence of medical composition of the present invention to rat body weight.
Technical staff's osseous tissue section that medical composition rat of the present invention is known this is taken in the 2nd figure demonstration.
3A-3B figure shows the influence of each composition on cell proliferation in the medical composition of the present invention.
The 4th figure shows that each composition is to the active influence of osteocyte alkali phosphatase (ALP) in the medical composition.
5A-5B figure shows that each composition is to the influence of osteocyte mineralising in the medical composition.
6A figure shows that each composition is to the influence of osteoclast differentiation in the medical composition.
6B figure shows that each composition is to the influence of osteoclast resorption in the medical composition.
7A figure shows the influence that imperatorin and bergaptene are survived to osteocyte.
7B figure shows the influence that imperatorin and bergaptene are bred osteocyte.
8A figure shows that imperatorin and bergaptene are to the active influence of osteocyte ALP.
8B figure shows imperatorin and bergaptene are secreted collagen protein (collagen) to osteocyte influence.
8C-8D figure demonstration imperatorin and bergaptene are to the influence of osteocyte mineralising.
9A-9B figure shows that imperatorin and bergaptene are to bone morphogenetic protein (bonemorphogenetic protein, BMP-2) expression of gene.
9C figure shows that imperatorin, bergaptene and bmp antagonist (noggin) are to the active influence of osteocyte ALP.
10A figure shows the influence of imperatorin to SMADs, p38 and EPK protein phosphorylation.
10B figure shows the influence of bergaptene to SMADs, p38 and EPK protein phosphorylation.
11A-11B figure demonstration p38 inhibitor (SB203580) and ERK inhibitor (PD98059) are to the influence of p38 and EPK protein phosphorylation.
11C figure shows that p38 inhibitor (SB203580) and ERK inhibitor (PD98059) are to the active influence of osteocyte ALP.
11D figure shows that p38 mutant (DN-p38) and ERK mutant (DN-ERK) are to the active influence of osteocyte ALP.
The 12nd figure shows that imperatorin and bergaptene carry out zoopery.
Can become apparent in order to allow the present invention know this technical staff above-mentioned and other purpose, feature and advantage, hereinafter enumerate preferred embodiment especially, and cooperate appended accompanying drawing, be described in detail below:
Embodiment
The present invention provides the medical composition of a kind of prevention and/or treatment bone-loss.In one embodiment, medical composition of the present invention comprises Radix Glycyrrhizae, Semen sojae atricolor, Fructus Cnidii, Cornu Cervi etc., and wherein the weight ratio of each composition is a Radix Glycyrrhizae: Semen sojae atricolor: Fructus Cnidii: Cornu Cervi=1-10: 2-10: 1-10: 1-10 is preferably 2-4: 4-6: 1-3: 1-3.And also comprise osthole (osthol), imperatorin (imperatorin) and bergaptene (bergapten) in the medical composition of the present invention.
The manufacture method of medical composition of the present invention is special procedure for processing, and at first, with the extract extraction, use therein extract can be water, ethanol etc. with the Radix Glycyrrhizae heating.Again with Semen sojae atricolor and Fructus Cnidii with the about 2-40 of soak with ethanol hour, preferably about 10-20 hour, the reheat extraction.Afterwards, Semen sojae atricolor, Fructus Cnidii extract and Radix Glycyrrhizae extract liquid are merged, volume ratio each other is a Radix Glycyrrhizae: Semen sojae atricolor+Fructus Cnidii=1: 2, behind concentrating under reduced pressure, get concentrated solution 10-100 litre, the harts horn carbon of 10-40 kilogram is added to back and dry in the concentrated solution, with dry thing pulverizing, pelletize and make dosage form, be medical composition of the present invention.
Medical composition of the present invention can be used for prevention and/or treatment bone-loss, and wherein bone-loss comprises osteoporosis or osteoporotic fracture.This medical composition can increase individual bone density (BMD), bone content (BMC) and osteocyte alkali phosphatase (ALP) activity.Generally speaking, medical composition of the present invention can increase by 5.15% bone density, 3.77% bone content and 30.0% osteocyte alkali phosphatase (ALP) activity, can increase by 10.30% bone density, 11.32% bone content and 68.0% osteocyte alkali phosphatase (ALP) activity in preferred enforcement.And this medical composition also can increase maximum stress (maximal load), fracture strength (ultimate loading), young's modulus (young ' s modulus) and the fracture strength (ultimate stress) of individual osseous tissue.Generally speaking, medical composition of the present invention can increase by 3.14% maximum stress, 5.23% fracture strength, 7.49% young's modulus and 15.65% fracture strength, can increase by 10.24% maximum stress, 13.15% fracture strength, 10.07% young's modulus and 37.39% fracture strength in preferred enforcement.
Medical composition of the present invention can be made into various multi-form dosage forms, for example lozenge, capsule, granule, powder, liquor or pill etc. according to actual needs.
In another kind of embodiment, medical composition of the present invention comprises imperatorin (imperatorin) and the pharmaceutically acceptable carrier or the excipient of effective dose, and it can be oral or injection type, and injection type can be intramuscular injection or subcutaneous injection.This medical composition can increase individual bone density, bone content and osteocyte alkaline phosphatase activities, generally speaking, medical composition of the present invention can increase by 12.22% bone density, 28.91% bone content and 6.0% osteocyte alkaline phosphatase activities, can increase by 90% osteocyte alkaline phosphatase activities in preferred enforcement.
In another kind of embodiment, medical composition of the present invention comprises bergaptene (bergapten) and the pharmaceutically acceptable carrier or the excipient of effective dose, and it can be oral or injection type, and injection type can be intramuscular injection or subcutaneous injection.This medical composition can increase individual bone density, bone content and osteocyte alkali phosphatase (ALP) activity, generally speaking, medical composition of the present invention can increase by 12.22% bone density, 33.73% bone content and 5% osteocyte alkaline phosphatase activities, can increase by 75% osteocyte alkali phosphatase (ALP) activity in preferred enforcement.
Individuality mentioned among the present invention is an animal, comprises human or inhuman animal, as Canis familiaris L., cat, mice, rat, cattle, sheep, pig, goat or the inhuman technical staff primates of knowing this.In one embodiment, mammal is behaved, and in another kind of embodiment, mammal is a rodent, as mice or rat.
By the following examples to illustrate further feature of the present invention and advantage.
Embodiment
1. medical composition of the present invention is known this technical staff's preparation
Medical composition of the present invention is made up of for 8 kilograms 30 kilograms in Radix Glycyrrhizae (available from gold security personnel trade Co., Ltd), 60 kilograms in Semen sojae atricolor (available from Di Tai trade Co., Ltd), 10 kilograms of Fructus Cnidiis (available from easily holding trade Co., Ltd), Cornu Cervi (available from Di Tai trade Co., Ltd).At first, Radix Glycyrrhizae is heated to 100 ℃,, gets 150 liters of extracts with the water extraction.Again with Semen sojae atricolor and Fructus Cnidii with 30% soak with ethanol 20 hours, reheat to 80 ℃ extraction merges with Radix Glycyrrhizae extract liquid, and volume ratio each other is a Radix Glycyrrhizae: Semen sojae atricolor+Fructus Cnidii=1: 2, get 30 liters of concentrated solutions through concentrating under reduced pressure, following dry 48 hours behind the adding harts horn carbon in concentrated solution in 60 ℃.Dry thing is pulverized the back pelletize and is made dosage form.
2. this technical staff's step is known in zoopery
Adopt laboratory animal breeding of country of National Science Council and research center, Sprague-Dawley kind, the female rats in 8 ages in week after raising for 4 weeks, are carried out the spay operation.Be divided into (1) sham operated rats at random, (2) oophorectomize control group and (3) oophorectomize give medical composition and treat three groups, with of Chloral (trichloroacetaldehyde) anesthesia of Sprague-Dawley rat with 400mg/kg, the sterile working, enter the rat abdominal cavity dorsal part through lumbar vertebra escribe mouth, the rat bilateral ovaries of removal ovary control group and the upright benefit group of oophorectomize+thigh is excised fully, and sham operated rats is not cut ovary, hemostatic suture.Postoperative ingest and drink water as before.Back medical treatment group begins per os and gavages medical composition every other day, per kilogram of body weight gram every days 1.2, every day 1 time, sham operated rats and ovariectomized group then gavage carboxy methyl cellulose (Carboxymethyl cellulose, CMC).Dosage is proofreaied and correct 1 time by body weight weekly, and altogether nursing, 6 weeks of administration, whole rats are put to death in 6 all backs.
3. medical composition is to the influence of bone length and weight
After above-mentioned rat is put to death, two side femurs (femur) and tibia (tibia) taking-up are stored in-80 ℃ of technical staff's refrigerators of knowing this, and muscle that femur and tibia is outer and connective tissue are shaved and are removed, utilize microbalance to measure the weight of bone, bone length then be utilize micro-rear sight measure (± 0.05mm), bone amount (BMD) and bone density (BMC) are with dual-energy X-ray absorptiometer (DEXA, XR-26; Norland, Fort Atkinson WI) measures.And femur fixed two days in 4% formalin after, place 4 ℃ EDTA (10%) two weeks of decalcification, the femur embedding is done the 5mm section after utilizing dehydration of alcohol to paraffin.Utilize Mayer ' s hematoxylin-eosin solvent dyeing, re-use the bone amount (bone volume) of the quantitative secondary spongy bone of Image-pro plus (3.0ed) software (secondary spongiosa).With reference to the 1st figure, the weight ratio sham operated rats of ovariectomized group is heavy, and takes the increase that medical composition of the present invention can't suppress rat body weight.With reference to the 2nd figure, by finding that medical composition of the present invention has the effect that prevents bone-loss in the osseous tissue section.Other data as shown in Table 1.
Table one, medical composition are to the influence of osseous tissue
4. the analysis of bone biomechanical
Use material testing system (MTS-858, MTS System Inc., Minneapolis, MN) 3 fracture (three-point bending test) measure the osseous tissue biomechanics.The distance of bone two ends is 20mm, and pressing the speed of falling that hangs down is per minute 1mm, can obtain young's modulus and fracture strength after gained speed and pressure are done the regression line and brought the below formula into.
5. osteoblastic cultivation of generation just
Be taken from conceived the 18th day Spraque-Dawley Mus embryo's parietal bone frame (parietal bone), after female Mus is put to death, whole uterus is taken off and is put in the aseptic culture dish, separate by uterus taking-up tire Mus and with its skull (calvarium), cut parietal bone and avoid getting sutura (suture), the periosteal layer of osteocomma both sides is removed, again osteocomma is cut into pieces, Collagenase with 0.1% (collagenase) is done totally 5 times sequentiality digestion in each 20 minutes with osteocomma, so that cell is discharged from osteocomma.Collect the cell that last three digestion are come out, the cell that mixes these three times digestion is the osteoblast suspension.Cell culture is in α-MEM, wherein contains 1% penicillin-streptomycin (penicillin-streptomycin) and 10% FBS, places 37 ℃ to contain 5%CO
2Incubator in, when cell covers with, carry out successive transfer culture, all experiments are only taken five generations of the first generation to the as experiment material.
6. the effect of each composition on cell proliferation in the analysis medical composition
Above-mentioned osteoblast is cultivated in 96 hole culture dishs, the concentrate that adds (1) Radix Glycyrrhizae, (2) Semen sojae atricolor+Fructus Cnidii, (3) Fructus Cnidii, (4) Semen sojae atricolor and (5) medical composition of 5,15,50,100 μ g/ml respectively, act on after 48 hours, culture fluid on the careful absorption cell, according to Cell Proliferation ELISA, the process of BrdU kit (Roche Applied Science) is measured cel l proliferation.Adding 0.1% DMSO, and be decided to be 100% with the cell proliferation rate of matched group as matched group.The result is shown in 3A-3B figure, and all compositions all can increase the propagation of osteocyte.
In the analysis medical composition each composition to the active influence of osteocyte alkali phosphatase (ALP)
After above-mentioned osteoblast handled with the concentrate of (1) Radix Glycyrrhizae of 5,15,50,100 μ g/ml, (2) Semen sojae atricolor+Fructus Cnidii, (3) Fructus Cnidii, (4) Semen sojae atricolor and (5) medical composition respectively, add 0.2%NP-40 with cytolysis, the centrifugal again 1500 * g of lysate process vibration five minutes utilizes ALP kit to measure the activity of ALP in the supernatant.Adding 0.1% DMSO, and be decided to be 100% with the activity of the ALP of matched group as matched group.The result is shown in the 4th figure, and except Semen sojae atricolor, other composition all can increase the activity of osteocyte ALP.
In the analysis medical composition each composition to the influence of osteocyte mineralising
Give differentiation culture liquid respectively to osteoblast and (contain the vitamin C of 50 μ g/ml and 10mM
Glycerophosphate (β-glycerophosphate)) and 5,15,50, (1) Radix Glycyrrhizae of 100 μ g/ml, (2) Semen sojae atricolor+Fructus Cnidii, (3) Fructus Cnidii, (4) concentrate of Semen sojae atricolor and (5) medical composition, culture fluid was changed once in per three days, cell adds 75% ethanol and fixes 30 minutes after 14 days, the alizarin red (Alizarin red-S) that adds 40mM more at room temperature acts on one hour, to remove not the stain with the calcium bond, add the dissolving of 10% cetylpyridinium chloride (cetylpyridinium chloride) at last, utilize the 550nm light absorption value to measure bone brief summary content.Adding 0.1% DMSO, and be decided to be 100% with the mineralization degree of matched group as matched group.The result is shown in 5A-5B figure, and except Semen sojae atricolor, other 4 kinds of compositions all can increase mineralization.
9. the influence that each composition breaks up osteoclast in the analysis medical composition
Utilize syringe to extract the bone marrow of 6-8 week Spraque-Dawley mouse out,, calculate buoyant cell (hematopoietic cell) 1 * 10 through one day sedimentation
6In culture dish, the concentrate that adds (1) Radix Glycyrrhizae, (2) Semen sojae atricolor+Fructus Cnidii, (3) Fructus Cnidii, (4) Semen sojae atricolor and (5) medical composition of differentiation agent (containing the RANKL of 50ng/ml and the M-CSF of 20ng/ml) and 150 μ g/ml respectively, changed differentiation agent in per three days, cultivate after 8 days, with anti-tartaric acid phosphatase ferment (tartrate-resistant acid phosphatase, TRAP) dyeing is calculated at microscopically and to be dyed TRAP and nucleus more than 3 cell.To add 0.1%DMSO as matched group.With reference to 6A figure, only there is the Semen sojae atricolor composition to understand the differentiation that a little ground suppresses osteoclast.
In the analysis medical composition each composition to the influence of osteoclast resorption
Five the biggest New Zealand rabbits are put to death, utilize shears to shred long bone and isolate sophisticated osteoclast on the bone wall, with cell harvesting and plant in OAAS dish, give the concentrate of (1) Radix Glycyrrhizae, (2) Semen sojae atricolor+Fructus Cnidii, (3) Fructus Cnidii, (4) Semen sojae atricolor and (5) medical composition of 150 μ g/ml respectively, after cultivating 3 days, the sodium hydroxide (NaOH) that adds 1N will clean 3 times behind the cytolysis, utilize digital camera to obtain image, use image analysing computer software (Image-Pro Plus 3.0) to calculate the region area that osteoclast absorbs at microscopically.Adding 0.1% DMSO, and be decided to be 100% with the osteoclast reabsorption rate of matched group as matched group.With reference to 6B figure, above-mentioned 5 kinds of portionings all can not influence the resorbent effect of osteoclast.
11. analyze imperatorin (imperatorin) and bergaptene (bergapten) shadow to the osteocyte survival
Ring
Osteocyte is layered in the 96 hole culture dishs, respectively with the imperatorin of 0.3,1,3,10 μ M and bergaptene effect after 48 hours, culture fluid on the careful absorption cell, add MTT (0.5mg/ml) solution and act on 30 minutes down in 37 ℃, then sop up solution, the inferior maple of dimethyl (DMSO) with 100 μ l comes dissolved cell, uses Bio-Kinetic Elisa Reader (BIO-TEK) to measure its light absorption value down in wavelength 550nm at last.Adding 0.1% DMSO, and be decided to be 100% with the cell number of matched group as matched group.With reference to 7A figure, imperatorin and bergaptene all can not influence the survival of osteocyte.
12. analyze the influence of imperatorin and bergaptene to osteocyte propagation
With above-mentioned osteoblast kind in 96 hole culture dishs, after handling 48 hours with the imperatorin of 0.3,1,3,10 μ M and bergaptene respectively, culture fluid on the careful absorption cell, according to Cell Proliferation ELISA, the process of BrdU kit (Roche Applied Science) is measured cel l proliferation.Adding 0.1% DMSO, and be decided to be 100% with the cell proliferation of matched group as matched group.With reference to 7B figure, imperatorin and bergaptene all can not influence the propagation of osteocyte.
13. analyze imperatorin and bergaptene to the active influence of osteocyte ALP
After above-mentioned osteoblast handled with the imperatorin of 0.3,1,3,10 μ M and bergaptene respectively, add 0.2%NP-40 with cytolysis, lysate utilizes ALP kit to measure the activity of ALP in the supernatant through the centrifugal again 1500 * g of vibration five minutes.Add 0.1% DMSO as matched group, and be decided to be 100% with the activity of the ALP of matched group.The result is shown in 8A figure, and imperatorin and bergaptene all can increase the activity of ALP.
14. analyze imperatorin and bergaptene are secreted collagen protein (collagen) to osteocyte influence
Utilize the amount that 4-hydroxyl proline (4-hydroxyproline) content is represented the osteoblast collagen protein synthesis of measuring.After above-mentioned osteoblast handled with the imperatorin of 0.3,1,3,10 μ M and bergaptene respectively, behind cell differentiation, give 6N HCl dissolved cell, place glass tubing 116 ℃ of following hydrolysis 16 hours lysate.The hydrolyzed solution vacuum is drained, added the water dissolution of equivalent, under the 550nm wavelength, measure the content of 4-hydroxyl proline (4-hydroxyproline).Adding 0.1% DMSO, and be decided to be 100% with the amount of the collagen protein of matched group as matched group.The result is shown in 8B figure, and imperatorin and bergaptene all can not influence the ability of osteocyte secretion collagen protein.
15. analyze the influence of imperatorin and bergaptene to the osteocyte mineralising
Osteoblast gives differentiation culture liquid and (contains the vitamin C of 50 μ g/ml and the β glycerophosphate (glycerophosphate)) of 10mM, culture fluid was changed once in per three days, and simultaneously with 0.3,1,3, the imperatorin of 10 μ M and bergaptene are handled, add 75% ethanol after 14 days and fix 30 minutes, the alizarin red (Alizarin red-S) that adds 40mM more at room temperature acts on one hour, remove not stain with the calcium bond, add the dissolving of 10% cetylpyridinium chloride (cetylpyridinium chloride) at last, utilize the 550nm light absorption value to measure bone brief summary content.Adding 0.1% DMSO, and be decided to be 100% with the mineralising of the bone brief summary of matched group as matched group.The result is shown in 8C-8D figure, and imperatorin and bergaptene all can increase mineralization.
16. analyze imperatorin and bergaptene to bone morphogenetic protein (bone morphogenetic
Protein, BMP-2) expression of gene
Osteocyte is cultivated among the culture dish in 6 holes, added 0.3,1,3,10 μ M respectively
Imperatorin and bergaptene handle 1,3,6, after 12 hours, adding TRIzol reagent leaves standstill in room temperature and made cytolysis in 5 minutes, cytolysis liquid is collected in centrifuge tube, chloroform (chloroform) jolting that adds 0.5ml becomes uniform milky liquid, in room temperature leave standstill made the solution layering in 5 minutes after, under 4 ℃ centrifugal 15 minutes with 14000 * g, carefully extract supernatant afterwards, the isopropyl alcohol (isopropanol) and the centrifuge tube that overturns that add 0.25ml make its mixing, then can obtain precipitating the RNA that separates out under 4 ℃ after centrifugal 15 minutes with 14000 * g, outwell behind the supernatant again with 75% alcohol wash precipitate, descended 14000 * g centrifugal 10 minutes in 4 ℃ again, at last that RNA is air-dry and add an amount of DEPC (diethyl pyrocarbonate) water dissolution, with OD
260/280Quantitatively.The process that RNA changes cDNA into is to utilize Supperscript
TMIII reverse transcription, quantitative PCR then are to use ABI Prism 7900, and the reagent of reaction is all from Applied Biosystems company.(glyceraldehydephosphate dehydrogenase GAPDH) is matched group, and is decided to be 100% with the expression of the BMP-2mRNA of matched group with Triose phosphate dehydrogenase.With reference to 9A figure, the expression of BMP-2mRNA can increase along with the increase of imperatorin and bergaptene, and with reference to 9B figure, under the concentration of fixedly imperatorin and bergaptene, the expression of BMP-2mRNA also can increase along with the increase of time.
17. analyzing imperatorin, bergaptene and bmp antagonist (noggin) lives to osteocyte ALP
The influence of property
After above-mentioned osteoblast handled 48 hours with the noggin of the bergaptene+noggin of imperatorin+noggin, (4) the 10 μ M of the bergaptene of the imperatorin of (1) 10 μ M, (2) 10 μ M, (3) 10 μ M and (5) 1 μ g/ml respectively, add 0.2%NP-40 with cytolysis, the centrifugal again 1500 * g of lysate process vibration five minutes utilizes ALP kit to measure the activity of ALP in the supernatant.Adding 0.1% DMSO, and be decided to be 100% with the ALP activity of matched group as matched group.The result is shown in 9C figure, and noggin can suppress by imperatorin and ALP activity that bergaptene promoted, and by the result of embodiment 17-18 as can be known, imperatorin and bergaptene may be the differentiation that promotes osteocyte via the amount that increases BMP-2.
18. analyze imperatorin and bergaptene shadow to SMADs, p38 and EPK protein phosphorylation
Ring
With cell culture after handling 1,3,6,12,24 hour with the imperatorin of 10 μ M and bergaptene respectively among the culture dish of 6 wells, culture medium is siphoned away, add suitable dissolving buffer (lysisbuffer) and (contain 50mM HEPES (PH 7.4), 150mM NaCl, 4mM EDTA, 10mM Na
4P
2O
7, 100mM NaF, 2mM Na
3VO
41% (v/v) Triton X-100,0.25% (w/v) oxycholic acid sodium (sodium deoxycholate), the 4-of 50mM (2-aminoethyl) benzene sulfonyl fluorine (4-(2-aminoethyl) benzene sulfonylfluoride), 50 μ g/ml leupeptin and 20 μ g/ml aprotiniies (aprotinin)), cell is scraped with burning-in knife, when being drawn to centrifuge tube after the extraction fully, with 13, centrifugal 15 minutes of 000rpm.The equal protein matter of 30 μ g is added 5 times of Laemmli buffer, boiled 5 minutes at 95 ℃, behind the 8%SDS-PAGE isolated protein, be transferred to PVDF membrane, add then and contain 4%BSA in room temperature after following 1 hour, clean three times with the PBS that contains 0.1%Tween-20 (PBST), add an antibody and (be respectively p-SMAD, p-ERK, P-38) room temperature reaction 1 hour, after PBST cleans three times, add the anti-ageing Mus of goat of labelling Wasabia japonic (Euterma Wasabi) peroxidase or the secondary antibodies of goat antirabbit, in room temperature reaction 1 hour, clean 3 times with PBST at last after, promptly detect and carry out ferment and connect plain cold light analysis with ECL reagent.To add
α-tubulineAs matched group.With reference to the 10th figure, imperatorin and bergaptene all can promote the proteic phosphorylation of SMADs, p38 and EPK.
19. analyze p38 inhibitor (SB203580) and ERK inhibitor (PD98059) to p38 and EPK
The influence of protein phosphorylation
Institute is identical with embodiment 17 in steps, only before handling with imperatorin and bergaptene, handles 30 minutes with p38 inhibitor (SB203580) and the ERK inhibitor (PD98059) of 10 μ M earlier.To add not phosphorylation p38 or ERK as matched group.The result is shown in 11A-11B figure, and p38 inhibitor (SB203580) and ERK inhibitor (PD98059) all can suppress by imperatorin and phosphorylation that bergaptene promoted.
20. analyze p38 inhibitor (SB203580) and ERK inhibitor (PD98059) to osteocyte ALP
Active influence
After above-mentioned osteoblast handled 48 hours with the bergaptene+PD98059 of bergaptene+SB203580, (7) the 10 μ M of imperatorin+SB203580, (6) the 10 μ M of the bergaptene of the imperatorin of PD98059, (3) the 10 μ M of SB203580, (2) the 10 μ M of (1) 10 μ M and (4) 10 μ M, (5) 10 μ M and (8) 10 μ M PD98059 respectively, add 0.2%NP-40 with cytolysis, the centrifugal again 1500 * g of lysate process vibration five minutes utilizes ALP kit to measure the activity of ALP in the supernatant.Adding 0.1%DMSO, and be decided to be 100% with the ALP activity of matched group as matched group.The result is shown in 11D figure, and p38 inhibitor (SB203580) and ERK inhibitor (PD98059) all can suppress by imperatorin and osteocyte ALP activity that bergaptene promoted.
21. analyzing p38 mutant (DN-p38) and ERK mutant (DN-ERK) lives to osteocyte ALP
The influence of property
Institute is identical with embodiment 19 in steps, only partly the cell material of experimental group changes p38 mutant (DN-p38 into, by Dr.J.Han (Scripps Research Institute, San Diego, CA) provide) and ERK mutant (DN-ERK, by Dr.M.Cobb (South-Western Medical Center, Dallas, TX) provide), as follows: (1) DN-p38 cell strain, (2) DN-ERK cell strain, (3) imperatorin of DN-p38 cell strain+10 μ M, (4) bergaptene of DN-p38 cell strain+10 μ M, (5) bergaptene of imperatorin (8) normal bone cell+10 μ M of bergaptene (7) normal bone cell+10 μ M of imperatorin (6) DN-ERK cell strain+10 μ M of DN-ERK cell strain+10 μ M.As matched group, and be decided to be 100% with 0.1%DMSO with the ALP activity of matched group.The result is shown in 11D figure, p38 mutant (DN-p38) and ERK mutant (DN-ERK) can suppress by imperatorin and osteocyte ALP activity that bergaptene promoted, and can be by embodiment 18-20 found that imperatorin and bergaptene are the effects that increases osteocyte via SMADs, p38 and EPK albumen.
22. carry out zoopery with imperatorin and bergaptene
Get three all macrandry children Mus (Sprague-Dawley), weight range 78 to 90 grams, single water Chloral (trichloroacetaldehyde monohydrate) anesthesia with 400mg/ml, the 22G needle tip is taken off, after the syringe needle sterilization it is imbedded tibia near knee, the next day, the syringe needle outer end just can be covered in the past by crust, the flexible pipe that utilizes the 30G syringe needle to be made is injected, the per injection amount is 10 μ l, give normal saline solution respectively, the imperatorin of 30 μ M or bergaptene after one week of injection, are had a rest a week continuously, mice is put to death the taking-up tibia, remove muscle and connective tissue, use DEXA to measure BMD and BMC, tibia is utilized specimens paraffin embedding slices dyeing after finishing said process.As shown in Table 3, imperatorin and bergaptene all can increase the BMD and the BMC of tibia, and the 12nd figure section statining result as can be known, and imperatorin and bergaptene all can promote the synthetic of sclerotin.
Table three, imperatorin and bergaptene are to the influence of BMD and BMC
Medical composition of the present invention can increase weight, the bone density of femur and tibia, and confirms in other biochemical test, and it can increase the activity of ALP and the content of collagen protein, and then reaches prevention and/or treatment bone-loss.
Though the present invention discloses as above with preferred embodiment; right its is not in order to limit the present invention; any technical staff who knows this; without departing from the spirit and scope of the present invention; can do a little change and retouching, so protection scope of the present invention is as the criterion when looking the scope that claim defined of enclosing.
Claims (8)
- One kind the prevention and/or the treatment bone-loss medical composition, comprising:The bergaptene of effective dose; AndPharmaceutically acceptable carrier or excipient.
- 2. the medical composition of prevention according to claim 1 and/or treatment bone-loss, wherein this bone-loss comprises osteoporosis or osteoporotic fracture.
- 3. the medical composition of prevention according to claim 2 and/or treatment bone-loss, wherein this medical composition increases the osteocyte alkaline phosphatase activities more than 80.0%.
- 4. the medical composition of prevention according to claim 3 and/or treatment bone-loss, wherein this medical composition increases the collagen content of osteocyte more than 100.0%.
- 5. the medical composition of prevention according to claim 4 and/or treatment bone-loss, wherein this medical composition increases the mineralization ability of osteocyte more than 105.0%.
- 6. the medical composition of prevention according to claim 5 and/or treatment bone-loss, wherein this medical composition increases the bone density more than 12.2%.
- 7. the medical composition of prevention according to claim 6 and/or treatment bone-loss, wherein this medical composition increases the bone content more than 33.7%.
- 8. the medical composition of prevention according to claim 7 and/or treatment bone-loss, wherein this medical composition comprises oral or injection type.
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