阅读说明：本技术 肉桂醛在制备用于治疗卵巢上皮癌药物中的应用及其药物制剂 (Application of cinnamaldehyde in preparing medicine for treating ovarian epithelial cancer and medicinal preparation thereof ) 是由 蓝婷 王悦 王忠诚 穆春艳 于 2021-08-25 设计创作，主要内容包括：本发明公开了肉桂醛在制备用于治疗卵巢上皮癌药物中的应用及其药物制剂,属于生物医药领域。实验结果显示,肉桂醛能通过逆转EMT过程抑制卵巢上皮癌细胞增殖、迁移和侵袭,从而抑制卵巢上皮癌的生长。肉桂醛对正常卵巢上皮细胞毒性较小,说明对正常人体细胞伤害较小；肉桂醛并非直接通过杀死卵巢上皮癌细胞,而是通过逆转EMT过程来抑制卵巢上皮癌细胞增殖、迁移和侵袭,因此不易产生耐药性；肉桂醛对裸鼠移植的卵巢上皮癌生长有明显的抑制作用,这在新型抗卵巢上皮癌药物的研发中具有很好的应用前景。本发明拓宽了肉桂醛的应用领域,为肉桂醛在制备治疗卵巢上皮癌药物方面的临床应用提供了实验依据,具有良好的应用前景和巨大的潜在价值。(The invention discloses application of cinnamaldehyde in preparation of a medicine for treating ovarian epithelial cancer and a medicinal preparation thereof, and belongs to the field of biological medicines. The experimental result shows that the cinnamaldehyde can inhibit the proliferation, migration and invasion of the ovarian epithelial cancer cells by reversing the EMT process, thereby inhibiting the growth of the ovarian epithelial cancer. Cinnamaldehyde has less toxicity to normal ovarian epithelial cells, which indicates that the cinnamaldehyde has less damage to normal human body cells; the cinnamaldehyde does not directly kill the ovarian epithelial cancer cells, but inhibits the proliferation, migration and invasion of the ovarian epithelial cancer cells by reversing the EMT process, so that drug resistance is not easy to generate; the cinnamaldehyde has an obvious inhibiting effect on the growth of the ovarian epithelial cancer transplanted to the nude mouse, and has a good application prospect in the research and development of novel anti-ovarian epithelial cancer drugs. The invention widens the application field of the cinnamaldehyde, provides experimental basis for the clinical application of the cinnamaldehyde in the aspect of preparing the medicine for treating the ovarian epithelial cancer, and has good application prospect and great potential value.)

1. Application of cinnamaldehyde in preparing medicine for treating ovarian epithelial cancer is provided.

2. Use of cinnamaldehyde in the preparation of a medicament for the treatment of ovarian epithelial cancer according to claim 1, wherein the medicament is for inhibiting the epithelial-mesenchymal transition process of ovarian epithelial cancer cells.

3. Use of cinnamaldehyde according to claim 1 or 2 in the manufacture of a medicament for the treatment of ovarian epithelial cancer, wherein the cinnamaldehyde is present in an amount of 20 mg/kg.

4. A pharmaceutical formulation for the treatment of ovarian epithelial cancer comprising (1) an effective amount of cinnamaldehyde as an active ingredient, and (2) optionally a pharmaceutically acceptable carrier.

5. The pharmaceutical preparation for treating ovarian epithelial cancer according to claim 4, wherein the pharmaceutical dosage form is capsule, oral liquid, pill, tincture, syrup or injection.

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of cinnamaldehyde in preparation of a medicine for treating ovarian epithelial cancer and a medicinal preparation thereof.

Background

Ovarian cancer, one of the most fatal gynecological tumors, is diagnosed as ovarian cancer at about 239000 every year worldwide, and 152000 people die of this disease and its complications, epithelial ovarian cancer being the major type of ovarian cancer. Most ovarian cancer patients are diagnosed at an advanced stage due to the lack of effective early screening means. The first-line treatment method for ovarian epithelial cancer clinically at present comprises surgical operation and auxiliary chemotherapy means, the five-year survival rate of patients diagnosed with stage III or stage IV ovarian epithelial cancer is less than 25%, and most patients are sensitive to treatment drugs in the early treatment stage but eventually develop chemotherapy resistance.

At present, the drugs clinically used for treating the ovarian epithelial cancer mainly comprise molecular targeted drugs such as platinum drugs, taxol drugs, bevacizumab and poly adenosine diphosphate ribose polymerase (PARP) inhibitors, but the drugs still have the defects of more adverse reactions, drug resistance, broad-spectrum anticancer, high price and the like, and the overall survival rate of the ovarian epithelial cancer in the world is not remarkably improved at present. Therefore, it is very important to explore a new natural drug for treating ovarian epithelial cancer.

Disclosure of Invention

The invention aims to provide application of cinnamaldehyde in preparation of a medicine for treating ovarian epithelial cancer and a medicinal preparation thereof, and provide important data for further developing a medicine for treating ovarian epithelial cancer.

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

provides the application of cinnamaldehyde in preparing the medicine for treating the ovarian epithelial cancer.

The Cinnamaldehyde (Cinnanaldehyde) is a natural product existing in cortex Cinnamomi stem bark, is an aldehyde organic compound, has CAS number of 104-55-2 and molecular formula of C₉H₈O, molecular weight 132.16, yellow viscosityThe thick liquid, poorly soluble in water, with a boiling point of 253 ℃ (atmospheric pressure), has the following structure:

preferably, the medicament is for inhibiting epithelial-mesenchymal transition processes of ovarian epithelial cancer cells.

Preferably, the amount of the cinnamaldehyde is 20 mg/kg.

In another aspect, the present invention provides a pharmaceutical preparation for treating ovarian epithelial cancer, comprising (1) an effective amount of cinnamaldehyde as an active ingredient, and (2) optionally a pharmaceutically acceptable carrier.

Preferably, the pharmaceutical dosage form is capsule, oral liquid, pill, tincture, syrup or injection.

Epithelial-mesenchymal transition (EMT): refers to a biological process in which Epithelial cells are transformed into cells having a mesenchymal phenotype by a specific process, specifically, a reversible differentiation transformation process occurring in embryogenesis, wound healing, inflammation, tumor progression, and fibrosis, EMT is considered to be closely related to tumor progression, including enhancement of invasion ability into extracellular matrix and metastatic ability to distant organs, to gradually transform cancer into high-grade malignant tumor, and it was experimentally found that reversal of the EMT-suppressing process in Epithelial tumors may effectively inhibit cancer progression.

Cell and animal experiment results show that the cinnamaldehyde can inhibit proliferation, migration and invasion of ovarian epithelial cancer cells by reversing the EMT process, so that the growth of ovarian epithelial cancer is inhibited, and the cinnamaldehyde can be used as an anti-cancer drug to treat the ovarian epithelial cancer. In addition, cinnamaldehyde has less toxicity to normal ovarian epithelial cells, which indicates that the cinnamaldehyde has less damage to normal human cells; in addition, the cinnamaldehyde does not directly kill the ovarian epithelial cancer cells, but inhibits the EMT process of the ovarian epithelial cancer cells under effective concentration dose to achieve the purpose of the invention, so that the drug resistance is not easy to generate; and the cinnamaldehyde has an obvious inhibiting effect on the growth of the ovarian epithelial cancer transplanted to the nude mouse, and has a good application prospect in the research and development of novel anti-ovarian epithelial cancer drugs.

The invention takes the process of inhibiting the EMT of the epithelial ovarian cancer cells as an entry point, and screens out a natural medicine which has low toxicity, is not easy to generate drug resistance and has the effect of inhibiting the EMT process of the epithelial ovarian cancer cells and the growth of the epithelial cancer cells. The cinnamaldehyde is a natural product existing in the stem bark of cinnamon, has no report in the treatment of the ovarian epithelial cancer, and has good potential clinical value when being used as a novel natural medicine for resisting the ovarian epithelial cancer. The invention widens the application field of the cinnamaldehyde, provides experimental basis for the clinical application of the cinnamaldehyde in the aspect of preparing the medicine for treating the ovarian epithelial cancer, and has good application prospect and great potential value.

Drawings

FIG. 1 is a graph showing the results of Western blot analysis of ovarian epithelial cancer cells (SKOV3, A2780) after adding cinnamaldehyde at different concentrations, respectively; (A) SKOV 3; (B) a2780; in the figure, E-cad represents E-cadherin, N-cad represents N-cadherin, Vimentin represents Vimentin, and Snail represents a transcription factor Snail, which are used as EMT marker proteins, beta-actin is actin used as an internal reference, and CA represents cinnamaldehyde;

FIG. 2 is a graph showing the results of immunofluorescence analysis of ovarian epithelial cancer cells (SKOV3, A2780) after adding cinnamaldehyde at different concentrations, respectively; (A) SKOV 3; (B) a2780; in the figure, E-cad represents EMT marker protein E-cadherin, N-cad represents EMT marker protein N-cadherin, DAPI represents 4',6-diamidino-2-phenylindole, Merge represents an image in which E-cad/N-cad and DAPI are fused together, and CA represents cinnamaldehyde;

FIG. 3 is a statistical graph of the proliferation potency of normal ovarian epithelial (IOSE80) and ovarian epithelial cancer cells (SKOV3, A2780) tested by CCK8 after adding cinnamaldehyde at different concentrations, respectively; (A) cell proliferation histogram of IOSE 80; (B) a cell proliferation profile of SKOV 3; (C) a cell proliferation histogram of a 2780; in the figure, CA represents cinnamaldehyde, and call viatility represents the proliferative capacity of the cell;

FIG. 4 is an appearance diagram and a statistical diagram of scratch area of a scratch board detected by a scratch test after adding cinnamaldehyde into ovarian epithelial cancer cells (SKOV3, A2780) at different concentrations respectively; (A) a2780 scratch board appearance; (B) a2780, a scratch area statistical chart; (C) a scratch board appearance map of SKOV 3; (D) a scratch area statistical plot of SKOV 3; in the figure, CA represents cinnamaldehyde, and real Cell viability represents the migration ability of cells;

FIG. 5 is a graph showing the result of staining and the number statistics of invasion cells detected by the Trans-well method after different concentrations of cinnamaldehyde were added to ovarian epithelial cancer cells (SKOV3, A2780), respectively; (A) a graph of invasive cell staining results for SKOV3 and a 2780; (B) a statistical plot of the number of invading cells of SKOV 3; (C) a2780 statistical chart of the number of invading cells; in the figure, CA represents cinnamaldehyde, and number of migratedcells represents the invasive ability of cells;

FIG. 6 is a statistical chart of apoptosis number detected by flow cytometry after different concentrations of cinnamaldehyde were added to ovarian epithelial cancer cells (SKOV3, A2780), respectively; (A) flow charts for SKOV3 and a 2780; (B) a2780 cell apoptosis number histogram; (C) statistical plots of the number of apoptosis in SKOV 3; in the figure, CA represents cinnamaldehyde, 7-AAD and Annexin-APC represent fluorescence channels, quadrant Q2 represents early apoptotic cells, quadrant Q4 represents late apoptotic or necrotic cells; apoptosis represents the percentage of apoptosis;

FIG. 7 is a graph showing the difference in body weight of nude mice among groups with time after treatment with cinnamaldehyde at different concentrations in nude mice in vivo tumor formation experiment; in the figure, weight represents the body weight of nude mice, and cinnanaldehyde represents Cinnamaldehyde;

FIG. 8 is a graph showing the difference in tumor weight among groups of nude mice treated with cinnamaldehyde at different concentrations in nude mice in vivo tumor formation experiment; (A) a mouse abdominal cavity tumor object map; (B) schematic comparison of tumor weight data; in the figure, tomorrweight represents tumor weight and CA represents cinnamaldehyde;

FIG. 9 is a representative graph of the metastasis of tumors in liver tissues of nude mice among groups after treatment with cinnamaldehyde at different concentrations in nude mice in vivo tumor formation experiments; in the figure, CA represents cinnamaldehyde.

Detailed Description

The invention is described in further detail below with reference to the figures and specific examples.

Unless otherwise indicated, reagents, methods and equipment used in the present invention are conventional in the art, and materials of kits used in the following examples are commercially available.

Western-blot detection method: western blotting is an experimental method frequently used in molecular biology, biochemistry and immunogenetics, and is an analytical method capable of performing qualitative and semi-quantitative analysis on proteins, which is to stain a cell or biological tissue sample treated by gel electrophoresis with a specific antibody, and obtain information on the expression of a specific protein in the analyzed cell or tissue by analyzing the location and depth of staining.

BCA reaction working solution: BCA is an abbreviation of bicinchoninic acid protein, and BCA is a widely used protein quantification method in which the BCA reaction working solution used is a mixture of a reagent a (BCA alkaline solution) and a reagent B (copper sulfate solution), and the ratio of the reagent a to the reagent B is 50: 1.

loadingbuffer: the Western-blot assay uses loading buffer to indicate the progress of electrophoresis and to allow the sample to sink into the wells without floating.

Acr-Bis: acrylamide, methylene bisacrylamide solution.

Tris-HCI buffer: tris-hydroxymethyl aminomethane-hydrochloric acid buffer.

SDS buffer: sodium dodecyl sulfate buffer.

ddH 20: the abbreviation of doubldistilledwater, double distilled water, is water obtained by redistilling water after primary distillation.

An EP pipe: a miniature centrifugal tube is a small centrifugal tube, is matched with a miniature centrifugal machine for use, and is used for separating a micro reagent.

TEMED: tetramethyl ethylenediamine, used for preparing SDS-PAGE glue.

Marker: protein labeling, prestained or non-prestained proteins of various molecular weights, used for labeling the size and tracing of the protein in electrophoresis.

PVDF film: polyvinylidene fluoride (pvdf) is a solid support commonly used in western blotting.

TBST: contains Tris-HCl, NaCl and Tween20, and is one kind of buffering liquid commonly used in Western blotting.

DAPI: 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole), a fluorescent dye capable of strongly binding to DNA, is commonly used for common nuclear staining and double-stranded DNA staining in some specific cases.

Cinnamaldehyde in the present invention was purchased from Aladdin.

Antibody: the primary antibody comprises: p-AKT, p-PI3K, p-mTOR, AKT, PI3K, mTOR, beta-actin, available from affinity; the secondary antibody comprises: HRP-labeled secondary goat anti-rabbit antibodies, HRP-labeled secondary goat anti-mouse antibodies, purchased from affinity; goat anti-rabbit fluorescent secondary antibody and goat anti-mouse fluorescent secondary antibody, which are purchased from Thermo company.

Normal human ovarian epithelial cells (IOSE80) and human ovarian epithelial cancer cells (SKOV3, A2780) were purchased from the cell resource center of Shanghai Life sciences institute of Chinese academy of sciences.

Female nude mice were purchased from Experimental animals technology, Inc. of Weitonglihua, Beijing under license number SCXK (Jing) 2007-0001. The mice are placed under the standard conditions of humidity of 50 +/-10% and temperature of 23 +/-2 ℃ for adapting to survival for 12 hours every day and night. Mice had free access to water and food. All animal management and treatment protocols were approved by the animal ethics committee of xu nationality medical university. All experiments were performed as recommended by the ethical guidelines for managing and using animal behavior.

The first embodiment is as follows: detection of cinnamaldehyde inhibition of EMT activity

A2780 and SKOV3 in the western-blot detection method are respectively divided into 4 groups, and the experimental groups are as follows:

experimental group 1: 0ug/ml cinnamic aldehyde

Experimental group 2: 5ug/ml cinnamic aldehyde

Experimental group 3: 10ug/ml cinnamic aldehyde

Experimental group 4: 20ug/ml cinnamic aldehyde

In the immunofluorescence assay, A2780 and SKOV3 are divided into 3 groups respectively, and the experimental groups are as follows:

experimental group 1: 0ug/ml cinnamic aldehyde

Experimental group 2: 10ug/ml cinnamic aldehyde

Experimental group 3: 20ug/ml cinnamic aldehyde

2. Cell processing

After A2780 and SKOV3 cells are attached to the wall, cinnamaldehyde with corresponding concentration is added into each group for treatment for 48 hours when the density is about 80%.

3. And (2) carrying out subsequent treatment and detection on each group of cells by adopting a Western-blot detection method (a protein immunoblotting method), wherein the specific method comprises the following steps:

A. protein extraction and concentration determination

(1) Cell: placing a six-hole plate for culturing cells on ice, sucking off a culture medium, adding 100ul of cell lysate into each hole, standing on ice for 3-5min, scraping the cells by using a cell scraper, collecting the cell lysate in a 1.5ml centrifuge tube, oscillating on a vortex mixer for 20s, and standing on ice for 30 min;

(2) placing the standing EP tube in a low-temperature centrifuge, and centrifuging at 4 ℃ (15000rpm is multiplied by 18 min);

(3) gently sucking the supernatant into another standard EP tube to obtain total protein solution;

(4) and (3) detecting the protein concentration: taking 5ul of protein solution from each sample, adding 20ul of ddH20 to dilute the protein solution by 5 times, taking 10ul of the protein solution, putting the protein solution into a 96-well plate, and making 2 multiple wells for each sample;

(5) adding protein standard substances A-H into the foremost column of a 96-well plate, wherein the concentration is from high to low;

(6) preparing BCA reaction working solution (A: B is 50: 1) under the condition of keeping out of the sun, mixing uniformly, adding 100ul of working solution into each hole, shaking gently, mixing uniformly, wrapping with tinfoil, and incubating in a incubator at 37 ℃ for 30min under the condition of keeping out of the sun;

(7) detecting the Optical Density (OD) value of each hole and the concentration of the protein sample under the wavelength of 562nm by using an enzyme-labeling instrument;

(8) packaging unused protein, and storing at-80 deg.C.

B. Denaturation of proteins

And calculating the volume of the added protein liquid according to the required protein amount and the experimental sample loading times. Taking the example that 30ug of total protein per well needs to be loaded 4 times to detect different test indexes for electrophoresis, the volume (ul) of the needed protein solution is calculated to be (30ug protein/protein concentration) × 5, then ddH20 is supplemented to 15ul × 5 to 75ul, 3ul × 5 to 15ul of 6 × loadingbuffer (6 × loadingbuffer: β -mercaptoethanol ═ 9: 1) is added, the mouth is sealed, the solution is boiled in boiling water for 5min, and the solution is stored at 4 ℃.

C. SDS-PAGE gel electrophoresis

(1) Preparation of 10% polyacrylamide separation gel (2 gel portions): taking a special bottle for preparing the glue, adding the following reagents respectively, and carefully and uniformly mixing: ddH204 ml; 30% Acr-Bis3.3ml; 1.5M Tris-HCl buffer (pH8.8)2.5 ml; 10% SDS buffer 0. lml; 0.1m1l of 10 percent Ammonium Persulfate (AP) and 0.004ml of TEMED0.004ml of the mixture are mixed evenly, 1ml of gun is used for sucking separation glue and injecting the separation glue into a glass plate interlayer of an electrophoresis tank, no air bubbles are formed, the glue is added with about 2/3 small glass plate height, about 1ml of isopropanol liquid is slowly added for sealing a pressing line, and the polymerization is carried out for about 30min at room temperature;

(2) preparation of 5% polyacrylamide concentrated gum (2 gum portions): taking a special bottle for preparing the glue, adding the following reagents respectively, and carefully and uniformly mixing: ddH202.7ml; 30% Acr-Bis0.67ml; 0.5ml of 1.0M Tris-HCl buffer (pH 6.8); 0.04ml of 10% SDS buffer; 0.04ml of 10% ammonium persulfate; TEMEDO.004ml and mixing evenly;

(3) pouring off isopropanol on top of the gel, sucking residual liquid with filter paper, injecting the concentrated gel on top of the interlayer, inserting into comb, and polymerizing at room temperature for 30 min;

(4) after the gel is solidified, fixing the gel plate in an upper buffer solution chamber of an electrophoresis device, adding electrophoresis solution, pulling out a comb, sucking 18ul of sample solution by using a microsyringe, adding the sample solution into a sample adding hole, adding a Marker into the first sample hole, adding 1 × loadingbuffer into the last sample hole, and putting the sample into the electrophoresis device with poured 1 × Tris-glycine electrophoresis buffer solution;

(5) switching on a power supply, wherein the voltage of the concentrated gel is 90V, the electrophoresis is carried out for about 30min, the voltage of the separation gel is 120V, and the voltage and the time for separating the gel are adjusted according to the molecular weight of the target protein in principle until the bromophenol blue reaches the bottom of the separation gel after the electrophoresis;

(6) and (3) turning off the power supply, taking down the gel, and cutting the protein gel containing the target protein according to a Marker for further film transfer.

D. Rotary film (Whole wet method)

(1) Cutting a PVDF membrane with proper size according to the size of the cut target glue, marking a cut corner on the membrane, soaking in methanol for 1-3min, and transferring to a 1 Xmembrane transfer buffer solution;

(2) installing a film transfer device according to the sequence of negative electrode side-porous filter cotton-thick filter paper-glue-PVDF film-thick filter paper-porous filter cotton-positive electrode side, then placing the film transfer device into a Bio-RAD (Bio-Rad electrophoresis) electrophoresis apparatus, pouring 1 multiplied by film transfer liquid, carrying out ice-bath film transfer, determining the film transfer time according to the molecular weight of target protein, generally using a constant current of 350mA for transfer for 2 hours or 400mA for transfer for 1.5 hours;

(3) after the film is rotated, cutting one corner of the PVDF film to mark the front side and the back side of the film, enabling a Marker to be on the left side and to be in an increasing sequence from bottom to top, cutting the corner to be on the upper left, and rinsing for 3 times and for 10min by using a 1 × TBST solution;

(4) a, B liquids in ECL were mixed in equal volumes, dropped onto the membrane (protected from light) and photographed using a chemiluminescent gel imaging system.

E. Immune response

(1) Putting the rinsed transfer printing film into 5% skimmed milk powder sealing liquid, sealing for 2h at room temperature, and slowly shaking on a shaking table;

(2) diluting primary antibody with 5% skimmed milk powder sealing solution to appropriate concentration, covering appropriate amount of antibody on the membrane, and incubating overnight at 4 deg.C;

(3) washing with 1 × TBST for 3 times × 15min, and shaking on shaking table;

(4) diluting the secondary antibody to a proper concentration by using 5% skimmed milk powder sealing solution, covering a proper amount of antibody on a membrane, and incubating for 1-2h at room temperature in a wet box;

(5) washing with 1 × TBST for 3 times × 15min, and shaking on shaking table;

(6) the two reagents A and B in ECL were mixed in equal volume, added to the membrane uniformly (protected from light), and photographed with a chemiluminescence gel imaging system.

4. Immunofluorescence

(1) Plate paving and dosing: the cells in good condition were selected, digested and counted, and the cell density was adjusted so that the total number of cells per well of the 24-well plate was 5X 104. After the cells adhere to the wall, starving overnight, treating DMSO according to different groups, respectively adding 10ug/ml and 20ug/ml cinnamaldehyde into each hole, and acting in an incubator for 1 h;

(2) fixing and penetrating: taking out 24-well plate, washing with PBS 3 times, fixing with 4% paraformaldehyde for 15min, washing with PBS 3 times, and penetrating with 0.3% Triton-x100 for 10 min;

(3) and (3) sealing and incubating: washing with PBS for 3 times, blocking with 3% BSA at room temperature for 1-2h, and incubating with pre-diluted primary antibody at 4 ℃ overnight; washing with PBS for 3 times, adding a pre-diluted fluorescent secondary antibody in a dark place, and standing at 37 ℃ for 1-2 h;

(4) nuclear counterstaining: washing with PBS for 3 times, dyeing with DAPI dye solution for 8-10 min, washing with PBS for 3 times, and dripping an anti-fluorescence quenching agent.

5. Results of the experiment

The experiment adopts western-blot and immunofluorescence to detect EMT marker protein: expression of E-cad, N-cad, Vimentin, Snail proteins after cinnamaldehyde treatment (0ug/ml, 10ug/ml, 5ug/ml, 20ug/ml) in ovarian epithelial carcinoma A2780 and SKOV 3. The results are shown in fig. 1-2, after the treatment of cinnamaldehyde, the expression level of E-cad in the cells is gradually increased along with the gradual increase of cinnamaldehyde concentration, and the expression levels of N-cad, Vimentin and Snail are gradually decreased, which indicates that cinnamaldehyde with proper concentration can reverse the EMT process of ovarian epithelial cancer cell lines. Therefore, cinnamaldehyde can reverse the EMT process of ovarian epithelial cell carcinoma A2780 and SKOV3, and can be used as an EMT inhibitor.

Example two: cinnamaldehyde can inhibit proliferation and invasion of ovarian epithelial cancer cells

A2780, SKOV3 and IOSE80 were divided into 6 groups, respectively, and the experimental groups were as follows:

experimental group 1: 0ug/ml cinnamic aldehyde

Experimental group 2: 0.5ug/ml cinnamic aldehyde

Experimental group 3: 1ug/ml cinnamic aldehyde

Experimental group 4: 5ug/ml cinnamic aldehyde

Experimental group 5: 10ug/ml cinnamic aldehyde

Experimental group 6: 20ug/ml cinnamic aldehyde

CCK8 experiment, the procedure was as follows:

(1) plate paving and dosing: selecting cells in logarithmic growth phase, digesting, counting, and adjusting cell concentration to 4 × 10⁴Perml, 100. mu.L/well in 96-well plates, and appropriate amount of PBS around the 96-well plates to prevent evaporation of water. After the cells are attached to the wall, starving the cells by using a serum-free culture medium overnight, respectively adding a corresponding amount of cinnamaldehyde into each hole according to different groups, and putting the holes into a culture box for culture;

(2) termination and measurement: culturing for 24h, 48h, and 72h under dark condition, adding CCK-8 reagent 10 μ L per well, mixing, standing at 37 deg.C and 5% CO₂Reading at 450nm of an enzyme-labeling instrument in the incubator for 1-2 h.

3. Scratch test, the procedure was as follows:

(1) plate paving: one day before the experiment, a straight ruler, a marking pen and a 10 mu L high-pressure gun head are subjected to ultraviolet irradiation in a biological safety cabinet for 30min, and the marking pen is used for marking a straight line crossing the through hole on the back of the 6-hole plate; digesting the cells and inoculating the cells into a 6-well plate, wherein the inoculation density is about 90% of the fusion degree of the adherent cells on the next day;

(2) scratching and dosing: during the experiment, a sterile 10-mu-L gun head is used to mark than a ruler, the same angle and force are used for marking, and each hole is marked with 3 straight lines. Washing with PBS for 2 times, adding 1ml DMEM medium containing 1% FBS, adding cinnamaldehyde into each well according to different groups, and placing 6-well plate into an incubator for continuous culture;

(3) photographing and analyzing: photographs were taken at the same positions at 0h, 24h, and 48h, respectively, and the wound distances were measured at each time point, and the experimental results were analyzed by Image J software.

Trans-well invasion assay

(1) Invasion assay coating basement membrane: the night before the experiment, Matrigel was removed from-20 ℃ and thawed overnight at 4 ℃. Diluting Matrigel matrix glue and a pre-cooled serum-free culture medium according to a ratio of 1: 5, adding 100 mu L of diluted glue into an upper chamber of a Trans-well chamber to avoid bubbles, putting the chamber into a 24-hole plate, and putting the chamber into an incubator for 4-6 hours;

(2) plate paving and dosing: digesting the cells and counting, adjusting the cell density to 2X 10 with serum-free culture⁵Per ml, aspirate 200. mu.L of cell suspension into the chamberAdding 600 mu L of complete culture medium into the lower layer of the small chamber, placing the small chamber into a 24-pore plate to avoid generating bubbles, respectively adding corresponding amount of cinnamaldehyde into each small chamber according to different groups, placing into an incubator, and culturing for 48 h;

(3) fixing and dyeing: taking out the small chamber, discarding the culture medium, fixing with 4% paraformaldehyde for 15min, washing with PBS for 3 times, dyeing with crystal violet stain for 10min, washing with PBS for 3 times, and slightly wiping off the cells which do not pass through the upper chamber with cotton swab;

(4) photographing and analyzing: the chamber was removed, photographed under a 200-fold inverted microscope, and five field cells were counted at random.

5. Results of the experiment

In the experiment, CCK8, a scratch experiment and a transwell invasion experiment are adopted to respectively detect the changes of the proliferation, migration and invasion capacities of cells after the ovarian epithelial cancer cell strains A2780, SKOV3 and the ovarian normal epithelial cell strain IOSE80 are treated by cinnamaldehyde (0ug/ml, 5ug/ml, 10ug/ml and 20ug/ml), and the results are shown in figures 3-5. As can be seen from fig. 3, at an appropriate cinnamaldehyde concentration, the proliferation potency of the cells was significantly decreased as the concentration increased, and the ovarian epithelial cancer cell line was more sensitive to cinnamaldehyde than the IOSE80 cell line, suggesting that cinnamaldehyde can inhibit the proliferation potency of ovarian epithelial cancer and is less harmful to normal cells at an appropriate concentration. As can be seen from fig. 4-5, at an appropriate cinnamaldehyde concentration, the migration ability and invasion ability of the ovarian epithelial cancer cells are significantly reduced with increasing concentration, suggesting that cinnamaldehyde at an appropriate concentration can inhibit the migration ability and invasion ability of the ovarian epithelial cancer cells.

According to the above analysis, cinnamaldehyde can inhibit the proliferation, migration and invasion capabilities of ovarian epithelial cancer cell lines (A2780 and SKOV3), and can be used as an anti-ovarian epithelial cancer drug.

Example three: cinnamic aldehyde can promote apoptosis of ovarian epithelial cancer cell

1, A2780 and SKOV3 were divided into 3 groups, respectively, and the experimental groups were as follows:

experimental group 1: 0ug/ml cinnamic aldehyde

Experimental group 2: 5ug/ml cinnamic aldehyde

Experimental group 3: 20ug/ml cinnamic aldehyde

2. Flow cytometry detection, the steps are as follows:

(1) inoculating cells to a six-hole plate, wherein the density is about 80%, adding corresponding doses of cinnamyl aldehyde according to different groups after the cells are attached to the wall, and treating for 48 hours;

(2) digesting cells with EDTA-free pancreatin (noting that the pancreatin digestion time cannot be too long), adding complete culture medium to stop reaction, transferring the cells into a centrifuge tube, centrifuging at 2000rpm for 5min, removing supernatant, adding a proper amount of complete culture medium to adjust the cell concentration to 1x 10%/ml;

(3) precooled PBS washes cells for 2 times, abandons the supernatant (abandons it as much as possible), adds 1 Xbuffer solution 100. mu.l to resuspend cells;

(4) and (5) transferring to a flow pipe, and setting a blank group, a single standard group, an NC group and an experimental group for carrying out the operation. Blank group: the ovarian epithelial cancer cells are not added with any reagent; single standard group (APC): NC group added 5 μ 1 APC; single label group (7-AAD): adding 5 μ l Annexin 7-AAD into NC group; respectively adding 5ul Annexin 7-AAD and 5ul APC into the NC group and the experimental group;

(5) incubating for 15min at room temperature in dark condition;

(6) add 400. mu.l of 1 Xbuffer to each tube and mix well, and detect with flow cytometer in 1 h.

3. Results of the experiment

The experiment detects apoptosis levels of ovarian epithelial cancer cells A2780 and SKOV3 after cinnamaldehyde treatment (0ug/ml, 5ug/ml and 20ug/ml) through flow cytometry, and fig. 6 shows that with increase of cinnamaldehyde concentration, the number of cells positioned in Q2 quadrant (7-AAD + APC +) and Q4 quadrant (7-AAD-APC +) gradually increases, which prompts that cinnamaldehyde can improve apoptosis levels of ovarian epithelial cancer cells and prompts that cinnamaldehyde can improve apoptosis levels of ovarian epithelial cancer cells, thereby indicating that cinnamaldehyde can promote apoptosis of ovarian epithelial cancer cells. Therefore, cinnamaldehyde can promote apoptosis of ovarian epithelial cancer cell lines A2780 and SKOV3, and can be used as an anticancer drug.

Example four: inhibitory effect of cinnamaldehyde on ovarian epithelial cancer in vivo

1. Nude mice were divided into three groups of 5 mice each, and the experiments were grouped as follows:

experimental group 1: PBS control

Experimental group 2: 50mg/kg

Experimental group 3: 100mg/kg

2. In vivo tumor formation experiment in nude mice:

A. abdominal inoculation of tumor cells in nude mice

(1) When the density of the A2780 cells reaches about 80-90%, replacing the fresh culture medium at night before collecting the cells;

(2) pancreatin the cells after digestion with pre-cooled PBS two times, in order to remove the cell serum;

(3) blowing and beating the cell sediment by PBS or serum-free medium to a proper concentration, wherein the cell quantity inoculated in the abdominal cavity is (1-5) multiplied by 10⁶The inoculation volume was 0.1ml per cell, so that the concentration of the cell suspension was (1-5). times.10⁷Individual cells/ml.

(4) After the cells are digested, the cells are inoculated to the abdominal cavity of a nude mouse as soon as possible and generally completed within half an hour, and the cell suspension is placed on ice in the process to reduce the metabolism of the cells;

(5) selecting nude mice with weight of about 18-20g at age of 5-8 weeks;

(6) before inoculation, the cell suspension is blown off fully by a gun, so that cell agglomeration is prevented, and the cell survival rate is reduced;

(7) during inoculation, a nude mouse lies on a flat part, holds the back of the nude mouse and inverts the nude mouse, then disinfects the skin, firstly inserts a needle for about 0.5cm under the skin and then inserts the needle for 45 degrees, injects 200ul of cells, and compresses 5-10S by a cotton ball;

B. injection of drugs after transplantation of tumor in nude mice

(8) One week after inoculation, nude mice were injected intraperitoneally with PBS or cinnamaldehyde at the corresponding concentration every two days for about 20 days, and body weights of the nude mice were recorded every day;

(9) approximately three weeks later, nude mice were sacrificed, tumor volume was measured and recorded, the presence or absence of peritoneal metastasis was observed and a portion of the tumor tissue was taken and soaked in 4% paraformaldehyde to prepare for subsequent experiments.

3. Results of the experiment

The experiment adopts a nude mouse in-vivo tumor formation experiment to detect the inhibition effect of cinnamaldehyde on the growth of an ovarian epithelial cancer cell strain A2780 in vivo. As shown in fig. 7, after the nude mice successfully tumorigenic, the weight of the mice slowly increased with time after the treatment of cinnamaldehyde, and no obvious difference exists between experimental groups, which suggests that cinnamaldehyde has no harm to the mice in vivo. As shown in fig. 8, the tumor volume gradually decreased with the increase of cinnamaldehyde concentration, suggesting that cinnamaldehyde has a significant inhibitory effect on the growth of ovarian epithelial cancer in vivo. As shown in FIG. 9, in the control group, three mice exhibited liver metastasis, in the 50mg/kg CA-treated group, only one mouse exhibited liver metastasis, and in the 100mg/kg CA-treated group, none of the mice exhibited liver cancer metastasis, suggesting that cinnamaldehyde can inhibit metastasis of ovarian epithelial cancer cells in vivo. Therefore, cinnamaldehyde inhibits the proliferation and metastasis of ovarian epithelial cancer cells in nude mice, and can be used as an anti-ovarian epithelial cancer drug.

The cell and animal experiment results show that the cinnamaldehyde can inhibit the proliferation, migration and invasion of the ovarian epithelial cancer cells by reversing the EMT process, so that the growth of the ovarian epithelial cancer is inhibited, and the cinnamaldehyde can be used as an anti-cancer drug to treat the ovarian epithelial cancer.