CN115671096A - Application of 6-methoxy angelicin and structural analogue thereof - Google Patents
Application of 6-methoxy angelicin and structural analogue thereof Download PDFInfo
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Abstract
The invention discloses application of 6-methoxy angelicin and structural analogues thereof in preparing agonists, inhibitors, medicaments, health-care products and functional foods for treating, preventing and relieving primary or secondary osteoporosis or bone loss. The in vitro experiments prove that the 6-methoxy angelicin and the structural analogs and derivatives thereof can up-regulate the expression of osteoblast differentiation specific genes ALP and OCN, promote osteoblast differentiation, and inhibit osteoclast differentiation and bone absorption at the same time. And has small toxic and side effect and high safety, and has potential to be developed into a reagent, a medicament, a health-care product or a functional food for resisting osteoporosis or preventing and treating bone loss related diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of 6-methoxy angelicin and structural analogues thereof in preparation of products for preventing and/or treating primary or secondary osteoporosis and/or bone loss related diseases.
Background
Osteoporosis is a degenerative bone disease in which decreased bone mass, which occurs with age and decreased postmenopausal estrogen levels in women, leads to increased bone fragility and is prone to fracture. In which a decrease in bone mineral mass (BMC) and bone density (BMD) has a correlation with a decrease in bone strength, making it easy for a patient to fracture, so that the degree of osteoporosis can be judged through bone density measurement. The disease is more female than male, and for female, within 5-10 years from menopause, the bone absorption is obvious, the bone loss is promoted to cause osteoporosis symptoms, patients mainly show soreness of waist and back, pain aggravation and limited activity when the load is increased, and serious patients can have spine deformation and brittle fracture. In addition, mechanical disuse osteoporosis, which is caused by reduction or disappearance of mechanical load, has also drawn more and more attention, such as reduction of stress to bones due to long-term bed rest or space flight, and the like, mainly due to increased bone resorption caused by lack of stimulation such as load bearing, muscle activity, and the like. The patient usually has symptoms such as reduced vertebral density and damaged bone trabecular structure due to movement and fixation, and serious symptoms such as bone tissue structure degeneration and neck and waist paralysis can occur. Osteoporosis can reduce the life quality of patients, spinal deformation and disability caused by fracture, so that the activity of the patients is limited, the death rate of the patients is increased, and economic burden is brought to the family society. With the increase of the population of the elderly in China, the incidence rate of osteoporosis is on the rising trend, and the osteoporosis becomes a health problem which is closely concerned.
At present, the occurrence mechanism of osteoporosis of postmenopausal women or mechanical disuse osteoporosis is complex, wherein the differentiation balance of osteoblasts and osteoclasts is one of the keys for maintaining normal bone mass. Osteoblast differentiation promotes bone formation and bone resorption by osteoclasts, a dynamic process that mediates bone formation and bone loss. The underlying mechanism lies in the balance of bone remodeling in the body, i.e., the imbalance between osteoclast removal of old bone (bone resorption) and osteoblast formation of new bone (bone formation).
Some existing medicines are widely applied to the aspect of controlling osteoporosis, including bone health supplements (calcium agents and vitamin D) and medicine interventions (bone formation medicines and bone resorption resisting medicines) and the like, the medicines mainly inhibit bone resorption or promote bone formation to play an anti-osteoporosis role, but most of the medicines have single treatment targets, the calcium supplements only serve as basic auxiliary medicines and cannot serve as treatment medicines for treating osteoporosis, and other western medicines have long treatment periods, high side effects and high prices. Compared with the traditional medicines for treating osteoporosis (such as bisphosphonate and hormone replacement therapy), the natural small molecular compound has the advantages of high safety, strong treatment targeting property, low price, outstanding treatment effect and the like. The 6-methoxy angelicin is a natural small molecular compound and has the effects of resisting inflammation, relieving fever and easing pain, and the effect of the 6-methoxy angelicin and structural analogues thereof in treating and relieving primary or secondary osteoporosis diseases is not reported at present.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a new use of 6-methoxy angelicin or a structural analogue or a pharmaceutically acceptable derivative thereof for the preparation of a medicament or a pharmaceutical composition for the treatment, prevention, alleviation of primary or secondary osteoporosis and/or bone loss related diseases, which solves the problems of the prior art.
To achieve the above objects and other related objects, a first object of the present invention is to provide a novel use of 6-methoxy angelicin and its structural analogs or pharmaceutically acceptable derivatives thereof in the preparation of a medicament or a pharmaceutical composition for treating, preventing, alleviating primary or secondary osteoporosis and/or bone loss related diseases.
The chemical structural formula of the 6-methoxy angelicin monomer is as follows:
the structural general formula of the 6-methoxy angelicin structural analogue is shown as a formula (I) or a formula (II):
in the formula (I), R 1 And R 2 Each independently selected from:
in the formula (II), R 1 And R 2 Each independently selected from:
preferably, the structural general formula of the 6-methoxy angelicin structural analogue is shown as the formula (II): and R is 1 And R 2 Wherein one of them is H.
The pharmaceutically acceptable derivative is any one of pharmaceutically acceptable salt, ester, ether, stereoisomer or prodrug molecules thereof. The substances can promote osteoblast differentiation, inhibit osteoclast bone resorption, promote osteoblast marker genes Alp and Ocn expression, and achieve the purpose of preventing, treating and relieving primary or secondary osteoporosis and/or bone loss related diseases.
Preferably, the medicament is a single substance with 6-methoxy angelicin or a structural analogue or a pharmaceutically acceptable derivative thereof as a main component, or a pharmaceutical composition with the single substance as the main component.
Preferably, when the medicament is a composition taking 6-methoxy angelicin or a structural analogue or a pharmaceutically acceptable derivative thereof as an active ingredient, the composition comprises the 6-methoxy angelicin or the structural analogue or the pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable auxiliary material.
The prepared medicament dosage form comprises injection, capsule, tablet, granule or solution.
The second purpose of the invention is also to provide the application of the 6-methoxy angelicin and the structural analogs thereof or the pharmaceutically acceptable derivatives thereof in preparing health care products for relieving diseases related to primary or secondary osteoporosis and/or bone loss.
The third purpose of the invention is to provide the application of the 6-methoxy angelicin and the structural analogue or the pharmaceutically acceptable derivative thereof in preparing functional food for relieving diseases related to primary or secondary osteoporosis and/or bone loss.
The fourth purpose of the invention is to provide the application of the 6-methoxy angelicin and the structural analogue or the pharmaceutically acceptable derivative thereof in preparing an osteoblast differentiation and/or osteoblast bone formation agonist.
Preferably, the osteoblast related gene is selected from at least one of ALP and OCN.
The fifth purpose of the invention is to provide the application of the 6-methoxy angelicin and the structural analogue or the pharmaceutically acceptable derivative thereof in preparing the osteoclast differentiation and/or osteoclast bone resorption inhibitor.
The sixth object of the present invention is also to provide a product for treating, preventing, alleviating primary or secondary osteoporosis and/or bone loss related diseases, which comprises 6-methoxy angelicin or a structural analogue thereof or a pharmaceutically acceptable derivative thereof.
Preferably, the product can be an agonist, an inhibitor, a drug, a health product or a functional food.
Compared with the prior art, the invention has the following beneficial effects:
a large number of experimental researches show that the 6-methoxy angelicin and the structural analogs and derivatives thereof can not only remarkably promote osteoblast differentiation and up-regulate the expression of osteoblast marker genes Alp and Ocn, but also inhibit osteoclast differentiation, thereby remarkably increasing the bone density of an osteoporosis model mouse, recovering bone microstructure and increasing bone mass. Therefore, a series of products, such as agents, drugs, health products or foods including 6-methoxy angelicin, can be developed, which can effectively treat and prevent primary or secondary osteoporosis and/or bone loss-related diseases.
Drawings
FIG. 1 is a graph showing the results of alkaline phosphatase and alizarin red staining after MC3T3-E1 cells were treated with 6-methoxy angelicin according to example 1 of the present invention;
FIG. 2 is a schematic diagram showing the results of detecting osteogenic differentiation marker genes ALP and OCN by using a qPCR detection method after MC3T3-E1 cells are treated by 6-methoxy angelicin of example 1 of the present invention;
FIG. 3 is a schematic diagram showing the result of alkaline phosphatase staining after MC3T3-E1 cells are treated with each structural analog of 6-methoxy angelicin provided by the present invention;
FIG. 4 is a schematic diagram showing the results of quantitative determination of alkaline phosphatase after MC3T3-E1 cells are treated with structural analogs of 6-methoxyangelicin provided by the present invention (the attached notes: 1: adefovir dipivoxil; 2: isobergaptan; 3: hydrated oxypeucedanin; 4: bergaptan; 5: isoimperatorin; 6: oxypeucedanin; 7: allopurin; 8: isocorallin; 9: blank; 10: bergapten; 11: notopterol; 12: xanthotoxin; 13: decursinol; 14: isoanenolide; 15: anisic lactone; 16: xanthotoxol; 17: bergaptol);
FIG. 5 is a schematic diagram showing the alizarin red staining result after MC3T3-E1 cells are treated by using each structural analog of 6-methoxy angelicin provided by the invention;
FIG. 6 is a TRAP staining result of osteoclasts after induction by treating primary myelomononuclear macrophages with 6-methoxy angelicin according to example 1 of the present invention;
FIG. 7 is a schematic diagram showing the results of femur bone density (FIG. 7 a) and micro CT scan analysis (FIG. 7 b) after the ovariectomized osteoporosis model mice are drenched with 6-methoxy angelicin or isoimperatorin;
FIG. 8 is a graph showing the results of quantitative analysis of femoral bone density (FIG. 8 a) and micro CT scan analysis (FIG. 8 b) after the hind limb unloading osteoporosis model mice are drenched with 6-methoxy angelicin or isoimperatorin;
FIG. 9 is a schematic diagram showing the results of measurement of three-point bending mechanical properties of tibia after 6-methoxy angelicin or isoimperatorin is drenched;
FIG. 10 is a schematic diagram of the results of the detection of the liver and kidney function indexes of an osteoporosis model animal after 6-methoxy angelicin or isoimperatorin is drenched.
Detailed Description
The following examples are further illustrative of the practice and advantages of the present invention, and are intended to facilitate a better understanding of the nature and spirit of the invention, and should not be construed as limiting the scope of the practice of the invention.
The 6-methoxy angelicin (also called as aegilops tauschii) is a 6-methoxy angelicin monomer, and the chemical structural formula is as follows:
Sphondin CAS:483-66-9
the inventor discovers the new application of the 6-methoxy angelicin through a large number of experimental researches, and the substance not only can remarkably promote osteoblast differentiation and up-regulate the expression of osteoblast marker genes Alp and Ocn, but also can inhibit the bone resorption of osteoclasts, remarkably increase the bone density of osteoporosis model mice, restore the bone microstructure and increase the bone mass. Through further analysis, the small molecular structural analogue containing the same or similar mother nucleus structure as the 6-methoxy angelicin also has the effect, and the structural analogue represented by the following formula I or formula II is screened out through experiments to have a very positive effect on osteoblast differentiation. In addition, based on 6-methoxy angelicin or the structural analogue shown in formula I or formula II, the pharmaceutically acceptable derivatives thereof are pharmaceutically acceptable salts, esters, ethers, stereoisomers or prodrug molecules thereof, and the like. The small molecular substances can effectively treat and prevent osteoporosis diseases and bone loss related diseases caused by ovariectomy or estrogen deficiency.
The structural general formula of the 6-methoxy angelicin structural analogue is shown as a formula (I) or a formula (II):
in the formula (I), R 1 And R 2 Each independently selected from:
in the formula (II), R 1 And R 2 Each independently selected from:
a representative 6-methoxy angelicin structural analog is shown in FIG. 3: specifically, it comprises isobergapten (CAS: 482-48-4); hydrated oxypeucedanin (CAS: 2643-85-8); bergapten (CAS: 484-20-8); isoimperatorin (CAS: 482-45-1); oxyprophorus (CAS: 737-52-0); pelargonium sidoides (CAS: 642-05-7); isophellopterin (CAS: 14348-22-2); bergamottin (CAS: 7380-40-7); notopterygii rhizoma alcohol (CAS: 88206-46-6); xanthotoxin (CAS: 298-81-7); decursinol (CAS: 23458-02-8), isoanisic acid lactone (CAS: 482-27-9), anisic acid lactone (CAS: 131-12-4), xanthotoxol (CAS: 2009-24-7), bergamot alcohol (CAS: 486-60-2), and imperatorin (CAS: 482-44-0).
Example 1 Effect of 6-methoxy-angelicin on osteoblast differentiation
1. Culture of mouse preosteoblastic cell line MC3T3-E1 cells
MC3T3-E1 preosteoporosisCulturing the cells in complete medium, standing the cells at 37 deg.C, 5% 2 The incubator of (2).
The complete medium consists of the following components:
per mL α -MEM liquid medium was added: 10% fetal bovine serum (volume percent), 10. Mu. Mol L-glutamine, 100. Mu.g penicillin, 100. Mu.g streptomycin sulfate.
Osteogenic differentiation Induction of MC3T3-E1 cells
After the MC3T3-E1 preosteoblasts are cultured until the cell density is 80%, the MC3T3-E1 preosteoblasts are subjected to osteogenic differentiation induction culture, and the experiment group is divided into a control group and an experiment group.
The control group adopts an osteogenic differentiation culture medium to carry out osteogenic differentiation induction culture, the experimental group adopts the osteogenic differentiation culture medium added with 6-methoxy angelicin to carry out osteogenic differentiation induction culture, the final concentration of the 6-methoxy angelicin contained in the osteogenic differentiation culture medium in the experimental group is 20 mu M, the liquid is changed every two days in the induction culture process, and the osteogenic differentiation culture medium needs to be prepared as it is.
The osteogenic differentiation medium consists of the following components:
alpha-MEM liquid culture medium contains per liter: 10% fetal bovine serum (volume percent), 10 mML-glutamine, 100U/mL penicillin, 100 mug/mL streptomycin sulfate, 0.005% vitamin C (mass volume percent), 10mM beta-sodium glycerophosphate, and 20 mug M6-methoxy angelicin.
Osteoblast differentiation analysis was performed on the experimental group of example 1 and the control group of comparative example 1 by using an alkaline phosphatase (ALP) staining detection method, an alizarin red staining detection method, and an osteogenic differentiation-related gene RT-PCR detection method, which are specifically as follows:
(1) Alkaline phosphatase (ALP) staining detection method
When the experimental group of example 1 and the control group of comparative example 1 both reached the induction of osteogenic differentiation of MC3T3-E1 pre-cells for 2 days, the level of osteogenic differentiation of the cells was examined by alkaline phosphatase staining, as follows:
the medium was decanted and washed 3 times with PBS, 5min each; adding 0.5mL of 4% paraformaldehyde solution, fixing at room temperature for 20min, and then continuously washing with PBS for 3 times, 5min each time; staining the cells with BCIP/NBT alkaline phosphatase staining kit (Shanghai Biyuntian Biotechnology Co., ltd.), and incubating at 37 deg.C for 30min; washing with deionized water for 5min for 2 times; drying at normal temperature, and scanning and storing by using a scanner. Results of the tests with particular reference to fig. 1, the depth of staining represents the differentiation level of osteoblasts. After treatment by the osteogenic differentiation medium added with the 6-methoxy angelicin, ALP staining is obviously deepened, which shows that the 6-methoxy angelicin can enhance ALP activity and promote osteoblast differentiation.
(2) Alizarin red staining detection method
When the experimental group of example 1 and the control group of comparative example 1 both reached the induction culture of osteogenic differentiation of MC3T3-E1 cells for 14 days, the level of osteogenic differentiation of the cells was detected by alizarin red staining, which was specifically performed as follows:
the medium was decanted and washed 3 times with PBS, 5min each; adding 0.5mL of 4% paraformaldehyde solution, fixing at room temperature for 20min, and then continuously washing with PBS for 3 times, 5min each time; cells were stained with 0.5% alizarin red solution (pH = 4.2) and incubated at room temperature for 2h; washing with distilled water for 5 times, each for 10min; drying at normal temperature, scanning with scanner and storing.
With specific reference to fig. 1, alizarin red staining detects calcified nodules in a culture medium deposited by extracellular matrix secreted by cells during osteoblast differentiation, and alizarin red is used to stain the cells, so that the calcified nodules can be stained red by alizarin red, while extracellular matrix without calcified nodules is not stained after being washed. The number of red nodules on the staining result may reflect the amount of calcium deposited during osteoblast differentiation, and the more calcium deposited, the higher the osteoblast differentiation. After the treatment of the osteogenic differentiation medium added with the 6-methoxy angelicin, the number of red calcified nodules is obviously increased, which indicates that the 6-methoxy angelicin can promote osteoblast differentiation.
(3) RT-PCR detection method for osteogenic differentiation related gene
When the experimental group of example 1 and the control group of comparative example 1 reached two days after osteoblasts were cultured for osteogenic differentiation induction before MC3T3-E1, total RNA of both was extracted using Trizol method, and concentration was measured using a micro uv spectrophotometer. Reverse transcription was performed using the One step PrimeScript RT reagent Kit and RT-PCR detection was performed using a Thermal Cycler C-1000 fluorescent quantitative PCR instrument, three duplicate wells were set for each set of sample experiments, and all loading was done on ice.
With specific reference to fig. 2, the results of the molecular biological assay and the osteogenic differentiation-related gene expression levels were significantly increased in the experimental group administered with 6-methoxyangelicin, as compared to the control group. The 6-methoxy angelicin can promote the differentiation of osteoblast and effectively improve the osteoporosis symptom.
Example 2 Effect of 6-methoxy angelicin structural analogs on osteoblast differentiation
This example uses serial 6-methoxy angelicin structural analogs to perform osteoblast differentiation experiments, and the procedures for culturing the mouse pre-osteoblast MC3T3-E1 cells, inducing osteogenic differentiation, and detecting alkaline phosphatase (ALP) staining were the same as those of example 1, except that the osteogenic differentiation medium used in the osteogenic differentiation induction process was different for each experimental group.
The differentiation component added to the experimental group of this example was a series of 6-methoxy angelicin structural analogs, including isobergapten; hydrated oxypeucedanin; bergapten; isoimperatorin; oxidizing decursin; bikunin; isophellopterin; bergamottin; notopterygii rhizoma alcohol; chinese prickly ash toxin; decursinol; isoanetholide; anisic lactone; xanthotoxol; bergamotol; imperatorin. The final concentration of each of the above substances contained in the osteogenic differentiation medium in the experimental group was still 20. Mu.M, and the osteogenic differentiation induction culture was performed using the osteogenic differentiation medium in the control group. Culturing the MC3T3-E1 preosteoblasts until the cell density is 80%, and then carrying out osteogenic differentiation induction culture on the MC3T3-E1 preosteoblasts, wherein liquid is changed once every two days in the induction culture process, and the osteogenic differentiation culture medium needs to be prepared at present.
After the experimental group and the control group reach the induction culture of the osteoblastic differentiation of the MC3T3-E1 anterior cells for 2 days, the osteoblastic differentiation level of the cells is detected by alkaline phosphatase staining, and the specific operation is performed in the same manner as in example 1. Results of the assay referring specifically to fig. 3, the depth of staining represents the differentiation level of osteoblasts. After being treated by the osteogenic differentiation medium added with the representative 6-methoxy angelicin structural analogue, ALP staining is changed to different degrees, which shows that the series of structural analogues change the activity of ALP to different degrees, thereby promoting or inhibiting osteoblast differentiation. Among them, 6-methoxyangelicin, isobergapten, oxypeucedanin hydrate, bergapten, isoimperatorin, oxypeucedanin, alloimperatorin, isophellandrin, bergamottin, notopterygium alcohol, isoanetholide and anetholide can deepen ALP staining, indicating that they have an osteoblast differentiation promoting effect.
Next, we quantitatively determined the ALP activity of MC3T3-E1 cells, a mouse preosteoblastic cell line, by the following method:
(1) Sample pretreatment
And (3) sucking supernatant of adherent cells after differentiation induction culture for 2 days, scraping the cells by a cell scraper, transferring the cells into an EP (EP) tube, then rotating the cells per minute at 1000 rpm, centrifuging the cells for 10 minutes, removing the supernatant, leaving cell precipitates, adding 1mL of PBS (phosphate buffer solution) for gentle blow beating, repeating the centrifugation, and leaving the cell precipitates for later use.
Adding a certain amount of lysate into an EP tube, cracking for 30-40 minutes, observing the cracking condition, and then sucking out the lysate to be detected by using a micropipette. The operating table is shown in the following table:
(2) Quantitative calculation
Definition of ALP Activity (King units/gprot) in cultured cells: one King-unit of phenol was produced at 1mg per gram of tissue protein by exposure to the matrix at 37 ℃ for 15 minutes. The formula is as follows:
FIG. 4 is a graph showing the results of quantitative determination of alkaline phosphatase in MC3T3-E1 cells treated with structural analogs of 6-methoxyangelicin, compared with a control group (blank solvent control), in which ALP activity in cells to which compounds Nos. 1 to 8 were added was stronger than that in the blank solvent control group. The compounds [1 to 8 are: 1, the bovine amethystin (namely 6-methoxy angelicin); 2, isobergapten; 3, hydrating and oxidizing the decursin; 4, bergapten; 5, isoimperatorin; oxidizing decursin; 7, bielespedezanin; and 8, isophellopterin.
Furthermore, by referring to fig. 4, it can be seen that 1: bovine hurricane oxide (6-methoxy angelicin) and 2: isobergapten have the strongest ALP activity, and have a very significant effect on the differentiation of mouse preosteoblastic cell line MC3T3-E1 cells, and the ALP activity value in cultured cells is more than 39 King units/gprot.
We analyze the influence of the compound structure on the ALP activity value in cultured cells through a structure-effect relationship, and the compound with the angle-type coumarin structure has stronger overall activity and strongest activity of 6-position single methoxyl substitution. 15: the anisic lactone belongs to a compound with an angular coumarin structure, but the compound activity is rather low due to the fact that the anisic lactone contains two substituent groups, and the fact that the number of the substituent groups on the angular coumarin structure is increased does not benefit the compound activity.
For furocoumarin, the 4-position substituent is more favorable for the activity of the compound, the size and the fat solubility of the substituent have great influence on the activity, the carbon chain length is moderate, and the fat solubility is moderate and has better activity. The substituent at the 8-position is not beneficial to the activity of the compound, wherein the inhibition effect of a larger substituent or a hydroxyl substituent on the activity is more obvious. The 4-position substituent has larger influence on the activity, the carbon chain length is moderate, and the adverse effect of the 8-position substituent can be partially reversed by the 4-position substituent with moderate fat solubility. The results further indicate that the activity on pyranocoumarin is weaker than that on furanocoumarin.
Then, the differentiation of the mouse preosteoblastic cell line MC3T3-E1 was further examined by alizarin red staining test method, and the procedures for culturing the mouse preosteoblastic cell line MC3T3-E1 and alizarin red staining test method were the same as in example 1.
With specific reference to fig. 5, the results of the tests were compared with those of the control group (blank), and after treatment with an osteogenic differentiation medium containing pralletin (6-methoxyangelicin), isobergapten, hydrated oxypeucedanin, bergapten, isoimperatorin, oxypeucedanin, alloimperatorin, isophellandrin, the number of red calcified nodules was significantly increased, indicating that the structural analogs of each of the above-mentioned pralletins can promote osteoblast differentiation.
Example 3 Effect of 6-methoxy angelicin on osteoclast differentiation
a. Bone marrow mononuclear macrophage in vitro induction of osteoclast differentiation
Bone marrow mononuclear macrophages (BMMs) were subjected to in vitro induction of osteoclastic differentiation using osteoclastogenesis induction medium (complete medium supplemented with 10ng/mL M-CSF (macrophage colony stimulating factor) and 10ng/mL RANKL (nuclear factor kappa B receptor activating factor ligand)). The specific induction method is as follows:
primary BMMs extracted from mouse bone marrow are divided into 1 × 10 5 The cells/well were seeded in 48-well plates and osteoclast induction was performed by observing that adherent BMMs account for more than 20% of the total cells. The original medium supernatant was discarded, and the osteoclast induction medium was replaced with the original medium, and induction was started. And continuously supplementing the osteoclast induction culture medium into the holes on the next day without discarding the culture medium, discarding the culture medium on the third day, replacing the fresh osteoclast induction culture medium, and continuously supplementing the osteoclast induction culture medium on the fourth day, namely circularly replacing the fresh osteoclast induction culture medium every two days.
The experiment is divided into a control group and an administration group, wherein the control group is that the BMMs cells are normally induced after being added with an osteoclast induction culture medium, and the administration group is that the primary BMMs cells are simultaneously added with the osteoclast induction culture medium and 20 mu M6-methoxy angelicin. Mature multinuclear osteoclast can be observed in the control group about 4 days after the osteoclast induction culture, and the sample collection and detection can be carried out.
TRAP staining
Osteoclast TRAP staining was performed on the control group and the administration group after osteoclast induction culture, and the specific process was as follows:
discarding the culture medium in the sample, and gently rinsing with PBS buffer solution for 1-2 times, 3-5 min each time; adding 4% paraformaldehyde solution, and fixing for 20min; removing the stationary liquid, rinsing with PBS buffer solution for 2-3 times, 3-5 min each time; discarding the PBS buffer solution, adding a proper amount of TRAP staining solution (adding 200 mu L of TRAP staining solution into each hole of a 48-hole plate), and incubating at 37 ℃ in the dark for 1h; after the incubation is finished, discarding the staining solution, and rinsing for 3 times by using PBS buffer solution to terminate the reaction; the results of cell staining were observed using an optical microscope and photographed.
The detection result specifically refers to fig. 6, after the 6-methoxy angelicin induction treatment, the activity of TRAP enzyme induced by RANKL is inhibited, so that staining positive cells of the TRAP enzyme are reduced, the number of mature multinuclear osteoclasts is reduced, and the 6-methoxy angelicin can inhibit osteoclast differentiation and bone resorption.
EXAMPLE 4 therapeutic Effect of 6-methoxy angelicin and isoimperatorin on ovariectomized osteoporotic mice
C57BL/6 mice of 2 months of age are selected to establish an ovariectomized osteoporosis animal model, and the treatment effect of 6-methoxy angelicin (Sphondin) and isoimperatorin (isohermiferin) on the osteoporosis animal model is detected. The 6-methoxy angelicin is administered to mice by gavage once a day for 8 weeks at doses of 10mg/kg and 30mg/kg, and isoimperatorin is administered in the same manner as 6-methoxy angelicin at doses of 10mg/kg, 30mg/kg and 50mg/kg.
1. Bone density detection in mice
After the mice were anesthetized with sodium pentobarbital, the bone density of the mice after administration was measured using a high resolution dual energy X-ray imaging system (MEDIKORS, inAlyzer,55KeV/80 KeV).
Referring to fig. 7a, the femoral bone density was significantly lower in the ovariectomized mice than in the normal healthy mice; after different doses of 6-methoxy angelicin and isoimperatorin are administrated, the bone density of femurs of mice without ovaries and with osteoporosis is obviously increased compared with that of mice without the ovaries and the effect of mice with a lower dose of a high dose group is better, which shows that the 6-methoxy angelicin and the isoimperatorin can obviously improve osteoporosis symptoms, wherein the effect of the 6-methoxy angelicin is better than that of the isoimperatorin.
Micro CT detection
The mice were sacrificed by removing the neck, the femur was removed, the soft tissue on the bone surface was removed, after fixation with 4% paraformaldehyde, scanning was performed with a micro CT scanner, and the following parameters were analyzed with special bone CT analysis software after reconstruction: bone density (BMD), bone volume fraction (BV/TV%), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular resolution (Tb.Sp), etc.
FIG. 7b is a femoral micro CT scan and the results of software quantitative analysis after femoral micro CT scan of mice dosed with different amounts of 6-methoxy angelicin and isoimperatorin in this example. Compared with the control group of mice, after the mice are drenched with the 6-methoxy angelicin and the isoimperatorin, the bone density, the bone volume fraction, the trabecular bone thickness, the trabecular bone number and the trabecular bone separation degree of the mice are obviously recovered, and the effect of the high-dose group and the low-dose group is better. The results show that the treatment of 6-methoxy angelicin and isoimperatorin can improve the femoral bone density loss and the bone microstructure of mice, wherein the effect of the 6-methoxy angelicin is better than that of the isoimperatorin.
EXAMPLE 5 therapeutic Effect of methoxy angelicin and Isoimperatorin on mechanically unloaded osteoporosis mice
C57BL/6 mice of 6 months old are selected to establish a hind limb tail suspension mechanical unloading osteoporosis animal model, and C57BL/6 ground feeding group mice of 6 months old are used as a control to detect the treatment effect of 6-methoxy angelicin and isoimperatorin on mechanical unloading osteoporosis. The 6-methoxy angelicin is administrated to the mice in a gastric lavage mode once a day for 4 weeks, and the dosage is 30mg/kg; the administration mode of isoimperatorin is the same as that of 6-methoxy angelicin, and the dosage is also 30mg/kg.
1. Bone density detection in mice
The detection method was the same as in example 4. Referring to fig. 8a, the density of femur bone of mechanically unloaded osteoporosis mouse is significantly lower than that of ground feeding control group; after the 6-methoxy angelicin and the isoimperatorin are drenched, the bone density of the femur of the mice with mechanical unloading osteoporosis is obviously increased compared with that of the mice without administration, which shows that the 6-methoxy angelicin and the isoimperatorin can improve the symptoms of the mechanical unloading osteoporosis, and the effect of the 6-methoxy angelicin is better than that of the isoimperatorin.
2. Mouse micro CT detection
The detection method was the same as in example 4. Fig. 8b is the result of quantitative analysis of the femur micro CT scan and the software after femur micro CT scan of the mice in this embodiment after the mice are drenched with 6-methoxy angelicin or isoimperatorin for different time. Compared with the mechanical unloading mouse, after the 6-methoxy angelicin or isoimperatorin is drenched for 4 weeks, the bone density, the bone volume fraction, the trabecular bone thickness, the trabecular bone number and the trabecular bone separation degree of the mouse are obviously recovered. The results show that the treatment of 6-methoxy angelicin and isoimperatorin can increase the femoral bone density of mice with mechanical unloading bone loss and improve the bone microstructure, wherein the effect of 6-methoxy angelicin is better than that of isoimperatorin.
Example 6 improvement of mechanical Properties of bone tissue of an osteoporosis model animal by methoxy angelicin and isoimperatorin
The establishment method of the ovariectomized osteoporosis mouse model and the administration scheme of the 6-methoxy angelicin and the isoimperatorin are the same as the embodiment 4; the establishment method of the hind limb tail suspension mechanics unloading osteoporosis mouse model and the administration scheme of the 6-methoxy angelicin and isoimperatorin are the same as example 5.
And (3) removing the neck of the mouse, killing the mouse, taking out the femur by stripping, removing soft tissues on the surface of the bone, washing and infiltrating by using a PBS (phosphate buffer solution), and detecting the mechanical property of the tibia of the mouse by three-point bending mechanical loading.
Referring to fig. 9, the mechanical property indexes of the mice, such as maximum load, elastic modulus and hardness coefficient, are significantly improved after treatment with 6-methoxy angelicin or isoimperatorin, compared with the control group. The results show that 6-methoxy angelicin and isoimperatorin can effectively recover the bone tissue mechanical property after continuous gastric lavage administration for a long time (8 weeks), wherein the effect of 6-methoxy angelicin is better than that of isoimperatorin.
Example 7 detection of hepatorenal toxicity of osteoporosis model animals by methoxy angelicin and isoimperatorin
The establishment method of the ovariectomized osteoporosis mouse model and the administration scheme of the 6-methoxy angelicin and the isoimperatorin are the same as the embodiment 4; the establishment method of the hind limb tail suspension mechanics unloading osteoporosis mouse model and the administration scheme of the 6-methoxy angelicin and isoimperatorin are the same as example 5.
Blood was drawn from the mice after the administration by cardiac puncture, serum was separated, and ALT, AST, r-GT and BUN levels in the serum were measured. Referring to FIG. 10, there was no significant change in the mean levels of liver and kidney function indices ALT, AST, r-GT, and BUN in mice treated with 6-methoxyangelicin or isoimperatorin compared to the control group. It is demonstrated that the application of the 6-methoxy angelicin or isoimperatorin for a long time (8 weeks) has no significant hepatorenal toxicity and is safe.
Through the experimental results, the research finds that the 6-methoxy angelicin and the structural analogue thereof can effectively treat and prevent the osteoporosis diseases, and by constructing a mouse model for simulating the symptoms of the postmenopausal climacteric diseases of women and a waste osteoporosis mouse model, the 6-methoxy angelicin can effectively relieve the symptoms of the osteoporosis, promote bone formation to inhibit bone absorption, remarkably increase the bone density of model animals, recover the bone microstructure and have no obvious toxic or side effect. The pharmaceutically acceptable derivatives thereof, such as pharmaceutically acceptable salts, esters, ethers, stereoisomers or prodrug molecules thereof, also can be used as medicaments for treating, preventing and relieving osteoporosis and/or bone loss due to the same acting groups.
In view of the outstanding effects of 6-methoxy angelicin and structural analogs thereof in the aspects, the substance can be used alone or used as a main component, and other medically acceptable auxiliary materials are added to form a pharmaceutical composition for use, so that the pharmaceutical composition can be used for treating, preventing and relieving primary or secondary osteoporosis and/or bone loss and other diseases. The dosage forms of the medicaments comprise injections, capsules, tablets, granules or solutions. The protein can remarkably promote osteoblast differentiation and up-regulate the expression of osteoblast marker genes Alp and Ocn, has wide application in preparing osteoblast differentiation and/or osteoblast bone formation agonists, osteoclast differentiation and/or osteoclast bone resorption inhibitors, and can be applied to health products or functional foods to prepare various products including agonists, inhibitors, medicaments, health products and functional foods.
In summary, the preferred embodiments of the present invention have significant efficacy, low cost, and low toxic and side effects, and any simple modifications, alterations, and equivalent structural changes made to the above embodiments according to the technical essence of the invention are still within the scope of the technical solution of the present invention, and can be used for treating disease symptoms caused by estrogen deficiency in female animals after menopause.
Claims (12)
- Use of 6-methoxy angelicin and 6-methoxy angelicin structural analogs or pharmaceutically acceptable derivatives thereof in the preparation of a medicament for treating, preventing, or alleviating primary or secondary osteoporosis and/or bone loss related diseases.
- 2. The use according to claim 1, wherein the structural general formula of the 6-methoxy angelicin structural analogue is represented by formula (I) or formula (II):
in the formula (I), R 1 And R 2 Each independently selected from:
in the formula (II), R 1 And R 2 Each independently selected from:
- 3. the use according to claim 1, wherein the pharmaceutically acceptable derivative is a pharmaceutically acceptable salt, ester, ether, stereoisomer or prodrug molecule thereof.
- 4. The use of claim 1, wherein the medicament is a single substance or composition comprising 6-methoxy angelicin or a 6-methoxy angelicin structural analogue or a pharmaceutically acceptable derivative thereof as an active ingredient.
- 5. The use according to claim 1, wherein when the medicament is a composition comprising 6-methoxy angelicin or a 6-methoxy angelicin structural analogue or a pharmaceutically acceptable derivative thereof as an active ingredient, the composition comprises 6-methoxy angelicin or a 6-methoxy angelicin structural analogue or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable adjuvant, and the dosage form of the medicament comprises injection, capsule, tablet, granule or solution.
- Use of 6-methoxy angelicin or a 6-methoxy angelicin structural analogue or a pharmaceutically acceptable derivative thereof for the preparation of a health product for alleviating diseases related to primary or secondary osteoporosis and/or bone loss.
- Use of 6-methoxy angelicin or a 6-methoxy angelicin structural analogue or a pharmaceutically acceptable derivative thereof for the preparation of a functional food for alleviating a disease associated with primary or secondary osteoporosis and/or bone loss.
- Use of 6-methoxy angelicin or a 6-methoxy angelicin structural analogue or a pharmaceutically acceptable derivative thereof for the preparation of an agonist for osteoblast differentiation and/or osteoblast bone formation.
- 9. The use according to claim 8, wherein the osteoblast associated genes are selected from at least one of ALP and OCN.
- Use of 6-methoxy angelicin or a 6-methoxy angelicin structural analogue or a pharmaceutically acceptable derivative thereof for preparing an osteoclast differentiation and/or osteoclast bone resorption inhibitor.
- 11. A product for the treatment, prevention, alleviation of primary or secondary osteoporosis and/or bone loss related disorders characterized by: comprises 6-methoxy angelicin or a 6-methoxy angelicin structural analogue or a pharmaceutically acceptable derivative thereof.
- 12. The product for use in the treatment, prevention, alleviation of primary or secondary osteoporosis and/or bone loss related diseases according to claim 11, characterized in that: the product is an agonist, an inhibitor, a medicament, a health product or a functional food.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110840882A (en) * | 2019-09-11 | 2020-02-28 | 西北工业大学 | Composition for treating osteoporosis |
| CN110898089A (en) * | 2019-12-27 | 2020-03-24 | 西北工业大学 | Traditional Chinese medicine extract composition for treating osteoporosis |
| CN113648306A (en) * | 2021-08-16 | 2021-11-16 | 西北工业大学 | Application of bergamottin in preventing or treating osteoporosis and/or bone loss |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110840882A (en) * | 2019-09-11 | 2020-02-28 | 西北工业大学 | Composition for treating osteoporosis |
| CN110898089A (en) * | 2019-12-27 | 2020-03-24 | 西北工业大学 | Traditional Chinese medicine extract composition for treating osteoporosis |
| CN113648306A (en) * | 2021-08-16 | 2021-11-16 | 西北工业大学 | Application of bergamottin in preventing or treating osteoporosis and/or bone loss |
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| Title |
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| MAHENDRA CK等: "Angelicin—A Furocoumarin Compound With Vast Biological Potential", FRONTIERS IN PHARMACOLOGY, vol. 11, pages 7 - 8 * |
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