Composition and application thereof in preparation of medicine for preventing and treating osteoporosis

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a composition and application thereof in preparation of medicines for preventing and treating osteoporosis. The composition comprises enterotoxin c and a traditional Chinese medicine extract, wherein the weight ratio of the enterotoxin c to the traditional Chinese medicine extract is 1: (10-100). Researches show that the composition has the effect of treating or preventing osteoporosis; therefore, the compound has important application value when being used as an effective component for preparing the medicine with the effect of treating or preventing the osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a composition and its use in the preparation of a medicament for the prevention and treatment of osteoporosis. Background Technology

[0002] Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and destruction of bone microstructure. Its main clinical manifestations are increased bone fragility and elevated fracture risk. With the increasing aging of the global population, osteoporosis has become a significant public health issue affecting the health of middle-aged and elderly people. Currently, the main drugs used clinically to treat osteoporosis include bisphosphonates, selective estrogen receptor modulators (SERMs), calcitonin, parathyroid hormone analogs, and RANKL inhibitors. Although these drugs can inhibit bone resorption or promote bone formation to some extent, they still have problems such as gastrointestinal adverse reactions, decreased efficacy with long-term use, and high cost, which limit their widespread application.

[0003] In recent years, with the deepening research on the relationship between gut microbiota and bone health, it has been found that certain gut microbes and their metabolites can regulate bone metabolism through the "gut-bone axis" pathway. Among them, enterotoxin C, as a superantigen protein secreted by certain Gram-positive bacteria, has been proven to have immunomodulatory functions. Studies have shown that enterotoxin C can indirectly affect osteoclast differentiation and activity by regulating T cell activation and cytokine release, thereby participating in the regulation of bone metabolism. However, systematic research on its direct role and mechanism in the prevention and treatment of osteoporosis is still lacking. On the other hand, traditional Chinese medicine has accumulated rich experience in the prevention and treatment of osteoporosis. Many Chinese herbs, such as Epimedium, Rehmannia glutinosa, Drynaria fortunei, and Astragalus membranaceus, are widely used in prescriptions for tonifying the kidneys and strengthening bones, promoting blood circulation and relieving pain, and have shown good bone-protective effects. Modern pharmacological studies have also confirmed that these Chinese herbs contain a variety of active ingredients that promote osteoblast activity and inhibit osteoclast function, such as flavonoids, saponins, and polysaccharides. Therefore, combining traditional Chinese medicine extracts with modern biotechnology to develop novel, highly effective, and low-toxicity anti-osteoporosis drugs has broad application prospects.

[0004] However, there are currently no reports or patent publications regarding the combined application of enterotoxin C and traditional Chinese medicine extracts in the prevention and treatment of osteoporosis. Therefore, developing a novel composition combining enterotoxin C and traditional Chinese medicine extracts is of great significance for expanding the treatment options for osteoporosis. Summary of the Invention

[0005] In order to overcome at least one of the technical problems existing in the prior art, the present invention first provides a composition and its use in the preparation of a medicament for the prevention and treatment of osteoporosis.

[0006] This invention provides the following technical solution: This invention first provides a composition comprising enterotoxin C and a traditional Chinese medicine extract; wherein, enterotoxin C and The weight ratio of the Chinese herbal extracts is 1:10~100.

[0007] This invention provides a novel composition containing enterotoxin C and traditional Chinese medicine extracts. Studies have shown that the composition has therapeutic or preventive effects on osteoporosis.

[0008] Preferably, the weight ratio of enterotoxin C to the traditional Chinese medicine extract is 1:30~60.

[0009] Most preferably, the weight ratio of enterotoxin C to the traditional Chinese medicine extract is 1:40.

[0010] Preferably, the herbal extract is prepared from the herbal extracts Epimedium, Pueraria lobata, and Aconitum carmichaelii.

[0011] Preferably, the weight ratio of the traditional Chinese medicines Epimedium, Pueraria lobata, and Aconitum carmichaelii is 3~6:1~3:1~3.

[0012] The most preferred ratio is 5:2:2 for the traditional Chinese medicines Epimedium, Pueraria lobata, and Aconitum carmichaelii.

[0013] Preferably, the herbal extract is prepared by the following method: The Chinese medicinal materials are mixed together, including Epimedium, Pueraria lobata, and Aconitum carmichaelii, to obtain a mixture of Chinese medicinal raw materials. Then, the mixture is extracted with a solvent. After the extraction is completed, the extract is separated, concentrated and dried to obtain a Chinese medicinal solvent extract. The Chinese medicinal extract is obtained by taking the Chinese medicinal solvent extract.

[0014] Preferably, the ratio of the mixture of Chinese herbal raw materials to the solvent is 1 kg: 4~10 L.

[0015] Preferably, the ratio of the mixture of Chinese herbal raw materials to the solvent is 1 kg: 8 L.

[0016] Preferably, the solvent is selected from one or a mixture of more than one of water, ethanol, acetone or diethyl ether.

[0017] Preferably, the solvent is selected from diethyl ether.

[0018] The inventors discovered in their research that the choice of extraction solvent is crucial in this invention. The combination of the herbal extract obtained by using ether as a solvent to extract Epimedium, Pueraria lobata, and Aconitum carmichaelii with enterotoxin C has a significantly higher therapeutic or preventive effect on osteoporosis than the combination obtained by using other solvents to extract Epimedium, Pueraria lobata, and Aconitum carmichaelii with enterotoxin C.

[0019] Preferably, the extraction refers to reflux extraction, and the extraction time is 1-3 hours.

[0020] Preferably, the method for preparing the traditional Chinese medicine extract further includes the following steps: The Chinese herbal extract was loaded onto a silica gel column and then eluted with a mixed organic solvent consisting of chloroform and acetone in a volume ratio of 93-95:7-5, which was 3-8 column volumes. The eluent was collected, concentrated, and dried to obtain the Chinese herbal extract.

[0021] Most preferably, the method for preparing the traditional Chinese medicine extract further includes the following steps: The Chinese herbal extract was loaded onto a silica gel column and then eluted with a mixed organic solvent consisting of chloroform and acetone in a volume ratio of 94:6 (5 column volumes). The eluent was collected, concentrated, and dried to obtain the Chinese herbal extract.

[0022] In further research, the inventors discovered that the combination of the Chinese herbal solvent extract obtained by further eluting it under the silica gel column elution conditions described in this invention with enterotoxin C has a significantly higher therapeutic or preventive effect on osteoporosis than the combination obtained by simply combining the Chinese herbal solvent extract with enterotoxin C.

[0023] The inventors also discovered in their research that the elution conditions of this invention are crucial; the combination of the Chinese herbal solvent extract obtained by eluting it under the silica gel column elution conditions described above with enterotoxin C can further significantly enhance its therapeutic or preventive effect on osteoporosis; however, the combination of the Chinese herbal solvent extract obtained by further eluting it under other silica gel column elution conditions with enterotoxin C does not further significantly enhance its therapeutic or preventive effect on osteoporosis.

[0024] The present invention also provides the use of the above composition in the preparation of a medicament for the prevention and / or treatment of osteoporosis.

[0025] Beneficial effects: This invention contains novel enterotoxin C and traditional Chinese medicine extracts. Studies have shown that the composition has therapeutic or preventive effects on osteoporosis. Therefore, using it as an active ingredient in the preparation of drugs with therapeutic or preventive effects on osteoporosis has important application value. Detailed Implementation

[0026] The present invention will be further explained below with reference to specific embodiments, but the embodiments do not limit the present invention in any way.

[0027] Example 1 Preparation of the composition The composition is obtained by mixing enterotoxin C and traditional Chinese medicine extract at a weight ratio of 1:40. The herbal extract is prepared by the following method: the herbal raw materials are mixed with Epimedium, Pueraria lobata and Aconitum carmichaelii. The mixture is then heated to boiling with a solvent and refluxed for 2 hours. After extraction, the extract is separated, concentrated and dried to obtain the herbal solvent extract. The herbal extract is obtained by taking the herbal solvent extract. The weight ratio of the traditional Chinese medicines Epimedium, Pueraria lobata, and Aconitum carmichaelii is 5:2:2; the ratio of the mixture of Chinese medicinal materials to the solvent is 1 kg: 8 L; and the solvent is water.

[0028] Example 2 Preparation of the composition The composition is obtained by mixing enterotoxin C and traditional Chinese medicine extract at a weight ratio of 1:40. The herbal extract is prepared by the following method: the herbal raw materials are mixed with Epimedium, Pueraria lobata and Aconitum carmichaelii. The mixture is then heated to boiling with a solvent and refluxed for 2 hours. After extraction, the extract is separated, concentrated and dried to obtain the herbal solvent extract. The herbal extract is obtained by taking the herbal solvent extract. The weight ratio of the traditional Chinese medicines Epimedium, Pueraria lobata, and Aconitum carmichaelii is 5:2:2; the ratio of the mixture of Chinese medicinal materials to the solvent is 1 kg: 8 L; the solvent is an aqueous solution of ethanol with a volume fraction of 70%.

[0029] Example 3 Preparation of the composition The composition is obtained by mixing enterotoxin C and traditional Chinese medicine extract at a weight ratio of 1:40. The herbal extract is prepared by the following method: the herbal raw materials are mixed with Epimedium, Pueraria lobata and Aconitum carmichaelii. The mixture is then heated to boiling with a solvent and refluxed for 2 hours. After extraction, the extract is separated, concentrated and dried to obtain the herbal solvent extract. The herbal extract is obtained by taking the herbal solvent extract. The weight ratio of the traditional Chinese medicine Epimedium, Pueraria lobata, and Aconitum carmichaelii is 5:2:2; the ratio of the mixture of Chinese medicine raw materials to the solvent is 1kg:8L; and the solvent is acetone.

[0030] Example 4 Preparation of the composition The composition is obtained by mixing enterotoxin C and traditional Chinese medicine extract at a weight ratio of 1:40. The herbal extract is prepared by the following method: the herbal raw materials are mixed with Epimedium, Pueraria lobata and Aconitum carmichaelii. The mixture is then heated to boiling with a solvent and refluxed for 2 hours. After extraction, the extract is separated, concentrated and dried to obtain the herbal solvent extract. The herbal extract is obtained by taking the herbal solvent extract. The weight ratio of the traditional Chinese medicines Epimedium, Pueraria lobata, and Aconitum carmichaelii is 5:2:2; the ratio of the mixture of Chinese medicinal materials to the solvent is 1 kg: 8 L; and the solvent is diethyl ether.

[0031] Example 5 Preparation of the composition The composition is obtained by mixing enterotoxin C and traditional Chinese medicine extract at a weight ratio of 1:40. (1) The Chinese herbal extract is prepared by the following method: the Chinese herbal raw materials mixture is obtained by mixing Epimedium, Pueraria lobata and Aconitum carmichaelii. Then, the mixture is heated to boiling with a solvent and refluxed for 2 hours. After the extraction is completed, the extract is separated and concentrated and dried to obtain the Chinese herbal solvent extract. The weight ratio of Epimedium, Pueraria lobata and Aconitum carmichaelii is 5:2:2. The ratio of the Chinese herbal raw material mixture to the solvent is 1 kg: 8 L. The solvent is ether. (2) Load the Chinese herbal solvent extract onto a silica gel column, and then elute it with a mixed organic solvent consisting of chloroform and acetone in a volume ratio of 94:6, which is 5 times the column volume; collect the eluent, concentrate and dry it to obtain the Chinese herbal extract.

[0032] Comparative Example 1: Preparation of the Composition The composition is obtained by mixing enterotoxin C and traditional Chinese medicine extract at a weight ratio of 1:40. (1) The Chinese herbal extract is prepared by the following method: the Chinese herbal raw materials mixture is obtained by mixing Epimedium, Pueraria lobata and Aconitum carmichaelii. Then, the mixture is heated to boiling with a solvent and refluxed for 2 hours. After the extraction is completed, the extract is separated and concentrated and dried to obtain the Chinese herbal solvent extract. The weight ratio of Epimedium, Pueraria lobata and Aconitum carmichaelii is 5:2:2. The ratio of the Chinese herbal raw material mixture to the solvent is 1 kg: 8 L. The solvent is ether. (2) The Chinese herbal solvent extract was loaded onto a silica gel column and then eluted with a mixed organic solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 94:6, which was 5 times the column volume; the eluent was collected, concentrated and dried to obtain the Chinese herbal extract.

[0033] Comparative Example 2: Preparation of the Composition The composition is obtained by mixing enterotoxin C and traditional Chinese medicine extract at a weight ratio of 1:40. (1) The Chinese herbal extract is prepared by the following method: the Chinese herbal raw materials mixture is obtained by mixing Epimedium, Pueraria lobata and Aconitum carmichaelii. Then, the mixture is heated to boiling with a solvent and refluxed for 2 hours. After the extraction is completed, the extract is separated and concentrated and dried to obtain the Chinese herbal solvent extract. The weight ratio of Epimedium, Pueraria lobata and Aconitum carmichaelii is 5:2:2. The ratio of the Chinese herbal raw material mixture to the solvent is 1 kg: 8 L. The solvent is ether. (2) Load the Chinese herbal solvent extract onto a silica gel column, and then elute it with a mixed organic solvent consisting of chloroform and acetone in a volume ratio of 85:15, which is 5 times the column volume; collect the eluent, concentrate and dry it to obtain the Chinese herbal extract.

[0034] Preparation of the composition in Comparative Example 3 The composition is obtained by mixing enterotoxin C and traditional Chinese medicine extract at a weight ratio of 1:40. (1) The Chinese herbal extract is prepared by the following method: the Chinese herbal raw materials mixture is obtained by mixing Epimedium, Pueraria lobata and Aconitum carmichaelii. Then, the mixture is heated to boiling with a solvent and refluxed for 2 hours. After the extraction is completed, the extract is separated and concentrated and dried to obtain the Chinese herbal solvent extract. The weight ratio of Epimedium, Pueraria lobata and Aconitum carmichaelii is 5:2:2. The ratio of the Chinese herbal raw material mixture to the solvent is 1 kg: 8 L. The solvent is ether. (2) Load the Chinese herbal solvent extract onto a silica gel column, and then elute it with a mixed organic solvent consisting of chloroform and acetone in a volume ratio of 60:40, which is 5 times the column volume; collect the eluent, concentrate and dry it to obtain the Chinese herbal extract.

[0035] Experimental Example 1 3dpf zebrafish were treated with 30mM prednisolone to construct an osteoporotic zebrafish model. Ninety osteoporotic zebrafish models were randomly divided into experimental groups 1-8 and a model group, with 10 zebrafish in each group. Zebrafish in experimental groups 1-8 were given 0.1mg / mL of the compositions prepared in Examples 1-5 and Comparative Examples 1-3 in their culture medium. No compositions were added to the culture medium of the model group. Ten normal zebrafish were taken as a normal control group and no compositions were added to the culture medium. After 5 days of culture, the zebrafish were sacrificed, stained with alizarin red, and photographed. The average optical density value of the zebrafish skull in each group was counted. The results are shown in Table 1.

[0036] Table 1. Effect of the composition of the present invention on the optical density value in zebrafish skull.

[0037] As can be seen from the experimental results in Table 1, the average optical density value of the zebrafish skull in the model group was significantly lower than that in the normal group; this indicates that the osteoporotic zebrafish model was successfully constructed.

[0038] As can be seen from the experimental results in Table 1, the average optical density value of the zebrafish skulls in experimental groups 1-3 was significantly higher than that in the model group; this indicates that the composition obtained by combining enterotoxin C and the traditional Chinese medicine extract described in this invention has the effect of treating or preventing osteoporosis.

[0039] As can be seen from the experimental results in Table 1, the average optical density value of the zebrafish skull in experimental group 4 was significantly higher than that in experimental groups 1-3. This indicates that the choice of extraction solvent in this invention is crucial. The combination of the herbal extract obtained by using ether as a solvent to extract Epimedium, Pueraria lobata, and Aconitum carmichaelii with enterotoxin C has a significantly higher therapeutic or preventive effect on osteoporosis than the combination of the herbal extract obtained by using other solvents to extract Epimedium, Pueraria lobata, and Aconitum carmichaelii with enterotoxin C.

[0040] As can be seen from the experimental results in Table 1, the average optical density value of the zebrafish skull in experimental group 5 was significantly higher than that in experimental group 4; its average optical density value was close to that of the normal group. This indicates that the combination of the traditional Chinese medicine solvent extract obtained by further eluting it under the silica gel column elution conditions described above and the combination with enterotoxin C has a significantly higher therapeutic or preventive effect on osteoporosis than the combination obtained by simply combining the traditional Chinese medicine solvent extract with enterotoxin C; it has a very excellent therapeutic or preventive effect on osteoporosis.

[0041] As can be seen from the experimental results in Table 1, although the average optical density value of the zebrafish skulls in experimental groups 6-8 was higher than that in experimental group 4, the increase was not significant and was far less than that in experimental group 5. This indicates that the elution conditions of this invention are crucial. The combination of the Chinese herbal solvent extract obtained by eluting the extract under the silica gel column elution conditions described above and the enterotoxin C composition can achieve a further significant improvement in its therapeutic or preventive effect on osteoporosis. However, the combination of the Chinese herbal solvent extract obtained by further eluting the extract under other silica gel column elution conditions and the enterotoxin C composition does not achieve a further significant improvement in its therapeutic or preventive effect on osteoporosis.

Claims

1. A composition characterized in that, The composition comprises enterotoxin C and a traditional Chinese medicine extract, wherein the weight ratio of enterotoxin C to the traditional Chinese medicine extract is 1:10-100.

2. The composition of claim 1, wherein, The weight ratio of enterotoxin C to the traditional Chinese medicine extract is 1:30-60.

3. The composition of claim 1, wherein, The traditional Chinese medicine extract is prepared from Epimedium, Pueraria and Aconitum.

4. The composition of claim 3 wherein, The weight ratio of Epimedium, Pueraria and Aconitum is 3-6:1-3:1-3.

5. The composition of claim 3, wherein, The traditional Chinese medicine extract is prepared by the following method: The traditional Chinese medicine raw material mixture is obtained by mixing Epimedium, 6. The composition of claim 5, wherein, Pueraria and Aconitum, and then extracted with a solvent.

7. The composition of claim 6, wherein, The traditional Chinese medicine solvent extract is obtained by separating the extract, concentrating and drying the extract.

8. The composition of claim 5, wherein, The dosage ratio of the traditional Chinese medicine raw material mixture to the solvent is 1kg:4-10L.

9. The composition of claim 5, wherein, The dosage ratio of the traditional Chinese medicine raw material mixture to the solvent is 1kg:8L. The solvent is selected from one or more than one of water, ethanol, acetone or diethyl ether. The extraction is heating reflux extraction, and the extraction time is 1-3h.

10. Use of the composition of any one of claims 1-9 in the preparation of a drug for preventing and / or treating osteoporosis.

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