Application of liquidambar formosana acid in preparation of medicine for treating and / or preventing osteoporosis

Abstract

The invention discloses an application of liquidambar formosana hance acid in preparation of a medicine for treating and / or preventing osteoporosis, and the liquidambar formosana hance acid can improve the problems of reduction of bone trabecula number, reduction of bone mineral density, increase of bone brittleness, increase of fracture probability and the like caused by osteoporosis. The pharmaceutical composition has the effects of reducing serum CTX-1, reducing serum TRACP-5b, increasing tibia BV / TV, increasing tibia Tb.Th, increasing tibia Tb.N, reducing tibia Tb.SP, reducing tibia SMI and other bone related parameters, and has the prospect of being developed into a medicine for treating and / or preventing osteoporosis caused by menopause.

Description

Technical Field

[0001] The invention relates to the application of lulutonic acid in preparing a medicine for treating and / or preventing osteoporosis. Background Art

[0002] Chlorogenic Acid Chlorogenic Acid is a pentacyclic triterpenoid compound extracted from the Chinese herbal medicine Rhizoma Lulutong. Its molecular formula is C 30 H 46 O 3 , the chemical structure is as follows. Lulutong acid is one of the main components of Lulutong, which is the dried mature fruit sequence of Liquidamtar formosana Hance, a plant of the Hamamelidaceae family. Its functions and indications are to dispel wind and activate collaterals, and to promote diuresis and menstruation. It is used to treat joint pain, numbness, cramps, edema, fullness, milk deficiency, amenorrhea, etc. Lulutong acid has multiple biological activities, including anti-inflammatory and treatment of rheumatoid arthritis.

[0003]

[0004] Osteoprosis (OP) is a systemic metabolic bone disease characterized by decreased bone mass, degenerative changes in bone tissue fiber structure, increased bone brittleness, and susceptibility to fractures. It is mainly manifested by back pain, decreased bone density, and susceptibility to fractures. With the advent of an aging society, the incidence of osteoporosis has also increased. After menopause, the estrogen level in women's bodies decreases significantly, bone loss accelerates, and the bone absorption rate is greater than the bone formation rate, eventually causing postmenopausal osteoprosis (PMOP), which has become a common disease in postmenopausal women.

[0005] Currently, the main treatments for postmenopausal osteoporosis include bisphosphonates, selective estrogen receptor modulators (SERMs), and estrogen replacement therapy. However, long-term use of bisphosphonates may lead to excessive inhibition of bone turnover, which may interfere with the normal mineralization process of bones and lead to decreased bone quality. SERMs and estrogen replacement therapy may be accompanied by many side effects, such as hot flashes, vaginal dryness and irritation, thrombosis, musculoskeletal pain, etc. Therefore, new osteoporosis treatment drugs are urgently needed to be developed. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide the use of lulutonic acid in the preparation of drugs for treating and / or preventing osteoporosis.

[0007] Technical solution: The present invention discloses the use of lulutonic acid in the preparation of drugs for treating and / or preventing osteoporosis.

[0008] Wherein, the osteoporosis is osteoporosis caused by estrogen deficiency.

[0009] The osteoporosis is a decrease in bone mass or a degeneration of bone microstructure.

[0010] Wherein, the treatment and / or prevention is to reduce serum type I collagen cross-linked carboxyl terminal peptide or reduce serum tartrate-resistant acid phosphatase.

[0011] Wherein, the treatment and / or prevention also includes increasing the tibial bone volume fraction, increasing the tibial trabecular thickness, increasing the number of tibial trabeculae, reducing the tibial trabecular separation or reducing the tibial structural model index.

[0012] Wherein, the daily dosage of lulutong acid is 30 to 90 mg / kg.

[0013] The dosage is specifically a low dose of 30 mg / kg, a medium dose of 60 mg / kg or a high dose of 90 mg / kg.

[0014] The present invention also discloses a pharmaceutical composition for treating and / or preventing osteoporosis, comprising lulutonic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, to prepare a pharmaceutically acceptable dosage form.

[0015] Among them, the pharmaceutically acceptable salts are specifically: salts formed by the reaction of passutonic acid with metal ions (such as sodium, potassium, calcium, magnesium, etc.); ester salts formed by the reaction of passutonic acid with alcohols; salts formed by the reaction of passutonic acid with ammonia or its derivatives; salts formed by the reaction of passutonic acid with hydrogen ions.

[0016] The pharmaceutically acceptable carrier or excipient includes one or more of solid, semisolid or liquid excipients.

[0017] Wherein, the pharmaceutically acceptable dosage forms include tablets, capsules, granules, injections, pills, syrups, powders or pastes.

[0018] Principle of invention: Osteoporosis is caused by the disruption of bone balance and the abnormal activity of osteoclasts. Osteoporosis can be divided into primary and secondary. Primary osteoporosis is the most common and usually related to age. It often occurs in postmenopausal women and elderly men. Among them, postmenopausal women are a high-risk group for osteoporosis.

[0019] The present invention discloses the effects of lulutong acid in improving osteoporosis, increasing bone density, and improving bone loss. It has been found through research that lulutong acid and a pharmaceutical composition containing its active ingredient can inhibit bone loss by inhibiting osteoclast growth, thereby effectively restoring the number of trabeculae in postmenopausal osteoporosis model mice caused by oophorectomy, improving bone strength, and reducing the risk of fractures. At the same time, as the main extract of the traditional Chinese medicine Lulutong, lulutong acid is mild and less irritating; and it is widely present in a variety of plants and fruits, and is safer, will not have an excessive inhibitory effect on the osteoclast process, and does not have the various side effects of hormone drugs.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: Lulutong acid and its pharmaceutical composition can effectively reduce the number of osteoclasts and reduce the activity of osteoclasts, thereby effectively improving the bone loss of postmenopausal osteoporosis model mice, and reduce the levels of CTX-1 and TRACP-5B in the serum of postmenopausal osteoporosis model mice, while increasing the number of trabeculae and bone mineral density of postmenopausal osteoporosis model mice, and are safer and milder in effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the changes of CTX-1 in mouse serum;

[0022] Figure 2 Schematic diagram of the changes of TRACP-5b in mouse serum;

[0023] Figure 3 Schematic diagram of the changes in tibia BV / TV of mice in each group;

[0024] Figure 4 Schematic diagram of the changes in Tb.N in the tibia of mice in each group;

[0025] Figure 5 Schematic diagram of the changes in Tb.Th in the tibia of mice in each group;

[0026] Figure 6 Schematic diagram of the changes in Tb.SP of the tibia of mice in each group;

[0027] Figure 7 Schematic diagram of the changes in tibia SMI of mice in each group;

[0028] Figure 8 Schematic diagram of the changes in tibia BMD of mice in each group;

[0029] Fig. 9 Schematic diagram of the changes in BS / TV of the tibia of mice in each group;

[0030] Fig.10 Schematic diagram of the changes in BS / BV of the tibia of mice in each group;

[0031] Fig.11 Schematic diagram of Micro-CT detection of tibiae of mice in each group;

[0032] Fig.12 Schematic diagram of HE staining results of femur of mice in each group;

[0033] Fig.13 Schematic diagram of TRAP staining results of femurs of mice in each group. DETAILED DESCRIPTION

[0034] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0035] Example 1

[0036] The experiments on the effect of lulutonic acid on osteoporosis were divided into groups:

[0037] The ovariectomy (OVX) model is the most commonly used model for studying osteoporosis and is often used to evaluate the efficacy of estrogen replacement therapy or anti-osteoporosis drugs. CTX-I is the C-terminal peptide of type I collagen, which is an important component of mature bones. In the normal metabolism of bones, bone formation and bone resorption are in dynamic balance. When bones are damaged or diseased (such as osteoporosis), bone resorption increases, causing CTX-I to be released into the blood. TRACP-5b is mainly expressed in osteoclasts, and the increase in TRACP-5b levels in the blood can reflect the increase in bone resorption, so it is used as one of the diagnostic and monitoring indicators for osteoporosis.

[0038] Fifty 7-week-old C57BL / 6 female mice were randomly divided into two groups according to their body weight: 10 mice in the sham operation group (Sham group) and 40 mice in the modeling group.

[0039] Ovariectomy was performed on mice in the modeling group: the mice were anesthetized by intraperitoneal injection of 0.5% sodium pentobarbital 0.1 mL / 10 g body weight, fixed in the lateral position, and a skin incision of about 0.5 cm long was made with ophthalmic scissors parallel to the spine at a level slightly above the thigh root of the mouse and less than 1 cm to the side of the spine. The lower psoas muscle was cut open, and the fat tissue wrapped around the ovary was found. When pink ovarian tissue appeared in the field of vision, it was pulled to the incision, and the ovaries on both sides were ligated with non-absorbable surgical thread, and then the ovaries were removed. The endothelial layer and the outer layer of the skin were sutured in turn, and iodine was applied with a cotton swab to prevent wound infection.

[0040] After one week of postoperative rest, the wound showed no abnormalities, and the modeling group was divided into a model group of 10 mice (OVX group), a low-dose group of 10 mice (OVX+BTA (30 mg / kg)), a medium-dose group of 10 mice (OVX+BTA (60 mg / kg)), and a high-dose group of 10 mice (OVX+BTA (90 mg / kg)). After the mice were grouped, each group was gavaged, and the low-, medium-, and high-dose groups of 30 mg / kg, 60 mg / kg, and 90 mg / kg were given respectively. The blank group and the OVX model group were gavaged with an equal volume of 0.5% CMC-Na solution. The mice in the above groups were given once a day for 16 consecutive weeks, and their body weights were recorded once a week.

[0041] After the administration, the mice in each group were eyeballed and blood was collected, and then the mice were killed. The femurs and tibias on both sides were removed, the soft tissues around the bone tissues were removed, and the bone tissues were fixed with 4% paraformaldehyde and reserved for decalcified paraffin sections and three-dimensional reconstruction analysis.

[0042] Example 2

[0043] Mouse serum index detection was performed on the samples treated in Example 1:

[0044] Sample preparation: Eyeballs of mice in each group were removed to obtain whole blood samples, which were placed at room temperature for 2 hours and then centrifuged at 4°C, 3000 g for 15 minutes. The upper serum was divided into 200 μL EP tubes and stored at -80°C. Before testing, the thawed samples were centrifuged again.

[0045] Experimental operation and detection: Mouse serum CTX-1 and serum TRACP-5b were tested using CTX-1 ELISA Kit and TRACP-5b ELISA Kit, respectively.

[0046] like Figure 1 As shown in the figure, compared with the sham operation group, the serum CTX-1 of mice in the model group was significantly increased, and the model was successfully established; compared with the model group, the CTX-1 of the low, medium and high dose groups of lulutonic acid was significantly reduced in a dose-dependent manner, indicating that lulutonic acid can effectively reduce the serum CTX-1 of osteoporosis model mice.

[0047] like Figure 2 As shown in the figure, compared with the sham operation group, the serum TRACP-5b of mice in the model group was significantly increased, and the model was successfully established; compared with the model group, the TRACP-5b of the low, medium and high dose groups of lulutonic acid was significantly reduced in a dose-dependent manner, indicating that lulutonic acid can effectively reduce the serum TRACP-5b of osteoporosis model mice.

[0048] Example 3

[0049] The samples treated in Example 1 were tested for mouse tibia tissue morphology indexes:

[0050] Before the test, the left tibia was removed and soaked in saline for 24 hours, and then the tissue morphology index was detected by Micro-CT: the tibia was placed in the specimen tube of micro-CT and fixed with paper towels to prevent dehydration and displacement of the specimen during scanning. The Scanner software of SkyScan 1176 micro-CT was used for scanning, and the parameters were set as follows: voltage 50kV, current 800μA, tomographic image resolution 1304pixel×1024pixel, scanning spatial resolution 9μm, rotation angle 0.8 degrees, and exposure time 5300ms.

[0051] After scanning, N-Recon software was used for three-dimensional reconstruction. The three-dimensional reconstruction images of mouse trabeculae in each group are shown in Fig.11 ,from Fig.11 It can be seen that compared with the sham operation group, the tibia bone density of the model group mice was significantly reduced, indicating that the mice had osteoporosis; after low, medium and high doses of lulutonic acid were given, the phenomenon of bone density loss was significantly improved, indicating that lulutonic acid has the effect of improving osteoporosis caused by OVX. Finally, CT-AN software was used for three-dimensional analysis (the above software is the software provided by Skyscan1176 micro-CT). The bone marrow cavity area with a thickness of 1.5 mm and less than 0.6 mm from the growth plate was selected. The following parameters were obtained by analysis: bone volume fraction (BV / TV): the total volume of trabeculae and the volume of the bone marrow cavity in the area of ​​interest; trabecular number (Tb.N): the number of trabeculae per 1 mm; trabecular thickness (Tb.Th): the average thickness of trabeculae; trabecular separation (Tb.SP): the average distance between trabeculae; structural model index (SMI): an indicator of whether trabeculae are "rod-shaped" or "plate-shaped", the closer to 3, the more rod-shaped the trabeculae are; bone density (BMD): the amount of bone mineral contained in a unit area of ​​bone; bone surface area density (BS / TV): the ratio of bone surface area to tissue volume; bone surface area to bone volume ratio (BS / BV): the ratio of bone surface area to bone volume.

[0052] like Figure 3 As shown in the figure, compared with the sham operation group, the BV / TV of the tibia of the mice in the model group was significantly reduced, and the model was successfully established; compared with the model group, the BV / TV of the low, medium and high dose groups of lulutonic acid increased significantly in a dose-dependent manner, indicating that lulutonic acid can effectively increase the BV / TV of the tibia of osteoporosis model mice and can restore it to a level similar to that of the sham operation group.

[0053] like Figure 4As shown in the figure, compared with the sham operation group, the Tb.N of the tibia of the mice in the model group was significantly reduced, indicating that the model was successful; compared with the model group, the Tb.N of the low, medium and high dose groups of lulutonic acid increased significantly in a dose-dependent manner, indicating that lulutonic acid can effectively increase the Tb.N of the tibia of osteoporosis model mice and can restore it to a level similar to that of the sham operation group.

[0054] like Figure 5 As shown, compared with the sham operation group, the Tb.Th in the tibia of mice in the model group was significantly reduced, indicating that the model was successful; compared with the model group, the Tb.Th in the low, medium and high dose groups of lulutonic acid significantly increased in a dose-dependent manner, indicating that lulutonic acid can effectively increase the Tb.Th in the tibia of osteoporosis model mice and can restore it to a level similar to that of the sham operation group.

[0055] like Figure 6 As shown in the figure, compared with the sham operation group, the Tb.SP of the tibia of the mice in the model group was significantly increased, indicating that the model was successful; compared with the model group, the Tb.SP of the low, medium and high dose groups of lulutong acid were all reduced, indicating that lulutong acid can reduce the Tb.SP of the tibia of osteoporosis model mice.

[0056] like Figure 7 As shown in the figure, compared with the sham operation group, the tibia SMI of mice in the model group was significantly increased, indicating that the model was successful; compared with the model group, the SMI of the medium-dose and high-dose groups of lulutonic acid were significantly reduced, indicating that lulutonic acid can reduce the tibia SMI of osteoporosis model mice.

[0057] like Figure 8 As shown, compared with the sham operation group, the tibia BMD of mice in the model group was significantly increased, indicating that the model was successful; compared with the model group, the BMD of the low, medium and high dose groups of lulutonic acid increased significantly in a dose-dependent manner, indicating that lulutonic acid can effectively increase the tibia BMD of osteoporosis model mice and can restore it to a level similar to that of the sham operation group.

[0058] like Fig. 9 As shown in the figure, compared with the sham operation group, the BS / TV of the tibia of the mice in the model group was significantly reduced, indicating that the model was successful; compared with the model group, the BS / TV of the low, medium and high dose groups of lulutonic acid increased significantly in a dose-dependent manner, indicating that lulutonic acid can effectively increase the BS / TV of the tibia of osteoporosis model mice.

[0059] like Fig.10 As shown in the figure, compared with the sham operation group, the BS / BV of the tibia of the mice in the model group was significantly increased, indicating that the model was successfully established; compared with the model group, the BS / BV of the low, medium and high dose groups of lulutonic acid were significantly reduced in a dose-dependent manner, indicating that lulutonic acid can effectively reduce the BS / TV of the tibia of osteoporosis model mice.

[0060] Example 4

[0061] The samples treated in Example 1 were subjected to mouse femoral tissue morphology test:

[0062] Preparation of decalcified bone paraffin sections: fix the tissue with 4% paraformaldehyde solution at 4°C for 24 hours, soak in distilled water for 4 hours, and then decalcify with 20% (10% by volume) EDTA solution (ethylenediaminetetraacetic acid). The needle can easily penetrate the tissue block, indicating that the decalcification is sufficient. After conventional dehydration, embed it in paraffin, and after corresponding treatment, slice along the horizontal tissue, and control the thickness to 10μm. Conventional staining.

[0063] HE staining steps: The prepared paraffin-containing sections were immersed in fresh xylene I and xylene II for dewaxing for 10 minutes each; the sections were immersed in pure alcohol I, pure alcohol II, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol for 3 minutes; washed with distilled water for 5 minutes; the sections were dropped into hematoxylin staining solution for 8 minutes; washed with tap water for 10 minutes; immersed in distilled water for 15 seconds; immersed in 95% alcohol for 5 seconds; immersed in acid eosin solution for 15 minutes; the sections were immersed in 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, pure alcohol I, and pure alcohol II for 3 minutes each; the sections were immersed in pure alcohol I, pure alcohol II, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol for 3 minutes each; the sections were immersed in xylene I and xylene II for 5 minutes each; the sections were sealed and the bone tissue morphology was observed under a microscope.

[0064] Result judgment: After the above treatment, the cytoplasm is stained red and the nucleus is stained blue. The bone substances such as trabeculae, osteocytes, and medullary adipocytes are observed under a light microscope. The corresponding field of view is selected for comparison and observation under a 20x microscope for each slice. Fig.12 It can be seen that compared with the other groups of mice, the model group had reduced bone matrix accumulation and larger spaces between the medulla. After intervention with low, medium and high doses of lulutong acid, this phenomenon was significantly improved, indicating that lulutong acid has a pharmacological effect of improving osteoporosis.

[0065] TRAP staining steps:

[0066] The prepared paraffin-containing sections were sequentially immersed in fresh xylene Ⅰ and xylene Ⅱ for dewaxing for 10 minutes each; the sections were sequentially immersed in pure alcohol Ⅰ, pure alcohol Ⅱ, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol for 3 minutes; washed with distilled water for 5 minutes; the sections were placed in TRAP incubation solution and incubated at 37°C for 50 minutes until the osteoclasts were wine red under the microscope; rinsed with distilled water; the sections were stained with Harris hematoxylin for 3-8 minutes; washed with tap water; differentiated with 1% hydrochloric acid alcohol for a few seconds; rinsed with tap water; blued with 0.6% ammonia water; rinsed with running water; the sections were sequentially immersed in pure alcohol Ⅰ, pure alcohol Ⅱ, 95%, 90%, 80%, and 70% alcohol for 3 minutes; the sections were immersed in xylene Ⅰ and Ⅱ for 5 minutes each; sealed and the bone tissue morphology was observed under a microscope.

[0067] Result judgment: After the above treatment, the cytoplasm of osteoclasts was wine red and the nucleus was blue. Each slice was selected under a 20x microscope for comparative observation. Fig.13 It can be seen that compared with the other groups of mice, the model group had a significantly increased number of osteoclasts around the trabeculae. After intervention with low, medium and high doses of lulutong acid, this phenomenon was significantly improved, indicating that lulutong acid has a pharmacological effect of improving osteoporosis.

[0068] It can be seen that by gavage of lumedric acid to ovariectomized mice for 4 months, it was found that the percentage of trabecular volume of the tibia in the drug-treated group was significantly higher than that in the model group, and the percentage of trabecular formation surface and trabecular absorption surface were lower than those in the model group, indicating that the absorption of osteoclasts was reduced, making bone formation greater than bone absorption, thereby preventing the occurrence of bone loss. Related experimental results showed that compared with the sham operation group, the BV / TV, Tb.Th, Tb.N, BMD and BS / TV of the tibia in the model group mice were significantly reduced, while Tb.SP, SMI and BS / BV were significantly increased. This result shows that the removal of the ovaries of mice can significantly reduce their tibia BV / TV, Tb.Th, Tb.N, BMD and BS / TV, and significantly increase Tb.SP, SMI and BS / BV, thereby leading to osteoporosis in mice. The results of related experiments further showed that lulutong acid can reverse the above indicators to a certain extent. Compared with the model group, the low, medium and high dose groups of lulutong acid significantly increased the BV / TV, Tb.Th, Tb.N, BMD and BS / TV of the mouse tibia, while BS / BV, Tb.SP and SMI decreased. The results showed that lulutong acid has a therapeutic and / or preventive effect on osteoporosis in mice caused by ovariectomy. Therefore, lulutong acid has a therapeutic and / or preventive effect on osteoporosis caused by estrogen deficiency.

Claims

1. Use of lulutonic acid in the preparation of drugs for treating and / or preventing osteoporosis.

2. The use according to claim 1, characterized in that: The osteoporosis is osteoporosis caused by estrogen deficiency.

3. The use according to claim 1, characterized in that: Osteoporosis is a decrease in bone mass or a deterioration in the microstructure of bones.

4. The use according to claim 1, characterized in that: The treatment and / or prevention is to reduce serum type I collagen cross-linked carboxyl terminal peptide or reduce serum tartrate-resistant acid phosphatase.

5. The use according to claim 1, characterized in that: The treatment and / or prevention also includes increasing tibial bone volume fraction, increasing tibial trabecular thickness, increasing tibial trabecular number, decreasing tibial trabecular separation, or decreasing tibial architectural model index.

6. The use according to claim 1, characterized in that: The daily dosage of lulutonic acid is 30 to 90 mg / kg.

7. The use according to claim 6, characterized in that: The dosage is specifically a low dose of 30 mg / kg, a medium dose of 60 mg / kg or a high dose of 90 mg / kg.

8. A pharmaceutical composition for treating and / or preventing osteoporosis, characterized in that: The composition comprises the lulutong acid or the pharmaceutically acceptable salt thereof as claimed in claim 1, and further comprises a pharmaceutically acceptable carrier or excipient to prepare a pharmaceutically acceptable dosage form.

9. The composition according to claim 8, characterized in that The pharmaceutically acceptable carrier or excipient includes one or more of solid, semisolid or liquid excipients.

10. The composition according to claim 8, characterized in that The pharmaceutically acceptable dosage forms include tablets, capsules, granules, injections, pills, syrups, powders or pastes.

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