CN106913877A - Application of the magnolol in antineoplastic sensitizer is prepared - Google Patents

Abstract

The invention belongs to cell biology and field of medicaments, it is related to application of the magnolol in antineoplastic sensitizer is prepared.Magnolol is natural products, the effect with reversing multiple medicine resistance of tumor cells, can be as the reversal agent of tumor multi-medicine drug-resistant；Magnolol also has increases the effect of tumor multi-medicine drug-resistant cells against neoplastic drug susceptibility, can be used as chemotherapeutic sensitizer.Present invention also offers the method that the pharmaceutical composition that antineoplastic and magnolol are used in combination suppresses tumor multi-medicine drug-resistant cell propagation.Micromolecular compound magnolol in the present invention is developed as new antineoplastic or its auxiliary element, and substantially, environmental protection will provide a kind of new approach and means to tumor killing effect to treat and curing tumour.

Description

Application of magnolan alcohol in the preparation of antitumor drug sensitizer

technical field

The invention belongs to the field of cell biology and medicine, in particular, the invention relates to a new application of magnolol. The invention also relates to the corresponding pharmaceutical composition and its application method.

Background technique

Tumor is an abnormal lesion formed by the body under the action of various carcinogenic factors when a certain cell in a local tissue loses its normal regulation of its growth at the gene level, resulting in its clonal abnormal proliferation. The number of cancer cases in our country is quite large, accounting for 55% of the total number of cases in the world.

Benign tumors have less impact on the body, mainly showing symptoms of local compression and obstruction. Malignant tumors have a serious impact on the body due to their immature differentiation, rapid growth, infiltration and damage to the structure and function of organs, and the possibility of metastasis. In addition to local compression and obstruction symptoms similar to those of benign tumors mentioned above, malignant tumors may also cause fever and intractable pain, and severe emaciation, fatigue, anemia, and systemic exhaustion may appear in the advanced stage.

Traditional Chinese medicine believes that the cause of cancer is the balance of yin and yang in the human body, and the tissue cells are caused by cell mutations under the long-term action of different carcinogenic factors. In fact, cancer tissue is also a part of the human body. Only when the balance of yin and yang is out of balance and the five elements change, the immune monitoring system of the human body will lose its monitoring and let it develop. Chinese herbal medicine can preserve life by regulating qi and blood, adjusting the balance of yin and yang, and maintaining normal vital signs; Therefore, Chinese herbal medicine extracts have become an important direction for the treatment of tumors.

Magnolol is a natural product with high bioavailability and relatively stable properties, and has clinical application value. With the deepening of its chemical and biological research, its molecular mechanism will be gradually clarified, which will further promote the chemical structure modification and structure-activity relationship research of these compounds.

Contents of the invention

The purpose of the present invention is to provide a new pharmaceutical application of magnolol.

The object of the present invention is to provide an antitumor drug sensitizer.

In one aspect, the present invention provides the application of magnolol in the preparation of antitumor drug sensitizers. Among them, the English full name of magnolan alcohol is magnolol, and its structure is shown in Figure 1.

Said tumors include oral cancer, breast cancer, liver cancer, lung cancer, cervical cancer, colon cancer or pancreatic cancer.

The antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil.

Magnolol can be used as a tumor multidrug resistance reversal agent.

On the other hand, the present invention provides an anti-tumor pharmaceutical composition, the active ingredients of which are anti-tumor drugs and magnolia alcohol.

The anti-tumor drugs are cell cycle specific drugs or cell cycle non-specific drugs.

The antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil.

The invention also provides a method for inhibiting the growth and proliferation of tumor cells in vitro, that is, adding magnolanol and antitumor drugs to the culture medium of tumor cells.

Wherein, the final concentration of adding magnolan alcohol is 1-20 μmol/L.

The tumor includes oral cancer, breast cancer, liver cancer, lung cancer, cervical cancer or pancreatic cancer.

When implementing the above method, first add magnolia alcohol, and then add antineoplastic drugs. It is also possible to add magnolia alcohol and antineoplastic drugs at the same time.

The invention provides a new application of magnolol, that is, its application in the preparation of antitumor drug sensitizers. Magnolol is a natural product that has the effect of reversing the multidrug resistance of tumor cells and can be used as a reversal agent for tumor multidrug resistance; it also has the effect of increasing the sensitivity of tumor multidrug resistant cells to antitumor drugs and can be used as a chemotherapy agent. Sensitizer use. The small molecule compound magnolanol in the present invention is developed as a new anti-tumor drug or its auxiliary component, has obvious tumor-suppressing effect, is green and environment-friendly, and will provide a new approach and means for treating and curing tumors.

Description of drawings

Figure 1 is the structure of magnolol.

detailed description

The invention provides an antineoplastic drug, the active ingredient of which is magnolia alcohol. The tumor can be liver cancer cells, gastric cancer cells, cervical cancer cells or blood cancer cells.

The small molecule compounds of the present invention can be prepared by various conventional preparation methods. For example, the method of artificial chemical synthesis is adopted.

Utilizing the small molecular compound of the present invention, substances interacting with magnolol, such as receptors, inhibitors or antagonists, can be screened out through various conventional screening methods.

When the present invention and its inhibitors, antagonists, etc. are administered (administered) therapeutically, various effects can be provided. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, and the pH value can be formulated according to Depending on the nature of the substance and the condition being treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

Taking magnolol of the present invention as an example, it can be used in combination with a suitable pharmaceutically acceptable carrier. Such pharmaceutical compositions contain a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The magnolanol of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, magnolol of the present invention can also be used together with other therapeutic agents. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

experimental method:

1. Cell recovery

1) Take out the cryotube from the liquid nitrogen tank, put it directly into warm water at 37°C, and shake it from time to time to melt it as soon as possible.

2) Take out the cryotube from the 37°C water bath, suck out the cell suspension with a pipette, pour into the centrifuge tube and add more than 10 times of culture medium, mix and centrifuge at low speed, discard the supernatant, and wash with culture medium again.

3) After properly diluting with the culture medium, inoculate the culture bottle, place it in a 37°C incubator for static culture, replace the culture medium the next day, and continue the culture. Subculture when cultured to a certain concentration. PANC-1 cells were cultured in DMEM high-glucose medium containing 10% Gibico fetal bovine serum; K562, HGC, QGY, MCF-7, PC-3 and other cells were cultured in 1640 medium containing 10% fetal bovine serum. SK-hep1, HeLa, HepG2, and other cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, and the medium contained 100 U/ml penicillin and 100 μg/ml streptomycin.

2. Subculture of cells

Observe the growth of the cells every day, and subculture when the cells grow to about 90% confluence in the culture flask, and subculture once every 2-4 days. Passage one bottle into three flasks, or one 25cm ² into one 75cm ² culture flask. method:

1) Wash the cells once with 1× phosphate buffer.

2) Add 2-3ml trypsin digestion solution for digestion, and place in a 37°C incubator for several minutes. Tap the cell culture flask with your hands to detach the cells.

3) Stop trypsinization with appropriate medium containing 10-15% Gibico fetal calf serum. Aliquot the cells into new culture flasks and continue culturing.

When the suspension cells are subcultured, they are directly collected in a centrifuge tube and centrifuged, and the old medium is discarded. Generally, the ratio of one bottle to three bottles is divided into new culture bottles, and fresh medium is added to continue the culture.

3. Cell cryopreservation

1) The cells cultured to the logarithmic growth phase were digested with trypsin, collected in a centrifuge tube, counted, and centrifuged.

2) Discard the trypsin and the old culture medium, add the prepared frozen culture medium (containing 10% DMSO, 40% DMEM and 50% Gibico fetal bovine serum), the final concentration of cells in the frozen medium is 0.5-1 ×10 ⁷ /ml. Gently pipette with a pipette to make the cells uniform, then divide into sterile cryopreservation tubes, add 1-1.5ml to each tube.

3) Put the cryopreservation tubes into the cryopreservation box and place them for quick freezing at -80°C, and transfer them to a liquid nitrogen tank for storage after 5 hours.

4. Experimental materials

Drug preparation:

Magnolol was dissolved in DMSO (dimethyl sulfoxide) and prepared as 100mM or 50mM mother solution for later use.

Magnolol can be purchased from Shanghai Yuanmu Biotechnology Co., Ltd., and its specific properties and parameters are as follows:

The structure of magnolol is shown in Figure 1.

Verapamil (VPL), vincristine (VCR) and doxorubicin (ADR) were purchased from Roche Chemical Company with a purity greater than 99%.

Cell source:

Human oral cancer cell line KB and its drug-resistant cell line KB/VCR were provided by the Chinese Academy of Sciences, human breast cancer cell line MCF-7 and its drug-resistant cell line MCF-7/ADR, human colon cancer HCT-8 and its drug-resistant cell line Drug cell line HCT-8/VCR were purchased from Nanjing Kaiji Biological Company.

Reagent test kit:

CCK-8 kit was purchased from Tongren Company.

CCK-8 experiment

KB and KB/VCR cells as well as MCF-7 and MCF-7/ADR cells were inoculated into 96-well plates at a density of 3500/well, and after 24 hours, different concentrations of ADR, VCR, magnolanol, and VCR and magnolanol were used together with α-MEM with 10% fetal bovine serum was added to each well. After culturing for 48 h, discard the culture medium, and add 90 μL serum-free medium and 10 μL CCK-8 reagent to each well. After reacting at 37°C for 2 h, the absorbance value (OD450) at a wavelength of 450 nm was read with a microplate reader. The cell proliferation ratio of the medication group relative to the control group was obtained by calculation. In the blank group, only the culture medium was added without cells, and in the control group, DMSO with the same volume as the drug was added, and the cell survival rate=(OD450 of the experimental group-OD450 of the blank group)/(OD450 of the control group-OD450 of the blank group). The resistance fold (Resistance Fold, RF) was calculated based on the IC ₅₀ value.

RF = IC ₅₀ value of drug-resistant cell line/IC ₅₀ value of parental cell line. Three replicate wells were set up for each concentration, and the experiment was repeated three times.

All drug-resistant cell lines were grown in drug-free medium for 3 days prior to growth inhibition experiments. Each value is the result of 3 independent experiments, and IC ₅₀ is expressed in the form of "mean ± standard deviation". VCR, vincristine; ADR, daunorubicin; RF, resistance fold.

Example 1

Experimental results:

KB/VCR and MCF-7/ADR are two commonly used multidrug-resistant cell lines, and in this example, these two kinds of cells also exhibit the characteristic of multidrug resistance. As shown in Table 1, compared with KB cells, the multiples of drug resistance of KB/VCR cells to VCR and ADR were 81.9 times and 94.4 times, respectively, while MCF-7/ADR cells were more resistant to VCR and ADR than MCF-7 cells. The drug multiples were 38.1 times and 20.9 times respectively, showing that the drug-resistant cells used in the experiment had multidrug resistance, and the MDR activity was similar to the results reported in the literature.

The _IC50 of magnolanol to the parental cell line KB and MCF-7 was 238.7 μM and 167.2 μM respectively, and the _IC50 to the drug-resistant cell line KB/VCR and MCF-7/ADR was 254.9 μM and 168.0 μM respectively, There was no significant difference between the two, and the multidrug-resistant cell lines KB/VCR and MCF-7/ADR did not show cross-resistance to the compound magnolanol, indicating that magnolanol can escape the multidrug resistance of drug-resistant cells . At a concentration of 10 μM and below, magnolol has no obvious inhibition on cell growth.

Conclusion: Magnolol can overcome the drug resistance of drug-resistant cell lines, and the sensitivity of drug-resistant cell lines to magnolial is basically the same.

Example 2: CCK-8 method detects the reversal effect of magnolanol on the multidrug resistance activity of tumor cells

KB/VCR, MCF-7/ADR and HCT-8/VCR cells were inoculated into 96-well plates at a density of 3500/well, and after 24 hours, different concentrations of VCR, as well as different concentrations of VCR and magnolanol were used together with 10% fetal α-MEM with bovine serum was added to each well after preparation. After cultivating for 48 hours, discard the culture medium, and use the CCK-8 kit to measure the activity of the drug-resistant cell line and its parental cells to VCR and VCR+magnolanol, and draw a curve with the method in Example 1. Three replicate wells were set up for each concentration, and the experiment was repeated three times.

Experimental results:

The multiplicity of drug resistance of KB/VCR cells to VCR was 81.9 times, and that of MCF-7/ADR cells to ADR was 20.9 times. When 10μM magnolanol was added, the sensitivity of KB/VCR cells to VCR was increased by 8.14 times, while the sensitivity of ADR to drug-resistant cells MCF-7/ADR was not significantly increased. Magnolol can reverse the resistance of tumor multidrug resistant cells to chemotherapy drugs.

in conclusion:

Magnolol can reverse the drug resistance of tumor multidrug-resistant cells KB/VCR, and can be used as a sensitizer for anti-tumor drugs, or made into a pharmaceutical composition with anti-tumor drugs.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. application of the magnolol in antineoplastic is prepared, described application is that magnolol is preparing antineoplastic sensitizer In application.

2. application as claimed in claim 1, it is characterised in that magnolol is tumor multi-drug resistance reversal agents.

3. application as claimed in claim 1, it is characterised in that described tumour includes carcinoma of mouth, nasopharyngeal carcinoma, breast cancer, liver Cancer, lung cancer, cervical carcinoma, colon cancer or cancer of pancreas.

4. application as claimed in claim 1, it is characterised in that described antineoplastic is vincristine, daunorubicin, purple China fir alcohol or 5 FU 5 fluorouracil.

5. a kind of antineoplastic pharmaceutical compositions, it is characterised in that the active ingredient of described antineoplastic pharmaceutical compositions is anti-swollen Tumor medicine and magnolol.

6. antineoplastic pharmaceutical compositions as claimed in claim 5, it is characterised in that described antineoplastic is the cell cycle Specific drug or cell cycle nonspecific agent (CCNSA).

7. antineoplastic pharmaceutical compositions as claimed in claim 5, it is characterised in that described antineoplastic is new Changchun Alkali, daunorubicin, taxol or 5 FU 5 fluorouracil.

8. it is a kind of suppress tumor cell in vitro growing multiplication method, it is characterised in that in the culture medium of tumour cell add Magnolol and antineoplastic.

9. method as claimed in claim 8, it is characterised in that described tumour includes carcinoma of mouth, breast cancer, nasopharyngeal carcinoma, liver Cancer, lung cancer, cervical carcinoma or cancer of pancreas.

10. method as claimed in claim 8, it is characterised in that first add magnolol, be subsequently adding antineoplastic.