WO2020191845A1

WO2020191845A1 - Drug for reversing resistance of tumor cells to anti-tumor drugs

Info

Publication number: WO2020191845A1
Application number: PCT/CN2019/083938
Authority: WO
Inventors: 袁诚; 许文东; 李菁; 蔡鸿飞; 蒋兆健; 刘菊妍; 陈亮; 王小妹; 张懿; 刘春芳
Original assignee: 广州白云山汉方现代药业有限公司
Priority date: 2019-03-25
Filing date: 2019-04-23
Publication date: 2020-10-01
Also published as: CN109758486A; JP2021519745A; JP7064589B2

Abstract

A drug for combating tumor multi-drug resistance, which comprises a Ganoderma lucidum extract, the Ganoderma lucidum extract being obtained by means of performing extraction by using alcohol.

Description

[Name of invention established by ISA according to Rule 37.2] 　A drug that reverses the resistance of tumor cells to anti-tumor drugs

Technical field

The invention relates to a method and a medicine for reversing the resistance of tumor cells to antitumor drugs, and belongs to the technical field of pharmacy.

Background technique

Cancer is one of the leading causes of death worldwide. In China, both in urban and rural areas, tumors are one of the main causes of death among residents. Tumor Multidrug Resistance (MDR) refers to the cross-resistance of cancer cells to many structurally unrelated chemotherapeutics. It is one of the main reasons why tumors are difficult to treat and easy to relapse. It has become an important obstacle to successful tumor chemotherapy. . According to estimates by the American Cancer Society, more than 90% of the deaths of cancer patients are affected by drug resistance to varying degrees. Therefore, overcoming MDR is a top priority for successful tumor chemotherapy. Finding drugs with anti-drug resistance has become a hot spot for tumor MDR research and development.

Although the mechanism of MDR production is complicated, the overexpression of P-glyco-protein (P-gp) encoded by the mdr1 gene is the main cause of MDR. Finding a low-toxic and effective MDR reversal agent is an important method to improve the efficacy of chemotherapy, and it is an urgent problem in the field of chemotherapy.

Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is one of the main reasons for the failure of cancer treatment. Many synthetic P-gp inhibitors have been discovered over the years, and they have been found to significantly resist the function of P-gp both in vivo and in vitro. However, due to drug toxicity and narrow therapeutic window, phase III trials of these drugs did not get the expected results. Therefore, it is particularly important to find new MDR modulators with high efficiency and low toxicity. Looking for anti-tumor drug resistance in traditional Chinese medicine with small side effects and unique pharmacological effects is one of the ways to solve the problem of tumor drug resistance.

Ganoderma lucidum is a treasure in the treasure house of traditional Chinese medicine in the motherland, and is known as the "fairy grass". Both ancient and modern pharmacology and clinical studies have proved that Ganoderma lucidum has the effects of preventing and curing diseases, strengthening the body, and prolonging life. It is used in the treatment of many chronic diseases, such as cancer, hepatitis, type II diabetes, and hypertension. Ganoderma contains triterpenes, polysaccharides, steroids, alkaloids, nucleosides, amino acids, proteins and other components. Among them, triterpenes and polysaccharides have the most significant biological activities.

The current reports on the anti-tumor drug resistance of Ganoderma include: (1) Fuzheng Xiaoliu Granules against MCF_7 drug-resistant cell proliferation and mechanism in vitro (Zhang Ruiqing, Master Thesis of Southern Medical University, May 20, 2011): Fuzheng Xiaoliu Granules It is a compound Chinese medicine containing Ganoderma lucidum, which can reduce the transcription of MDR1 and the expression of P-gp protein in MCF-7 resistant cells to exert its anti-drug resistance effect on breast cancer resistant cells MCF/ADM; (2) Ganoderma polysaccharide reversal Study on the effect and mechanism of cisplatin-resistant ovarian cancer cells (Qu Hongguang, Doctoral Dissertation of Jilin University, June 2012): Ganoderma lucidum polysaccharide has a reversal effect on DDP-resistant ovarian cancer cells SKOV-3/DDP, and its mechanism of action, It may be achieved by reducing the expression of drug resistance genes and antioxidant genes, reducing oxygen free radical scavenging and inducing lipid peroxidation, and promoting cell apoptosis; (3) The extraction and separation of Ganoderma lucidum triterpenoids and their anti-tumor activity ( Shen Cui'e, 2015 Medical Frontier Forum and the 14th National Cancer Pharmacology and Chemotherapy Academic Conference): It is suggested that the triterpenoid chemical components of Ganoderma lucidum can improve the multidrug resistance of tumor cells K562/A02 (leukemia multidrug resistant cells), KBv200( The potential activity of oral epithelial cancer multidrug resistance cells to adriamycin sensitivity; (4) Ganoderma enoic acid B reversal of ABCB1-mediated tumor multidrug resistance activity evaluation and mechanism of action (Liu Dao, Master's thesis of Jinan University , May 31, 2015): The lanostane-type triterpene ganoderic acid B isolated from Ganoderma lucidum reversing ABCB1 (P-gp, or P-glycoprotein)-mediated tumor multidrug resistance activity evaluation and Mechanism study.

The current research on the anti-tumor multi-drug resistance of Ganoderma lucidum is the study of Ganoderma lucidum extract monomers, and there is no report on the reversal effect of Ganoderma lucidum extract on drug-resistant tumors.

Summary of the invention

The purpose of the present invention is to overcome the above shortcomings of the prior art and provide a method for reversing the resistance of tumor cells to antitumor drugs.

In order to achieve the above objective, the technical solution adopted by the present invention is: a method for reversing the resistance of tumor cells to antitumor drugs, the method comprising administering Ganoderma lucidum extract to the patient.

The Ganoderma lucidum extract of the present invention has no toxic and side effects, has an obvious inhibitory effect on the P-gp function of multi-drug resistant tumor cells, can significantly reverse the multi-drug resistance of tumor cells, and restore the efficacy of anti-tumor chemotherapy drugs.

As a preferred embodiment of the application of the present invention, the Ganoderma lucidum extract is obtained by alcohol extraction.

As a preferred embodiment of the application of the present invention, the preparation method of the Ganoderma lucidum extract is:

Ganoderma lucidum fruiting bodies are dried and crushed into coarse powder, extracted with ethanol under reflux, filtered, and the extracts are combined to obtain an ethanol extract, which is concentrated and dried to obtain an alcohol extract of Ganoderma lucidum fruiting bodies, that is, Ganoderma lucidum extract.

As a preferred embodiment of the application of the present invention, in the steps, the volume fraction of ethanol is 5 to 99%, the number of extractions is 1 to 3, and the time for each extraction is 3 h.

As a preferred embodiment of the application of the present invention, the ganoderma lucidum extract contains ganoderic acid A, ganoderic acid B, ganoderic acid C, ganoderic acid C2, ganoderic acid G, ganoderic acid LM2, ganoderic acid B, ganoderic acid H , Ganoderma Acid D, Ganoderma Acid F, Ganoderma Acid, Ganoderma Acid D, Ergosterol and Ganoderma Polysaccharides.

As a preferred embodiment of the application of the present invention, in the Ganoderma lucidum extract, the mass percentage of ergosterol is 0.1 to 0.25%, the mass percentage of Ganoderma lucidum polysaccharide is 0.5 to 20%, ganoderic acid A, ganoderic acid B. The total mass percentage of ganoderic acid C2, ganoderic acid G, ganoderic acid LM2, ganoderic acid H, ganoderic acid F and gibberellic acid is 0.1-11%.

As a preferred embodiment of the application of the present invention, said reversing the resistance of tumor cells to antitumor drugs is reversing the resistance of HepG2/ADM cells to paclitaxel, vincristine, and doxorubicin, and said reversing the resistance of tumor cells against tumors The drug resistance is to reverse the resistance of MCF-7/ADR cells to paclitaxel and doxorubicin, and the reversal of the resistance of tumor cells to antitumor drugs is to inhibit the function of P-glycoprotein.

The present invention also provides an anti-tumor multi-drug resistance drug, the drug contains Ganoderma lucidum extract.

As a preferred embodiment of the application of the present invention, the medicine also contains a pharmaceutically acceptable carrier.

As a preferred embodiment of the application of the present invention, the dosage forms of the drug are tablets, capsules, oral lozenges, granules, granules, pills, powders, ointments, elixirs, suspensions, solutions, injections, Suppositories, creams, sprays, drops or patches.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The Ganoderma lucidum extract of the present invention has an anti-tumor multidrug resistance effect; at the same dosage, the Ganoderma lucidum extract of the present invention has a much stronger effect than other ganoderma lucidum triterpene monomers and ganoderma lucidum polysaccharides, etc. Ganoderma lucidum extracts The characteristic active ingredients in Ganoderma lucidum have a synergistic effect, and the Ganoderma lucidum extract does not increase liver toxicity and has good safety, indicating that the Ganoderma lucidum extract obtained by the present invention has achieved the beneficial effect of increasing efficiency and reducing toxicity;

(2) The Ganoderma lucidum extract of the present invention has no cytotoxicity and can significantly increase the accumulation of rhodamine 123 (Rh123) in multidrug resistant cells. Its effect is stronger than other Ganoderma lucidum monomer components, indicating that it has an inhibitory effect on P-gp function ; And it can reverse the resistance of HepG2/ADM (human liver cancer drug-resistant tumor cells) cells to paclitaxel, vincristine, and doxorubicin, and it can reverse the resistance of MCF-7/ADR (human breast cancer drug-resistant tumor cells) cells to paclitaxel , Doxorubicin resistance, has an inhibitory effect on the function of P-glycoprotein;

(3) Under the action of the Ganoderma lucidum extract of the present invention, the transplanted tumor restores its sensitivity to paclitaxel and vincristine without enhancing the in vivo side effects of paclitaxel.

Description of the drawings

Figure 1 is a high performance liquid chromatogram of the Ganoderma lucidum extract in Example 2;

Figure 2 is a statistical diagram of the results of the cytotoxicity test in Example 4;

Figure 3 is a statistical diagram of the effect of Ganoderma lucidum extract on the accumulation of Rh-123 in HepG-2/ADM cells in Example 5; wherein, A is the control group and B is the sample group;

Figure 4 is a statistical diagram of the results of the MDR reversal effect of the Ganoderma lucidum extract on the HepG-2/ADM xenograft tumor model in Example 6; where A is the 16th day after the treatment, and the HepG-2/ADM of each group of mice was removed The representative graph of tumor size, B is the statistical graph of the tumor volume change with the experimental time in the P-gp overexpression HepG-2/ADM xenograft tumor model, C is the HepG-2/ADM tumor resection of each group of mice The statistical graph of the average weight of the tumor, D is the statistical graph of the average weight of the P-gp overexpression HepG-2/ADM xenograft tumor model with the experimental time;

Figure 5 is a statistical diagram showing the effect of Ganoderma lucidum extract on the expression level of P-gp protein in different groups of xenograft tumor tissues in Example 7; where A is sample 6 (Ganoderma lucidum extract) and its effect on P-gp in combination with paclitaxel The effect of expression, the left is the electropherogram of three tumor samples, the right is the electropherogram of the other three tumor samples, B is the quantitative effect of sample 6 and its combined use with paclitaxel on P-gp expression (for the three tumor samples on the left) Quantification of electrophoresis), C is the quantitative effect of sample 6 and its combination with paclitaxel on P-gp expression (quantification of electrophoresis of the three tumor samples on the right);

Figure 6 is a representative pathological tissue section of each group in Example 8;

Figure 7 is a statistical diagram of the effect of Ganoderma lucidum extract on the levels of ALT and AST in mice in Example 9.

detailed description

In order to better illustrate the objectives, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Example 1 Preparation of Ganoderma lucidum extract

After the Ganoderma lucidum fruit body is dried and crushed into coarse powder, it is heated and refluxed with 10 times the amount of 5% ethanol for 3 times, each time for 3 hours, filtered, and the three extracts are combined to obtain the ethanol extract, concentrated and dried to obtain the alcohol extraction of the Ganoderma lucidum fruit body. Material, namely Ganoderma lucidum extract.

Example 2 Preparation of Ganoderma lucidum extract

After the Ganoderma lucidum fruit body is dried and crushed into coarse powder, it is heated and refluxed for 3 times with 5 times the amount of 95% ethanol for 3 hours, filtered, and the three extracts are combined to obtain the ethanol extract, which is concentrated and dried to obtain the Ganoderma lucidum fruit body alcohol extraction Material, namely Ganoderma lucidum extract.

The components of the Ganoderma lucidum extract prepared in this example were analyzed by high performance liquid chromatography. Among them, 8 kinds of triterpenes (Ganoderma acid A, Ganoderma acid B, Ganoderma acid C2, Ganoderma acid G, Ganoderma acid LM2, Ganoderma acid H, Ganoderma acid F The total content of Ganoderma lucidum is 9.45%, the content of ergosterol is 0.21%, and the content of Ganoderma lucidum polysaccharide is 2.8%. The HPLC chart is shown in Figure 1, and the contents of the 8 triterpenes are shown in Table 1. The test drugs used in the pharmacological experiments in the following examples of the present invention are all the Ganoderma lucidum extract obtained in this example. The effect of the Ganoderma lucidum extract in Example 1 is similar to that of this example, and will not be detailed here.

Table 1

Example 3 The composition range of Ganoderma lucidum extract

The inventors used the preparation method of the present invention to prepare Ganoderma lucidum extract under different extraction parameters, and summarized the composition range of the Ganoderma lucidum extract prepared. The preparation method of the Ganoderma lucidum extract with this content ratio is shown in Example 2.

The composition range of the Ganoderma lucidum extract obtained by the preparation method of the present invention is shown in Table 2-1 and Table 2-2.

table 2-1

Table 2-2

组分名称Component name	本实施例含量比例The content ratio of this example	含量比例范围Content ratio range
麦角甾醇Ergosterol	0.21％0.21%	0.1～0.25％0.1～0.25%
灵芝多糖Ganoderma Polysaccharide	2.8％2.8%	0.5～20％0.5～20%

Example 4 Tumor cell proliferation experiment

The test drug used in the tumor cell proliferation experiment in this example is the Ganoderma lucidum ethanol (95%) extract obtained in Example 2. Among them, 8 kinds of triterpenes (Ganoderma acid A, Ganoderma acid B, Ganoderma acid C2, Ganoderma acid G, The total content of ganoderic acid LM2, ganoderic acid H, ganoderic acid F, gibberellic acid) is 9.45%, ergosterol content is 0.21%, and polysaccharide content is 2.8%.

To evaluate cell proliferation by MTT method, human hepatoma cell line (HepG-2) or human breast cancer cell line (MCF-7) is seeded in 96-well plate with 4×10 ³ cells per well and cultured overnight, and then used Cells were treated with samples of different concentrations (31.25-1000.00μg/mL) for 48h. Then, 20μl MTT was added to each well, and the cells were incubated for 4h. Finally, the formed purple formazan crystals were dissolved in 150 μl DMSO, and their light absorption values were measured at a wavelength of 490 nm with a multi-mode microplate reader (Bio-Rad laboratories, USA). Use Graph Pad Prism 5.0 to calculate IC ₅₀ value.

The toxic effect of the samples on the drug-resistant human liver cancer cells HepG-2/ADM was tested by the MTT test. The results are shown in Figure 2, where the abscissa is the sample concentration and the ordinate is the cell viability. The MTT cytotoxicity test was evaluated in HepG-2/ADM cells. The cells were treated with samples of different concentrations for 48 hours to determine cell viability. The inhibition of cell proliferation by the sample is calculated based on the absorbance ratio between the sample and the control. The calculated value represents the mean ± standard deviation of three independent experiments (n=6). It can be seen from Figure 2 that treatment with samples with increasing concentrations between 0-1000.00 μg/mL for 48 hours did not inhibit the proliferation of these cells. When treated with 1000.00 μg/mL sample, more than 90% of the cells were alive. The results show that the Ganoderma lucidum extract of the present invention has no cytotoxicity to human liver cancer drug-resistant cells, and the multidrug resistance reversal effect of the Ganoderma lucidum extract will be further studied.

Example 5 Tumor drug resistance reversal experiment

The test drug used in the tumor drug resistance reversal experiment of this example is the Ganoderma lucidum ethanol (95%) extract obtained in Example 2. Among them, 8 kinds of triterpenes (Ganoderma acid A, Ganoderma acid B, Ganoderma acid C2, Ganoderma acid G The total content of ganoderic acid LM2, ganoderic acid H, ganoderic acid F, gibberellic acid) is 9.45%, ergosterol content is 0.21%, and polysaccharide content is 2.8%.

1. In vitro anti-drug resistance activity test

Choose 2 resistant/sensitive cell lines to tumors: human liver cancer sensitive cell line HepG-2 and drug resistant cell line HepG-2/ADM, human breast cancer sensitive cell MCF-7 and drug resistant cell MCF-7/ADR.

The positive control drug is verapamil, and the anti-tumor positive drugs are paclitaxel (paclitaxel), vincristine (VCR), and doxorubicin (DOX).

Among the tested drugs, Sample 1 (Sample 1) is Ganoderma Acid A, Sample 2 (Sample 2) is Ganoderma Acid B, Sample 3 (Sample 3) is Ganoderma Acid B, and Sample 4 (Sample 4) is Ganoderma Alcohol F, Sample 5 (Sample 5) is Ganoderma lucidum polysaccharide, and Sample 6 (Sample 6) is the Ganoderma lucidum extract of Example 2 of the present invention.

Because resistance reversal agents need to be evaluated at non-toxic doses, the MTT method is used to determine cytotoxicity. Set three doses at a non-toxic dose or a dose equivalent to IC ₁₀ , and set up corresponding positive and negative control groups. The positive control drug is verapamil.

Take the cells in the logarithmic growth phase and inoculate them in a 96-well plate. When the cells are fully attached, the original medium is discarded, and the medium containing both the test drug and different concentrations of anti-tumor drugs is added. Verapamil (VRP) is used as Positive control. After 72h incubation, 30μL of 5mg/mL MTT solution was added to each well for 4h. Discard the waste solution, add 100μL of DMSO to dissolve the formazan, detect the OD value with a microplate reader, and calculate the IC ₅₀ by Prism 4.0 software, calculate the reversal factor of the reversal agent by IC ₅₀ , and evaluate the reversal effect with fold reversal , FR) represents: reversal multiple drug alone = IC ₅₀ / combination IC _50. The reversal effect of Ganoderma lucidum extract on drug-resistant human liver cancer cell line HepG-2/ADM cells is shown in Table 3. The reversal effect of Ganoderma lucidum extract on drug-resistant human breast cancer cell line MCF-7/ADR cells is shown in Table 4. Show.

table 3

Table 4

It can be seen from Table 3 and Table 4 that the Ganoderma lucidum extract of the present invention can significantly reverse the resistance of HepG2/ADM cells to paclitaxel, vincristine, and doxorubicin, and can significantly reverse the resistance of MCF-7/ADR cells to paclitaxel and doxorubicin. The resistance of rubicin enhances the sensitivity to chemotherapy drugs.

The Ganoderma lucidum extract of the present invention has a significant effect of reversing tumor drug resistance, can reverse the drug resistance of various tumor cells to various antitumor drugs, and is stronger than other Ganoderma lucidum components. At the same dosage, the Ganoderma lucidum extract of the present invention has a much stronger action intensity than other Ganoderma lucidum triterpene monomers and Ganoderma lucidum polysaccharides, which suggests that the characteristic active ingredients in the Ganoderma lucidum extract have synergistic effects.

2. The effect of Ganoderma lucidum extract on the accumulation of Rh-123 in HepG-2/ADM cells

Rhodamine 123 (Rh-123) is a specific fluorescent probe substrate for glycoprotein, used to detect the function of glycoprotein.

(1) Rh-123 accumulation test

Cells were seeded in a 6-well plate at 1×10 ⁵ cells per well and incubated overnight. The sample solution with a cell concentration of 0.5mg/mL was treated at 37°C for 48h, and then incubated with 10.0μM Rh-123 dye solution at constant temperature and protected from light for 4h. After the above operation, the cells were collected and washed 3 times with cold PBS solution. Finally, the cells were resuspended in PBS buffer, the fluorescence intensity of Rh-123 in the cells was detected by a flow cytometer, and the data obtained was analyzed in FlowJo 7.6.1 software.

(2) Ganoderma lucidum extract increased the accumulation of Rh-123 in HepG-2/ADM cells

In this study, it was detected that the sample had an effect on the accumulation of Rh-123 and P-glycoprotein overexpression HepG-2/ADM cells. The cells were treated with 500 μg/mL sample for 4 hours and then analyzed by flow cytometry. The experimental results are shown in Figure 3. Human liver cancer sensitive cells (Rh-123 accumulation in HepG-2) that do not express P-protein accumulate higher than human liver cancer resistant cells (Rh-123 accumulation in HepG-2/ADM) The level of Rh-123 indicates that this human liver cancer cell line is extremely resistant to most P-protein substrate anticancer drugs; flow cytometry analysis of Rh- in control group and sample group HepG-2/ADM cells The accumulation of 123 (10.0μM), the fluorescence intensity in the cell was detected by flow cytometry to reflect the content of Rh-123 in the cell, among which Ganoderma lucidum extract (Sample6) has the strongest effect, and Ganoderma lucidum below 0.5mg/mL The extract can significantly increase the accumulation of Rh-123 in multidrug resistant cells. Both results indicate that Ganoderma lucidum extract has inhibitory effect on P-gp function, and its effect is the strongest.

Example 6 MDR reversal effect test of Ganoderma lucidum extract on HepG-2/ADM and MCF-7/ADR xenograft tumor models

1. Construction of human cancer xenograft tumor model in nude mice

In the study of P-gp-mediated multidrug resistance, HepG-2/ADM and MCF-7/ADR are suitable target cells for research. This experiment uses HepG-2/ADM and MCF-7/ADR human cancer nude mouse xenograft tumor models, and found that they maintain the multidrug resistance phenotype in vivo and are resistant to paclitaxel and vincristine.

In the overall experiment, the positive anti-tumor drugs were paclitaxel and vincristine. HepG-2/ADM and MCF-7/ADR cells were cultured and collected in vitro, and then HepG2/ADM or MCF-7/ADR cells (200μl phosphate buffer containing 25% Matrigel) were subcutaneously injected into the flanks of nude mice. 1×10 ⁷ cells in the liquid). When the tumor is visible, measure the length and width of the tumor to calculate the volume. Once the size of the tumor is about 100mm ³ , nude mice will be randomly divided into 4 groups (a group of 5 nude mice), and then treated with different treatments: (a) control: normal saline (0.9%, ip, q2d×8) ); (b) only paclitaxel (18mg/kg, ip, q2d×8) or vincristine (5mg/kg, ip, q2d×8); (c) sample (400mg/kg, ig, q1d×16); (d) Paclitaxel (18mg/kg, ip, q2d×8) or vincristine (5mg/kg, ip, q2d×8) and samples (400mg/kg, ig, q1d×16). Record the weight and length of the mouse every two days, and calculate the volume of the tumor according to the following formula:

Draw a tumor growth curve based on tumor volume and treatment time. When the average tumor volume of the control group reached 1300 mm ³ (due to individual differences in mice), the mice were immediately euthanized by exposure to CO ₂ . The tumor tissue was excised from the mouse and the weight was measured. The growth inhibition rate (IR) was calculated according to the following formula: IR=1-(average tumor weight of the experimental group/average tumor weight of the control group)×100%.

At the end of each group of experiments, the animals were euthanized, and the mouse body weight and tumor weight were recorded. Xenograft tumor tissues were collected, 3 tumors in each group were frozen in liquid nitrogen for Western blot analysis, and other tumors were collected and fixed in formalin for HE staining study.

2. The overall anti-cancer effect of Ganoderma lucidum extract on drug-resistant cancer cells

(1) Nude mouse HepG-2/ADM cell xenograft model is used to evaluate the effect of Ganoderma lucidum samples in reversing paclitaxel resistance in vivo

The results of the MDR reversal effect of the HepG-2/ADM xenograft tumor model on nude mice treated with the combination of the sample and paclitaxel are shown in Figure 4. In Figure 4, A is a representative picture of the size of HepG-2/ADM tumors in each group of mice resected on the 16th day after treatment; B is a P-gp overexpression HepG-2/ADM xenograft tumor model along with the experiment Changes in tumor volume over time, the treatment process is as follows: (a) control: saline (ip, q2d×8); (b) paclitaxel: only paclitaxel (18 mg/kg, ip, q2d×8); (c) Sample 6: only Ganoderma lucidum extract (400mg/kg, ig, q1d×16); (d) Sample 6+paclitaxel: paclitaxel (18mg/kg, ip, q2d×8) and Ganoderma lucidum extract (400mg/kg, ig, q1d×16) , Each point on the line graph represents the average tumor volume (mm ³ ) on a specific day after implantation (n=5), and each bar represents SD/100; compared with the control group and paclitaxel alone, * Means significant difference (P<0.05), ** means extremely significant difference (P<0.01); C is the average tumor weight of the HepG-2/ADM tumor removed from each group of mice (mice, n=5), Each column represents the measured mean, and the bar represents SD; compared with the untreated control group, * represents a significant difference (P<0.05), ** represents a very significant difference (P<0.01); D is overexpression in P-gp In the HepG-2/ADM transplanted tumor model, the average body weight changes with the experimental time.

It can be seen from Figure 4 that compared with the normal saline group, paclitaxel at a dose of 18 mg/kg can only slightly inhibit tumor growth in nude mice, indicating that the transplanted tumor maintains the MDR phenotype and is resistant to paclitaxel treatment. In addition, there was no significant difference in tumor size between animals treated with saline (negative control), paclitaxel (positive control), and Ganoderma lucidum extract. The tumor growth inhibition rate of mice treated with pure paclitaxel was 18.56%. The combination of Ganoderma lucidum extract and paclitaxel significantly inhibited tumor growth compared with the paclitaxel alone group and the Ganoderma lucidum extract treatment group. The tumor growth inhibition rate of the combination group of Ganoderma lucidum extract and paclitaxel was 68.18%, which was higher than the 18.56% of the paclitaxel alone group. The results show that Ganoderma lucidum extract has a significant reversal effect on P-gp-mediated MDR in vivo. In addition, in the dose test, it was observed that the Ganoderma lucidum extract and paclitaxel combination group had no death or significant weight loss, indicating that the combination regimen did not increase the occurrence of toxic and side effects.

(2) Human cancer nude mice MCF-7/ADR cell xenograft model is used to evaluate the effect of Ganoderma lucidum samples in reversing vincristine resistance in vivo

In the human cancer nude mouse MCF-7/ADR cell xenograft model, the tumor growth inhibition rate of the Ganoderma lucidum extract and vincristine group was 76.52%, which was higher than that of the vincristine alone group by 24.31%, indicating that the Ganoderma lucidum extract reversed The resistance of human breast cancer MCF-7/ADR cells to vincristine.

Therefore, in the nude mouse xenograft model of human cancer, Ganoderma lucidum extract reversed the glycoprotein (P-gp)-mediated tumor multidrug resistance (MDR).

Example 7 The effect of Ganoderma lucidum extract on the level of P-gp protein

1.Western blot analysis

Place the tumor tissue in the lysis buffer (20mM Tris-HCl pH 7.4, 2mM EDTA, 500mM sodium vanadate, 1% Triton X-100, 0.1% SDS, 10mM NaF, 10mg/mL leupeptin, and 1mM PMSF) , Homogenization under CoolBox ^TM XT system (Sigma). The Bradford method was used to quantify the protein concentration. The 40mg protein solution was separated by 4-12% SDS-PAGE method, and then transferred to PVDF membrane, and blocked with 5% skimmed milk powder on a decolorizing shaker for 2 hours. The blocking solution was discarded and the primary antibody (1:1000) 4 Incubate overnight at ℃, wash with PBST, add secondary antibody at a ratio of 1:2000 and incubate at 37℃ for 1 hour, and analyze with enhanced chemiluminescence detection system (Thermo Fisher).

2. Ganoderma lucidum extract did not change the protein level of P-gp

The P-gp-mediated reversal of MDR can be achieved by inhibiting its function or reducing its expression. In this study, three tumors in each group were analyzed for protein expression levels. The expression level of P-gp was analyzed by Western blotting. GAPDH is used for dosing control. The results of three independent experiments are shown under the Western blot image.

The effect of Ganoderma lucidum extract on the expression level of P-gp protein in xenograft tumor tissues of different groups is shown in Figure 5, where A is sample 6 (Ganoderma lucidum extract) and its effect on P-gp expression in combination with paclitaxel , The left is the electropherogram of three tumor samples, the right is the electropherogram of the other three tumor samples, B is the quantitative effect of sample 6 and its combination with paclitaxel on P-gp expression (the electropherogram of the three tumor samples on the left Quantitative), C is the quantitative effect of sample 6 and its combination with paclitaxel on P-gp expression (quantification of the electrophoresis of the three tumor samples on the right). It can be seen from Figure 5 that the 400mg/kg Ganoderma extract does not Too much change the expression of P-pg in the transplanted tumor tissue. In addition, in this study, the paclitaxel treatment group can slightly increase the expression of P-pg in tumor tissues. It shows that the reversal of MDR is probably achieved by directly inhibiting the efflux function of P-gp, rather than the down-regulation of its mRNA or protein levels.

Example 8 Pathological examination of tumor tissue-HE staining experiment

Fix the xenograft tissue in 4% polyformaldehyde, embed it in paraffin according to the standard operation, cut out 5μm sections with a rotary microtome and stain with hematoxylin and eosin, use the attached digital camera (Leica ICC50) in Leica DM750 Check the slide under the microscope. The representative pathological tissue sections of each group are shown in Figure 6, where control is the control group, paclitaxel is the paclitaxel group, Sample 6 is the Ganoderma lucidum extract group, and Sample 6+paclitaxel is the Ganoderma lucidum extract and paclitaxel group.

It can be seen from Figure 6 that under the combined action of Ganoderma lucidum extract and paclitaxel, a large area of nucleus shrinkage, tumor cell cytoplasm and necrotic tumor cells appear in the transplanted tumor tissue, accompanied by lymphocyte and plasma cell infiltration. The hyperemia of the tumor tissue suggests that the HepG-2/ADM transplanted tumor restored its sensitivity to paclitaxel under the action of Ganoderma lucidum extract.

Example 9 Study on the side effects of Ganoderma lucidum extract

1. Blood Chemistry Analysis

Drew Trilogy Analyzer (Diamond Diagnostics, Holliston, MA, USA) was used to measure plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

2. Ganoderma lucidum extract does not enhance the in vivo side effects of paclitaxel

Hepatotoxicity and cardiomyopathy are two dangerous side effects of paclitaxel, leading to congestive heart failure and liver failure. The statistical diagram of the effect of Ganoderma lucidum extract on the levels of ALT and AST in mice is shown in Figure 7. Compared with the saline group, paclitaxel can slightly increase the concentration of AST in the blood. The combined treatment of paclitaxel and Ganoderma lucidum extract in nude mice did not increase the concentration of ALT or AST in these animals, which indicates that the combined program did not increase the incidence of side effects.

In summary, the Ganoderma lucidum extract of the present invention has no cytotoxicity to MDR tumor cells, and can significantly increase the accumulation of the dye rhodamine Rh123 by multidrug resistant tumor cells, and its effect is stronger than other Ganoderma monomer components; in vitro and in vivo experiments Among them, the Ganoderma lucidum extract of the present invention significantly reverses the multidrug resistance of various tumors to paclitaxel, vincristine and doxorubicin, and restores the anti-tumor effects of paclitaxel, vincristine and doxorubicin; At doses, the Ganoderma lucidum extract of the present invention has much stronger action intensity than other Ganoderma triterpene monomers and Ganoderma lucidum polysaccharides, suggesting that the characteristic active ingredients in the Ganoderma lucidum extract have a synergistic effect; the animals in the Ganoderma lucidum extract combination group of the present invention did not die or Significant weight loss, no enhancement of the in vivo hepatotoxic side effects of paclitaxel, and no change in the liver ALT or AST levels of experimental animals, suggesting that the Ganoderma lucidum extract does not increase liver toxicity and has good safety; the Ganoderma lucidum extract of the present invention does not change the tumor resistance protein The expression of P-gp, the mechanism of reversing drug resistance may be obtained by directly inhibiting the efflux function of drug-resistant protein instead of lowering its mRNA or protein level; the Ganoderma lucidum extract of the present invention has achieved the beneficial effect of increasing efficiency and reducing toxicity .

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those of ordinary skill in the art should understand that, The technical solution of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solution of the present invention.

Claims

A method for reversing the resistance of tumor cells to antitumor drugs is characterized in that the method comprises administering Ganoderma lucidum extract to a patient.
The method of claim 1, wherein the Ganoderma lucidum extract is obtained by alcohol extraction.
The method of claim 2, wherein the preparation method of the Ganoderma lucidum extract is:

Ganoderma lucidum fruiting bodies are dried and crushed into coarse powder, extracted with ethanol under reflux, filtered, and the extracts are combined to obtain an ethanol extract, which is concentrated and dried to obtain an alcohol extract of Ganoderma lucidum fruiting bodies, namely, Ganoderma lucidum extract.
The method of claim 3, wherein the volume fraction of the ethanol is 5 to 99%, the number of extractions is 1 to 3, and the time for each extraction is 3 hours.
The method of claim 1, wherein the ganoderma lucidum extract contains ganoderic acid A, ganoderic acid B, ganoderic acid C, ganoderic acid C2, ganoderic acid G, ganoderic acid LM2, ganoderic acid B, ganoderma lucidum Acid H, Ganoderma Acid D, Ganoderma Acid F, Ganoderma Acid, Ganoderma Acid D, Ergosterol and Ganoderma Polysaccharide.
The method according to claim 5, characterized in that in the Ganoderma lucidum extract, the mass percentage of ergosterol is 0.1 to 0.25%, the mass percentage of Ganoderma lucidum polysaccharide is 0.5 to 20%, and the ganoderic acid A, The sum of the mass percentages of ganoderic acid B, ganoderic acid C2, ganoderic acid G, ganoderic acid LM2, ganoderic acid H, ganoderic acid F and gibberellic acid is 0.1-11%.
The method of claim 1, wherein said reversing the resistance of tumor cells to antitumor drugs is reversing the resistance of HepG2/ADM cells to paclitaxel, vincristine, and doxorubicin, and said reversing the resistance of tumor cells Antitumor drug resistance is to reverse the resistance of MCF-7/ADR cells to paclitaxel and doxorubicin, and the reversal of tumor cell resistance to antitumor drugs is to inhibit P-glycoprotein function.
An anti-tumor multi-drug resistant drug, characterized in that the drug contains Ganoderma lucidum extract.
The medicine according to claim 8, wherein the medicine further contains a pharmaceutically acceptable carrier.
The medicine according to claim 8 or 9, characterized in that the dosage form of the medicine is a tablet, capsule, mouthpiece, granule, granule, pill, powder, ointment, pill, suspension, solution Preparations, injections, suppositories, creams, sprays, drops or patches.