Application of combination of mangiferin aglycone and steviosin E in preparation of medicine for treating cervical cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of combination of mangiferin aglycone and steviosin E in preparation of a medicine for treating cervical cancer. Through cervical cancer cell inhibition and cell apoptosis tests, it is found that the combination of mangiferin aglycone and steviosin E can obviously inhibit cervical cancer cell proliferation and promote cervical cancer cell apoptosis, it is proved that the combination of mangiferin aglycone and steviosin E has good anti-cervical cancer activity, and the combination of mangiferin aglycone and steviosin E has a synergistic effect in the anti-cervical cancer aspect. Furthermore, by constructing a cervical cancer mouse model, it is found that the combination of mangiferin aglycone and steviosin E can significantly reduce expression of an HPV16E6 oncogene and inhibit tumor growth, and it is indicated that the combination of mangiferin aglycone and steviosin E can effectively delay the cervical cancer process and has a good anti-cervical cancer effect. Therefore, the invention can provide a new research and development direction and technical support for the development of cervical cancer chemotherapy drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to the application of mangiferin aglycone combined with stevioside E in the preparation of a medicine for treating cervical cancer. Background Art

[0002] Cervical cancer is one of the most common and most prevalent malignant tumors in women, and one of the leading causes of death in women, posing a great threat to the lives and public health of women worldwide. The occurrence and development of cervical cancer is a complex process regulated by multiple stages and multiple genes. Existing studies have shown that persistent high-risk human papillomavirus infection is the main factor causing cervical cancer.

[0003] Human papilloma virus (HPV) is a type of DNA virus and a common pathogen that causes skin and mucous membrane infections. HPV is divided into more than 200 gene types, which are further divided into high-risk and low-risk types according to their carcinogenic potential. Among them, HPV-16 and HPV-18 are the most common high-risk types. Generally speaking, most HPV infections can be eliminated by the body's own immune system. However, when the body's immune system is low and the virus cannot be cleared, persistent HPV infection is prone to occur, leading to abnormal changes in cervical epithelial cells and eventually developing into cervical cancer.

[0004] At present, HPV vaccination and lesion screening can reduce the incidence and mortality of cervical cancer to a certain extent, but most vaccines only work for disease prevention or early stages, and the popularity of vaccines is limited, which is not very helpful in delaying the progression of cervical cancer caused by HPV infection. At present, the main methods for clinical treatment of clinical symptoms of cervical cancer are surgical resection, chemotherapy, radiotherapy and other therapies, but the above treatment methods often face defects such as difficult handling, poor treatment effect, repeated attacks of patients, and large side effects. How to safely and effectively enhance the inhibitory and killing activity of cervical cancer cells is an important intervention method to delay the development and metastasis of cervical cancer, and it is also of great significance for the treatment of cervical malignancies.

[0005] In recent years, natural plant extracts have become a hot spot for the research and development of new chemotherapy drugs due to their advantages of high safety, few side effects and low toxicity, and have gradually become a new idea to alleviate the development of cervical cancer. However, the current research on plant-derived chemotherapy drugs in the prevention and treatment of cervical cancer is still insufficient. Therefore, developing more safe, mild and easy-to-use drugs to improve the treatment effect of cervical cancer can better provide a favorable theoretical basis and application basis for the prevention and treatment of cervical cancer. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide the use of mangiferin aglycone combined with stevioside E in the preparation of a drug for the treatment of cervical cancer. The experiment of the present invention found that mangiferin aglycone combined with stevioside E has obvious synergistic effect in inhibiting the proliferation of cervical cancer cells and promoting the apoptosis of cervical cancer cells, and is suitable for development and application as a cervical cancer drug.

[0007] The second object of the present invention is to provide a medicine for treating cervical cancer.

[0008] One of the purposes of the present invention is to adopt the technical solution:

[0009] Application of mangiferin combined with stevioside E in the preparation of drugs for treating cervical cancer.

[0010] As a preferred embodiment, the mass ratio of mangiferin aglycone to stevioside E is (1-7):1.

[0011] As a more preferred embodiment, the mass ratio of mangiferin aglycone to stevioside E is 3:1.

[0012] The second object of the present invention is to adopt the following technical solution:

[0013] A medicine for treating cervical cancer, comprising mangiferin aglycone, stevioside E and pharmaceutically acceptable excipients.

[0014] As a preferred embodiment, the mass ratio of mangiferin aglycone to stevioside E is (1-7):1.

[0015] As a more preferred embodiment, the mass ratio of mangiferin aglycone to stevioside E is 3:1.

[0016] As a preferred embodiment, the dosage form of the drug is one of tablets, capsules, pills, granules and solutions.

[0017] In the present invention, when the drug for treating cervical cancer is a tablet, capsule, pill, or granule, the drug can be prepared by the molding method and pharmaceutical excipients commonly used in the art for preparing the above dosage forms, and the present invention does not specifically limit it. The pharmaceutical excipients used can be, for example, corn starch, magnesium stearate, dextrin, hydroxypropyl cellulose, talc, silicon dioxide, gelatin, sucrose, etc. Further, when the drug is a solution, the excipient used can be a common pharmaceutical solvent in the art, such as medical corn oil, Tween-80, ethanol, etc.

[0018] The present invention has the following beneficial effects:

[0019] At present, there are many kinds of plant extracts with anti-cancer activity, but there are few plant raw materials suitable for the development of cervical cancer drugs. The present invention found that mangiferin combined with stevioside E can significantly inhibit the proliferation of cervical cancer cells and promote the apoptosis of cervical cancer cells through cervical cancer cell inhibition and cell apoptosis experiments, confirming that mangiferin combined with stevioside E has good anti-cervical cancer activity, and the two have a synergistic effect in anti-cervical cancer.

[0020] Furthermore, by constructing a cervical cancer mouse model, the present invention found that mangiferin combined with stevioside E can significantly reduce the expression of HPV16 E6 oncogenes and inhibit tumor growth, indicating that mangiferin combined with stevioside E can effectively delay the progression of cervical cancer and has a significant anti-cervical cancer effect. Therefore, the present invention can provide a new research and development direction and technical support for the development of cervical cancer chemotherapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1a This is the effect of the blank control group in Experimental Example 2 of the present invention on apoptosis of human cervical cancer SiHa cells;

[0022] Figure 1b This is the effect of mangiferin aglycone combined with stevioside E (ratio 1:0) on apoptosis of human cervical cancer SiHa cells in Experimental Example 2 of the present invention;

[0023] Figure 1c The effect of mangiferin combined with stevioside E (ratio 3:1) on apoptosis of human cervical cancer SiHa cells in Experimental Example 2 of the present invention;

[0024] Figure 1d The effect of mangiferin aglycone combined with stevioside E (ratio 1:1) on apoptosis of human cervical cancer SiHa cells in Experimental Example 2 of the present invention;

[0025] Figure 1e The effect of mangiferin aglycone combined with stevioside E (ratio 1:3) on apoptosis of human cervical cancer SiHa cells in Experimental Example 2 of the present invention;

[0026] Figure 1f The effect of mangiferin aglycone combined with stevioside E (ratio 0:1) on apoptosis of human cervical cancer SiHa cells in Experimental Example 2 of the present invention;

[0027] Figure 2 Effect of mangiferin combined with stevioside E on tumor mass in mice with cervical cancer in Experimental Example 3 of the present invention;

[0028] Figure 3 The effect of mangiferin combined with stevioside E on the expression of HPV16 E6 protein in cervical cancer mice in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0029] In order to make the technical purpose and technical solution of the present invention more clear, the present invention is described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not used to limit the protection scope of the present invention.

[0030] In the present invention, the raw materials or reagents involved are conventional materials or reagents that can be obtained through commercial channels unless otherwise specified. Among them, in the experimental examples of the present invention, the raw materials mangiferin, stevioside E, stevioside A, and mangiferin involved are all purchased standard products with a purity greater than 98%. During the experiment, RPMI-1640 culture medium was used to dilute them to the required concentration. Among them, mangiferin is 7-O-methylmangiferin. Mangiferin CAS No. 3542-72-1, structural formula is

[0031] In other embodiments, the above raw materials can also be prepared by technicians using well-known extraction and separation techniques (such as alcohol extraction, microwave extraction, column chromatography, resin adsorption, etc.) to obtain high-purity products. The present invention does not impose too many restrictions on the sources of the above raw materials. In the embodiment of the present invention, the human cervical cancer SiHa cell line (containing the HPV-16 genome) and the HeLa cell line (containing the HPV-18 genome) are from a domestic biological company.

[0032] Example 1

[0033] This embodiment provides the use of mangiferin aglycone combined with stevioside E in the preparation of a drug for treating cervical cancer. The mass ratio of mangiferin aglycone to stevioside E is 7:1.

[0034] This embodiment also provides a drug for treating cervical cancer, the drug comprising mangiferin, stevioside E and pharmaceutically acceptable excipients. The mass ratio of mangiferin to stevioside E is 7:1.

[0035] Example 2

[0036] This embodiment provides the use of mangiferin combined with stevioside E in the preparation of a drug for treating cervical cancer. The mass ratio of mangiferin to stevioside E is 3:1.

[0037] This embodiment also provides a drug for treating cervical cancer, the drug comprising mangiferin aglycone, stevioside E and pharmaceutically acceptable excipients. The mass ratio of mangiferin aglycone to stevioside E is 3:1.

[0038] Example 3

[0039] This embodiment provides the use of mangiferin aglycone combined with stevioside E in the preparation of a drug for treating cervical cancer. The mass ratio of mangiferin aglycone to stevioside E is 1:1.

[0040] This embodiment also provides a drug for treating cervical cancer, the drug comprising mangiferin, stevioside E and pharmaceutically acceptable excipients. The mass ratio of mangiferin to stevioside E is 1:1.

[0041] In the above embodiment of the present invention, when the drug for treating cervical cancer is a tablet, capsule, pill, or granule, the drug can be prepared by using the molding method and pharmaceutical excipients commonly used in the art for preparing the above dosage forms, and the pharmaceutical excipients used can be corn starch, magnesium stearate, dextrin, hydroxypropyl cellulose, talc, silicon dioxide, gelatin, sucrose, etc. Further, when the drug is a solution, the excipient used can be a common pharmaceutical solvent in the art, such as medical corn oil, Tween-80, ethanol, etc.

[0042] Experimental Example 1: Effects of mangiferin combined with stevioside on proliferation of cervical cancer cells

[0043] In this experiment, SiHa cells or HeLa cells were used as experimental cells to investigate the inhibitory effects of different ratios of mangiferin combined with stevioside E on cell proliferation. At the same time, mangiferin and stevioside A were used as comparative components to investigate the differences in the inhibitory effects of each component on cell proliferation. Among them, mangiferin, stevioside E, mangiferin, and stevioside A were prepared into mother solutions (2 mg / mL) using RPMI-1640 culture medium and diluted to the corresponding concentrations when used.

[0044] During the experiment, cells (SiHa cells or HeLa cells) in the logarithmic growth phase were obtained, digested with trypsin, and diluted with RPMI-1640 medium to prepare 5×10 4 cells / mL of cell suspension, then inoculated in a 96-well plate at 200 μL / well, cultured in a 37°C, 5% CO2 incubator for 24 hours, and then replaced with fresh culture medium containing different ratios of mangiferin and stevioside E (ratio 1:0, 7:1, 3:1, 1:1, 1:3, 1:7, 0:1) for drug intervention, and the final drug concentrations in each well were controlled to be 50, 100, and 200 μg / mL, respectively. Five replicate wells were set for each concentration test, and five control wells without drug were set at the same time. After 48 hours of drug addition, 20 μL of MTT solution was added to each well and cultured for 2 hours, then the supernatant was discarded, 200 μL of DMSO was added to each well, shaken and mixed for 5 minutes, and the absorbance value at a wavelength of 570 nm was measured using an enzyme reader, and then the average inhibition rate of each test group was calculated. In addition, mangiferin was used to replace mangiferin for the above experiment, and stevioside A was used to replace stevioside E for the above experiment. Cell inhibition rate = (1 - absorbance value of drug intervention group / absorbance value of control group) × 100%. The results are shown in Tables 1 to 4.

[0045] Table 1. Effects of mangiferin combined with stevioside E on the proliferation of SiHa cells

[0046]

[0047]

[0048] Table 2 Effects of mangiferin combined with stevioside E on the proliferation of SiHa cells

[0049]

[0050] Table 3 Effects of mangiferin combined with stevioside A on the proliferation of SiHa cells

[0051]

[0052] Table 4 Effects of mangiferin combined with stevioside E on HeLa cell proliferation

[0053]

[0054]

[0055] It can be seen from Tables 1 to 4 that different drug combinations have an inhibitory effect on the growth of cervical cancer SiHa and HeLa cells, and within different drug concentration ranges, as the drug concentration increases, the inhibition rate increases, showing a concentration-dependent cytotoxic effect.

[0056] As shown in Table 1, when mangiferin and stevioside E were used alone, the inhibition rate on SiHa cells was low (41.3% and 31.8% (drug concentration 200 μg / mL), respectively, while the combination of mangiferin and stevioside E mostly showed a synergistic relationship. Among them, when the mass ratio of mangiferin to stevioside E was 3:1, the synergistic effect was the strongest, and the inhibition rate on SiHa cells was the best, reaching 78.5% (concentration 200 μg / mL), and the inhibition rates when the mass ratios were 1:1 and 7:1 were second.

[0057] As shown in Tables 2 and 3, the inhibitory activity of the mangiferin combined with stevioside E experimental group and the mangiferin combined with stevioside A experimental group on SiHa cells reached the best when the mass ratio was 1:1.

[0058] From the comparison of the results in Table 1, Table 2 and Table 3, it can be seen that compared with mangiferin combined with stevioside E (Table 2) and mangiferin combined with stevioside A (Table 3), the present invention adopts mangiferin combined with stevioside E (Table 1), which shows the best inhibitory activity on cervical cancer SiHa cells. The best inhibition rates of the three are 52.4% (mangiferin combined with stevioside E), 48.2% (mangiferin combined with stevioside A), and 78.5% (mangiferin combined with stevioside E), respectively.

[0059] As shown in Table 4, mangiferin combined with stevioside E also has significant inhibitory activity against HeLa cells, and the inhibitory activity is optimal when the ratio of the two is 3:1. Comparison of the results in Table 1 and Table 4 shows that the inhibition rate of the drug combination on SiHa cells is lower than that on HeLa cells, which may be due to the different tolerance of SiHa cells and HeLa cells to drugs.

[0060] Experimental Example 2: Effect of mangiferin combined with stevioside E on apoptosis of cervical cancer cells

[0061] SiHa cells were seeded into 6-well plates (5×10 5 cells / well), and cultured in a 37°C, 5% CO2 incubator for 24 hours. Then, the old culture medium was aspirated, and the culture medium containing mangiferin and stevioside E was added for drug intervention (the ratio of the two was 1:0, 3:1, 1:1, 1:3, 0:1). The final drug concentration was 100 μg / mL, and a blank control group without drug was set up. After 48 hours of continuous culture, the intervention was terminated, the cells were washed with PBS, and then trypsin was added to digest the cells, centrifuged, the supernatant was discarded, the cells were collected, resuspended with PBS and counted. The resuspended cells (1×10 5 cells), centrifuged and discarded the supernatant, then added Annexin V-FITC binding solution (195 μL) and gently blown the cells, then added 5 μL Annexin V-FITC binding solution to resuspend the cells, further added 10 μL of propidium iodide staining solution, resuspended again, incubated in an ice bath away from light for 20 minutes, and the apoptosis of cervical cancer cells was detected by flow cytometry. The flow cytometric detection results are shown in Figure 1a to Figure 1f The calculation results of cell apoptosis rate are shown in Table 5. In the table, * indicates significant difference compared with the blank control group, P < 0.05.

[0062] Table 5 Effects of mangiferin combined with stevioside E on apoptosis of SiHa cells

[0063] Grouping Apoptosis rate (%) Blank control group 8.73±1.32 Mangiferin: Stevioside E=1:0 26.57±3.36* Mangiferin: Stevioside E=3:1 58.78±2.21* Mangiferin: Stevioside E=1:1 43.50±3.78* Mangiferin: Stevioside E=1:3 32.83±2.45* Mangiferin: Stevioside E=0:1 17.26±0.69*

[0064] Depend on Figure 1a to Figure 1fCombined with Table 5, it can be seen that compared with the experimental group using mangiferin or stevioside E alone, the drug combination group of mangiferin combined with stevioside E (ratio 3:1, 1:1, 1:3) can effectively promote the apoptosis of SiHa cells, and the difference in apoptosis rate between the combination group and the blank control group and the single drug group is statistically significant (P < 0.05). Among them, when the mass ratio of mangiferin to stevioside E is 3:1, the apoptosis rate of SiHa cells is the highest, reaching 58.78%, indicating that mangiferin combined with stevioside E has a significant synergistic effect in promoting apoptosis of cervical cancer cells.

[0065] Experimental Example 3: Effects of mangiferin combined with stevioside E on tumor growth and HPV16 E6 protein expression in cervical cancer mice

[0066] BALB / c female nude mice aged 6-8 weeks and weighing 15-20 g were selected. The SPF level was maintained at 20-25°C, 40%-70% relative humidity, and a 12-h light and dark cycle. After one week of standard feed, they were marked and grouped and raised in cages, with 5 mice per group. SiHa cells in the logarithmic growth phase were prepared into 1×10 7 cells / mL of cell suspension, and then 200μL of cell suspension was injected subcutaneously in the right axilla of each nude mouse to construct a mouse solid tumor model. After the inoculation, the mice were raised conventionally, and the tumor formation of the mice was observed. After the tumor was formed, they were divided into groups, and the experimental groups were given drug intervention. Experimental group I: intraperitoneal injection of single mangiferin aglycone every 4 days; Experimental group II: intraperitoneal injection of single stevioside E every 4 days; Experimental group III: intraperitoneal injection of mangiferin aglycone + stevioside E (mass ratio 3:1) every 4 days. The injection dose of each group was 20μL / g, and the drug concentration was 100μg / mL. A total of 5 times of administration were given, and the control group was injected with an equal dose of normal saline. 24 hours after the last administration, the nude mice were anesthetized and killed, the subcutaneous tumor was peeled off, and the mass of the tumor was measured. The results are as follows Figure 2 ** indicates that there is a significant difference between the two groups, P < 0.01; *** indicates that there is a significant difference between the two groups, P < 0.001.

[0067] Depend on Figure 2 It can be seen that mangiferin combined with stevioside E can significantly reduce the tumor mass of cervical cancer mice. In addition, by monitoring the weight of mice during the drug administration process, it was found that the drug administration process had no significant effect on the overall weight of mice, indicating that the drug has good biosafety.

[0068] Furthermore, HPV16 E6 is the most important high-risk HPV for cervical cancer infection, and it plays a very important role in the occurrence and development of cervical cancer. The present invention further measures the expression of HPV16 E6 protein in tumor tissue. Specifically, after the above-mentioned tumor mass measurement, the tumor tissue is placed in a cryopreservation tube and quickly placed in liquid nitrogen for cryopreservation. After cryopreservation, the tumor tissue is taken out and ground, and the cell RNA is extracted using an RNA extraction kit, and then reverse transcribed into cDNA, and amplified using an RT-PCR instrument. GAPDH is used as an internal reference to detect the relative expression of HPV16 E6 protein. The results are as follows Figure 3 shown.

[0069] Depend on Figure 3 It can be seen that the expression of HPV16 E6 protein in group III was the lowest, followed by group I and group II, and the control group was the highest. It can be seen that mangiferin combined with stevioside E can effectively reduce the expression of the oncogene HPV16 E6, which is beneficial to inhibit the malignant proliferation of cervical tumor cells.

[0070] In summary, the present invention, through cervical cancer cell inhibition and apoptosis experiments, found that mangiferin combined with stevioside E can significantly inhibit the proliferation of cervical cancer cells and promote the apoptosis of cervical cancer cells, confirming that mangiferin combined with stevioside E has good anti-cervical cancer activity, and the two have a synergistic effect in anti-cervical cancer. Further, by constructing a cervical cancer mouse model, the present invention found that mangiferin combined with stevioside E can significantly reduce the expression of HPV16 E6 oncogenes and inhibit tumor growth, indicating that mangiferin combined with stevioside E can effectively delay the progression of cervical cancer and has a significant anti-cervical cancer effect. Therefore, the present invention can provide new research and development directions and technical support for the development of cervical cancer chemotherapy drugs.

Claims

1. Application of mangiferin combined with stevioside E in the preparation of drugs for the treatment of cervical cancer.

2. The use of mangiferin aglycone combined with stevioside E in the preparation of a drug for treating cervical cancer according to claim 1, characterized in that: The mass ratio of mangiferin aglycone to stevioside E is (1-7):

1.

3. The use of mangiferin aglycone combined with stevioside E in the preparation of a drug for treating cervical cancer according to claim 2, characterized in that: The mass ratio of mangiferin aglycone to stevioside E is 3:

1.

4. A drug for treating cervical cancer, characterized in that: The medicine comprises mangiferin aglycone, stevioside E and pharmaceutically acceptable excipients.

5. The drug for treating cervical cancer according to claim 4, characterized in that: The mass ratio of mangiferin aglycone to stevioside E is (1-7):

1.

6. The drug for treating cervical cancer according to claim 5, characterized in that: The mass ratio of mangiferin aglycone to stevioside E is 3:

1.

7. The drug for treating cervical cancer according to claim 4, characterized in that: The dosage form of the medicine is one of tablets, capsules, pills, granules and solutions.

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