Application of oroxylum indicum flavone extract in tumor-induced bone destruction

Abstract

The invention relates to application of an oroxylum indicum flavone extract in tumor-induced bone destruction, and relates to the field of biological medicines. The oroxylum indicum flavone extract comprises at least one of oroxylum indicum A and oroxylum indicum B, can inhibit tumor-induced osteoclast generation, can be used as a tumor-induced osteoclast differentiation inhibitor drug, and is used for treating tumor-induced bone destruction osteoclast activity abnormity diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of the flavonoid extract of Oroxylum indicum in tumor-induced bone destruction. Background Art

[0002] Multiple tumors have the characteristics of inducing osteoclastogenesis and osteolysis. Among them, breast cancer is the leading malignant tumor threatening women's health globally. The annual number of new breast cancer cases in Chinese women is about 420,000, and the number of death cases is 120,000. Advanced breast cancer is prone to metastasis, and the bone is the most common metastatic site of advanced breast cancer. Approximately 80% of patients with advanced breast cancer will develop distant bone metastases, leading to skeletal-related events (SREs).

[0003] Osteoclasts are multinucleated giant cells with bone-eroding effects and directly participate in and affect the processes of bone development and remodeling. In normal bone metabolism, the bone resorption function mediated by RANKL-induced osteoclast differentiation and the bone formation function mediated by osteoblasts maintain a dynamic balance. However, when malignant cancer cells enter the bone marrow microenvironment, they secrete a large amount of non-RANKL active mediators. For example, in triple-negative breast cancer (such as MDA-MB-231 cells), RANKL is not highly expressed, and such cancer cells will induce osteoclastogenesis through non-RANKL-dependent mechanisms by secreting PTH-rP and other pathways. Compared with other types of breast cancer, the bone destruction associated with triple-negative breast cancer is more difficult to treat. A large number of non-RANKL-dependent factors in tumors will induce the overactivation of osteoclasts, resulting in osteolytic destruction, thereby triggering a series of SREs, such as pathological fractures, severe pain, hypercalcemia, and nerve damage, which seriously affect the quality of life of cancer patients.

[0004] Currently, bisphosphonates and human monoclonal RANKL antibody (denosumab) are used to target and inhibit osteoclasts to inhibit osteolysis, but they affect the physiological bone formation mediated by RANKL. Bisphosphonates may bring potential side effects such as acute-phase reactions, renal insufficiency, and osteonecrosis of the jaw. Denosumab can cause osteonecrosis and exacerbate the risk of fractures due to rapid bone loss after drug withdrawal. Therefore, novel safe and effective drugs for tumor-induced overactivation of osteoclasts, especially for bone destruction caused by RANKL-independent breast cancer, are highly attractive.

[0005] Chinese herbal medicines are precious traditional resources in our country. Screening new active ingredients from them that can treat breast cancer-induced bone destruction and deeply exploring their mechanisms of action have important research and application values. Oroxylum indicum, as a traditional Chinese herbal medicine, has a medication history of hundreds of years and is commonly used in clinical practice to treat inflammatory diseases such as acute and chronic bronchitis, pulmonary tuberculosis, cough, sore throat, pharyngitis, tonsillitis, etc. The main components of Oroxylum indicum are flavonoids and their glycoside compounds. The flavonoid active compounds with the highest content in the flavonoid extract of Oroxylum indicum, namely oroxylin A and oroxylin B, have significant antibacterial, anti-inflammatory, antioxidant, antitumor and other pharmacological activities. However, so far, there have been no reports on the inhibition of tumor-induced osteoclast differentiation and function by the flavonoid extract of Oroxylum indicum, oroxylin A, or oroxylin B, and even less reports on the total flavonoid extract of Oroxylum indicum as a drug for the preparation of treating tumor-induced bone destruction. Summary of the Invention

[0006] In view of the above problems, the present invention provides the application of the flavonoid extract of Oroxylum indicum in tumor-induced bone destruction. This flavonoid extract of Oroxylum indicum can inhibit tumor-induced osteoclastogenesis and can be used as a drug for inhibiting tumor-induced osteoclast differentiation to treat diseases with abnormal osteoclast activity caused by tumor-induced bone destruction.

[0007] To achieve the above object, the present invention provides the application of the flavonoid extract of Oroxylum indicum in tumor-induced bone destruction. This flavonoid extract of Oroxylum indicum includes at least one of oroxylin A and oroxylin B.

[0008] In one embodiment, the chemical structure of oroxylin A is shown in Formula I:

[0009]

[0010] The chemical structure of oroxylin B is shown in Formula II:

[0011]

[0012] In one embodiment, the tumor-induced bone destruction is bone destruction caused by tumor bone metastasis, and the tumor includes at least one of breast cancer, prostate cancer, lung cancer, kidney cancer, multiple myeloma.

[0013] In one embodiment, the administration routes of the drug include oral administration, sublingual administration, intravenous injection or transdermal administration.

[0014] In one embodiment, the working concentration of the flavonoid extract of Oroxylum indicum is 0.1 - 20 μM.

[0015] In the present invention, the working concentration is the concentration at which the component can achieve the expected effect when in use. It is understandable that those skilled in the art can prepare a mother liquor or a stock solution with a higher concentration and dilute it when in use. The mother liquor or stock solution and their concentrations are within the protection scope of the present invention.

[0016] In one embodiment, when the flavonoid extract of Oroxylum indicum includes oroxylin A, the working concentration of the flavonoid extract of Oroxylum indicum is 0.1 - 10 μM.

[0017] In one embodiment, when the flavonoid extract of Oroxylum indicum includes oroxylin B, the working concentration of the flavonoid extract of Oroxylum indicum is 0.2 - 20 μM.

[0018] The present invention also provides a drug for treating tumor-induced bone destruction, which comprises a drug active ingredient and a pharmaceutically acceptable excipient. The drug active ingredient includes a flavonoid extract of Oroxylum indicum, and the flavonoid extract of Oroxylum indicum includes at least one of oroxylin A and oroxylin B.

[0019] In one embodiment, the excipient includes at least one of a shaping agent, a lubricant, an antioxidant, a preservative, a binder, a filler, or a thickening agent.

[0020] In one embodiment, the dosage form of the drug is a capsule, a tablet, an oral liquid, a granule, a pill, an injection, or an ointment.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The application of the flavonoid extract of Oroxylum indicum in the present invention in tumor-induced bone destruction. The flavonoid extract of Oroxylum indicum includes at least one of oroxylin A and oroxylin B. It has been experimentally confirmed that oroxylin A and oroxylin B can significantly inhibit the generation of mouse osteoclasts induced by the supernatant of breast cancer cells, so that it can be used as a drug for inhibiting osteoclast differentiation induced by tumors to treat diseases with abnormal osteoclast activity in tumor-induced bone destruction. Description of the Drawings

[0023] Figure 1 It is the test result of the cell survival effect of oroxylin A on mouse bone marrow macrophages in Example 2 of the present invention; abscissa: Oroxin A (oroxylin A), ordinate: cell percentage;

[0024] Figure 2 It is the test result of the cell survival effect of oroxylin B on mouse bone marrow macrophages in Example 2 of the present invention; abscissa: Oroxin B (oroxylin B), ordinate: cell percentage.

[0025] Figure 3 It is a tartrate-resistant acid phosphatase (TRAP) staining result graph of the differentiation of mouse bone marrow macrophages (BMMs) induced by different concentrations of baicalein A in the supernatant of triple-negative breast cancer MDA-MB-231 cells in Example 3;

[0026] Figure 4 It is a tartrate-resistant acid phosphatase (TRAP) staining result graph of the differentiation of mouse bone marrow macrophages (BMMs) induced by different concentrations of baicalin B in the supernatant of triple-negative breast cancer MDA-MB-231 cells in Example 3;

[0027] Figure 5 It is a statistical graph of detecting the effect of different concentrations of baicalein A on the differentiation of mouse bone marrow macrophages (BMMs) induced by the supernatant of triple-negative breast cancer MDA-MB-231 cells into osteoclasts in Example 3;

[0028] Figure 6 It is a statistical graph of detecting the effect of different concentrations of baicalin B on the differentiation of mouse bone marrow macrophages (BMMs) induced by the supernatant of triple-negative breast cancer MDA-MB-231 cells into osteoclasts in Example 3;

[0029] Wherein: 231CM is the culture supernatant of MDA-MB-231 human breast cancer cells (Note: Compared with the 231CM group, *P<0.05, **P<0.01, ***P<0.001). Detailed implementation manners

[0030] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0032] Source:

[0033] Human breast cancer MDA-MB-231 cells (purchased from the Cell Bank of the Chinese Academy of Sciences).

[0034] Mouse bone marrow macrophages (Source: C57 / BL6 female mice);

[0035] TRAP staining kit (purchased from Sigma, USA, 387A-1kT);

[0036] Australian blood-derived fetal bovine serum (purchased from Gibco, USA, 10099141).

[0037] Unless otherwise specified, all reagents, materials, and equipment used in this example are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0038] Example 1

[0039] In this example, the preparation method of the drug for treating tumor-induced bone destruction includes the following steps:

[0040] Dissolve oroxylin A and oroxylin B in dimethyl sulfoxide (DMSO), and then dilute with culture medium or PBS. After uniform mixing, it is obtained.

[0041] Example 2

[0042] Effects of the main active components of oroxylum extract, oroxylin A and oroxylin B, on the cell viability of mouse bone marrow macrophages (BMMs):

[0043] (1) Isolation of mouse bone marrow macrophages: After decapitating 8-12-week-old female C57BL / 6 mice, take the femurs and tibias, wash the bone marrow cavities to collect bone marrow, filter, lyse red blood cells, resuspend, and culture overnight. Collect the supernatant to prepare bone marrow cells. Add 30 ng / mL M-CSF to the cells, discard the supernatant after 2 days of culture, wash with PBS, and then digest and purify the cells with trypsin to obtain bone marrow macrophages (BMMs).

[0044] (2) Effects of different concentrations of oroxylin A and oroxylin B on the cell viability of mouse bone marrow macrophages (BMMs): Inoculate BMMs in 96-well plates. After overnight incubation, add 0.4-40 μM oroxylin A and oroxylin B. Culture in a 37 °C, 5% CO2 incubator for 96 h, then discard the culture medium, add 100 μl / well MTT solution and incubate for 4 h. Add 150 μl / well DMSO, mix thoroughly with a micro oscillator at low speed for 10 min, and measure the OD value at 570 nm with a TECAN GENios Pro multifunctional microplate reader.

[0045] The experimental results prove that within 96 h, oroxylin A and oroxylin B at an in vitro concentration of 0.4-4 μM have no cytotoxic effect on mouse bone marrow macrophages (BMMs). The results are as shown in Figure 1 、 Figure 2 shown.

[0046] Example 3

[0047] Effect of the main active components of Oroxylum indicum extract, oroxylin A and oroxylin B, on the differentiation of mouse bone marrow macrophages (BMMs) into osteoclasts induced by breast cancer supernatant: BMMs were seeded in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator in a medium containing 30 ng / ml M-CSF. After adherent growth, different concentrations of oroxylin A and oroxylin B were added to the drug groups and incubated for 2 h. Except for the negative control group (Blank group), an osteoclastogenesis stimulator (5% breast cancer cell conditioned medium) was added to the positive control group (231CM group) and the drug groups, and the cells were further cultured for 3 - 4 d. The cells were fixed and stained with a TRAP staining kit, observed and photographed under a microscope, and the number of osteoclasts with ≥3 nuclei (TRAP-positive cells) was counted and statistically analyzed.

[0048] The experimental results showed that oroxylin A at concentrations of 0.1 - 10 μM and oroxylin B at concentrations of 0.2 - 20 μM could significantly inhibit the differentiation of mouse bone marrow macrophages (BMMs) into osteoclasts induced by 231CM, suggesting that oroxylin A and oroxylin B could inhibit the generation of osteoclasts induced by breast cancer in vitro. The results are as Figures 3 - 6 shown.

[0049] Based on the above results, the main active components of Oroxylum indicum extract, oroxylin A and oroxylin B, can significantly inhibit the generation of mouse osteoclasts induced by breast cancer cell supernatant.

[0050] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as falling within the scope described in this specification.

[0051] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. An application of an Oroxylum flavonoid extract in the preparation of a drug for treating tumor-induced bone destruction, wherein the Oroxylum flavonoid extract comprises at least one of Oroxylum glycoside A and Oroxylum glycoside B.

2. The use according to claim 1, characterized in that: The chemical structure of oroxylin A is shown in Formula I: The chemical structure of oroxylin B is shown in Formula II:

3. The use according to claim 1, characterized in that: The tumor-induced bone destruction is bone destruction caused by tumor bone metastasis, and the tumor includes at least one of breast cancer, prostate cancer, lung cancer, kidney cancer, and multiple myeloma.

4. The use according to any one of claims 1 to 3, characterized in that: The administration routes of the drug include oral administration, sublingual administration, intravenous injection or transdermal administration.

5. The use according to claim 4, characterized in that: The working concentration of the Oroxylum indica flavonoids extract is 0.1-20 μM.

6. The use according to claim 5, characterized in that: When the Oroxylum indica flavonoid extract comprises Oroxylin A, the working concentration of the Oroxylum indica flavonoid extract is 0.1-10 μM.

7. The use according to claim 5, characterized in that: When the Oroxylum indica flavonoid extract includes Oroxylin B, the working concentration of the Oroxylum indica flavonoid extract is 0.2-20 μM.

8. A drug for treating tumor-induced bone destruction, characterized in that: The invention comprises active pharmaceutical ingredients and pharmaceutically acceptable excipients. The active pharmaceutical ingredients comprise oroxylum flavonoid extracts. The oroxylum flavonoid extracts comprise at least one of oroxylin A and oroxylin B.

9. The drug according to claim 8, characterized in that The auxiliary material includes at least one of an excipient, a lubricant, an antioxidant, a preservative, a binder, a filler or a thickener.

10. The drug according to claim 8, characterized in that The dosage form of the medicine is capsule, tablet, oral liquid, granule, pill, injection or ointment.

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