CN110711201A - Application of punicalagin in preparing medicine for preventing and treating postmenopausal osteoporosis - Google Patents

Abstract

The invention discloses an application of punicalagin in preparing a medicine for preventing and treating postmenopausal osteoporosis, and also discloses a pharmaceutical preparation for preventing and treating postmenopausal osteoporosis. According to the invention, by applying the ovariectomized mouse osteoporosis model, the treatment effect of Punicalagin (PUN) on ovariectomized mouse osteoporosis is observed, the changes of main indicators of osteoclast activation RANKL and CTSK are evaluated, and various indicators of osteoporosis are analyzed, so that the treatment effect mechanism of PUN on estrogen-deficient osteoporosis is clarified. After menopause, due to estrogen deficiency, the bone resorption factor can up-regulate the expression of RANKL, activate an RANKL pathway and cause osteoclast activation, and is currently considered as an important mechanism for causing postmenopausal osteoporosis, and PUN inhibits osteoclast activation by inhibiting the pathway, so that PUN provides a new way for preventing and treating postmenopausal osteoporosis.

Description

Application of punicalagin in preparing medicine for preventing and treating postmenopausal osteoporosis

Technical Field

The invention belongs to the technical field of new application of chemical medicines, and particularly relates to application of punicalagin in preparing a medicine for preventing and treating postmenopausal osteoporosis.

Background

Postmenopausal Osteoporosis (PMO) is a common metabolic bone disease, which is a bone disease with decreased bone mass and changed bone tissue microstructure due to osteoclast hyperfunction and bone metabolism imbalance caused by estrogen deficiency of Postmenopausal women, and is manifested by increased bone fragility and easy fracture. Osteoporosis has become a chronic disease second only to cardiovascular disease, the most dangerous and incurable worldwide today. It is statistically estimated that postmenopausal osteoporosis affects more than 2 billion women worldwide, and one third of women over the age of 50 experience osteoporotic fractures, which causes great suffering and heavy economic loss to the patient. Although scholars experts at home and abroad attach great importance to the prevention and treatment of the disease, the disease is still lack of effective treatment medicines.

The current clinically used medicines for preventing and treating osteoporosis comprise estrogen substitute medicines, bisphosphates, selective estrogen receptor modulators, vitamin D and the like. Unfortunately, bisphosphonates are effective against osteoporosis, but also risk complications of jaw necrosis and renal failure. Hormone replacement therapy, while effective in reducing osteoporotic fractures, results in an increased risk of breast cancer, myocardial ischemia and thromboembolic disease. Other drugs are either not effective for the long term or are expensive. Since osteoporosis is a chronic disease and needs to be taken for a long time, finding a natural compound with low price and small toxic and side effects has become one of the hot spots for researching and treating osteoporosis.

Punicalagin (PUN) with molecular formula of C₄₈H₂₈O₃₀The relative molecular mass is 1084.72, the CASNo is 65995-63-3, and the structure is shown in formula (I):

formula (I)

Punicalagin (PUN) is the most main active component (about 60-70%) in pomegranate peel polyphenol, is in the form of brown-yellow indefinite powder, has strong polarity, is easily soluble in water, can be dissolved in organic solvents such as ethanol and the like to form colloidal solution, can perform color reaction with various chemical reagents, is one of gallic acid tannins, and is a hydrolyzable polyphenol compound with relatively large molecular mass which is specific to pomegranate. PUN has many important pharmacological effects, such as anti-inflammatory, anti-tumor, antibacterial and antioxidant properties. In recent years, due to the wide attention on the bioactivity, the compound is increasingly applied to the prevention and treatment of cardiovascular diseases, diabetes, fatty liver and other diseases.

Although it has been reported in the prior art that PUN alone has an inhibitory effect on osteoclast differentiation from RAW264.7 cells in vitro, it has been shown in the prior art that even if a certain drug has an inhibitory effect on osteoclast differentiation in vitro, the effect of preventing and treating osteoporosis after application to the body is not ideal.

At present, effective drug treatment for postmenopausal osteoporosis (PMO) is lacked, and no report that PUN prevents and treats PMO is provided, and the invention also comes from the report.

Disclosure of Invention

The invention aims to provide a new application of Punicalagin (PUN), in particular to an application of PUN in treating PMO, and provides a new way for preventing and treating postmenopausal osteoporosis.

In order to achieve the purpose, the invention provides the following technical scheme:

application of punicalagin in preparing medicine for preventing and treating postmenopausal osteoporosis mainly refers to osteoporosis caused by postmenopausal estrogen deficiency. The structural formula of the punicalagin is shown as a formula (I),

formula (I).

A pharmaceutical preparation for preventing and treating postmenopausal osteoporosis comprises punicalagin with effective treatment amount and pharmaceutically acceptable adjuvants.

Further, the content of the punicalagin is 1-99% by mass.

Further, the pharmaceutical preparation is suitable for pharmaceutical preparations for gastrointestinal or parenteral administration.

Furthermore, the pharmaceutical preparation is a freeze-dried preparation, an injection, a tablet, a granule or a capsule.

According to the technical scheme, whether the PUN has a treatment effect on the osteoporosis of the mice caused by estrogen deficiency after ovariectomy is researched by a gastric lavage administration method, and indexes such as nuclear factor kappa B receptor activator ligand (RANKL), cathepsin K (CTSK) and the like are measured to research the relation between the effect and osteoclast activation, so that the mechanism and the theoretical basis of the PUN for treating the postmenopausal osteoporosis are discussed.

In the present invention, 60 10-week C57BL/J6 female mice were randomly divided into a Sham-operated group (Sham group), an osteoporosis model group (OVX group), a low-dose PUN-treated group (20 mg/kg/d), and a high-dose PUN-treated group (50 mg/kg/d), and 15 mice were each group. All animals in each group were subjected to general surgical procedures under general anesthesia, with Sham resecting only a portion of the adipose tissue surrounding the ovaries, and the remaining three groups being subjected to bilateral ovariectomy. The PUN treatment group was injected with 200. mu.l of sterile PBS solution containing PUN by gavage every day from the 3 rd week after the operation (wherein the administration doses of the low dose group and the high dose treatment group were 20 mg/kg/d and 50 mg/kg/d, respectively), and the Sham group and the OVX group were administered with 200. mu.l of sterile PBS solution every day by gavage. After continuously administering for four weeks (5 times per week), the animals are sacrificed, the thighbone is taken for Micro-CT and histological examination, and the thighbone osteoporosis degree is analyzed by measuring the thighbone bone density, the bone volume fraction, the trabecular number, the trabecular separation degree, the mature osteoclast number and the like; meanwhile, an immunohistochemical method is applied to detect the expression quantity of RANKL, CTSK and the like in bone tissues. Statistical analysis was performed on each group of data using one-way anova.

Has the advantages that: the invention provides an application of punicalagin in preparing a medicine for preventing and treating postmenopausal osteoporosis. According to the invention, by applying the ovariectomized mouse osteoporosis model, the treatment effect of Punicalagin (PUN) on ovariectomized mouse osteoporosis is observed, the changes of main indicators of osteoclast activation RANKL and CTSK are evaluated, and various indicators of osteoporosis are analyzed, so that the treatment effect mechanism of PUN on estrogen-deficient osteoporosis is clarified. After menopause, due to estrogen deficiency, the bone resorption factor can up-regulate the expression of RANKL, activate an RANKL pathway and cause osteoclast activation, and is currently considered as an important mechanism for causing postmenopausal osteoporosis, and PUN inhibits osteoclast activation by inhibiting the pathway, so that PUN provides a new way for preventing and treating postmenopausal osteoporosis.

Drawings

The invention will be further described with reference to the accompanying drawings and examples

FIG. 1 is a three-dimensional model diagram of femurs of mice of each experimental group scanned by Micro-CT. A is Sham group, B is OVX group, C is PUN low dose treatment group, and D is PUN high dose treatment group.

FIG. 2 is a graph showing the measurement values of bone density (BMD) of femurs of mice in each experimental group.

FIG. 3 is a graph showing the measurement values of femoral bone volume fraction (BV/TV) of mice in each experimental group.

Fig. 4 is a graph showing the detection values of the number of femoral cancellous bone trabeculae (tb.n) in each experimental group of mice.

Fig. 5 is a graph showing the results of the mean bone trabecular separation (tb.sp) of cancellous bone of femur in mice of each experimental group.

FIG. 6 is a graph showing the results of HE staining of femurs of mice in each experimental group. A is Sham group, B is OVX group, C is PUN low dose treatment group, and D is PUN high dose treatment group.

FIG. 7 is a graph showing the detection values of bone volume fraction (BV/TV) of HE stained femur of mice in each experimental group.

FIG. 8 is a graph showing the results of TRAP staining of femurs of mice in each experimental group. A is Sham group, B is OVX group, C is PUN low dose treatment group, and D is PUN high dose treatment group.

FIG. 9 is a graph showing the results of TRAP staining positive cells in mice of each experimental group.

Fig. 10 is a graph of RANKL immunohistochemical staining results of the femurs of mice in each experimental group. A is Sham group, B is OVX group, C is PUN low dose treatment group, and D is PUN high dose treatment group.

Fig. 11 is a graph showing the results of counting RANKL-expressing positive cells in the mice femur immunohistochemistry of each experimental group.

Fig. 12 is a graph of CTSK immunohistochemical staining results of femurs of mice in each experimental group. A is Sham group, B is OVX group, C is PUN low dose treatment group, and D is PUN high dose treatment group.

Fig. 13 is a graph showing the counting results of the CTSK positive cells expressed by the femoral immunohistochemistry of the mice in each experimental group.

Detailed Description

The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Materials and methods

1. Material

1.1 reagent and Experimental Equipment

1.1.1 major drugs and reagents

Punicalagin (PUN), available from Sigma-Aldrich, USA; TRAP staining kit, purchased from Sigma-Aldrich, USA; paraformaldehyde, sterile PBS, DAB color developing agent, hematoxylin, eosin, absolute ethyl alcohol, distilled water and 10% chloral hydrate. The RANKL, CTSK antibodies were purchased from Abcam, uk.

1.1.2 Main Instrument

Micro-CT (SkyScan 1176, belgium), paraffin microtome (Leica 2135, germany), bake microtome (Leica 1120, germany), paraffin embedding machine (Leica 1150, germany), Axiovert 40C optical microscope (Zeiss, germany), surgical instrument suite, etc.

1.2 Experimental animals

60 healthy C57BL/J6 mice, female, 21-24 g in weight, 10 weeks old, SPF grade, were provided by Suzhou university animal laboratories. The breeding conditions were as follows: five cages are used, the room temperature is 18-20 ℃, the humidity is 50-60%, the ventilation is good, and the water can be freely taken.

2. Experimental methods

2.1 grouping of Experimental animals

60C 57BL/J6 mice, randomized into the following 4 groups:

(1) sham group: 15 patients are subjected to castration sham operation, the operation is the same as that of the OVX group, but the operation is limited to cutting off partial adipose tissues around the ovary without cutting off the ovary, 200 mu l of sterile PBS solution is injected by a gavage method from the 3 rd week after the operation, the PBS solution is fed for 5 days every week, and the patients are killed after 4 weeks of continuous gavage;

(2) OVX group: 15 mice, an osteoporosis model group, were subjected to a posterior median incision of the lumbar vertebrae to expose ovaries on both sides. Ligating the junction of the ovary and the uterus on two sides by silk threads respectively, cutting off the ovary, injecting 200 mul of sterile PBS solution by a gavage administration method from the 3 rd week after operation, feeding the PBS solution for 5 days every week, and killing the ovary after 4 weeks of continuous gavage;

(3) Low-PUN group: for the PUN low dose treatment group, mice were incised at the posterior midline of the lumbar spine and the ovaries on both sides were exposed. Ligating the junction of the ovary and the uterus at two sides by silk threads respectively, removing the ovary, injecting 200 microliter sterile PBS solution containing 20mg/kg PUN by a gavage administration method from the 3 rd week after operation, administering the solution for 5 days every week, and killing the ovaries and the uterus after 4 weeks of continuous gavage;

(4) High-PUN group: for the PUN high dose treatment group, mice were incised in the posterior median of the lumbar spine to expose both ovaries. Ligation was performed by silk thread at the junction of both ovaries and uterus, and ovariectomy was performed from the 3 rd week after operation by intragastric administration of 200 μ l sterile PBS solution containing 50mg/kg PUN administered for 5 days per week and sacrificed after 4 weeks of continuous intragastric administration.

2.2 preparation of mouse ovariectomized osteoporosis model

The invention adopts an ovariectomy castration osteoporosis model to simulate the pathological process of postmenopausal osteoporosis (Kinoshita M, et al. Change in micro-CT 3D Bone parameters reflex effector a post-Bone cement K inhibitor (SB-553484) on Bone preservation and corticobalamin formation in acquired osteoporosis. Bone 2007; 40(5): 1231-7.). The experimental mice were anesthetized by intraperitoneal injection of 0.1ml/10g of 4% chloral hydrate. Fixing the mouse in a supine position on an animal operation table, unhairing the skin of the lower abdomen of the mouse, sterilizing for three times by using an iodine solution, laying a sterile hole towel, taking a central incision behind the lumbar vertebra of the mouse, exposing and separating the ovaries on two sides, ligating the root line of the connection between the ovaries and the uterus by using an operation suture line, and excising the ovaries on two sides. Following successful ovariectomy, the skin was sutured with 4-0 sutures. To prevent post-operative infection, penicillin was injected intramuscularly once after the operation. All procedures were completed on the same day.

2.3 specimen Collection

Taking out bilateral femurs of the sacrificed mice, and stripping peripheral periosteum, muscles and other soft tissues; fixing the right femur with 4% paraformaldehyde solution for 24h, decalcifying with 10% EDTA for 3 weeks, and paraffin embedding for HE staining, TRAP staining evaluation and immunohistochemical detection; left femoral specimens were stored in 10% cold PBS for Micro-CT for cancellous bone density and related bone morphological parameters.

2.4 Micro-CT detection

After the femur of the mouse is fixed for 24h, Micro-CT scanning is carried out. Scanning parameters are as follows: the resolution is 9 mu m, and the voltage is 80 kV; current is 100 muA; each exposure time is 100 ms; 0.9 °/8 images. Selecting a cylindrical region of interest (ROI; diameter 3mm and height 1 mm), performing 3D analysis on the image by using Micro-CT image analysis software, and recording the bone density (BMD, mg/mm) of the ROI femur by using a Wedemeyer C method (Wedemeyer C, et al²) Bone volume to tissue volume ratio (BV/TV), average trabecular number (Tb.N), average trabecular resolution of cancellous bone (Tb.Sp), and the like.

2.5 histological staining

After the femoral bone specimen is decalcified by EDTA decalcification solution, trimming the specimen, removing proximal tissue of the femoral bone, and reserving 1/3 in the middle and lower part of the femoral bone so as to facilitate histological observation of metaphysis of the femoral bone; finally, the paraffin-embedded specimen was sliced with a microtome to finally prepare a paraffin section having a thickness of 5 μm.

2.5.1 HE staining procedure:

(1) dewaxing the paraffin section by dimethylbenzene (10 min multiplied by 3 times), and then sequentially passing 100%, 95%, 90% and 85% ethanol to water, wherein each pass lasts for 5 min;

(2) washing with distilled water for 3min, staining with hematoxylin solution for 5min, and washing with tap water for 5 min;

(3) differentiating with 1% hydrochloric acid alcoholic solution for 60s, washing with tap water for 1 min;

(4) carrying out anti-blue reaction in 10% ammonia water solution for 60s, and washing with tap water for 1 min;

(5) counterstaining with l% eosin solution for 3min, and washing with tap water for 1 min;

(6) and (5) performing conventional dehydration, transparency and sealing.

The morphological changes of the femur were observed under a light microscope. Bone density (BMD) and the number of bone body integrals (BV/TV) were calculated using a microscope computer Image analysis system (Image-Proplus 6.0) with reference to von Knoch M method (ob/ob) biomaterials 2004; 25: 4675-81).

2.5.2 anti-tartaric acid phosphatase staining:

tartrate-resistant acid phosphatase (TRAP) is characteristic of osteoclasts and is distributed in the osteoclast cytoplasm. Under acidic conditions with tartrate, TRAP is able to hydrolyze naphthol ASBI phosphate to produce naphthol ASB1, which immediately binds to hexaazo-parafuchsin in the dye liquor to form an insoluble red dye at the enzyme active site. The acid phosphatase activity can be indirectly understood by observing this dye. TRAP staining was used to identify osteoclasts. Staining was performed using TRAP staining kit (Sigma 387A).

2.5.2.1 reagent preparation:

preparing 2 test tubes, adding 0.5ml fast Garnet GBC Base Solution (parafuchsin) to one test tube and 0.5ml sodium Nitrite Solution to the other test tube, mixing for 30s, and standing for 2 min; 2 100ml beakers are prepared, marked with A and B, and TRAP dye solution (pH5.2) is prepared:

2.5.2.2 dyeing step:

(1) paraffin sections were deparaffinized and hydrated and washed 3 times for 3min with PBS

(2) Fixing the prepared specimen slices in an acetone solution for 30 s;

(3) washing with distilled water without drying;

(4) incubating TRAP dye liquor for 1 h at 37 ℃, and keeping out of the sun;

(5) distilled water was washed 3 times.

The positive TRAP staining results were purple red spots and lamellar areas, and the number of mature osteoclasts was counted under a 20 Xlight microscope field, with reference to the Nich C method (Nich C, et al, Roleof direct estrogen receptor signalling in near particulate-induced osteoclastogenesis. biomaterials.2013; 34(3): 641-50.).

2.6 immunohistochemical detection of RANKL, CTSK 1, deparaffinization and Hydrodeparaffinization sections were baked at room temperature for 60 minutes or in a 60 ℃ incubator for 30 minutes.

1) Soaking the slices in xylene for 10 minutes, and then soaking for 10 minutes after replacing the xylene; 2) Soaking in absolute ethyl alcohol for 5 minutes; 3) Soaking in 95% ethanol for 5 min; 4) Soaking in 70% ethanol for 5 min; 2. antigen retrieval

The enzyme digestion method comprises the steps of using 0.1% trypsin commonly, preheating the trypsin to 37 ℃ before use, preheating slices to 37 ℃, dripping 0.2ml of digestion solution into each slice to cover complete tissues, digesting the slices in an incubator at 37 ℃ for about 5 ~ 30 minutes, keeping out of the sun, 3, performing immunohistochemical staining, 1), washing the slices with PBS for 2-3 times and 5 minutes respectively, 2), dripping normal goat serum blocking solution, removing redundant liquid at room temperature for 20 minutes, 3), dripping 100 mu l of I antibody, standing the slices at room temperature for 1 hour or 4 ℃ overnight or 37 ℃ for 1 hour, 4), re-warming the slices at 37 ℃ for 45 minutes after standing the slices at 4 ℃, 5) washing the slices with PBS for 3 times and 5 minutes respectively, 6), dripping 40-50 mu l of II antibody, standing the slices at room temperature or 37 ℃ for 1 hour, 7) washing the slices with PBS for 3 times and 5 minutes respectively, 8) developing the slices for 5-10 minutes, performing microscopic staining, 9) washing with tap water or tap water for 10 minutes, performing hematoxylin re-staining for 3 minutes, performing differentiation, 11) and performing microscopic examination on the slices with tap water and 12-15 minutes, and performing microscopic examination and.

2.7 statistical analysis

And analyzing result data by adopting SPSS11.0 statistical software, expressing the data by using a mean +/-standard deviation (), comparing a plurality of groups by adopting one-way ANOVA (one-way ANOVA), and analyzing by adopting LSD (least squares) and Dunnett-t methods under the condition of uniform overall variance.p<A difference of 0.05 is statistically significant.

Second, result in

1. General conditions of the laboratory animals

All animals revive within 30-60 min after operation, can freely move in the cage, normally eat and have no obvious change in mental state. No incision, no inflammation reaction such as red swelling and exudation, and uniform healing. No animal was dead during the experiment.

Micro-CT detection

The Micro-CT scanning experiment mouse femur is used for three-dimensional image reconstruction and quantitative analysis, so that the bone mass and the bone microstructure can be accurately described, and the osteoporosis degree can be judged. Wherein, A is Sham group, B is OVX group, C is OVX + Low-PUN group (20 mg/kg PUN), and D is OVX + High-PUN group (50 mg/kg PUN). The three-dimensional image shows that the number of the bone trabeculae of the Sham group is large, the gap between the bone trabeculae is small, and the bone tissues are compact; compared with the Sham group, the OVX group can see a smaller amount of trabeculae, the gap between the trabeculae is large, and the bone tissues are obviously sparse; after PUN administration, trabecular bone number was increased and trabecular bone gap was decreased as compared with OVX group. (FIG. 1).

Bone density (BMD) change: following ovariectomy, the bone density near the femoral growth plate was significantly reduced in mice, and the difference was statistically significant compared to Sham groupp<0.01); significantly increased bone density of femurs in treated mice compared to OVX group, wherein low concentration of PUN treated mice were compared to OVX groupp<0.05 PUN high dose treatment groups compared to OVX groupsp<0.01. See fig. 2.

Bone bodyIntegral number (BV/TV): the bone integration number of OVX group is obviously reduced, compared with Sham groupp<0.01. And the number of bone volume increases significantly after the PUN treatment, wherein the high dose PUN treated group was compared to the OVX groupp<0.01, low dose PUN treated group compared to OVX groupp<0.01. See fig. 3.

Trabecular number of cancellous bone (tb.n): the number of distal trabeculae of femur was significantly reduced in the OVX group mice, compared to Sham groupp<0.01. After the PUN treatment, the number of trabeculae in cancellous bone is obviously increased, wherein the high-dose PUN treatment group is compared with the OVX groupp<0.01, low dose PUN treated group compared to OVX groupp<0.01. See fig. 4.

Cancellous bone average trabecular resolution (tb.sp): the mean trabecular bone resolution at distal femur of mice in OVX group was significantly increased compared to Sham groupp<0.01. And the mean trabecular resolution at distal femur was significantly reduced after PUN treatment, wherein the high dose PUN treated group was compared to the OVX groupp<0.05, low dose PUN treated group compared to OVX groupp<0.01. See fig. 5.

3. Histological examination

3.1 HE staining results:

under the optical lens, compared with the Sham group, the OVX group has the advantages that the number of trabeculae is reduced, the arrangement is loose, most trabeculae can not be connected into a net shape, the trabeculae become thin, narrow and sparse, the separation degree is increased, the gap is widened, the broken free tail ends of the trabeculae can be seen, the structure distortion is incomplete, and the change is typical osteoporosis-like; the damaged trabecular bone structure of the PUN-treated group was repaired to varying degrees, as indicated by increased trabecular bone number, decreased resolution, and narrowed gap. See FIG. 6, where A is Sham group, B is OVX group, C is OVX + Low-PUN group (20 mg/kg PUN), and D is OVX + High-PUN group (50 mg/kg PUN).

Bone volume fraction (BV/TV): compared with the Sham (20.351 +/-2.288) group, the OVX group (10.835 +/-2.626) has obviously smaller integral number of the bone bodies, and the difference has statistical significance (. + -.)p<0.01). After PUN treatment, the bone volume scores were 17.042 + -1.969 (Low-PUN group)20.008 + -2.274 (High-PUN group), the bone body score was significantly greater compared to the OVX group. Wherein the low concentration of PUN treatment group is compared with the OVX groupp<0.05, the PUN high dose treatment group showed statistical differences compared to the OVX group (. multidot.p<0.01). See fig. 7.

3.2 TRAP staining results

The TRAP staining positive area is purple red, and a large purple red area can be seen in the OVX group, which indicates that a large number of mature osteoclasts exist in the bone tissues of the OVX group; only a very small number of purple-red positive areas were seen in the Sham group; after administration (OVX + Low-PUN group, OVX + High PUN group) dark-stained areas were significantly reduced, and only a small number of purple-red positive areas were visible. See fig. 8.

Under-light-microscopy counting results showed that the OVX group TRAP-positive cells were 96. + -.14, compared to the Sham group (25. + -.6)p<0.01; and TRAP cell numbers of the low-concentration and high-concentration PUN treatment groups are 58 +/-13 and 36 +/-9 respectively. The differences between the high dose treatment groups of PUN and the OVX groups were statistically significant (. multidot.. sup.)p<0.01), the differences between the low dose treatment groups of PUN and the OVX groups were statistically significant (. about.p<0.01). See fig. 9.

4 immunohistochemical detection

The immunohistochemistry results showed that the expression amount of RANKL was observed under an optical microscope, and see fig. 10. Compared with the Sham group (9 +/-5), the expression level of RANKL of the OVX group (52 +/-12) is obviously increased, and the difference has statistical significancep<0.01). After the PUN treatment, the expression quantity of RANKL is 32 plus or minus 7 (Low-PUN group) and 13 plus or minus 6 (High-PUN group), and compared with the OVX group, the difference of the PUN High-dose treatment group has statistical significance (x)p<0.01), the differences between the low dose treatment groups of PUN and the OVX groups were statistically significant (.)p<0.05). See fig. 11.

Further measurement of CTSK expression level was carried out, and as shown in FIG. 12, the OVX group (61. + -. 9) showed a significant increase in CTSK expression level compared to the Sham group (14. + -.6), and the difference was statistically significant (. multidot.p<0.01). After PUN treatment, the expression quantity of CTSK is 39 plus or minus 7 (Low-PUN group) and 21 plus or minus 6 (High-PUN group), and compared with the OVX group, the PUN High-dose treatment group has statistical significance (x)p<0.01), PUN Low dose treatment groupThe differences were statistically significant compared to the OVX groupp<0.01). See fig. 13.

The above test results are summarized as follows:

the results of the invention show that compared with the Sham group, the OVX group has obviously reduced femur bone density, bone volume fraction and cancellous bone trabecula number (p is less than 0.01); the trabecular resolution of the average cancellous bone is obviously increased (p < 0.01), and the number of mature osteoclasts is increased (p < 0.01); the separation degree of cancellous bone trabeculae in the PUN treatment group is obviously reduced, the bone density, the bone volume fraction and the number of cancellous bone trabeculae are obviously increased, the osteoporosis degree is obviously reduced, the number of mature osteoclasts is reduced, and compared with the OVX group, the difference has statistical significance (p is less than 0.05), and the effect is especially obvious in the high-dose treatment group (p is less than 0.01).

The immunohistochemical result shows that the concentration of RANKL and the concentration of CTSK are lower in the Sham group; in OVX group, RANKL content and CTSK content increased dramatically, with × p <0.01 compared to Sham group; in the PUN treatment group, RANKL and CTSK levels decreased significantly with increasing concentration of PUN.

In summary, the following conclusions can be drawn through the above test results:

the experiment intervenes in a mouse ovariectomy osteoporosis model in a Punicalagin (PUN) intragastric administration mode, and the result shows that the femoral bone loss degree of a treatment group is reduced, the number of mature osteoclasts is reduced, the expression quantity of RANKL and CTSK is reduced, particularly, the effect of a high-dose treatment group is more obvious, the number of mature osteoclasts can be obviously reduced, the osteoporosis caused by estrogen deficiency is inhibited, and the expression of RANKL and CTSK in bone tissues is reduced. The PUN is proved to have a certain treatment effect on the osteoporosis caused by estrogen deficiency, and the effect of high dosage is more obvious.

The inventors speculate that the action mechanism of PUN on osteoporosis caused by estrogen deficiency may be related to inhibition of RANKL and osteoclast activation. RANKL is secreted from osteoblasts, plays an important role in osteoclast activation, and is a key factor that regulates osteoclast activation. In this experiment, PUN was able to potently inhibit the expression of RANKL, consistent with the decreased expression of RANKL in the bone tissue of the treated mice in this experiment. In recent years, the activation of osteoclast induced by RANKL due to estrogen deficiency after menopause is considered to play a main role in the occurrence and development of osteoporosis, the expression of RANKL content in the bone tissue of mice in a treatment group in the experiment is reduced, compared with an OVX group, the number of mature osteoclast in bone tissue of a PUN treatment group is also obviously reduced, and the inhibition of the activation of osteoclast by the intervention of the RANKL pathway by PUN is indicated, so the inventor speculates that the mechanism of the PUN for treating postmenopausal osteoporosis is probably to reduce the RANKL through inhibiting the expression of the RANKL, so that the activation of osteoclast is inhibited, the osteoporosis is reduced, the treatment effect is achieved, and the pharmaceutical intervention on the postmenopausal osteoporosis can be realized.

Claims (5)

1. Application of punicalagin in preparing medicine for preventing and treating postmenopausal osteoporosis is disclosed, wherein the structural formula of punicalagin is shown as formula (I),

formula (I).

2. A pharmaceutical preparation for preventing and treating postmenopausal osteoporosis is characterized by comprising therapeutically effective amount of punicalagin and pharmaceutically acceptable auxiliary materials.

3. The pharmaceutical preparation according to claim 2, wherein the punicalagin is contained in an amount of 1 to 99% by mass.

4. A pharmaceutical formulation according to claim 2 or 3, wherein the pharmaceutical formulation is suitable for parenteral administration.

5. The pharmaceutical preparation according to claim 2 or 3, wherein the pharmaceutical preparation is a lyophilized preparation, an injection, a tablet, a granule or a capsule.

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