CN103356607A - Application of cajanolactone A in preparation of medicine used for treating osteoporosis - Google Patents
Application of cajanolactone A in preparation of medicine used for treating osteoporosis Download PDFInfo
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Abstract
The invention discloses an application of cajanolactone A in preparation of a medicine used for treating osteoporosis. Ob/ob mouse in vivo pharmacodynamic test data shows that firstly no obvious influence is produced on weight of a fat ob/ob mouse and secondly bone density of the ob/ob mouse can be obviously increased, ALP (alkaline phosphatase) level and and TRACP (tartrate resistant acid phosphatase) level in blood serum can be obviously reduced, blood calcium concentration can be increased after cajanolactone A is continuously and orally taken for 21 days (20mg/kg/d), so that cajanolactone A has positive significance on treatment of osteoporosis caused by fat and hyperglycaemia, and cajanolactone A has a good curative effect on treatment of osteoporosis, especially has a definite treatment effect on osteoporosis caused by fat and hyperglycaemia, can improve blood lipid metabolism and also can avoid gradual disease transformation of fat, diabetes, diabetes chronic complication and osteoporosis in certain degree.
Description
Technical field
The present invention relates to a kind of novel medical use of chemical compound, particularly tree bean lactone A is at preparation treatment osteoporosis agents, or is of value to the application in osteoporosis crowd's the health product.
Background technology
Osteoporosis (osteoporosis) is a kind of commonly encountered diseases, mainly involves middle-aged and elderly people.Along with the arrival of aging society, osteoporosis has become a public health problem that can not be ignored that threatens middle-aged and elderly people health, has a strong impact on its quality of life.Osteoporosis is a kind of metabolic bone disease, can be divided into constitutional and Secondary cases two classes, primary osteoporosis wherein accounts for 90% of osteoporosis, can be divided into again 2 kinds of hypotypes, I type wherein claims postmenopausal osteoporosis, and the II type is senile osteoporosis.Secondary osteoporosis can be secondary to other diseases or be caused by medicine.
The characteristics of osteoporosis are that the bone amount reduces, the osseous tissue fine structure is impaired and the risk of fragility fractures increases.The pathogenic factors of osteoporosis is complicated, and endocrinopathy, metabolic disease and inflammation disease all have the probability that causes osteoporosis.The appearance of osteoporosis is the result who affects the h and E factor interaction of bone conversion, bone amount and skeleton geometry and the risk of falling, and also can be the various complication that have influence on heredity, endocrine, metabolism and the inflammation disease of bone conversion.
The control medicine of osteoporosis uses the vitamin D that comprises calcium preparation and promote calcium absorption usually, and the calcitonin class medicine, the bis phosphoric acid salt medicine that suppress bone resorption, the parathyroid hormone medicine of promoting bone growing, the estrogen of prevention bone loss or selective estrogen receptor modulators class medicine etc.The curative effect of these medicines is limited, has certain safety issue.
Tree bean lactone A is a kind of native compound that the inventor finds, its chemical constitution characterizes (chemical compound 3) at CN101422450A, at WO/2013/013417Al it has been carried out the elaboration in a step.
Summary of the invention
The object of the present invention is to provide the application of tree bean lactone A in preparing the health food for the treatment of osteoporosis agents or being of value to the osteoporosis crowd.
Mesenchymal stem cells MSCs (BMSCs) is a kind of pluripotent stem cell, can be divided into osteoblast and reflect that testing drug is to the therapeutic effect of osteoporosis.The inventor selects the BMSCs of SD rat as the cell model of test osteoporosis drug effect.Cellular level test of pesticide effectiveness data show, tree bean lactone A(CLA) can promote propagation and the Osteoblast Differentiation of SD rat BMSCs.
The inventor selects the ob/ob mice at three monthly ages as fat osteoporosis animal model, with the heterozygote ob mice retinue contrast of the birth same period, confirms that there is pathological changes really in the ob/ob animal pattern simultaneously.Ob/ob mice and ob mice of the same age relatively show as that blood calcium concentration is basically identical, the serum levels of ALP level is significantly higher, serum T RACP level is significantly higher, bone density is significantly on the low side in experiment, and ob/ob Mouse Bone Developmental and Metabolic Disorder be described, have obvious bone loss.The drug effect test data shows continuous 21 days oral tree bean lactone A(20mg/kg/d in the ob/ob Mice Body): (1) is on the not obviously impact of ob/ob obesity mice body weight; (2) can significantly increase the bone density of ob/ob mice, significantly reduce serum levels of ALP level and TRACP level, improve blood calcium concentration, prompting tree bean lactone A has positive effect to the treatment of fat and hyperglycemia induced osteoporosis.Specification tree bean lactone A has good curative effect in the treatment osteoporosis, particularly to fat and hyperglycemia are caused osteoporosis clear and definite therapeutic effect is arranged, simultaneously blood lipid metabolism can be improved, fat---the osteoporotic metabasis of diabetes---chronic complicating diseases of diabetes---can be avoided again to a certain extent.
Description of drawings
Fig. 1, tree bean lactone A (CLA) is on the impact (* 100) (Alizarin red staining method) of SD rat bone marrow mesenchymal stem cells (BMSCs) Osteoblast Differentiation.
The specific embodiment
Below in conjunction with experiment and experimental data, further specify the present invention.
The function of resisting osteoporosis of tree bean lactone A
Tree bean lactone A promotes rat bone marrow mesenchymal stem cells propagation and Osteoblast Differentiation
(1) experimental technique
1, cell and cultivation: SD rat bone marrow mesenchymal stem cells (BMSCs), available from Zhongshan University.Growth of Cells is in the low sugar DMEM culture medium that contains 10% hyclone, at 37 ℃, 5%CO
2, 95% humidity incubator cultivate, experimental technique carries out passage routinely, cell cryopreservation and recovery.Observation of cell form and taking pictures under optical microscope.
2, medicine to be measured: tree bean lactone A(English name cajanolactone A), code name is CLA, and purity＞99%(detects through HPLC).
3, CLA is on the impact of SD rat BMSCs propagation
Get the SD rat BMSCs that is in exponential phase, with blowing and beating with complete medium behind 0.25% trypsinization, be made into 5 * 10
4The single cell suspension of individual/mL.With every hole 100 μ L volumes cell is inoculated in 96 orifice plates, puts 37 ℃, 5%CO
2, 95% humidity incubator cultivate.Discard culture medium after Growth of Cells to 70% merges, again add the fresh medium 100 μ L that contain variable concentrations CLA, 6 parallel holes are established in each hole, and cell control well and the solvent blank control wells of not dosing are set.Behind the drug effect 48, every hole adds the freshly prepared MTT dye liquors of 10 μ L (5g/L), continues to hatch 3-4h in 37 ℃ of incubators, stops cultivating.Discard upper strata liquid, every hole adds the DMSO of 100 μ L, and low speed concussion 2min fully dissolves the pigment crystal of generation.Measure each hole absorbance (A) at microplate reader 492nm wavelength place, calculate cell proliferation rate under the CLA effect by following formula, data with
Expression:
Cell proliferation rate %=(drug effect hole A value-cell control well A value)/(cell control well A value-blank hole A value).
4, CLA is on the impact of SD rat BMSCs Osteoblast Differentiation
Be 5 * 10 with density
4The SD rat BMSCs single cell suspension of individual/mL is inoculated in 96 orifice plates, cellar culture by every hole 100 μ L.Contact inhibition is 3 days behind cell fusion, uses instead to contain differentiating inducer (50mg/L vitamin C+10
-2Mol/L β-phosphoglycerol+10
-8The mol/L dexamethasone) culture fluid breaks up with induced osteogenesis, changes liquid once every 3 days, continuous culture 9 days.Differentiation is carried out cell dyeing with alizarin red after finishing, and concrete operations are carried out according to the test kit explanation.Examine under a microscope after the dyeing, take pictures.In order to observe CLA to the impact of Osteoblast Differentiation, culture fluid also adds the CLA that contains variable concentrations simultaneously in the induction process.Experiment setting is not added the noble cells contrast (negative control) of CLA and is not added the undifferentiated cell contrast of CLA and differentiating inducer (conventional contrast).
(2) experimental result and conclusion
1, CLA is on the impact of SD rat BMSCs propagation
The rate of increase of BMSCs sees Table 1 under variable concentrations CLA effect.Can find out, CLA in BMSCs48h, all shows certain facilitation to the propagation of cell at 50-1.56 μ M mass action.When concentration 25-1.56 μ M, CLA has dose-effect relationship to the Effect of promoting growth of BMSCs.
Table 1CLA effect 48h is on the impact of the SD rat BMSCs rate of increase
2, CLA is on the impact of SD rat BMSCs Osteoblast Differentiation
BMSCs does not induce, the results are shown in accompanying drawing 1(A～D) behind the Alizarin red staining joint knot through Osteoblast Differentiation on the same group.The conventional contrast of the BMSCs(of cellar culture) can not be divided into osteoblast, thus the joint knot can not appear, can not be by Alizarin red staining (Figure 1A); Adopt derivant to carry out the BMSCs (negative control) that Osteoblast Differentiation is induced, the part cell has been finished Osteoblast Differentiation, has generated the joint knot (Figure 1B) that can be dyed by alizarin red on a small quantity Chinese red; Fig. 1 C and Fig. 1 D are respectively that derivant carries out the result that Osteoblast Differentiation is induced to BMSCs in the presence of the CLA of 12.5 μ M and 6.25 μ M.As seen the joint knot that generates illustrates that more than negative control CLA has promoted the Osteoblast Differentiation of BMSCs.
Conclusion: CLA can promote propagation and the Osteoblast Differentiation of SD rat BMSCs.
Tree bean lactone A increases the congenital fat hyperglycemia Mouse Bone density of ob/ob, improves blood lipid metabolism
1, test method
Reagent and medicine: triglyceride (TG) is measured test kit, T-CHOL (TC) is measured test kit, HDL-C (HDL-C) mensuration test kit, low-density lipoprotein cholesterol (LDL-C) mensuration test kit all available from Changchun remittance power Bioisystech Co., Ltd; Calcium, alkaline phosphatase enzyme reagent kit and Tartrate resistant acid phosphatase test kit build up bio-engineering research institute available from Nanjing.CLA purity＞99%(HPLC detects, area normalization method).
Animal feeding: test the congenital fat hyperglycemia mice of used ob/ob, retinue ob mice, be the ob mice that is purchased from U.S. Jax Lab and breed and obtain breeding at SPF level laboratory.Experiment obtains the license [experiment credit number: SYXY(Guangdong) 2008-001] of zoopery Ethics Committee of Traditional Chinese Medicine University Of Guangzhou, and carries out in strict accordance with the zoopery pertinent regulations.
Animal grouping and treatment: chose for three monthly ages, body weight is divided into ob/ob model group and ob/ob treatment group at random at 28 ob/ob obesity mices (♀ 14, and ♂ 14) of 42.3 ± 3.6g, 14 every group (7 ♀, 7 ♂).Alternative and ob/ob are of the same age, and body weight is divided into ob model group and ob treatment group at random 16 heterozygote ob mices of the same age (♀ 8, and ♂ 8) of 20.3 ± 0.8g, and 8 every group (4 ♀, 4 ♂) are as the retinue contrast of this experiment.Treat by following dosage regimen respectively, treated continuously 21 days.
1. model group ob/ob mice and ob mice: tween 80-DMSO (1:1) mixed liquor are used 10 times of dilutions of ultra-pure water of autoclave sterilization before use, by every 10g body weight 100 μ L gavages.
2. CLA treatment group ob/ob mice and ob mice: CLA are dissolved in first tween 80-DMSO (1:1) mixed liquor, are mixed with the storing solution of 20mg/mL, use 10 times of dilutions of sterilized water again, by every 10g body weight 100 μ L gavages, and dosage 20mg/kg/d.
Observe feed, drinking-water, voided volume, hair color, the mental status and the active situation of mice during the treatment every day; Every 4d measures the non-empty stomach body weight of a mice.
Biochemical markers of bone metabolism and blood fat Biochemical Indexes: water 12h was can't help in the mice fasting after treatment finished, pluck the eyeball blood sampling, the centrifugal 10min of 4000r/min gets serum, carries out the mensuration of part biochemical markers of bone metabolism and blood fat biochemical indicator according to the operating instruction of test kit.
Bone densitometry: after the blood sampling of mice last, the cervical vertebra dislocation is put to death, and dissects and gets the both sides tibia, separates and removes residual muscle, places 60 ℃ of baking ovens, heat drying 24h, and (DahoMeter DE-120M) measures its bone density with solid density measuring instrument.
Data analysis: adopt the SPSS19.0 statistical software to process.(Mean ± SD) expression relatively uses variance analysis between many groups to experimental data, and two means relatively adopt the t check with mean+SD.Significance level is made as P＜0.05.
2, experimental result
Each is organized the mice general status and observes: experimental session organizes respectively that the mice fur is glossy, and the mental status is good, and feed drinking-water is all normal, and the urine amount is normal, and body weight is stable to rise, without animal dead.
(1) CLA is on the impact of Mouse Weight
During drug treatment, each is organized, and Mouse Weight is stable to rise, and concrete data are as shown in table 2.
As can be seen from Table 2, begin to finish to experiment from experiment, the body weight gain rate of the model group of ob/ob mice and CLA treatment group is respectively 13.9% and 14.8%, and the model group of ob mice and CLA treatment group body weight gain rate are respectively 11.2% and 9.3%.Illustrate that CLA does not exert an influence to the ob/ob obesity mice of homology and the body weight of heterozygote ob mice, that is can the normal growth of ob/ob obesity mice and heterozygote ob mice not had a negative impact.Data by two kinds of isogenic animals compare, and can find out that the body weight of ob/ob mice is all significantly heavy than its heterozygote ob mice of the same age, and this situation are more serious with advancing age, illustrates that its pathological data and obesity have substantial connection.
Table 2CLA is on the impact (g) of Mouse Weight
(2) CLA is on the impact of mouse tibia density and bone metabolism index
Testing result to Bone metabolite in the measurement result of mouse tibia bone density and the serum sample is as shown in table 3.Compare with the model group ob mice of retinue contrast, model group ob/ob obesity mice tibial bone density of the same age is (P＜0.05) on the low side obviously, serum AKP level and TRACP level then significantly raise (P＜0.001), illustrate that the ob/ob obesity mice exists the on the low side and abnormal bone metabolism of obvious bone density really.Continuously 21d takes the tibial bone density that CLA can make the ob/ob mice obviously increases (P＜0.05); Blood calcium concentration significantly rises (P＜0.001), and serum AKP level and TRACP level then significantly reduce (P＜0.001).But for accompanying ob mice, take CLA and its tibia density and bone metabolism index are produced obviously impact, the related data there was no significant difference (P〉0.05) of comparing with the ob model group of accompanying.
Table 3CLA is on the impact of mouse tibia density and bone metabolism index
Annotate: compare * P＜0.05 with the ob/ob model group; * P＜0.01; * * P＜0.001; Compare with retinue ob model group,
#P＜0.05;
##P＜0.01;
###P＜0.001.
(3) CLA is on the impact of lipid of mice level
Serum sample carries out the detection of lipid metabolism index, and the result is as shown in table 4.Can find out: with the contrast of retinue ob model group, TC, TG and LDL-C level all significantly higher (P＜0.001) illustrate that the ob/ob obesity mice exists obvious metabolism disorder of blood lipid in the ob/ob model group mice serum.The ob/ob mice is behind process CLA drug treatment, and TC, TG and LDL-C level be significantly decline (P＜0.001) all.Illustrate that CLA has clear and definite curative effect aspect the lipid metabolism of obesity mice improving.For accompanying ob mice, CLA treatment produces obviously impact to its blood lipid level, the related data there was no significant difference (P〉0.05) of comparing with the ob model group.
Table 4CLA is on the impact of lipid of mice level
Annotate: compare * P＜0.05 with the ob/ob model group; * P＜0.01; * * P＜0.001; Compare with retinue ob model group,
#P＜0.05;
##P＜0.01;
###P＜0.001.
3, conclusion
The CLA of continuous 21 days oral 20mg/kg makes the bone metabolism disturbance of fat companion's osteoporosis ob/ob mice obtain obviously to improve, and tibial bone density significantly increases; And can significantly improve the unusual blood lipid metabolism of ob/ob mice.But for bone density and the comparatively normal ob mice of blood lipid metabolism, the CLA treatment does not have anomalous effects to its bone density level, bone metabolism or blood lipid metabolism aspect.
To sum up, CLA has clear and definite therapeutical effect to fat and hyperglycemia induced osteoporosis.
Claims (3)
1. the application of tree bean lactone A in preparation treatment or prevention of osteoporosis disease drug.
2. the application of tree bean lactone A in being prepared with the health food that benefits the osteoporosis crowd.
3. application according to claim 1 and 2 is characterized in that: osteoporosis is obesity and/or diabetes osteoporosis disease that cause and/or that follow.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109105894A (en) * | 2017-06-22 | 2019-01-01 | 宋祖莹 | Set beans extracting process and its application |
| CN110563690A (en) * | 2016-05-13 | 2019-12-13 | 贵州医科大学 | Tree ketonic acid A structural analogue, composition thereof and application thereof in medicines |
| WO2022011914A1 (en) * | 2020-07-17 | 2022-01-20 | 广州中医药大学(广州中医药研究院) | Use of cajanolactone a in preparing formulation for treating acquired obesity and concomitant diseases thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101422450A (en) * | 2008-10-09 | 2009-05-06 | 广州市允中投资发展有限公司 | Cajanus cajan L. natural medicine with blood sugar reduction and weight reduction function |
| WO2013013417A1 (en) * | 2011-07-28 | 2013-01-31 | 广州市允中投资发展有限公司 | Medicament useful for reducing blood sugar and body weight having the structure of stilbene compounds |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101422450A (en) * | 2008-10-09 | 2009-05-06 | 广州市允中投资发展有限公司 | Cajanus cajan L. natural medicine with blood sugar reduction and weight reduction function |
| WO2013013417A1 (en) * | 2011-07-28 | 2013-01-31 | 广州市允中投资发展有限公司 | Medicament useful for reducing blood sugar and body weight having the structure of stilbene compounds |
Non-Patent Citations (1)
| Title |
|---|
| 李宜融: "树豆叶的HPLC指纹图谱研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110563690A (en) * | 2016-05-13 | 2019-12-13 | 贵州医科大学 | Tree ketonic acid A structural analogue, composition thereof and application thereof in medicines |
| CN109105894A (en) * | 2017-06-22 | 2019-01-01 | 宋祖莹 | Set beans extracting process and its application |
| WO2022011914A1 (en) * | 2020-07-17 | 2022-01-20 | 广州中医药大学(广州中医药研究院) | Use of cajanolactone a in preparing formulation for treating acquired obesity and concomitant diseases thereof |
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