CN108030779A - Carnosic acid is used to prepare ERR alpha expressions inhibitor and treats the purposes of the medicine of osteoporosis - Google Patents
Carnosic acid is used to prepare ERR alpha expressions inhibitor and treats the purposes of the medicine of osteoporosis Download PDFInfo
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Abstract
ERR alpha expressions inhibitor is used to prepare the invention discloses carnosic acid and treats the purposes of the medicine of osteoporosis.Inventor has found effective inhibitor that carnosic acid is ERR alpha expressions by vitro test, can suppress the expression of osteoporosis Research of predicting markers in osteoporosis cell model；The physiological phenomenon (such as shin bone BV/TV is reduced, shin bone Tb.Th is reduced, shin bone Tb.N reduces shin bone Tb.SP increases, shin bone SMI increases) that in vivo studies finds carnosic acid and can to cut off caused by mouse ovarian takes a turn for the worse to a certain extent, so as to treat the bone defect (such as bone mass the reduces and/or microstructure degeneration of bone) caused by estrogen deficiency, there is prospect of the exploitation into the medicine for treating the osteoporosis that menopause triggers.
Description
Technical field
The invention belongs to field of medicaments, it is related to the new application of known compound, and in particular to carnosic acid is used to prepare ERR
The purposes of alpha expression inhibitor and the medicine for the treatment of osteoporosis.
Background technology
Osteoporosis (Osteoprosis, OP) be reduced using bone mass, bone tissue fibre structure degenerative change as spy
Sign, bone brittleness increase, be prone to fracture a kind of general metabolism osteopathy, be mainly shown as pain in back and loin, bone density reduce,
Easily fracture.With the arrival of aging society, the incidence of osteoporosis is also with increasing, women post menopausal, in body
Estrogen level significantly reduces, and bone loss is accelerated, and causes bone resorption rate to be more than bon e formation speed, ultimately causes post menopausal sclerotin
Osteoporosis (Postmenopausal osteoprosis, PMOP), become a kind of common disease of postmenopausal women.
ERR points are tri- kinds of hypotypes of α, β, γ, and the functional study of wherein ERR α is relatively more, it is in tumour, coronary heart disease, diabetes
It is subject to larger concern (bibliography etc. the effect in disease：Constitutive activities ofestrogen-
relatedreceptors:Transcriptional regulation of metabolism by the ERR pathways
in health and disease.Biochim BiophysActa,2015).Some researches show that ERR α participate in estrogen receptor
Regulation and control and bone cell function activity is regulated and controled, there is good therapeutic effect (bibliography to Postmenopausal Osteoporosis：
The ERR α study on mechanism of the anti-postmenopausal osteoporosis of Zhuanggu Zhitong capsule, Chinese Journal of Modern Applied Pharmacy June the 33rd in 2016
Rolled up for the 6th phase).
Inventor has found that carnosic acid is effective inhibitor of ERR alpha expressions, can be used for treating postmenopausal osteoporosis
Disease.The above-mentioned effect of open carnosic acid is not yet had been reported that at present.
The content of the invention
ERR alpha expressions inhibitor is used to prepare it is an object of the present invention to provide carnosic acid and treats the medicine of osteoporosis
Purposes.
The above-mentioned purpose of the present invention is achieved by following technical solution：
Carnosic acid is used to prepare the purposes of ERR alpha expression inhibitor.
Carnosic acid is used to prepare the purposes of the medicine of prevention osteoporosis.
Further, the osteoporosis is the osteoporosis that menopause triggers.
A kind of pharmaceutical composition for being used to prevent osteoporosis, containing the carnosic acid of effective content or its pharmaceutical salts, goes back
Containing pharmaceutically acceptable carrier or excipient, pharmaceutically acceptable formulation is made.
Further, pharmaceutically acceptable carrier or excipient include one or more solid, semisolid or liquid
Auxiliary material.
Further, the pharmaceutically acceptable formulation include tablet, capsule, granule, injection, pill,
Syrup, powder, paste.
Further, the osteoporosis is the osteoporosis that menopause triggers.
Inventor has found effective inhibitor that carnosic acid is ERR alpha expressions by vitro test, can suppress osteoporosis
The expression of osteoporosis Research of predicting markers in cell model；In vivo studies finds that carnosic acid can to cut off mouse ovarian institute
(such as shin bone BV/TV is reduced caused physiological phenomenon, shin bone Tb.Th is reduced, shin bone Tb.N reduces shin bone Tb.SP increases, shin bone
SMI increases etc.) take a turn for the worse to a certain extent, so as to treat bone defect (such as bone mass reduction caused by estrogen deficiency
And/or microstructure degeneration of bone etc.), there is prospect of the exploitation into the medicine of the osteoporosis for the treatment of menopause initiation.
Brief description of the drawings
Fig. 1 is the expression that carnosic acid suppresses osteoporosis correlation target gene, wherein (A) is CtsK gene relative expression's tables
Up to amount, (B) is TRAP gene relative expression's expression quantity, and (C) is NFATc1 gene relative expression's expression quantity, and (D) is Acp5 genes
Relative expression's expression quantity；It can be seen from the figure that RANKL can cause the high expression of related gene of osteoporosis, the high table of these genes
Up to osteoclast being promoted to generate, and carnosic acid can significantly inhibit the high expression of these genes.
Fig. 2 can significantly inhibit ERR α high caused by RANKL for carnosic acid in osteoporosis cell model and express, from
It can be seen from the figure that, after giving stimulated in vitro factor R ANKL, the expression of ERR α albumen is significantly increased, after giving carnosic acid
It was found that carnosic acid can significantly suppress the high expression of ERR α；Wherein, (A) is the result figure of Western Blot, and (B) is A
The gray-scale statistical figure of figure.
Fig. 3 can suppress the expression of ERR α for carnosic acid as the inhibitor of ERR α；It can be seen from the figure that Salvia japonica
Acid is identical with the micromolecular inhibitor effect XCT790 effects of ERR α, can suppress the high expression of the ERR α caused by RANKL, this
Explanation carnosic acid can be used as the micromolecular inhibitor of ERR α；Wherein, (A) is the result of Western Blot
Figure, (B) are the gray-scale statistical figure of A figures.
Fig. 4 is the high expression that carnosic acid can inhibit ERR α correlation target genes caused by RANKL, it can be seen from the figure that
These genes belong to the relevant genes of ERR α, because RANKL can cause the high expression of ERR α, the table of its related target gene
Up to will also be raised, after giving carnosic acid, it has been found that it is related that carnosic acid can effectively suppress ERR α caused by RANKL
The high expression of target gene.
Fig. 5 is the change schematic diagram of each group mouse tibia BV/TV；Compared with sham-operation group, model group mouse tibia BV/TV
Substantially reduce, modeling success；Compared with model group, the BV/TV of carnosic acid high and low dose group and positive controls significantly increases
Add, illustrate that carnosic acid can be effectively increased osteoporosis model mouse tibia BV/TV, have the function that similar with positive drug.
Fig. 6 is the change schematic diagram of each group mouse tibia Tb.N；Compared with sham-operation group, model group mouse tibia Tb.N is shown
Write and reduce, illustrate modeling success；Compared with model group, the Tb.N of carnosic acid high dose group and positive controls significantly increases
Add, illustrate that carnosic acid can be effectively increased osteoporosis model mouse tibia Tb.N, have the function that similar with positive drug.
Fig. 7 is the change schematic diagram of each group mouse tibia Tb.Th；Compared with sham-operation group, model group mouse tibia Tb.Th
Substantially reduce, illustrate modeling success；Compared with model group, the Tb.Th of carnosic acid high dose group and positive controls is notable
Increase, illustrates that carnosic acid can be effectively increased osteoporosis model mouse tibia Tb.Th, has the work similar with positive drug
With.
Fig. 8 is the change schematic diagram of each group mouse tibia Tb.SP；Compared with sham-operation group, model group mouse tibia Tb.SP
Dramatically increase, illustrate modeling success；Compared with model group, the Tb.SP of carnosic acid high dose group and positive controls is notable
Reduce, illustrate that carnosic acid can effectively reduce osteoporosis model mouse tibia Tb.SP, there is the work similar with positive drug
With.
Fig. 9 is the change schematic diagram of each group mouse tibia SMI；Compared with sham-operation group, model group mouse tibia SMI is notable
Increase, illustrates modeling success；Compared with model group, carnosic acid is low, the SMI of high dose group and positive controls is reduced,
Illustrate that carnosic acid can reduce osteoporosis model mouse tibia SMI, have the function that similar with positive drug.
Figure 10 is each group mouse tibia Micro-CT detects schematic diagrams, it can be seen from the figure that compared with sham-operation group, mould
Type group mouse tibia bone density significantly reduces, and illustrates that osteoporosis phenomenon occurs in mouse；It is high and low to give positive drug, carnosic acid
After dosage, hence it is evident that improve the phenomenon that bone density is lost, illustrate that carnosic acid has the function that osteoporosis caused by improving OVX；
Wherein, (A) is sham-operation group；(B) it is model group；(C) it is positive drug group；(D) it is carnosic acid low dose group (30mg/kg)；
(E) it is carnosic acid high dose group (60mg/kg).
Figure 11 is the result schematic diagram of each group mouse femur HE dyeing, it can be seen from the figure that model group and other each groups
Mouse is compared, and bone matrix accumulation occurs and reduces, the gap between medullary substance becomes larger, by positive drug and carnosic acid high and low dose
Intervention after, which is obviously improved, and illustrates that carnosic acid has the pharmacological action consistent with positive drug；Wherein, (A) is to do evil through another person
Art group；(B) it is model group；(C) it is positive drug group；(D) it is carnosic acid low dose group (30mg/kg)；(E) it is high for carnosic acid
Dosage group (60mg/kg).
Embodiment
The specific guarantor for introducing essentiality content of the present invention, but the present invention not being limited with this with reference to the accompanying drawings and examples
Protect scope.
Embodiment 1：Inhibitory action of the carnosic acid to ERR alpha expressions in cell
First, experiment material
RAW264.7 cells：From ATCC companies；
MEM α culture mediums (Gibco, the U.S.)；Hyclone (Gibco, the U.S.)；Trypsase (Gibco, the U.S.)；Blue or green chain
Mycin mixed liquor (Gibco, the U.S.)；Nonessential amino acid glutamine (Gibco, the U.S.)；Inhibitors of phosphatases (Roche, it is auspicious
Scholar)；Protease inhibitors (Roche, Switzerland)；(the green skies biotechnology in Shanghai has SDS-PAGE albumen sample-loading buffer (5x)
Limit company)；30% polyacrylamide (the green skies Bioisystech Co., Ltd in Shanghai)；1.5MTris-HCl (pH 8.8) (Shanghai
Green skies Bioisystech Co., Ltd)；10% dodecyl sodium sulfate (SDS) (the green skies Bioisystech Co., Ltd in Shanghai)；
Ammonium persulfate (APS) (Biosharp bio tech ltd)；Tetramethylethylenediamine (TEMED) (Amresco, the U.S.)；
0.5M Tris-HCl (pH6.8) (the green skies Bioisystech Co., Ltd in Shanghai)；Tris (gives birth to the emerging limited public affairs of biotechnology in Nanjing
Department)；Glycine (Nanjing Sheng Xing Bioisystech Co., Ltd)；NaCl (Chinasun Specialty Products Co., Ltd)；Tween
(Tween-20) (Nanjing Chemistry Reagent Co., Ltd.)；Analyze pure methanol (Nanjing chemical reagent limited company)；Nitric acid is fine
The plain sodium (NC films) (Pall Corparation, the U.S.) of dimension；Skimmed milk power (Inner Mongol willpower industry Group Plc)；
Primary antibody dilution (the green skies Bioisystech Co., Ltd in Shanghai)；ERR alpha monoclonal antibodies (abcam)；Horseradish enzyme marks China fir goat-anti
Rabbit igg (H+L) (the green skies Bioisystech Co., Ltd in Shanghai)；Horseradish enzyme marks business's halter strap anti-mouse IgG (green skies biology skills in Shanghai
Art Co., Ltd)；Chloroform (Shanghai Ling Feng chemical reagent Co., Ltd)；Isopropanol (the limited public affairs of Ai Likai Dehua, Nanjing work
Department)；Absolute ethyl alcohol (the upper smooth Science and Technology Co., Ltd. of Haitai)；SuperMix (Nanjing Vazyme Biotechnology Co., Ltd.)；
RNase free H2O (Nanjing Vazyme Biotechnology Co., Ltd.)；(it is limited that biotechnology is only praised in Nanjing promise to SYBR Green
Company).Carnosic acid (CA) (Chengdu Puffy spy Bioisystech Co., Ltd, HPLC >=99.51%), XCT790 (the white members in Shanghai
Bio tech ltd).
2nd, experimental method
1st, cell culture
Mouse source RAW264.7 cells are containing 1% mycillin mixed liquor (100KU/L penicillin, 100mg/L strepto-s
Element), 1% glutamine, in the MEM α culture mediums of 5% hyclone, in 5%CO2, training is passed in 37 DEG C of cell incubator
Support.
Cell propagating method：
(1) cell is taken out, cell density reaches more than 80% in micro- Microscopic observation culture dish, removes nutrient solution,
PBS is added to wash 1 time；
(2) PBS is discarded, adds pancreatin, digests 30s, observes the removable pancreatin of the separated state of cell, addition contains 5%
MEM α culture mediums, cell gently is blown and beaten；
(3) cell is centrifuged into 3min in 500rpm；
(4) supernatant is sucked, 2ml fresh mediums is added, gently blows even and fine born of the same parents, cell suspension is made；
(5) 0.5ml suspensions are taken, are added in 10ml culture mediums, it is uniform to cell for several times that cross rocks culture dish, then is placed in
Culture is maintained in cell incubator.
2nd, experiment packet
RAW264.7 cell secondary cultures, culture medium are MEM α+5%FBS+1%Gln+1% mycillins, life of taking the logarithm
Long-term RAW264.7 cell inoculations are 12.8 × 10 per hole cell number in 6 orifice plates4A, packet transaction is as follows：
Solvent control group：DMSO solvent cultures are added in the medium；
Model group：The RANKL cultures of final concentration 100ng/mL are added in the medium；
CA intervention groups：The carnosic acid culture of 7.5 μM of final concentration is added on model group medium base；
Positive controls：The XCT790 cultures of 5 μM of final concentration are added on model group medium base.
3rd, Western blot methods detect intracellular ERR α protein expressions
RAW264.7 cells are put into incubator according to above-mentioned experiment packet and cultivate 48h；Culture medium is discarded after 48h, is used
PBS washs cell；Add cell pyrolysis liquid (containing 200 μ l of SDS 800ul, Loading buffer in 1ml lysates, 100
× PMSF10 μ l, 50 × inhibitors of phosphatases, 20 μ l), 5min is stood, attached cell is gently scraped with cell scraper, is drawn to EP pipes
In, 4 DEG C, 12000rpm centrifugation 1min, 99 DEG C, 20min is high-temperature denatured.SDSPAGE is carried out per 20 μ g loadings of hole with total protein concentration
Electrophoresis, transferring film；5% skimmed milk power is prepared with TBST, NC films is closed, is positioned over 1h in room temperature shaker；NC films are added and press 1:
In 1000 diluted GAPDH, ERR Alpha antibodies, 4 DEG C of shaking tables are incubated overnight；TBST cleaning item bands, 10min/ times, totally 3 times, addition is pressed
1:Secondary antibody after 2000 dilution proportions, is incubated at room temperature 1h；TBST cleaning item bands, 15min/ times, totally 3 times；ECL developer solutions is uniform
Band relevant position on film is coated on, is exposed in chemiluminescence imaging analyzer；Carried out using Image J image analysis softwares
Interpretation of result, measures the gray value of band, calculates the purpose band of each group and the ratio of internal reference, compares ERR α albumen phases between each group
To the difference of expression quantity.
4th, qRT-PCR methods detect the expression of osteoporosis marker in RAW264.7 cells
RAW264.7 cells are put into incubator according to above-mentioned experiment packet and cultivate 5d；Culture medium is discarded after 5d, uses PBS
Wash cell.TRIzol methods extract each group cell total rna respectively, and reverse transcription is cDNA.Set reaction condition be：95 DEG C of pre- changes
Property 30s；95 DEG C of denaturation 5s, 60 DEG C of annealing 30s, totally 40 circulate.The target gene and reference gene of each sample are expanded at the same time.
3 repeating holes of every group of cell design.Using 2-△CtMethod calculate each group cell in osteoporosis marker CtsK, TRAP, NFATc1,
The ratio of Acp5, Aco2, IDH3 α, VLCAD and internal reference are the relative expression quantity of osteoporosis marker in each group cell.
3rd, experimental result
Compared with solvent control group, ERR α protein expression levels significantly raise in model group cell, osteoporosis marker
Expression significantly raise, illustrate that RANKL can induce ERR α albumen is high to express and replicate osteoporosis model；With model group
Compare, ERR α protein expression levels significantly reduce in CA intervention group cells, have to ERR α protein expressions similar with positive drug
Inhibitory action；Compared with model group, the expression of osteoporosis marker significantly reduces in CA intervention group cells, alleviates sclerotin
It is loose.As a result as shown in Fig. 1-4 and table 1-2 (wherein, compared with DMSO+RANKL groups, * P<0.05, * * P<0.01, * * * P<
0.001, * * * * P<0.0001 similarly hereinafter).
Table 1
Table 2
RANKL is osteoclast differentiation factor, and RAW264.7 cells can be promoted to break up to osteoclast, and osteoclast
Activity it is directly related with the formation of osteoporosis, therefore, RANKL be it is commonly used in the art in duplication osteoporosis model modeling
Agent.Found using the cell model, carnosic acid is effective inhibitor of ERR alpha expressions, can suppress osteoporosis cell model
The expression of middle osteoporosis Research of predicting markers, alleviates osteoporosis.
Embodiment 2：Inhibitory action of the carnosic acid to ovariectomized mice osteoporosis
First, experiment material
Mouse is purchased from model animal institute of Nanjing University；
Main agents and kit：Sodium cellulose glycolate (Sinopharm Chemical Reagent Co., Ltd.)；PBS buffer
(Jiangsu Kai Ji Biotechnology Ltd.)；Wood shavings bedding and padding (collaboration bioengineering Co., Ltd of Jiangsu Province)；Commonly
Feed (Jiangsu Province cooperates with the engineering finite responsible company that wins a lawsuit)；Alkaline phosphatase (ALP) detection kit (magnificent biological work in Wuhan
Journey Co., Ltd)；Calcium (Ca) detection kit (Bioengineering Research Institute is built up in Nanjing)；Phos (P) detection kit (Nanjing
Build up Bioengineering Research Institute)；(the magnificent bioengineering in Wuhan is limited by mice serum 1 Collagen Type VI C-terminal peptide (CTX-1) ELISAKit
Company)；Mice serum Tartrate resistant acid phosphatase (strACP) detection kit (Bioengineering Research Institute is built up in Nanjing)；
4% neutral paraformaldehyde (Jiangsu Kai Ji Biotechnology Ltd.)；(Anhui Double-Crane Pharmaceutical Co., Ltd Limited Liability is public for physiological saline
Department).
2nd, experimental method and result
1st, it is grouped and handles
57 C57BL/6 female mices of 5 weeks or so are randomly divided into 2 groups by weight：Sham-operation group 10 (Sham groups),
Modeling group 47, carries out ovariectomy operation to modeling group mouse, abdominal cavity note is carried out to mouse with chloraldurate 400mg/kg weight
Anesthesia is penetrated, lateral position is fixed, horizontal just above in mouse thigh root, and place of the backbone side less than 1cm, one is cut off with ophthalmology
A and skin incision of the about 5mm long of spine parallel, carefully cuts off lower section psoas, rakes about the adipose tissue of wrapping ovary,
Notch is pulled to after seeing the ovary of pink, is ligatured in ovary both sides, extracts ovary.Then sew up the incision, and be coated with green grass or young crops
Mycin powder is to prevent infection.Same method extracts other side ovary.Postoperative 1 week, then by modeling component be model group 15
(OVX groups), positive drug group 12 (OVX+E2 groups), carnosic acid low dose group 10 (CA low dose groups), the high agent of carnosic acid
Amount group 10 (CA high doses group).Start to carry out gastric infusion to each group after packet, carnosic acid high and low dose group is given respectively
Concentration is 60mg/kg, 30mg/kg.Positive controls gavage gives the Estradiol Valerate solution that concentration is 0.01mg/kg.Blank
Gavage gives the CMC-Na solution of isometric 0.5% respectively for group and model group.Above each group mouse is administered once a day, continuously
16 weeks, record weight once weekly.
After administration, carry out plucking eyeball taking blood to each group mouse, put to death.Bilateral femur and shin bone are taken, rejects bone tissue
The soft tissue of surrounding, formalin are fixed, and give over to decalcification paraffin section respectively and three-dimensional reconstruction analysis is used.
2nd, bone tissue morphological index detects
Before detection, left tibias is taken out, after soaking 24h in physiological saline, tissue is carried out to it using Micro-CT
Morphological index detects：Shin bone is positioned in the specimen tube of micro-CT, and is fastened with hygenic towelette, is prevented in scanning process
The dehydration and displacement of sample.It is scanned using the Scanner softwares of Skyscan1174 types micro-CT, parameter setting is：Electricity
Press 50kV, electric current 800 μ A, faultage image resolution ratio 1304pixel × 1024pixel, 12 μm of scanning space resolution ratio, the anglec of rotation
0.8 degree of degree, time for exposure 5300ms.
After the completion of scanning, three-dimensionalreconstruction is carried out with N-Recon softwares, the rat bone trabecula three-dimensional reconstruction image of each group referring to
Figure 10, it can be seen from fig. 10 that compared with sham-operation group, model group mouse tibia bone density significantly reduces, and illustrates that mouse goes out
Existing osteoporosis phenomenon；After giving positive drug, carnosic acid high and low dose, hence it is evident that improve the phenomenon that bone density is lost, illustrate mouse
Tail oxalic acid has the function that osteoporosis caused by improving OVX.
Finally carry out three dimensional analysis with CT-AN softwares (above software carries software for Skyscan1174 types micro-CT).
Away from below growth plate 0.6mm, thickness is the marrow cavity region of 1.5mm for area-of-interest selection.Analyze following
Parameter：
Diaphysis fraction (BV/TV)：The cumulative volume of bone trabecula and the marrow cavity volume of area-of-interest；
Bone trabecula quantity (Tb.N)：Represent the bone trabecula quantity in per 1mm；
Bone trabecula thickness (Tb.Th)：The average thickness of bone trabecula；
Bone trabecula separating degree (Tb.SP)：Average distance between bone trabecula；
Structure model index (SMI)：Index of the bone trabecula in " bar-shaped " still " tabular " is reacted, represents that bones are small closer to 3
Liang Yue is in bar-shaped.
(1) change of each group mouse tibia BV/TV as shown in table 3 and Fig. 5 (compared with OVX groups, * P<0.05, * * P<
0.01, * * * P<0.001, * * * * P<0.0001, similarly hereinafter)：
Table 3
It can be seen that from table 3 and Fig. 5 compared with sham-operation group, model group mouse tibia BV/TV is substantially reduced, and explanation is made
Mould success；Compared with model group, the BV/TV of carnosic acid high and low dose group and positive controls is dramatically increased, and illustrates rat-tail
Oxalic acid can be effectively increased osteoporosis model mouse tibia BV/TV, have the function that similar with positive drug.
(2) change of each group mouse tibia Tb.N is as shown in table 4 and Fig. 6：
Table 4
It can be seen that from table 4 and Fig. 6 compared with sham-operation group, model group mouse tibia Tb.N is substantially reduced, and illustrates modeling
Success；Compared with model group, the Tb.N of carnosic acid high dose group and positive controls is dramatically increased, and illustrates that carnosic acid can
To be effectively increased osteoporosis model mouse tibia Tb.N, have the function that similar with positive drug.
(3) change of each group mouse tibia Tb.Th is as shown in table 5 and Fig. 7：
Table 5
It can be seen that from table 5 and Fig. 7 compared with sham-operation group, model group mouse tibia Tb.Th is substantially reduced, and explanation is made
Mould success；Compared with model group, the Tb.Th of carnosic acid high dose group and positive controls is dramatically increased, and illustrates Salvia japonica
Acid can be effectively increased osteoporosis model mouse tibia Tb.Th, have the function that similar with positive drug.
(4) change of each group mouse tibia Tb.SP is as shown in table 6 and Fig. 8：
Table 6
It can be seen that from table 6 and Fig. 8 compared with sham-operation group, model group mouse tibia Tb.SP is dramatically increased, and explanation is made
Mould success；Compared with model group, the Tb.SP of carnosic acid high dose group and positive controls is substantially reduced, and illustrates Salvia japonica
Acid can effectively reduce osteoporosis model mouse tibia Tb.SP, have the function that similar with positive drug.
(5) change of each group mouse tibia SMI is as shown in table 7 and Fig. 9：
Table 7
It can be seen that from table 7 and Fig. 9 compared with sham-operation group, model group mouse tibia SMI is dramatically increased, and illustrates modeling
Success；Compared with model group, carnosic acid is low, the SMI of high dose group and positive controls is reduced, and illustrates that carnosic acid can
To reduce osteoporosis model mouse tibia SMI, have the function that similar with positive drug.
3rd, mouse femur tectology detects
The making of paraffin sections of decalcified bone：It will be taken to be organized at 4 DEG C with 4% paraformaldehyde solution and fix 24h, distilled
Water soaks 4h, then through EDTA liquid (ethylenediamine tetra-acetic acid) decalcification that mass fraction is 20% (volume fraction 10%), syringe needle energy
Tissue block is penetrated easily illustrates that decalcification is abundant.Paraffin embedding is used after routinely dehydration etc., along row histotomy after respective handling,
It is 10 μm to control thickness.Normal dyeing.
HE staining procedures：
The section containing paraffin will be prepared and immerse in fresh dimethylbenzene I, II each 10min that dewaxes successively；
3min in absolute alcohol I, II, 95%, 90%, 80%, 70% alcohol of absolute alcohol is immersed in section successively；
Distillation washing 5min；
Section instills haematoxylin dyeing liquid dyeing 8min；
10min is originally washed in immersion；
Immerse distilled water 15s；
Immerse 95% alcohol 5s；
15min in the solution of acid Yihong is immersed in section；
It is each 3min in 70%, 80%, 90%, 95%, 100% I, 100% II alcohol that volume fraction is immersed in section successively；
Each 5min in dimethylbenzene I, II is immersed in section；
Mounting, Microscopic observation bone tissue form.
As a result judge：After above-mentioned processing, endochylema dyeing takes on a red color, and karyon dyeing is observed in blueness in light Microscopic observation
Material such as bone trabecula, osteocyte, pulp cavity adipocyte in bone.Every section chooses corresponding visual field under 20 times of mirrors and carries out contrast sight
Examine, reduced it can be seen from fig. 11 that bone matrix accumulation compared with other each group mouse, occurs in model group, the sky between medullary substance
Gap becomes larger, and after the intervention by positive drug and carnosic acid high and low dose, which is obviously improved, and illustrates that carnosic acid has
The pharmacological action consistent with positive drug.
As it can be seen that by gavaging carnosic acid 3 months to ovariectomized mouse, administration group mouse tibia trabecular volume hundred is found
Divide lower than apparently higher than model group, bone trabecula mineralization rate, bone trabecula formation surface percentage, bone trabecula sorbent surface percentage
In model group, this is because the absorption of osteoclast is reduced, bon e formation is set to be more than bone information, so as to prevent the hair that bone amount lowers
It is raw.The results show that compared with sham-operation group, model group mouse tibia BV/TV, Tb.Th and Tb.N are substantially reduced related experiment, and
Tb.SP is dramatically increased.This result shows that, excision mouse ovarian can substantially reduce its shin bone BV/TV, Tb.Th and Tb.N, make
Tb.SP is dramatically increased, so as to cause mouse that osteoporosis occurs.Related experiment result further shows that carnosic acid can make
Above index takes a turn for the worse to a certain extent, and carnosic acid high dose group is compared with model group, mouse tibia BV/TV, Tb.Th
Dramatically increased with Tb.N, and Tb.SP is significantly reduced, this result shows that, carnosic acid dredges the mouse sclerotin caused by removal ovary
Loose disease has therapeutic effect.
The effect of above-described embodiment is the essentiality content for specifically introducing the present invention, but those skilled in the art should know
Protection scope of the present invention, should not be confined to the specific embodiment by road.
Claims (7)
1. carnosic acid is used to prepare the purposes of ERR alpha expression inhibitor.
2. carnosic acid is used to prepare the purposes of the medicine of prevention osteoporosis.
3. purposes according to claim 2, it is characterised in that：The osteoporosis is the osteoporosis that menopause triggers.
A kind of 4. pharmaceutical composition for being used to prevent osteoporosis, it is characterised in that：Containing the carnosic acid of effective content or its
Pharmaceutical salts, also containing pharmaceutically acceptable carrier or excipient, are made pharmaceutically acceptable formulation.
5. pharmaceutical composition according to claim 4, it is characterised in that：The pharmaceutically acceptable carrier or figuration
Agent includes one or more solid, semisolid or Auxiliary Liquid Materials.
6. pharmaceutical composition according to claim 4, it is characterised in that：The pharmaceutically acceptable formulation includes piece
Agent, capsule, granule, injection, pill, syrup, powder, paste.
7. according to any pharmaceutical compositions of claim 4-6, it is characterised in that：The osteoporosis triggers for menopause
Osteoporosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810001062.2A CN108030779A (en) | 2018-01-02 | 2018-01-02 | Carnosic acid is used to prepare ERR alpha expressions inhibitor and treats the purposes of the medicine of osteoporosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810001062.2A CN108030779A (en) | 2018-01-02 | 2018-01-02 | Carnosic acid is used to prepare ERR alpha expressions inhibitor and treats the purposes of the medicine of osteoporosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108030779A true CN108030779A (en) | 2018-05-15 |
Family
ID=62098302
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810001062.2A Pending CN108030779A (en) | 2018-01-02 | 2018-01-02 | Carnosic acid is used to prepare ERR alpha expressions inhibitor and treats the purposes of the medicine of osteoporosis |
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| Country | Link |
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| CN (1) | CN108030779A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111840112A (en) * | 2020-07-23 | 2020-10-30 | 中南大学 | Application of carnosic acid or derivatives thereof in preparing medicine for treating diabetic complications |
-
2018
- 2018-01-02 CN CN201810001062.2A patent/CN108030779A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| HIROMI HAGIWARA，ET AL.: "Carnosic acid inhibits the formation of osteoclasts through attenuation of expression of RANKL", 《PHARMANUTRITION》 * |
| 李劲平等: "壮骨止痛胶囊抗绝经后骨质疏松的ERRα作用机制研究", 《中国现代应用药学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111840112A (en) * | 2020-07-23 | 2020-10-30 | 中南大学 | Application of carnosic acid or derivatives thereof in preparing medicine for treating diabetic complications |
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