Application of icaritin in preparation of anti-glioma medicine

Abstract

The invention discloses an application of icaritin in preparation of an anti-glioma drug. It is found for the first time that icaritin has a good inhibition effect on proliferation of glioma cells and can remarkably induce early apoptosis of the glioma cells, and a new basis is provided for developing new application of icaritin in resisting glioma.

Description

Technical Field

[0001] The invention relates to the field of pharmacy, and in particular to application of icariin in preparing anti-glioma drugs. Background Art

[0002] The Chinese medicine Epimedium, also known as Xianlingpi, is the dried aerial part of the Brachiaceae plant Epimedium. It was first recorded in the "Shennong Bencao Jing". It tastes spicy and sweet, is warm in nature, and belongs to the liver and kidney meridians. It has the effects of tonifying kidney yang, strengthening tendons and bones, and removing rheumatism. It is a commonly used kidney-tonifying Chinese medicine. Icariin is one of the main active monomer components of the natural product Epimedium. It is a light yellow powder with the effects of protecting nerves, enhancing osteoblasts and inhibiting the differentiation and activity of osteoclasts, inducing embryonic stem cells to differentiate into cardiomyocytes, and anti-tumor. Icariin (Acoradine) soft capsules have completed phase III clinical trials (NCT02496949, NCT01972672, NCT03236636) and have been approved by the National Medical Products Administration as a new class of Chinese medicine for the treatment of liver cancer in my country. It will be launched in 2022.

[0003] Glioma is the most common primary tumor of the central nervous system, originating from glial cells, with a high degree of malignancy and poor prognosis. According to the classification of the World Health Organization (WHO), gliomas are divided into grades I to IV, of which grades III and IV are high-grade gliomas (HGG), such as glioblastoma (GBM), which are characterized by high incidence, high recurrence rate, high mortality rate and low self-healing rate. At present, the standard treatment for gliomas includes maximal surgical resection followed by early radiotherapy and synchronous temozolomide chemotherapy, followed by temozolomide adjuvant chemotherapy. However, even after standard treatment, the prognosis of gliomas is still poor, with a low 5-year survival rate. Therefore, there is an urgent need to seek drugs that can treat gliomas. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes the use of icariin in the preparation of anti-glioma drugs.

[0005] The present invention provides application of icariin in preparing medicine for treating glioma.

[0006] Furthermore, the structural formula of icariin is as follows:

[0007]

[0008] Furthermore, the drug is used alone or in the form of a pharmaceutical composition; the pharmaceutical composition comprises a therapeutically effective amount of icariin and a pharmaceutically acceptable excipient and / or carrier.

[0009] The present invention also provides a pharmaceutical composition for treating glioma, which comprises a therapeutically effective amount of icariin, and pharmaceutically acceptable adjuvants and / or conventional chemotherapeutic drugs for glioma.

[0010] Furthermore, the pharmaceutical composition is in the form of an oral preparation, an injection, and a topical administration preparation.

[0011] Furthermore, the oral preparation includes tablets, capsules, pills, and granules; the injection includes injection solution and lyophilized powder injection; the topical administration agent includes pellets, sprays, and patches.

[0012] In summary, compared with the prior art, the present invention has achieved the following technical effects: the present invention first discovered that icariin has a good inhibitory effect on the proliferation of glioma cells both in vitro and in vivo, and can significantly induce early apoptosis of glioma cells. The above results show the new use of icariin in the preparation of drugs for treating gliomas, and icariin is a marketed drug for liver cancer, which can be directly applied to the human body without the need for clinical safety evaluation. Icariin can be quickly applied to the treatment of gliomas, greatly saving time and money costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0014] Figure 1 The figure is a detection diagram of the inhibitory activity of icariin of the present invention on the proliferation of different glioma cells; A represents the molecular structure of icariin; B represents the growth inhibition rate of icariin of different concentrations (10 μM, 20 μM) on glioma cells U87, U251, U118, and A172; C represents the inhibition rate curve of icariin of different concentration gradients acting on U87 cells for 24, 48, and 72 hours; D represents the effect of icariin of different concentrations (10 μM, 20 μM) on the cell morphology of glioma cells U87, U251, U118, and A172;

[0015] Figure 2 This is a graph showing the effect of icariin at different concentrations (10 μM, 20 μM, 40 μM) on apoptosis of glioma cells U87;

[0016] Figure 3The figure is a detection diagram of the inhibition of mouse glioma proliferation by icariin in vivo of the present invention; A represents the difference in tumor volume between the experimental group (200 mg / kg, 400 mg / kg icariin) and the model group after treatment; B represents the difference in tumor weight between the gavage group (200 mg / kg, 400 mg / kg icariin) and the model group after treatment; C represents the change in tumor volume during the treatment of each group; D represents the change in nude mouse body weight during the treatment of each group; E represents the difference in relative organ weight during the treatment of each group; F represents the pathological results of heart, liver, spleen, lung and kidney of each group shown by HE staining. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0018] The cells used in the examples of the present invention are human astroglioma cells U87, U251, U118, and A172, all of which are from the Natural Products Research Center of the Chinese Academy of Sciences in Guizhou Province. DMEM culture medium and trypsin were purchased from Esscience; fetal bovine serum (FBS) was purchased from Sijiqing; tetramethylthiazolyl salt (MTT) was purchased from Solarbio LifeScience; dimethyl sulfoxide (DMSO) and penicillin-streptomycin mixture was purchased from Shanghai Yishan Technology Co., Ltd.; PBS buffer was purchased from Wuhan Sewell Company; AnnexinV-FITC / PI apoptosis kit was purchased from Esscience. Example 1 Detection of the activity of icariin in inhibiting the proliferation of glioma cells by tetrazolium nitroblue method

[0019] Glioma cell lines U87, U251, U118, and A172 were taken out of the liquid nitrogen tank for recovery and cultured in a 37°C, 5% CO2 incubator using dimethyl sulfoxide (DMEM) medium containing 10% FBS. Cells in the logarithmic growth phase were taken and cultured at 5×10 3The cells were inoculated in a 96-well plate at a density of 1 / mL, with 190 μL of cell suspension in each well. The drug was added after the cells adhered to the wall. The inhibitory effect of icariin at concentrations of 10 μM and 20 μM on the proliferation of four types of glioma cells was detected. 10 μL of drug was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 48 hours. After taking pictures of the cell morphology under a microscope, 20 μL of MTT solution was added (light-proof operation), and the cells were placed in a CO2 incubator for further culture for 4 hours. The supernatant was discarded, 150 μL of DMSO was added to each well, and the cells were incubated in a light-proof shaker (20 min, 150 rpm, 37°C). The OD value was detected at a wavelength of 490 nm using an enzyme marker, and the inhibition rate was calculated. Each cell group was paralleled with 5 cells.

[0020] The above-mentioned MTT solution was prepared as follows: weigh 0.25 g of MTT powder, add 50 mL of sterilized PBS, dissolve at 60° C., filter through a 0.22 μm filter membrane for sterilization, and store at 4° C. for later use.

[0021] Figure 1 The results showed that 10μM and 20μM icariin had a significant inhibitory effect on the proliferation of four glioma cells, U87, U251, U118, and A172, and the cell morphology was significantly changed. However, at a concentration of 20μM, the inhibition rate of icariin on U87 cells was higher than that on U251 cells. Secondly, the half inhibitory concentration (IC 50 ) value. The results show that the IC value of icariin on glioma cells U8748 h 50 The value is 23.432±1.664.

[0022] Example 2 Flow cytometry detection of icariin inducing apoptosis of glioma cells

[0023] The glioma cell line U87 was taken out from the liquid nitrogen tank for recovery and cultured in a 37°C 5% CO2 incubator using DMEM medium containing 10% FBS.

[0024] Cells in the logarithmic growth phase were taken at 3×10 5The cells were inoculated at a density of 10 μM / mL in a small cell culture dish, with 3 mL of system per dish. The drug was added after the cells adhered to the wall. The effect of icariin at concentrations of 10 μM, 20 μM, and 40 μM on the apoptosis of glioma cells U87 was detected. After adding the drug, the cells were cultured in a 37°C, 5% CO2 incubator for 48 hours, the supernatant was collected, and the cells were digested in an incubator with trypsin without EDTA for 20 minutes. The DMEM culture medium terminated the digestion, the cell suspension was collected, centrifuged at 800 rpm for 3 minutes, and the washed cells were resuspended in 300 μL 1×Binding buffer and transferred to a 1.5 mL EP tube. The control group was set up with a FITC single staining group, a PI single staining group, and a double staining group. The drug-added group was only set up with a double staining group. Each group was added with 5 μL FITC or / and 10 μLPI to avoid light and stain for 30 minutes. Screened and tested on the machine.

[0025] Figure 2 The results showed that icariin at concentrations of 10 μM, 20 μM, and 40 μM all induced apoptosis of U87 cells, among which early apoptosis was more significant and showed a concentration-dependent manner (P < 0.001).

[0026] Combining the experimental results of Example 1 and Example 2, it can be seen that icariin has the potential value of being developed into an anti-glioma drug.

[0027] Example 3 Animal Experiment

[0028] In this example, BALB / c nude mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. and were adaptively raised in an SPF-grade experimental animal room for about one week. U87 glioma cells with good growth status were collected and prepared with PBS buffer to 1×10 7 / mL cell suspension, stored at 4℃ for future use. Each nude mouse was subcutaneously injected with 100μL of cell suspension in the left upper limb. Five days after inoculation, the nude mice that failed to grow tumors or were in poor physical condition were discarded, and oral administration began. They were divided into a model group and an experimental group. The model group was fed normally, and the experimental group was fed with 200mg / kg and 400mg / kg icariin mixed with feed. During this period, the changes in nude mouse body weight and tumor volume were recorded. After 14 days of feeding, the nude mice were dissected, the weight of their five internal organs and subcutaneous tumors was weighed, and HE staining was performed to analyze the therapeutic effect and toxicity. All animal studies followed institutional guidelines.

[0029] The weight changes of nude mice were analyzed, and the weights of heart, liver, spleen, lung, and kidney as well as HE staining of nude mice in the experimental group and the model group were compared. Figure 3The results showed that icariin had no significant toxic effects on the heart, liver, spleen, lungs, and kidneys (P>0.05), indicating that icariin had low toxicity. Analysis of the changes in nude mouse tumor volume and comparison of tumor weight between the experimental group and the model group showed that the tumor volume and weight of the experimental group were significantly smaller than those of the model group (P<0.001), indicating that feeding icariin to the experimental group had a significant therapeutic effect on nude mouse glioma.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of icariin in the preparation of drugs for treating glioma.

2. The use according to claim 1, characterized in that: The structural formula of icariin is as follows:

3. The use according to claim 1, characterized in that: The drug is used alone or in the form of a pharmaceutical composition; the pharmaceutical composition comprises a therapeutically effective amount of icariin and a pharmaceutically acceptable excipient and / or carrier.

4. A pharmaceutical composition for treating glioma, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of icariin, and pharmaceutically acceptable adjuvants and / or conventional chemotherapeutic drugs for glioma.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is in the form of oral preparation, injection and topical administration.

6. The pharmaceutical composition according to claim 5, characterized in that The oral preparations include tablets, capsules, pills, and granules; the injections include injection solutions and freeze-dried powder injections; and the topical administration agents include pellets, sprays, and patches.

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