阅读说明：本技术 6-甲氧基二氢血根碱在制备抗乳腺癌的药物中的应用 (Application of 6-methoxy dihydrosanguinarine in preparation of medicine for resisting breast cancer ) 是由 郭增军 张蕾 张卉 张新新 张富鑫 张笑颜 于 2021-09-13 设计创作，主要内容包括：本发明公开了6-甲氧基二氢血根碱在制备抗乳腺癌的药物中的应用,属于生物医药技术领域。拐枣七中的苯并菲啶类生物碱即6-甲氧基二氢血根碱6-ME对乳腺癌细胞具有增殖抑制作用,能够诱导乳腺癌细胞凋亡,从而发挥抗肿瘤作用。使用一定浓度的6-ME作用于人乳腺癌细胞系MDA-MB-231,可产生增殖抑制作用以及诱导凋亡作用,因此说明6-ME有发展成为治疗乳腺癌药物的潜力。(The invention discloses application of 6-methoxy dihydrosanguinarine in preparation of a medicine for resisting breast cancer, and belongs to the technical field of biological medicines. Benzophenanthridine alkaloid (6-methoxy dihydrosanguinarine 6-ME) in Hovenia dulcis has proliferation inhibiting effect on breast cancer cells, and can induce apoptosis of breast cancer cells, thereby playing an anti-tumor role. 6-ME with a certain concentration acts on a human breast cancer cell line MDA-MB-231, and can generate proliferation inhibition effect and apoptosis induction effect, so that the potential of developing a drug for treating breast cancer by 6-ME is shown.)

Application of 6-methoxy dihydrosanguinarine in preparing medicine for treating breast cancer is disclosed.

2. The use of claim 1, wherein the agent is an agent that inhibits proliferation of triple negative breast cancer cells MDA-MB-231.

3. The use of claim 2, wherein the agent that inhibits the proliferation of triple negative breast cancer cells MDA-MB-231 is dose-and time-dependent.

4. The use of claim 1, wherein the agent is an agent that induces apoptosis in triple negative breast cancer cells MDA-MB-231.

5. The use of claim 4, wherein the agent that induces apoptosis of triple negative breast cancer cells MDA-MB-231 is concentration dependent.

6.6 application of 6-methoxy dihydrosanguinarine in preparing anti-breast cancer chemotherapeutic drugs.

7. The use of claim 6, wherein the chemotherapeutic agent is an agent capable of inhibiting tumor volume growth.

8. The use of claim 6, wherein the chemotherapeutic agent is an agent capable of inhibiting tumor weight gain.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a novel pharmaceutical application of benzophenanthridine alkaloids with antitumor activity, in particular to an application of 6-methoxy dihydrosanguinarine in preparation of medicines for resisting breast cancer.

Background

The breast cancer is the most common malignant tumor disease of women, the incidence rate of the breast cancer is on the trend of increasing year by year, and the health of the women is seriously threatened. Although the incidence of breast cancer increases with the increase in age, the incidence of young women is increasing year by year through data statistics in recent years, and the trend toward youth is severe. At present, the treatment of breast cancer is based on the biological behavior and clinical stage of breast cancer and the physical condition of patients, and adopts a comprehensive treatment mode of mainly surgery and combining radiotherapy, chemotherapy and endocrine treatment. However, surgical treatment and radiotherapy are mainly suitable for local treatment, and drugs for breast cancer chemotherapy, such as adriamycin, cyclophosphamide, 5-fluorouracil and the like, are widely applied to clinical treatment, but the treatment effects of the drugs are limited due to excessive toxic and side effects and the generation of multidrug resistance. With the wide application of natural antitumor drugs such as paclitaxel, vinblastine and the like, the search of antitumor lead compounds with definite action targets from natural products becomes one of the most promising schemes in the development of antitumor drugs. Among the natural products with antitumor activity, isoquinoline alkaloid-benzophenanthridine alkaloids (benzophenanthridine alkaloids) are of great interest, and the parent nucleus structure of the alkaloids has four ABCD rings, as shown in the following structure:

isoquinoline alkaloid-benzophenanthridine alkaloid is widely existed in Papaveraceae and Rutaceae plants, and has antitumor, antiviral, antiinflammatory and antibacterial effects, wherein antitumor activity has been widely paid attention. Benzophenanthridine alkaloids, sanguinarine, chelerythrine and the like show cytotoxic activity to various tumors (colon cancer, brain cancer, ovarian cancer, melanoma, lymphoma and the like), can reverse multidrug resistance, and have the possibility of being developed into ideal tumor chemotherapy drugs, so the benzophenanthridine alkaloids are potential components for searching lead compounds with antitumor activity, and more advantageous, efficient and low-toxicity breast cancer treatment drugs or lead compounds can be found from the potential components.

The hovenia acerba is the root of the Hylomecon japonica (Hylomecon japonica) Prantl of the Hemsleya of the Papaveraceae, also named as the Heqing Hua and the big leaf radix seu caulis Parvifolii, is a commonly used folk medicine, is one of the seven medicines in Shanxi, has the effects of relaxing tendons and activating collaterals, dissipating blood stasis and reducing swelling, dispelling wind and removing dampness, relieving pain and stopping bleeding, and is used for treating symptoms such as rheumatic arthritis, traumatic injury, limb weakness, dysentery, epigastric pain and the like.

A benzophenanthridine alkaloid compound 6-methoxy dihydrosanguinarine (6-ME) separated from Hovenia dulcis has the following structural formula:

there has been no report on the treatment of breast cancer.

Disclosure of Invention

In order to overcome the disadvantages of the prior art, the invention aims to provide the application of 6-methoxydihydrosanguinarine in preparing the medicine for resisting breast cancer.

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

the invention discloses application of 6-methoxy dihydrosanguinarine in preparing a medicine for resisting breast cancer.

Preferably, the medicament is a medicament for inhibiting the proliferation of triple negative breast cancer cells MDA-MB-231.

Further preferably, the agent that inhibits the proliferation of triple negative breast cancer cells MDA-MB-231 is dose-and time-dependent.

Preferably, the medicament is a medicament for inducing the apoptosis of triple negative breast cancer cells MDA-MB-231.

Further preferably, the drug inducing the triple negative breast cancer cell MDA-MB-231 to die is concentration-dependent.

The invention also discloses application of the 6-methoxy dihydrosanguinarine in preparing anti-breast cancer chemotherapeutic drugs.

Preferably, the chemotherapeutic agent is an agent capable of inhibiting tumor volume growth.

Preferably, the chemotherapeutic agent is an agent capable of inhibiting tumor weight growth.

Compared with the prior art, the invention has the following beneficial effects:

the benzo-phenanthridine alkaloid 6-methoxy dihydrosanguinarine (6-ME) is extracted from seven hovenia dulcis thunb of the genus lotaceae, and the research finds that the benzo-phenanthridine alkaloid in the seven hovenia dulcis thunb, namely the 6-methoxy dihydrosanguinarine 6-ME, has the proliferation inhibition effect on breast cancer cells and can induce the apoptosis of the breast cancer cells so as to play the anti-tumor effect. 6-ME with a certain concentration acts on a human breast cancer cell line MDA-MB-231, and can generate proliferation inhibition effect and apoptosis induction effect, so that the potential of developing a drug for treating breast cancer by 6-ME is shown.

Drawings

FIG. 1 is a graph showing the effect of 6-ME on the relative proliferation rate of MDA-MB-231 cells;

FIG. 2 is a graph showing the MDA-MB-231 apoptosis rate after the flow cytometry detects that 6-ME with different concentrations acts for 24 hours;

FIG. 3 is a statistical graph of the apoptosis rate of MDA-MB-231 cells after 24 hours of action of 6-ME at different concentrations;

FIG. 4 is a photograph of the whole mouse of each group at the end of the experiment;

FIG. 5 shows the groups of tumors at the end of the experiment;

FIG. 6 is a curve showing the change in tumor volume in tumor-bearing mice (n-6);

FIG. 7 is a graph showing the change in body weight of tumor-bearing mice (n-6);

fig. 8 shows the volume of the tumor at the end of the experiment (n-6) compared to the control group; wherein, P <0.05 and P < 0.01;

fig. 9 shows the weight of the tumor at the end of the experiment (n-6) compared to the control group; wherein P < 0.01;

FIG. 10 shows HE staining of liver tissue of nude mice (x 100);

FIG. 11 shows HE staining of renal tissue of nude mice (x 100).

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

EXAMPLE 1 detection of 6-ME proliferation inhibition Using MTT method

Screening the anti-tumor activity of the benzophenanthridine alkaloids by adopting an MTT method. Pancreatin digests the cells in log phase, after termination, centrifugally collects, blows into cell suspension, counts the cells and adjusts their concentration. The prepared cell suspension was seeded in 96-well plates, 100. mu.L per well and the marginal wells were filled with sterile PBS. And (3) placing the inoculated cell culture plate into an incubator for culture, and adding the prepared medicine with gradient concentration after the cells adhere to the wall. 100 μ L of sample solution per well, 3 replicates per gradient. 5% CO₂After incubation at 37 ℃ for 48 hours, add each wellmu.L of MTT solution (5mg/mL, i.e., 0.5% MTT) was incubated for 4 h. After that, the culture was terminated, the supernatant was carefully aspirated off by a pipette gun, 150. mu.L of dimethyl sulfoxide was added to each well to dissolve crystals, and the absorbance of each well was measured at 490nm using an ELISA detector. Each 96-well plate is provided with a negative control group and a positive control group.

Referring to fig. 1, the results show that 6-ME has a significant proliferation inhibitory effect on triple negative breast cancer cell MDA-MB-231, presenting significant dose and time dependence.

Example 2 Annexin v/PI double staining method for detecting apoptosis-inducing effect of 6-ME on cells

Cells were seeded in 6-well plates at approximately 2X 10 per well⁵Putting the cells into a thermostat for overnight culture, treating the cells with drugs with different concentrations respectively after the tumor cells are attached to the wall, and culturing for 24 or 48 hours respectively; after the culture was completed, the cell culture broth was aspirated into the centrifuge tube, washed once with sterile PBS, and an appropriate amount of 0.25% trypsin was added to digest the cells. Adding a culture medium, lightly blowing and uniformly beating, transferring into a centrifugal tube, and centrifuging for 5 minutes; the supernatant was discarded, and the cells were gently resuspended in 1ml of PBS and counted, centrifuged for 5 minutes, and collected. This operation can be repeated twice; 400ul of 1 XBinding Buffer was added to gently resuspend the cells. 5ul Annexin V-FITC was added, mixed gently, and incubated for 15 minutes at room temperature in the absence of light. 10ul of PI staining solution was added, mixed gently, and placed in ice bath for 5 minutes in the dark. Flow cytometry detection was performed within 30 minutes.

The apoptosis ratio is detected by a flow cytometer by using an Annexin-V/PI method. This approach can distinguish between normal cells, early apoptotic cells, and late apoptotic and necrotic cells. Results referring to fig. 2, after double staining, the cells were detected by flow cytometry, and four cell populations were represented: namely normal cells, early apoptotic cells, late apoptotic and necrotic cells.

The statistical graph of the apoptosis rate of MDA-MB-231 cells after 6-ME at different concentrations acts for 24h is shown in FIG. 3, and the result shows that after 6-ME acts on MDA-MB-231 cells for 24h, compared with the Control group, the apoptosis rate has significant difference (P < 0.05X, P < 0.01X), which indicates that 6-ME has the effect of inducing MDA-MB-231 cells to undergo apoptosis and is concentration-dependent.

Example 36 study of tumor-inhibiting Effect of ME on tumor-bearing mice

Tumor cells were routinely cultured in culture medium containing 10% fetal bovine serum, digested and resuspended, 0.2mL of cell suspension, 10% per nude mouse⁷And (4) cells. After about 2 weeks, the right armpit of the nude mouse has a hard knot with the diameter of 5mm-7mm, and the model is successfully made. In the experiment, nude mice were randomly divided into: model group, low, medium and high dose group, positive drug group (adriamycin DOX), six of each group. The medicine is injected into abdominal cavity once every other day, and is added with medicine seven times for fourteen days. The body mass of nude mice was measured and recorded every 2 days from the day of administration, and the long diameter (a) and the short diameter (b) perpendicular thereto of the transplanted tumor were measured with a vernier caliper to calculate the tumor volume (V ═ a × b)²And/2), drawing a growth curve of the transplanted tumor. The next day after the treatment, nude mice were sacrificed by dislocation of cervical vertebrae, tumor mass was measured, and tumor inhibition rate was calculated, and HE staining was performed on main organs, and whether there was systemic damage was analyzed.

Photographs of the whole mice of each group at the end of the experiment are shown in FIG. 4, and photographs of the tumors of each group at the end of the experiment are shown in FIG. 5, and the tumor volume gradually decreases with increasing 6-ME dose.

The body weight change curves of the tumor-bearing mice are shown in fig. 6 and 7, and the results show that the body weight of the nude mice has no significant difference compared with the model group after the drug treatment, which indicates that 6-ME has no significant influence on the body weight of the nude mice.

The result of comparing the volume (n ═ 6) of the tumor at the end of the experiment with the control group is shown in fig. 8, and the result of comparing the weight (n ═ 6) of the tumor with the control group is shown in fig. 9, and the result shows that compared with the model group, the volume and the weight of the tumor after the treatment of the 6-ME with different doses are all significantly different, and in conclusion, the 6-ME can significantly inhibit the tumor growth of the breast cancer tumor-bearing mice, the tumor inhibition rate of the 6-ME group drug with the medium dose to the tumor is basically equivalent to that of the positive drug group, and the liver and kidney injury of the mice is not caused by the medium dose and the low dose of the drug, which indicates that the 6-ME has the potential of developing into the breast cancer chemotherapeutic drug.

The results of HE staining of liver tissues of nude mice are shown in fig. 10, and compared with the blank group, the 6-ME group did not cause damage to the liver at each dose, while the cells of the DOX group shriveled, indicating that DOX caused liver damage to some extent. HE staining was performed on the naked kidney tissue, and the results are shown in fig. 11, which shows that each dose of the 6-ME group did not cause significant kidney injury compared to the blank group, while tubular vacuolation of the DOX group indicates that DOX caused kidney injury to some extent.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.